lørdag den 17. august 2013

Immunological findings - overview

Immunological findings

Examined CFS patients:
11% autoimmune thyroiditis
31% immunoglobulin deficiency, including:
·         5%   IgA deficiency
·         6%   IgG main group deficiency
·         10% selective IgG3 deficiency
·         10% selective IgG4 deficiency
·         8%   elevated IgM
32% T-cell-activation at an increased expression of activation markers CD11a, CD28 og CD57 på CD8 + - T-lymphocytes.
5% had an elevation in CD11a and a decrease in CD28 at CD8 + T-lymphocytes.
33% had elevated CD57 + T-cell-activity
CFS patients could be divided into 4 groups:
·         high IL-2
·         high IL-5
·         decreased cytokine respons and predominantly T-cell activation and lymphopenia
·         26% no annormal cytokine production
22% had EBNA IgG deficiency
15% had leukopenia
25% had lymphopenia
6%   had low hemoglobin
25% had elevated MCHC niveau

Severely affected CFS patients characterized by significant decreases in NK lysis and increases in KIR3DL1, IL4, TNF-α, and IFN-γ

·         Increased levels of T regulatory cells (CD25+/FOXP3+) CD4 T cells.
·         CD8 T cells showed significantly lower activation and frequency of effector memory cells.
·         NK cells displayed higher expression of NKp46 and CD69 but lower expression of CD25 in all NK subsets defined.
·         Overall, T cell and NK cell features clearly clustered CFS individuals.

·         cytotoxic activity of Natural Killer Cells
·         cytotoxic activity of af CD8+T celler 
·         number of CD56bright NK cells
Upregulation of
  • IL-10
  • IFN- γ
  • TNF-α
  • CD4CD25 T-cells
  • FoxP3 expression
  • VPACR2 expression

baseline  = T1,     6 months = T2,      12 months = T3
·         NK cytotoxic activity was significantly decreased at T1, T2 and T3.
·         Significantly lower numbers of CD56brightCD16- NK cells at both T1 and T2.
·         Cytokine secretion revealed significant increases in IL-10, IFN-γ and TNF-α at T1.
·         Overall cytotoxic activity significantly decreased at T3 compared to T1 and T2.
·         CD56brightCD16- NK cells were much lower at T2 compared to T1 and T3.

CFS patients had:
·         greater numbers of naive B cells as a percentage of lymphocytes: 6·3 versus 3·9% in HC (P=0·034)
·         greater numbers of naive B cells as a percentage of B cells: 65 versus 47% in controls (P=0·003)
·         greater numbers of transitional B cells: 1·8 versus 0·8% in controls (P=0·025)
·         reduced numbers of plasmablasts: 0·5 versus 0·9% in controls (P=0·013)

·         Serum IL-1, TNFα, neopterin and elastase are significantly higher in patients with ME/CFS than in Chronic Fatigue  patients.
·         There are significant and positive associations between the IgA responses to LPS and serum IL-1, TNFα, neopterin and elastase.
·         Patients with an abnormally high IgA response show increased serum IL-1, TNFα and neopterin levels, and higher ratings on irritable bowel syndrome (IBS) than subjects with a normal IgA response.
·         Serum IL-1, TNFα and neopterin are significantly related to fatigue, a flu-like malaise, autonomic symptoms, neurocognitive disorders, sadness and irritability.

There was a significant reduction in the expression levels of miR-21, in both the NK and CD8(+)T cells in the CFS/ME sufferers. Additionally, the expression of miR-17-5p, miR-10a, miR-103, miR-152, miR-146a, miR-106, miR-223 and miR-191 was significantly decreased in NK cells of CFS/ME patients in comparison to the non-fatigued controls

Future research should examine p53 signaling in ME/CFS:
·         Inactivation of p53 impairs aerobic mitochondrial functions and causes greater dependence on anaerobic glycolysis, elevates lactate levels, reduces mitochondrial density in skeletal muscle and reduces endurance during physical exercise.
·         Lowered p53 and increased NF-κB are associated with elevated reactive oxygen species.
·         Increased NF-κB induces the production of pro-inflammatory cytokines, which increase glycolysis and further compromise mitochondrial functions.
·         All these factors together may contribute to mitochondrial exhaustion and indicate that the demand for extra ATP upon the commencement of increased activity cannot be met.
·         In conditions of chronic inflammation and oxidative stress, high NF-κB and low p53 may conspire to promote neuron and glial cell survival at a price of severely compromised metabolic brain function.
Chronic Fatigue and/or Fibromyalgia have long been diseases without definition. An explanatory model of coagulation activation has been demonstrated through use of the ISAC panel of five tests, including, Fibrinogen, Prothrombin Fragment 1+2, Thrombin/ AntiThrombin Complexes, Soluble Fibrin Monomer, and Platelet Activation by flow cytometry. These tests show low level coagulation activation from immunoglobulins (Igs) as demonstrated by Anti-B2GPI antibodies, which allows classification of these diseases as a type of antiphospholipid antibody syndrome. The ISAC panel allows testing for diagnosis as well as monitoring for anticoagulation protocols in these patients.

Complement activation resulting in significant increases of C4a split product may be a marker of postexertional malaise in individuals with chronic fatigue syndrome (CFS).

Exercise challenge induced significant increases of the complement split product C4a, but not C3a or C5a, at 6 hours after exercise only in the CFS group (P <.01), regardless of allergy status.

These preliminary findings suggest an abnormal or defective adaptive response to oxidative stress in CFS, and raise the possibility that HSP profiling may provide a more objective biologic marker for this illness

The response of CFS patients to incremental exercise associates a lengthened and accentuated oxidative stress, which might result from delayed and insufficient Hsp production.

Antibodies in ME/CFS
ME/CFS Antibodies

·         The incidence of positive autoimmune activity against 5-HT was significantly higher (p<0.001) in ME/CFS (61.5%) than in patients with Chronic Fatigue (13.9%) and controls (5.7%).
·         ME/CFS patients with 5-HT autoimmune activity displayed higher TNFα, IL-1 and neopterin and increased IgA responses against LPS of commensal bacteria than those without 5-HT autoimmune activity.
·         Anti-5-HT antibody positivity was significantly associated with increased scores on hyperalgesia, fatigue, neurocognitive and autonomic symptoms, sadness and a flu-like malaise.

Human muscarinic cholinergic receptor 1 (CHRM1), mu-opioid receptor (OPRM1),
5-hydroxytryptamine receptor 1A (HTR1A)
dopamine receptor D2 (DRD2)

·         The mean anti-CHRM1 antibody index was significantly higher in patients with CFS (p<0.0001) and autoimmune disease (p<0.05) than that in healthy controls, and positive reaction was found in 53.3% of patients with CFS.
·         Anti-OPRM1 antibodies, anti-HTR1A antibodies, and anti-DRD2 antibodies were found in 15.2, 1.7, and 5.0% of patients with CFS, respectively.
·         Anti-nuclear antibodies were found in 56.7% (34/60) of patients with CFS, but anti-nuclear antibody titers did not correlate with the activities of the above four autoantibodies

The present results demonstrate that serum autoantibody against the muscarinic cholinergic receptor (mAChR) can affect the brain mAChR without altering acetylcholinesterase activity and cognitive functions in CFS patients.

Anticardiolipin antibodies in the sera of patients with diagnosed chronic fatigue syndrome

Anticardiolipin antibodies (ACAs):
·         Immunoglobulin M isotypes in 95% of CFS serum samples tested.
·         The presence of immunoglobulin G and immunoglobulin A isotypes were also detected in a subset of the samples.
·         Future studies will focus on elucidating whether alterations to mitochondrial inner membranes and/or metabolic functions play a possible role in the expression of ACAs.

Antibodies testet in CFS.
Antibodies in AIDs
·         Anti-dsDNA No
·         Anti-Sm No
·         Anti-RNP No
·         Anti-SS-A/Ro No
·         Anti-SS-B/LA No
·         Anti-Scl-70 No
·         ANA Yes
Nuclear components
·         Laminin B1 Yes
·         68/48-kDa nuclear proteina Yes
·         p80-coilin nuclear proteinb Yes
Phospholipid antibodies
·         Antiphosphatidylinositolc Yes
Antibodies to CNS components
·         Serotonin Yes
·         ACTH Yes
·         CHMR1 Yes
·         MAP2 Yes
Antibodies to diverse microorganisms
·         Gram-negative  enterobacteriaYes
Coxiella burnetii (Q fever) Yes 

Serum IgM antibodies to the three anchorage molecules (palmitic and myristic acid and S-farnesyl-L-cysteine) and NO-phenylalanine were significantly higher in ME/CFS than in depression. The autoimmune responses to oxidatively, but not nitrosatively, modified self-epitopes were significantly higher in ME/CFS than in depression and were associated with fatigue and physio-somatic symptoms.

Antibodies to EBV EA(D) and neutralizing antibodies against the encoded-proteins EBV DNA polymerase and deoxyuridine triphosphate nucleotidohydrolase (dUTPase) were present in the EBV subset CFS patients. Of the sera samples obtained from patients with CFS 93.9% were positive for EA(D), while 31.6% of the control patients were positive for EBV EA(D). Serum samples were positive for neutralizing antibodies against the EBV-encoded dUTPase (23/52; 44.2%) and DNA polymerase (41/52; 78.8%) in EBV subset CFS patients, but negative in sera of controls.

Postural Orthostatic Tacycardia Syndrome, Orthostatic Intolerance, dysautonomia autoantibodies
POTS, OI, dysautonomi autoantibodies

Forty unique proteins were identified and these include proteins that are associated with cardiac hypertrophy (mimecan, myozenin), cardiac remodeling (periostin), cardiomyopathy (desmin, desmoplakin), cell survival (laminin), structural integrity (filamin), chaperone proteins (crystalline, HSP70), mitochondrial enzymes, and channel proteins.

The targets of autoimmunoreactive IgGs include proteins associated with caveolae structure, adrenergic signaling, calcium signaling, cytostructures, chaperone and energy metabolism. Multiple pathways were involved including those that regulate caveolae-mediated signaling, oxidative phosphorylation, fatty acid metabolism, protein ubiquitination, and cardiac β-adrenergic signaling.
·         Desmin
·         HSP70
·         Cavin-1

Activating autoantibodies to β1/2-adrenergic (AAβ1/2AR) and M2/3 muscarinic receptors (AAM2/3R) are present in some patients with idiopathic OH compatible with an in vivo effect.

We have identified the presence of autoantibodies to β2-adrenergic and/or M3 muscarinic receptors by ELISA in 75% (15 of 20) of patients with significant orthostatic hypotension. Antibody activation of vascular β2 and/or M3 receptors may contribute to systemic vasodilation.

Among the epitopes targeted are first and second extracellular loops of the β-adrenergic (β-adrenoceptor) and M2 muscarinic receptor. β(1)-adrenoceptor autoantibodies affect radioligand binding and cardiomyocyte function similar to agonists.
In conclusion, such autoantibodies (Antibodies against cholinergic and adrenergic receptors) seem to cause or promote chronic human left ventricular dysfunction by acting on their receptor targets in a drug-like fashion.

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