Some striking abnormalities are found in the 2-5A synthetase/RNase L pathway, which is normally involved in antiviral defense. In the early 90’s researchers at Temple University, USA, observed an abnormal low molecular weight form of RNase L in the blood cells of CFS patients. Scientists at R.E.D Laboratories later demonstrated that this low molecular weight form was produced upon cleavage by apoptotic and inflammatory proteases such as elastase. The low molecular weight form of RNase L shows a dysregulated enzymatic activity, and may thereby interfere with various cellular functions. Other frequently observed immune dysregulations include low NK cells activity, low CD57+ cells counts, altered cytokine profiles (Th1/Th2 imbalance and production of inflammatory cytokines), abnormal levels of soluble CD14 and C4a.
Useful tests include the following:
- RNase L cleavage assay
- Perforin expression assay (Natural Killer cell activation)
- Elastase expression assay
- CD57+/CD3- absolute cell count
- Soluble CD14 serum level
- C4a serum level
For a complete list of tests we can perform, please download our request form or contact us.
RNASE L CLEAVAGE ASSAY
Ribonuclease L (RNase L) is one of the key enzymes induced by interferon and is responsible for many of the anti-viral effects observed. The native RNase L has a molecular weight of 80 kDa and is activated by binding a small effector molecule known as 2-5A. Once activated, RNase L destroys viral RNA (stopping the infectious process) and at the same time triggers the removal of infected cells by inducing programmed cell death (apoptosis).
In the immune cells of CFS patients, RNase L is cleaved by the action of proteases. This generates low molecular weight (LMW) species of RNase L which are acting in an uncontrolled manner and interfere with the proper function of immune cells.
The increased presence of lower molecular weight forms can be detected by using molecular biology tequniques. The reported value corresponds to the ratio of LMW form to the native 80 kDa form.
PERFORIN EXPRESSION ASSAY
Natural Killer cells (NK) cells are cytotoxic cells that mediate the immune response against certain cancer and virus-infected cells. NK cells are normally found in the peripheral blood, and are classified by their cell surface markers as CD3-/CD56+cells. NK cells activity is altered in several disorders such as multiple sclerosis, lupus, and CFS, and is very sensitive to various environmental pollutants. Since NK cells play a central role in the defense against viruses, decreased NK activity can lead to the development of opportunistic viral infections. NK cells exert their cytotoxic effect by releasing perforin, a protein that will destroy the cytoplasmic membrane of target cells and finally kill them. Expression of perforin mRNA can be measured as a mean to evaluate NK cell activation.
ELASTASE EXPRESSION ASSAY
Elastase is an inflammatory protease expressed in immune cells (monocytes, neutrophils). Elastase contributes to immune defense by inactivating foreign bacteria but at the same time it causes damage to connective tissue, breaks down cytokines, immunoglobulins and immune cells receptors. An excess, chronic production of elastase is therefore detrimental. In CFS patients, elastase is responsible for the cleavage of RNase L. Expression of elastase mRNA can be quantified as a specific marker of inflammation.
CD57+/CD3- ABSOLUTE CELL COUNT
CD57+/CD3- cells are a subset of NK cells. Their exact function, and what differentiates them from CD56+ NK cells, is not well understood. The absolute number of CD57+/CD3- cells is low in patients suffering from chronic Lyme disease (a disease that follows an infection by a bacteria called Borrelia). Patients with very low CD57 have significantly more co-infections and persistent immunologic defects than patients with higher counts. In patients that respond to antibiotic therapy, the number of cells come back to normal, hence this is a useful marker to follow the effect of a therapy.
SOLUBLE CD14
CD14 is expressed in monocytes/macrophages and plays a critical role in the recognition of bacterial cell wall components (LPS). The extracellular part of CD14 can be cleaved and released in the plasma, where it will inactivate circulating LPS. Serum soluble CD14 levels are significantly elevated in patients with inflammatory bowel disease, Crohn's disease, but also in patients suffering from Brucellosis or Lyme disease.
C4a SERUM LEVEL
C4a is an anaphylatoxin generated by cleavage of complement component 4 (C4), upon activation of the complement system. C4a increase causes local inflammatory response and symptoms of hypersensitivity. C4a levels are elevated following exercise in CFS patients. A US study has reported that elevated complement C4a was an early marker for Lyme disease in tick bite patients.