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.
For assessment of immune function we offer the following assays:
- RNase L cleavage assay
- Serum cytokine profile
- Th1/Th2 cytokine expression assay
- Pro-inflammatory cytokine expression assay
- Perforin expression assay (Natural Killer cell activity)
- Elastase expression assay
- CD57+/CD3- absolute cell count
- Soluble CD14 serum level
- C4a serum level
RNASE L CLEAVAGE ASSAY (Code: RNAE)
Background
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.
Method
Radiolabeled ligand-receptor assay. Radiolabeled 2-5A is bound to the enzyme in cellular extracts, and the amounts of different 2-5A binding proteins (native or LMW forms of RNase L) are quantified following gel electrophoresis.
Result
Quantitative. The increased presence of lower molecular weight forms indicates a progressively disrupted and dysfunctional immune system. The reported value corresponds to the ratio of LMW form to the native 80 kDa form; this ratio is useful to classify the disease and monitor its progression. Ratios over 0.5 are considered positive.
Sample requirement
3x8ml whole blood in CPT-heparin tubes. Centrifuge within 2 hours. Gently invert 5-10 times. Sample is then stable for 24 hours at room temperature.
SERUM CYTOKINE PROFILE (Code: CYTS)
More information available soon
Th1/Th2 CYTOKINE EXPRESSION ASSAY (Code: CYTH)
More information available soon
PRO-INFLAMMATORY CYTOKINE EXPRESSION ASSAY (Code: CYTI)
More information available soon
PERFORIN EXPRESSION ASSAY (Code: PERF)
Background
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.
Method
Gene expression assay. Expression of perforin mRNA in peripheral blood cells is quantified by a PCR procedure.
Result
Quantitative. The reported value represents the level of perforin mRNA, normalized to the level of a "housekeeping" mRNA (produced by a gene wich is consistently expressed in all cells).
Sample requirement
2-3 ml blood in a Paxgene tube. The sample is stable for up to three days at ambient temperature; for longer periods freeze and ship at -20°C.
ELASTASE EXPRESSION ASSAY (Code: ELAS)
Background
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.
Method
Gene expression assay. Expression of elastase mRNA in peripheral blood cells is quantified by a PCR procedure.
Result
Quantitative. The reported value represents the level of elastase mRNA, normalized to the level of a "housekeeping" mRNA (produced by a gene wich is consistently expressed in all cells).
Sample requirement
2-3 ml blood in a Paxgene tube. The sample is stable for up to three days at ambient temperature; for longer periods freeze and ship at -20°C.
CD57+/CD3- ABSOLUTE CELL COUNT (Code: CD57)
Background
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.
Method
Three color flow cytometry. Whole blood cells are stained with anti-CD57, anti-CD3, anti-CD45 fluorescence-labeled antibodies. Absolute number of CD45+/CD57+/CD3- cells is determined by flow cytometry.
Result
Quantitative. The result gives the absolute number of CD57 positive, CD3 negative cells per µl of whole blood. The normal range is 60-360 cells/µl.
Sample requirement
2 ml whole blood in anticoagulant tube (EDTA, heparin or citrate), received within 24 hours. Tube can be kept at ambient temperature or 4°C; sample cannot be frozen.
SOLUBLE CD14 (Code: SCD14)
Background
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.
Method
Quantification of serum sCD14 by flow cytometry (Cytometric Bead Array assay).
Result
Quantitative. Result is expressed in ng/ml serum; normal range 2800-5000 ng/ml.
Sample requirement
2 ml serum. Stable for 24 hours. For longer periods aliquot serum, store and ship at -20°C/-80°C.
C4a SERUM LEVEL (Code: C4AS)
Background
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.
Method
ELISA.
Result
Quantitative. Result is expressed in ng/ml serum; normal range 20-1400 ng/ml.
Sample requirement
2 ml serum. Stable for 24 hours. For longer periods aliquot serum, store and ship at -20°C/-80°C.