Development Service of Apoptotic Markers

Apoptosis is a physiological process that occurs in cells during development and normal cellular processes. Furthermore, the degree of apoptosis is related to the severity of steatohepatitis and the stage of fibrosis. Liver damage and apoptosis that occur in adults are often caused by dysregulation and accompanied by accumulation of immune cells, which produce cytokines and growth factors that drive chronic inflammation and may lead to fibrosis. Creative Biolabs aims to create and validate non-invasive predictive models for NASH diagnosis based on specific cyclic markers of apoptosis.

Molecular Mechanisms of Apoptosis

The increase of cell death in the liver has emerged as an important mechanism leading to disease progression to NASH. Apoptosis is a normal physiological process, which is also a highly organized and genetically controlled form of cell death. During apoptosis, cells are fragmented into small membrane-bound apoptotic bodies containing cleaved DNA and proteolytic fragments. Apoptotic bodies are subsequently cleared by phagocytosis. Apoptosis can occur via 2 fundamental pathways: extrinsic and intrinsic molecular pathways. The extrinsic pathway involves activation of a death ligand-induced receptor (such as Fas), resulting in the formation of a death complex. The intrinsic pathway is activated by cellular stress and mitochondrial dysfunction. Both apoptotic pathways activate intracellular proteases, mainly the caspases 3, which cleave cellular components in an organized manner. Apoptotic bodies produced by Kupffer cell phagocytosis promote liver fibrosis and inflammation through activation of stellate cells and release of cytokines.

Fig.1 Apoptotic pathways in nonalcoholic fatty liver disease. (Alkhouri, 2011)

Biomarkers of Apoptosis for NASH Diagnosis

The extrinsic death receptor-mediated apoptotic pathway is involved in the development of NASH, and increased expression of death receptors such as Fas and TNF-R is found in most NASH patients. Caspase-3, 7 are activated during disease progression, followed by cleavage of cytokeratin-18 (CK-18), which is a major intermediate filament protein in hepatocytes, leading to characteristic morphological changes noted in apoptotic cells. Therefore, it is hypothesized that blood measurements of CK-18 provide a viable method for monitoring apoptotic cell death in epithelial cells. Serum CK-18 levels are significantly increased in NASH patients compared with patients with steatosis or normal biopsy.

The full length CK-18 can be cleaved by caspase-6, 3 and 7, yielding fragments of approximately 30 kDa and 45 kDa, respectively. The 30 kDa fragment can be measured in plasma using the M30 monoclonal antibody enzyme-linked immunosorbent assay (ELISA) and it is found to be significantly higher in NASH patients than in patients with simple steatosis (SS), while the full length and fragments are detected by different antibodies (M65). This test identifies the risk of NASH presence and associated fibrosis and allows disease progression to be monitored over time. The CK-18 test has multiple functions to meet NASH's ideal biomarker requirements, including simple testing, ease of measurement, and repeatability. In addition, serum soluble Fas and soluble FasL levels in NASH patients confirmed by biopsy are significantly higher than those in SS patients. Therefore, there is a predictive model for the diagnosis of NASH, including CK-18, soluble Fas and age. Particularly, this trial also shows some prospects for detecting steatosis, steatohepatitis and liver fibrosis in a population of patients with chronic liver disease.

Fig.2 Signaling pathways for death ligands/receptors and CK18. (Eguchi, 2014)

Based on our comprehensive platforms (e.g. Phage Display & Antibody Library, Antibody Analysis, Antibody Engineering), Creative Biolabs offers one-stop services in the development of biomarkers for NASH diagnosis. If you have any special needs in NASH services, please feel free to contact us for more details.

References

1. Alkhouri, N.; et al. Apoptosis in nonalcoholic fatty liver disease: diagnostic and therapeutic implications. Expert review of gastroenterology & hepatology. 2011, 5(2): 201-212.
2. Eguchi, A.; et al. Biomarkers of liver cell death. Journal of hepatology. 2014, 60(5): 1063-1074.