Apoc2 loss-of-function zebrafish mutant as a genetic model of hyperlipidemia.

Apolipoprotein C-II (APOC2) is an obligatory activator of lipoprotein lipase. Human patients with APOC2 deficiency display severe hypertriglyceridemia while consuming a normal diet, often manifesting xanthomas, lipemia retinalis and pancreatitis. Hypertriglyceridemia is also an important risk factor for development of cardiovascular disease. Animal models to study hypertriglyceridemia are limited, with no Apoc2-knockout mouse reported. To develop a genetic model of hypertriglyceridemia, we generated an apoc2 mutant zebrafish characterized by the loss of Apoc2 function. apoc2 mutants show decreased plasma lipase activity and display chylomicronemia and severe hypertriglyceridemia, which closely resemble the phenotype observed in human patients with APOC2 deficiency. The hypertriglyceridemia in apoc2 mutants is rescued by injection of plasma from wild-type zebrafish or by injection of a human APOC2 mimetic peptide. Consistent with a previous report of a transient apoc2 knockdown, apoc2 mutant larvae have a minor delay in yolk consumption and angiogenesis. Furthermore, apoc2 mutants fed a normal diet accumulate lipid and lipid-laden macrophages in the vasculature, which resemble early events in the development of human atherosclerotic lesions. In addition, apoc2 mutant embryos show ectopic overgrowth of pancreas. Taken together, our data suggest that the apoc2 mutant zebrafish is a robust and versatile animal model to study hypertriglyceridemia and the mechanisms involved in the pathogenesis of associated human diseases.

Influence of immunoglobulin light chain dimers on the results of the quantitative nephelometric assay.

BACKGROUND: Some polymeric forms of immunoglobulin free light chains (FLCs) have been identified, such as dimer, trimer and tetramer, and the quantitative nephelometric assay of kappa and lambda light chains is very important to the diagnosis and management of multiple myeloma (MM), primary systemic amyloidosis (AL), etc. But, whether polymeric molecules could affect the results of the nephelometric assay, is rarely reported. METHODS: 12 urinary samples from patients with light-chain MM were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and every sample was run with reduced and nonreduced sample buffer in one gel. Urinary kappa and lambda light chains were measured on the BNII nephelometer. We then added 3 microL beta-mercaptoethanol (2-ME) to the samples to transform the dimers into monomeric forms and measured them again. RESULTS: 10 samples showed obvious dimers by SDS-PAGE, of which 2 samples were kappa light chain dimers. Six samples had decreased quantitative results contrasted with the former values. The two samples of kappa light chain dimers decreased more obviously. CONCLUSIONS: The light chain dimer combined with more antibodies than the monomer and a larger antigen-antibody complex was formed. The larger complex could magnify the reaction between antigen and antibody of the nephelometric assay and therefore false elevated results could measured.

The effects of ox-LDL in human atherosclerosis may be mediated in part via the toll-like receptor 4 pathway.

Toll-like receptor 4 (TLR4) may provide a potential pathophysiological link between lipids and infection/inflammation and atherosclerosis. Oxidized low-density lipoprotein (ox-LDL) makes it more atherogenic than its native form. The purpose of the study was to investigate the relationship between the effects of ox-LDL in human atherosclerosis and the expression of TLR4. We studied the relationship between TLR4 and ox-LDL, pro and con, using both real-time quantitative RT-PCR and RNA interference technology through in vitro cell culture. Nuclear factor kappa B (NF-kappa B) activity and the concentrations of monocyte chemoattractant protein-1(MCP-1) and interleukin-8 (IL-8) were detected by ELISA. The results showed that the expression of TLR4 increased in response to ox-LDL. Simultaneously, NF-kappa B relative activity and the concentrations of MCP-1 and IL-8 in cell supernatant were upregulated by ox-LDL in a dose-dependent manner. TLR4 expression was inhibited by small interference RNA(siRNA) plasmid expression vectors; NF-kappa B activity and the secretions of MCP-1 and IL-8 in response to ox-LDL were significantly lower in the group wherein TLR4 expression has been inhibited than that in the group wherein TLR4 expression has not been inhibited. We suggest that the atherogenic effects of ox-LDL could be mediated in part via the TLR4 pathway. Furthermore, inhibition of TLR4 expression may downregulate the NF-kappa B activity and secretions of MCP-1 and IL-8 in monocytes due to oxidized LDL, resulting in the alleviation of the progress of atherosclerosis.

Siglec-1 on monocytes is a potential risk marker for monitoring disease severity in coronary artery disease.

OBJECTIVES: Siglec-1 has long been considered as an important biomarker of the activation of monocyte/macrophage and a type I interferon-specific imprint, but its role in atherosclerosis has not been elucidated. METHODS: We examined the expression of Siglec-1 by flow cytometry and RT-PCR in 83 CAD patients and 38 healthy controls. In addition, the levels of serum lipids, Gensini score, hs-CRP and homocysteine were determined. RESULTS: The transcriptional and protein levels of Siglec-1 on monocytes in CAD patients were significantly increased compared with healthy controls [3.17 versus 1.0, P<0.01; (11.5+/-3.9)% versus (1.8+/-2.0)%, P<0.01], but the increased Siglec-1 had no correlation with the level of native serum lipids. Interestingly, the expression of Siglec-1 was positively correlated with Gensini score (r=0.338, P=0.015), hs-CRP (r=0.316, P=0.016) and homocysteine level (r=0.224, P=0.042). CONCLUSION: Siglec-1 may be considered as a potential non-invasive indicator for monitoring disease severity and a biomarker for predicting the relative risk of cardiovascular events.

[A study on the relativity between La protein and the stability of HBV mRNA and the expression of HBV protein].

OBJECTIVE: To study the relativity between La protein and the stability of HBV mRNA and the expression of HBV protein. METHODS: Four specific siRNAs were obtained by transcription in vitro. After transfection with the siRNAs into HepG2.2.15 cells for 3 days, the inhibitive effects of La protein were analyzed by Western blot; the content changes of HBsAg, HBeAg and HBV-DNA were detected by ECL and RT-PCR. RESULTS: In comparison to normal cells, La protein was less in the cells. There was less La protein in the cells trans-infected with siRNAs. HBsAg, the HBeAg and HBV-DNA secreted by the cells transfected with siRNA were also less than that in the normal cells. CONCLUSION: There is a correlation between La protein and HBV mRNA and the expression of HBV protein.

[Quantitation of antigen specific T lymphocytes in peripheral blood of patients with primary biliary cirrhosis using tetramer technology].

AIM: To quantitate antigen specific T lymphocytes in peripheral blood from patients with primary biliary cirrhosis(PBC) and study the role of antigen specific T lymphocytes in the development of PBC. METHODS: Using tetramers and CD8 monoclonal antibody staining, PDC-E2 159-167aa and PDC-E2 165-174aa specific CD8(+) T lymphocytes were determined respectively in the peptide-induced cytotoxic T cell lines prepared from peripheral blood mononuclear cells(PBMC) of 15 PBC patients. The frequencies of these two kinds of antigen specific T lymphocytes in HLA-A*0201 positive (A2(+)) PBC were compared with those in A2(-) PBC patients, patients with other A2(+) chronic liver diseases and healthy controls. RESULTS: PDC-E2 159-167aa/HLA-A*0201 and PDC-E2 165-174aa/HLA-A*0201 tetramer positive CD8(+) T lymphocytes were detected in all of A2(+) PBC patients with average percentages of 0.42%+/-0.24% (0.17%-1.08%) and 0.27%+/-0.17% (0.05%-0.56%), respectively. The frequencies of the two kinds of antigen specific CD8(+) T lymphocytes from peripheral blood were significantly higher in earlier stages I and II of PBC as compared with stage III (P<0.001), while no difference was found between PDC-E2 159-167aa and PDC-E2 165-174aa specific CD8(+) T lymphocytes at the same stages. In addition, there existed no statistical difference between frequencies of antigen specific T lymphocytes in AMA or anti-PDC positive and negative PBC patients (P>0.05). CONCLUSION: This study suggests that HLA-A*0201 restricted PDC-E2 165-174aa and PDC-E2 159-167aa specific CTL play important roles in the development of PBC, and there might be a similar mechanism of T cell-mediated damage between AMA or anti-PDC positive and negative PBC patients.