Soluble TNF-R1, VEGF and other cytokines as markers of disease activity in systemic lupus erythematosus and lupus nephritis.

BACKGROUND: Current non-invasive methods of assessing disease activity in systemic lupus erythematosus (SLE) are of limited sensitivity and specificity. Testing includes acute phase markers, autoantibodies and complement levels. Although measurements of dsDNA antibodies and complement C3/C4 levels are routine, they remain of limited value. Improved blood and urine markers may help in early detection of flare, distinction between flare and chronic damage, and monitoring response to therapy. METHODS: A total of 87 patients with SLE were tested for the following cytokines in serum and urine: monocyte chemoattractant protein 1 (MCP-1), regulated upon activation, normal T cell expressed and secreted (RANTES), soluble tumour necrosis factor receptor 1 (sTNF-R1), interferon-inducible protein 10 (IP-10), monocyte inhibitory protein 1α (MIP-1α) and vascular endothelial growth factor (VEGF). Patients attending the Lupus Unit at St Thomas' Hospital, London, UK were divided into active lupus nephritis (LN), inactive LN and non-renal SLE groups based on their renal pathology and SLE disease activity index (SLEDAI). Cytokine testing was performed using the FIDIS multiplex bead assay. RESULTS: The mean level of serum sTNF-R1 was higher in the active LN group compared with both inactive LN and non-renal SLE groups ( p < 0.001). For urine measurements there were significant differences between active LN and non-renal SLE for VEGF ( p = 0.016), after statistical correction for multiple testing. Both urinary and serum sTNF-R1 and IP-10 levels correlated with SLEDAI scores ( p < 0.001), while serum VEGF correlated weakly with SLEDAI ( p = 0.025). The optimum combination for differentiating active from inactive LN patients was serum VEGF, sTNF-R1, MCP-1 and glomerular filtration rate plus urinary sTNF-R1 and protein-creatinine ratio. CONCLUSION: These results indicate that for active LN, sTNF-R1 could be a useful serum cytokine marker, with potential for VEGF in the urine. This study has confirmed the ability of the multiplex bead technique to detect cytokines in a good analytical range, including very low and high levels, in both serum and urine. Combining serum and urine markers provided additional sensitivity in distinguishing active from inactive LN.

Molecular characterization of breast cancer cell lines by expression profiling.

PURPOSE: Gene expression patterns provide detailed insights into cellular regulation that reflect minor differences of cellular capacity not accessible by standard descriptions of the cellular phenotype or origin. METHODS: To identify fundamental differences and similarities we analyzed the gene expression patterns of four breast cancer cell lines: MCF-7, SK-BR-3, T-47D, and BT-474. RESULTS: Although only a small subset of genes (597) is represented on the Atlas-cDNA-Array (Clontech) used, clear differences in the expression of a number of genes could be detected. For example, unique high levels of expressions were found for the HLH-protein ID-1 (MCF-7) and the receptor tyrosine kinase erbB2 (SK-BR-3 and T-47D). Most genes analyzed were expressed at comparable levels in all cell lines studied. CONCLUSIONS: For interpretation of the results sets of genes that show similar variation of expression among the cells were grouped together. Furthermore, our analysis allows the assignment of similarity values that lead to a relation profile of the cell lines. How these results correlate with known biological properties of the cell lines is discussed. Additionally, we demonstrate that results obtained by cDNA-Array hybridization for expression of the ErbB receptor family correlate well with competitive RT-PCR, thus confirming the reliability of the cDNA-Array analysis.

Functionality of aquaporin-2 missense mutants in recessive nephrogenic diabetes insipidus.

Aquaporin-2 (AQP2) missense mutants in recessive nephrogenic diabetes insipidus (NDI) are all retained in the endoplasmic reticulum (ER), but some could function as water channels. No conclusions could be drawn about the water permeability (Pf) of others, because there was no method for quantifying AQP2 expression in the plasma membrane. We recently developed such a method, which has allowed us to study the functionality of these AQP2 mutants. Immunoblot analysis of membranes of injected oocytes revealed that all mutants (AQP2-G64R, AQP2-N68S, AQP2 T126M, AQP2-A147T, AQP2-R187C, AQP2-S216P) are expressed as unglycosylated and high-mannose glycosylated AQP2. The level of the high-mannose form of AQP2-A147T in the plasma membranes was low, indicating that this mutation has a less severe effect on proper folding. Analysis of Pf values and plasma membrane expression levels reveals that AQP2-N68S, AQP2-R187C and AQP2-S216P are non-functional, AQP2-A147T is as functional as wt-AQP2, while AQP2-T126M and AQP2-G64R retain 20% of the permeability of wt-AQP2. Since G64 is highly conserved between AQPs and expected to form essential interactions with other amino acids within AQP1, the residual functionality of AQP2-G64R is surprising. Our data furthermore indicate that an eventual therapy with chemical chaperones that restores the routing of AQP2 mutants to the apical membrane of collecting ducts cells might relieve NDI in patients encoding AQP2-A147T, and to a lesser extent AQP2-T126M and AQP2-G64R, but not in patients encoding AQP2-N68S, AQP2-R187C or AQP2-S216P.

Phsyiological relevance of aquaporins: luxury or necessity?

Aquaporins are members of a large family of pore-forming intrinsic membrane proteins, the MIP family. Based on their permeability properties they are now further subdivided into aquaporins, with real water-selective pores, and aquaglyceroporins with slightly less selective pores. Aquaporins are expressed in a large variety of tissues throughout the body but in most situations it is not clear whether their presence is necessary for the proper physiological function of these tissues. This review focuses on recent insight into the physiological relevance of aquaporins gained from studying aquaporin knockout mouse models and from diseases, on new surprising findings related to gating and selectivity, and on the consequences of tetramerization for routing and the genetics of nephrogenic diabetes insipidus. The active fluid transport in proximal tubules and in salivary glands is seriously compromised by aquaporin deletion. This is in contrast to lung, airways and stomach, where active fluid transport proceeds unhindered in the face of greatly reduced water permeabilities due to aquaporin deletion. Therefore, aquaporins seem to be a necessity at extreme high rates of active fluid transport but appear to be more of a luxury at medium or low fluid transport rates.

Bone morphogenetic protein 2 (BMP-2) induces sequential changes of Id gene expression in the breast cancer cell line MCF-7.

Bone morphogenetic proteins (BMPs) are involved in the development of various organs including the mammary gland. They are well-regulated and act in a time-, concentration- and cell-type-specific manner. We found that BMP-2 is expressed in primary breast tumor tissue samples and in breast cancer cell lines. Hybridization of labeled cDNA, obtained from the breast cancer cell line MCF-7, against the Atlas human cDNA expression array revealed differential gene expression depending on BMP-2 treatment. The most prominent changes were observed for the helix-loop-helix proteins Id-1, Id-2 and Id-3. Id-1 expression had increased severalfold after 4 h and was even higher after 24 h. Id-2 and Id-3 were more strongly induced after 4 h and showed no further significant change after 24 h. Analysis of cell-cycle distribution revealed a marked increase of the sub-G1 phase after 48 h in serum-deprived cells. In the presence of BMP-2 no change was observed over 48 h indicating that BMP-2 does not induce apoptosis. In addition, expression of caspase-3 was reduced in BMP-2-treated cells after 24 h. In summary, our results clearly indicate that BMP-2 is a susceptibility factor keeping the cells ready for the integration of various other signals for cell progression.