Do-it-yourself: construction of a custom cDNA macroarray platform with high sensitivity and linear range

Background: Research involving gene expression profiling and clinical applications, such as diagnostics and prognostics, often require a DNA array platform that is flexibly customisable and cost-effective, but at the same time is highly sensitive and capable of accurately and reproducibly quantifying the transcriptional expression of a vast number of genes over the whole transcriptome dynamic range using low amounts of RNA sample. Hereto, a set of easy-to-implement practical optimisations to the design of cDNA-based nylon macroarrays as well as sample (33)P-labeling, hybridisation protocols and phosphor screen image processing were analysed for macroarray performance. Results: The here proposed custom macroarray platform had an absolute sensitivity as low as 50,000 transcripts and a linear range of over 5 log-orders. Its quality of identifying differentially expressed genes was at least comparable to commercially available microchips. Interestingly, the quantitative accuracy was found to correlate significantly with corresponding reversed transcriptase - quantitative PCR values, the gold standard gene expression measure (Pearson's correlation test p < 0.0001). Furthermore, the assay has low cost and input RNA requirements (0.5 mu g and less) and has a sound reproducibility. Conclusions: Results presented here, demonstrate for the first time that self-made cDNA-based nylon macroarrays can produce highly reliable gene expression data with high sensitivity and covering the entire mammalian dynamic range of mRNA abundances. Starting off from minimal amounts of unamplified total RNA per sample, a reasonable amount of samples can be assayed simultaneously for the quantitative expression of hundreds of genes in an easily customisable and cost-effective manner

Transcriptome analysis of monocytes and macrophages in mycobacterial infection and chronic kidney disease

Tissue macrophages and their circulating counterpart, the monocytes, constitute a key component of the innate immune system. Different triggers evoke different macrophage responses leading to different states of macrophage activation. The activation states of the following in vivo elicited macrophages were characterized by transcriptome profiling: (i) granuloma macrophages from mice after infection with Mycobacterium bovis BCG, and (ii) monocytes from patients with chronic kidney disease. Granuloma macrophages constitute an important component of granulomas, which are cellular aggregates of T cells and activated macrophages, and which are hallmarks of tuberculosis. By genome-wide expression profiling, we identified a specific molecular marker SynCAM1 for Th1 cytokine-related granuloma macrophages within the mononuclear phagocyte family. Moreover, we identified the differential regulation in granuloma macrophages of genes not previously associated with the in vivo macrophage response during mycobacterial infection. These included several chemotaxis-related genes and adhesion molecules. The regulation of SynCAM1 and some of the other newly identified molecules was dependent on TNF-derived signals, indicating that TNF might contribute to granuloma formation and/or maintenance not only by regulating expression of chemokines, but also expression of adhesion molecules in the granuloma. Thus, we identified several valuable targets for future research that might improve our understanding of the involvement of macrophages and of TNF in the granulomatous response during mycobacterial infection. Another part of the study was dedicated to the evaluation of monocytes from patients with chronic kidney disease (CKD). Monocytes in CKD are dysfunctional, and their particular activation state is believed to contribute to the major complications observed in CKD: (i) the slight pro-inflammatory activation state might contribute to accelerated atherosclerosis in CKD, and (ii) the blunted immune response upon further stimulation might contribute to increased susceptibility to infections. The transcriptome of monocyte/macrophage-related genes was analyzed in monocytes from CKD patients and controls, however, hardly any differences in gene expression were observed in the different study populations. Our results, together with previously described observations, indicate that it is only the CD16+ monocyte subset that is affected in CKD, and that post-transcriptional processes might be involved in the dysfunctional activation state

Transcriptome analysis of monocyte-HIV interactions

BACKGROUND: During HIV infection and/or antiretroviral therapy (ART), monocytes and macrophages exhibit a wide range of dysfunctions which contribute significantly to HIV pathogenesis and therapy-associated complications. Nevertheless, the molecular components which contribute to these dysfunctions remain elusive. We therefore applied a parallel approach of genome-wide microarray analysis and focused gene expression profiling on monocytes from patients in different stages of HIV infection and/or ART to further characterise these dysfunctions. RESULTS: Processes involved in apoptosis, cell cycle, lipid metabolism, proteasome function, protein trafficking and transcriptional regulation were identified as areas of monocyte dysfunction during HIV infection. Individual genes potentially contributing to these monocyte dysfunctions included several novel factors. One of these is the adipocytokine NAMPT/visfatin, which we show to be capable of inhibiting HIV at an early step in its life cycle. Roughly half of all genes identified were restored to control levels under ART, while the others represented a persistent dysregulation. Additionally, several candidate biomarkers (in particular CCL1 and CYP2C19) for the development of the abacavir hypersensitivity reaction were suggested. CONCLUSIONS: Previously described areas of monocyte dysfunction during HIV infection were confirmed, and novel themes were identified. Furthermore, individual genes associated with these dysfunctions and with ART-associated disorders were pinpointed. These genes form a useful basis for further functional studies concerning the contribution of monocytes/macrophages to HIV pathogenesis. One such gene, NAMPT/visfatin, represents a possible novel restriction factor for HIV

GlycoDelete engineering of mammalian cells simplifies N-glycosylation of recombinant proteins

Heterogeneity in the N-glycans on therapeutic proteins causes difficulties for protein purification and process reproducibility and can lead to variable therapeutic efficacy. This heterogeneity arises from the multistep process of mammalian complex-type N-glycan synthesis. Here we report a glycoengineering strategy—which we call GlycoDelete—that shortens the Golgi N-glycosylation pathway in mammalian cells. This shortening results in the expression of proteins with small, sialylated trisaccharide N-glycans and reduced complexity compared to native mammalian cell glycoproteins. GlycoDelete engineering does not interfere with the functioning of N-glycans in protein folding, and the physiology of cells modified by GlycoDelete is similar to that of wild-type cells. A therapeutic human IgG expressed in GlycoDelete cells had properties, such as reduced initial clearance, that might be beneficial when the therapeutic goal is antigen neutralization. This strategy for reducing N-glycan heterogeneity on mammalian protins could lead to more consistent performance of therapeutic proteins and modulation of biopharmaceutical functions

Quantitative RT-PCR data for CD16 mRNA expression (A) and CX3CR1 mRNA expression (B) in the 4 study groups; control (n = 9), CVE (n = 10), CKD5HD (n = 10), CKD5HD/CVE (n = 10).

<p>* p < 0.05, ** p < 0.01 vs. control; ° p < 0.05, °° p < 0.01 vs. CVE.</p

Representative sample of flow cytometric evaluation of the CD14 and CD16 expression on isolated monocytes from a healthy control (left panel) and a hemodialysis patient (right panel).

<p>According to the CD14 and CD16 fluorescence intensity three gates are drawn containing the CD14⁺⁺CD16^-, the CD14⁺⁺CD16⁺ and the CD14⁺CD16⁺⁺ monocyte subpopulations respectively. Hemodialysis patients show a decreased proportion of CD14⁺⁺CD16^- and an increase of CD14⁺⁺CD16⁺ and CD14⁺CD16⁺⁺ monocytes.</p