Hassan Dihazi

Comprehensive human urine standards for comparability and standardization in clinical proteome analysis

Purpose: Urine proteomics is emerging as a powerful tool for biomarker discovery. The purpose of this study is the development of a well‐characterized “real life” sample that can be used as reference standard in urine clinical proteomics studies.

Proteomic Analysis of Cellular Response to Osmotic Stress in Thick Ascending Limb of Henle’s Loop (TALH) Cells

Epithelial cells of the thick ascending limb of Henle’s loop (TALH cells) play a major role in the urinary concentrating mechanism. They are normally exposed to variable and often very high osmotic stress, which is particularly due to high sodium and chloride reabsorption and very low water permeability of the luminal membrane. It is already established that elevation of the activity of aldose reductase and hence an increase in intracellular sorbitol are indispensable for the osmotic adaptation and stability of the TALH cells. To identify new molecular factors potentially associated with the osmotic stress-resistant phenotype in kidney cells, TALH cells exhibiting low or high levels of resistance to osmotic stress were characterized using proteomic tools. Two-dimensional gel analysis showed a total number of 40 proteins that were differentially expressed in TALH cells under osmotic stress. Twenty-five proteins were overexpressed, whereas 15 proteins showed a down-regulation. Besides the sorbitol pathway enzyme aldose reductase, whose expression was 15 times increased, many other metabolic enzymes like glutathione S-transferase, malate dehydrogenase, lactate dehydrogenase, α enolase, glyceraldehyde-3-phosphate dehydrogenase, and triose-phosphate isomerase were up-regulated. Among the cytoskeleton proteins and cytoskeleton-associated proteins vimentin, cytokeratin, tropomyosin 4, and annexins I, II, and V were up-regulated, whereas tubulin and tropomyosins 1, 2, and 3 were down-regulated. The heat shock proteins α-crystallin chain B, HSP70, and HSP90 were found to be overexpressed. In contrast to the results in oxidative stress the endoplasmic reticulum stress proteins like glucose-regulated proteins (GRP78, GRP94, and GRP96), calreticulin, and protein-disulfide isomerase were down-regulated under hypertonic stress.

Immuno-MALDI-TOF MS: new perspectives for clinical applications of mass spectrometry.

The discovery of novel biomarkers by means of advanced detection tools based on proteomic analysis technologies necessitates the development of improved diagnostic methods for application in clinical routine. On the basis of three different application examples, this review presents the limitations of conventional routine diagnostic assays and illustrates the advantages of immunoaffinity enrichment combined with MALDI‐TOF MS. Applying this approach increases the specificity of the analysis supporting a better diagnostic recognition, sensitivity, and differentiation of certain diseases. The use of MALDI‐TOF MS as detection method facilitates the identification of modified peptides and proteins providing additional information. Further, employing respective internal standard peptides allows for relative and absolute quantitation which is mandatory in the clinical context. Although MALDI‐TOF MS is not yet established for clinical routine diagnostics this technology has a high potential for improvement of clinical diagnostics and monitoring therapeutic efficacy.

Impaired Chromatin Remodelling at STAT1-Regulated Promoters Leads to Global Unresponsiveness of Toxoplasma gondii-Infected Macrophages to IFN-γ

Intracellular pathogens including the apicomplexan and opportunistic parasite Toxoplasma gondii profoundly modify their host cells in order to establish infection. We have shown previously that intracellular T. gondii inhibit up-regulation of regulatory and effector functions in murine macrophages (MΦ) stimulated with interferon (IFN)-γ, which is the cytokine crucial for controlling the parasites replication. Using genome-wide transcriptome analysis we show herein that infection with T. gondii leads to global unresponsiveness of murine macrophages to IFN-γ. More than 61% and 89% of the transcripts, which were induced or repressed by IFN-γ in non-infected MΦ, respectively, were not altered after stimulation of T. gondii-infected cells with IFN-γ. These genes are involved in a variety of biological processes, which are mostly but not exclusively related to immune responses. Analyses of the underlying mechanisms revealed that IFN-γ-triggered nuclear translocation of STAT1 still occurred in Toxoplasma-infected MΦ. However, STAT1 bound aberrantly to oligonucleotides containing the IFN-γ-responsive gamma-activated site (GAS) consensus sequence. Conversely, IFN-γ did not induce formation of active GAS-STAT1 complexes in nuclear extracts from infected MΦ. Mass spectrometry of protein complexes bound to GAS oligonucleotides showed that T. gondii-infected MΦ are unable to recruit non-muscle actin to IFN-γ-responsive DNA sequences, which appeared to be independent of stimulation with IFN-γ and of STAT1 binding. IFN-γ-induced recruitment of BRG-1 and acetylation of core histones at the IFN-γ-regulated CIITA promoter IV, but not β-actin was diminished by >90% in Toxoplasma-infected MΦ as compared to non-infected control cells. Remarkably, treatment with histone deacetylase inhibitors restored the ability of infected macrophages to express the IFN-γ regulated genes H2-A/E and CIITA. Taken together, these results indicate that Toxoplasma-infected MΦ are unable to respond to IFN-γ due to disturbed chromatin remodelling, but can be rescued using histone deacetylase inhibitors.

Immunoadsorption therapy in patients with multiple sclerosis with steroid-refractory optical neuritis

BackgroundIn multiple sclerosis relapses refractory to intravenous corticosteroid therapy, plasma exchange is recommended. Immunoadsorption (IA) is regarded as an alternative therapy, but its efficacy and putative mechanism of action still needs to be established.MethodsWe prospectively treated 11 patients with multiple sclerosis who had optical neuritis and fulfilled the indications for apheresis therapy (Trial registration DE/CA25/00007080-00). In total, five IA treatments were performed using tryptophan-IA. Clinical activity (visual acuity, Expanded Disability Status Scale, Incapacity Status Scale), laboratory values and visual evoked potentials were measured before, during and after IA, with a follow-up of six months. Moreover, proteomic analyses were performed to analyze column-bound proteins as well as corresponding changes in patients’ sera.ResultsAfter the third IA, we detected an improvement of vision in eight of eleven patients, whom we termed responders. Amongst these, the mean visual acuity improved from 0.15u2009±u20090.12 at baseline to 0.47u2009±u20090.32 after the third IA (Pu2009=u20090.0252) up to 0.89u2009±u20090.15 (Pu2009<u20090.0001) at day 180u2009±u200910 after IA. Soluble interleukin-2 receptor decreased in responders (Pu2009=u20090.03), whereas in non-responders it did not. Proteomic analyses of proteins adsorbed to IA columns revealed that several significant immunological proteins as well as central nervous system protein fragments, including myelin basic protein, had been removed by IA.ConclusionsIA was effective in the treatment of corticosteroid-refractory optic neuritis. IA influenced the humoral immune response. Strikingly, however, we found strong evidence that demyelination products and immunological mediators were also cleared from plasma by IA.

Secretion of ERP57 is important for extracellular matrix accumulation and progression of renal fibrosis, and is an early sign of disease onset.

Summary Renal fibrosis is characterized by excessive accumulation of extracellular matrix (ECM), which compromises organ function by replacing normal organ tissue. The molecular mechanisms leading to renal fibrosis are not fully understood. Here we demonstrated that TGF&bgr;1, AGT or PDGF stimulation of renal cells resulted in endoplasmic reticulum (ER) stress followed by activation of the protective unfolded protein response pathway and a high secretory level of protein disulfide isomerase ERP57 (also known as PDIA3). The TGF&bgr;1-induced impairment of ER function could be reversed by treatment with BMP7, suggesting a specific involvement in renal fibrosis. A clear correlation between the degree of fibrosis, ER stress and the level of ERP57 could be seen in fibrosis animal models and in biopsies of renal fibrosis patients. Protein interaction studies revealed that secreted ERP57 exhibits a strong interaction with ECM proteins. Knockdown of ERP57 or antibody-targeted inhibition of the secreted form significantly impaired the secretion and accumulation of ECM. Moreover, ERP57 was excreted in the early stages of chronic kidney disease, and its level in urine correlated with the degree of renal fibrosis, suggesting that the secretion of ERP57 represents one of the first signs of renal fibrosis onset and progression.

Apolipoprotein A-I as a candidate serum marker for the response to lithium treatment in bipolar disorder

The molecular basis of bipolar disorder (BD) is still unknown as is the mechanism through which lithium, the therapy of choice, exerts its effects in treatment of BD. So far, no biomarkers exist to facilitate diagnosis of BD or treatment evaluation. To investigate whether BD and its treatment with lithium leaves a characteristic signature in the serum proteome, we used SELDI‐TOF MS to analyze individual serum samples from BD patients treated with lithium (BD‐plus‐Li, n=15) or other drugs (BD‐minus‐Li, n=10) and from healthy controls (n=15). Interestingly, features of 28u2009kDa (one peak) and 14u2009kDa (three peaks) showed a decreased level in the BD‐minus‐Li group and a level restored to that of the control group in the BD‐plus‐Li group. To reveal the identity of these features, we subjected pooled serum samples from both BD groups to the 2‐D DIGE technology and identified 28u2009kDa apolipoprotein A‐I (apo A‐I) and three 14u2009kDa fragments thereof as upregulated in the BD‐plus‐Li group. Immunoturbidimetry, a routine clinical assay, verified the characteristic apo A‐I signature in individual serum samples. In conclusion, we propose apo A‐I as a candidate marker that can visualize response to lithium treatment at the serum protein level.

Urinary proteomics: a tool to discover biomarkers of kidney diseases.

There is intense interest in applying proteomics to urine analysis in order to promote a better understanding of kidney disease processes, develop new biomarkers for diagnosis and detect early factors that contribute to end–stage renal diseases. This interest creates numerous opportunities as well as challenges. To fulfill this task, proteomics requires, in its different stages of realization, various technological platforms with high sensitivity, high throughput and large automation ability. In this review, we will give an overview of promising proteomic methods that can be used for analyzing urinary proteome and detecting biomarkers for different kidney diseases. Furthermore, we will focus on the current status and future directions in investigating kidney diseases using urinary proteomics.

Serum peptide reactivities may distinguish neuromyelitis optica subgroups and multiple sclerosis

Objective: To assess in an observational study whether serum peptide antibody reactivities may distinguish aquaporin-4 (AQP4) antibody (Ab)–positive and -negative neuromyelitis optica spectrum disorders (NMOSD) and relapsing-remitting multiple sclerosis (RRMS). Methods: We screened 8,700 peptides that included human and viral antigens of potential relevance for inflammatory demyelinating diseases and random peptides with pooled sera from different patient groups and healthy controls to set up a customized microarray with 700 peptides. With this microarray, we tested sera from 66 patients with AQP4-Ab-positive (n = 16) and AQP4-Ab-negative (n = 19) NMOSD, RRMS (n = 11), and healthy controls (n = 20). Results: Differential peptide reactivities distinguished NMOSD subgroups from RRMS in 80% of patients. However, the 2 NMOSD subgroups were not well-discriminated, although those patients are clearly separated by their antibody reactivities against AQP4 in cell-based assays. Elevated reactivities to myelin and Epstein-Barr virus peptides were present in RRMS and to AQP4 and AQP1 peptides in AQP4-Ab-positive NMOSD. Conclusions: While AQP4-Ab-positive and -negative NMOSD subgroups are not well-discriminated by peptide antibody reactivities, our findings suggest that peptide antibody reactivities may have the potential to distinguish between both NMOSD subgroups and MS. Future studies should thus concentrate on evaluating peptide antibody reactivities for the differentiation of AQP4-Ab-negative NMOSD and MS.

Calreticulin is crucial for calcium homeostasis mediated adaptation and survival of thick ascending limb of Henle's loop cells under osmotic stress

The thick ascending limb of Henles loop (TALH) is normally exposed to variable and often very high osmotic stress and involves different mechanisms to counteract this stress. ER resident calcium binding proteins especially calreticulin (CALR) play an important role in different stress balance mechanisms. To investigate the role of CALR in renal epithelial cells adaptation and survival under osmotic stress, two-dimensional fluorescence difference gel electrophoresis combined with mass spectrometry and functional proteomics were performed. CALR expression was significantly altered in TALH cells exposed to osmotic stress, whereas renal inner medullary collecting duct cells and interstitial cells exposed to hyperosmotic stress showed no significant changes in CALR expression. Moreover, a time dependent downregulation of CALR was accompanied with continuous change in the level of free intracellular calcium. Inhibition of the calcium release, through IP3R antagonist, prevented CALR expression alteration under hyperosmotic stress, whereas the cell viability was significantly impaired. Overexpression of wild type CALR in TALH cells resulted in significant decrease in cell viability under hyperosmotic stress. In contrast, the hyperosmotic stress did not have any effect on cells overexpressing the CALR mutant, lacking the calcium-binding domain. Silencing CALR with siRNA significantly improved the cell survival under osmotic stress conditions. Taken together, our data clearly highlight the crucial role of CALR and its calcium-binding role in TALH adaptation and survival under osmotic stress.