Analysis of complex protein elution behavior in preparative ion exchange processes using a colloidal particle adsorption model.

A fundamental understanding of the protein retention mechanism in preparative ion exchange (IEX) chromatography columns is essential for a model-based process development approach. For the past three decades, the mechanistic description of protein retention has been based predominantly on the steric mass action (SMA) model. In recent years, however, retention profiles of proteins have been reported more frequently for preparative processes that are not consistent with the mechanistic understanding relying on the SMA model. In this work, complex elution behavior of proteins in preparative IEX processes is analyzed using a colloidal particle adsorption (CPA) model. The CPA model is found to be capable of reproducing elution profiles that cannot be described by the traditional SMA model. According to the CPA model, the reported complex behavior can be ascribed to a strong compression and concentration of the elution front in the lower unsaturated part of the chromatography column. As the unsaturated part of the column decreases with increasing protein load density, exceeding a critical load density can lead to the formation of a shoulder in the peak front. The general applicability of the model in describing preparative IEX processes is demonstrated using several industrial case studies including multiple monoclonal antibodies on different IEX adsorber systems. In this context, the work covers both salt controlled and pH-controlled protein elution.

Bioinformatic Identification of Chinese Hamster Ovary (CHO) Cold-Shock Genes and Biological Evidence of their Cold-Inducible Promoters.

Lower cultivation temperature dramatically affects cell growth and cellular productivity in Chinese hamster ovary cells (CHO) and is often used in industrial applications with the aim to enhance productivity. Cold-inducible proteins whose activity is induced at lower temperature play an important role in understanding the mechanisms of cold-induced changes in gene expression. One of these mechanisms is increased transcription of specific target genes controlled by sequence elements in cold-inducible promoters. This study provides a list of cold-inducible genes and endogenous cold-inducible promoters of CHO cells. Transcriptome data before and after a temperature shift from 37 to 33 °C are analyzed to identify 94 cold-inducible genes, which are highly expressed and have a high fold change in expression after the temperature shift. Cold-inducible promoters are identified from the top ten cold-inducible genes, showing up to 11-fold increased luciferase expression at lowered temperature in transient transfections. Additionally, several common transcription factor binding sites are identified in the top cold-inducible promoter sequences and expression of their corresponding transcription factors over temperature-shift cultivation is evaluated. These may be responsible for enhanced promoter activity under lower temperature and can be used as engineering targets.

Novel Promoters Derived from Chinese Hamster Ovary Cells via In Silico and In Vitro Analysis.

For the industrial production of recombinant proteins in mammalian cell lines, a high rate of gene expression is desired. Therefore, strong viral promoters are commonly used. However, these have several drawbacks as they override cellular responses, are not integrated into the cellular network, and thus can induce stress and potentially epigenetic silencing. Endogenous promoters potentially have the advantage of a better response to cellular state and thus a lower stress level by uncontrolled overexpression of the transgene. Such fine-tuning is typically achieved by endogenous enhancers and other regulatory elements, which are difficult to identify purely based on the genomic sequence. Here, Chinese hamster ovary (CHO) endogenous promoters and enhancers are identified using histone marks and chromatin states, ranked based on expression level and tested for normalized promoter strength. Successive truncation of these promoters at the 5'- and 3'-end as well as the combination with enhancers are identified in the vicinity of the promoter sequence further enhance promoter activity up to threefold. In an initial screen within stable cell lines, the strongest CHO promoter appears to be more stable than the human cytomegalovirus promoter with enhancer, making it a promising candidate for recombinant protein production and cell engineering applications. A deeper understanding of promoter functionality and response elements will be required to take full advantage of such promoters for cell engineering, in particular, for multigene network engineering applications.

Affinity of FVIII-specific antibodies reveals major differences between neutralizing and nonneutralizing antibodies in humans.

Recently, we reported that distinct immunoglobulin (Ig) isotypes and IgG subclasses of factor VIII (FVIII)-specific antibodies are found in different cohorts of patients with hemophilia A and in healthy individuals. Prompted by these findings, we further investigated the distinguishing properties among the different populations of FVIII-specific antibodies. We hypothesized that the affinity of antibodies would discriminate between the neutralizing and nonneutralizing antibodies found in different study cohorts. To test this idea, we established a competition-based enzyme-linked immunosorbent assay technology to assess the apparent affinities for each isotype and IgG subclass of FVIII-specific antibodies without the need for antibody purification. We present a unique data set of apparent affinities of FVIII-specific antibodies found in healthy individuals, patients with congenital hemophilia A with and without FVIII inhibitors, and patients with acquired hemophilia A. Our data indicate that FVIII-specific antibodies found in patients with FVIII inhibitors have an up to 100-fold higher apparent affinity than that of antibodies found in patients without inhibitors and in healthy individuals. High-affinity FVIII-specific antibodies could be retrospectively detected in longitudinal samples of an individual patient with FVIII inhibitors 543 days before the first positive Bethesda assay. This finding suggests that these antibodies might serve as potential biomarkers for evolving FVIII inhibitor responses.

Improved prognostic diagnosis of systemic lupus erythematosus in an early stage of disease by a combination of different predictive biomarkers identified by proteome analysis.

BACKGROUND: Since the original characterizations of the pathological features defining glomerulonephritis in systemic lupus erythematosus (SLE) were reported, numerous studies have linked the development of pathology to the abnormal expression of protein in urine. The determination of proteinuria is important and necessary; however, this alone is not predictive enough to confirm a suspected diagnosis, especially in an early state of disease when symptoms are not yet observed. Furthermore, several studies have already highlighted the pitfalls of proteinuria both as a clinical prognostic marker and as a factor predicting the progressive loss of renal function. Therefore, the identification of more accurate and predictive biomarkers is urgently needed. To address this, comparative urinary and kidney profiling was performed in the MRL-lpr/lpr mouse as a model of lupus tubulointerstitial nephritis and lupus glomerulonephritis corresponding to SLE in humans. RESULTS: Tamm-Horsfall glycoprotein (THG; uromodulin) and beta2-microglubulin (ß2M) were identified as immune process-related molecules in the urine and kidney of the MRL-lpr/lpr mouse model. Furthermore, we show that the combinatory expression profile of THG and ß2M as biomarkers, normalized by the proteinuria level, is more predictive than proteinuria determination alone. Data were confirmed by comparative urinary profiling of SLE in mice by Western blot and quantitative polymerase chain reaction (qPCR) analysis. CONCLUSION: Based on our results, we are able to diagnose SLE in the MRL-lpr/lpr mouse in a very early state of disease, when the proteinuria level alone is not able to confirm a suspected diagnosis. The pre-validation of our urinary biomarkers is associated with clinical outcomes of glomerulonephritis in humans and merits additional investigation. Further conformations of our predictive biomarkers in the urine of SLE patients in the course of a clinical study are still ongoing.

Advanced proteomics procedure as a detection tool for predictive screening in type 2 pre-Diabetes.

It has been suggested that a more precise selection of predictive biomarkers may prove useful in the early diagnosis of type 2 diabetes (T2D), even when glucose tolerance is normal. This is vital since many T2D cases may be preventable by avoiding those factors that trigger the disease process (primary prevention) or by use of therapy that modulates the disease process before the onset of clinical symptoms (secondary prevention) occurs. The selection of predictive markers must be carefully assessed and depends mainly on three important parameters: sensitivity, specificity and positive predictive value. Unfortunately, biomarkers with ideal specificity and sensitivity are difficult to find. One potential solution is to use the combinatorial power of different biomarkers, each of which alone may not offer satisfactory specificity and sensitivity. Recent technological advances in proteomics and bioinformatics offer a great opportunity for the discovery of different potential predictive markers. In this review, we described a cellular T2D model as an example with the intent of providing specific enrichment and new identification strategies, which might have the potential to improve predictive biomarker identification and to bring accuracy in disease diagnosis and classification, as well as therapeutic monitoring in the early phase of T2D.

Effect of different biomaterials on the expression pattern of the transcription factor Ets2 in bone-like constructs.

Tissue engineering offers an exciting prospect for reconstructive surgery by replacing missing natural scaffolds with artificial ones. For optimal success the artificial scaffold should provide an environment closely resembling the natural tissue. Little is known about the direct influence of the scaffold on the expression of regulators of bone development, such as transcription factors. The aim of this study was to investigate the influence of the scaffold material on the expression of V-ets erythroblastosis virus E26 oncogene homolog 2 (avian) (Ets2), a key transcription factor in bone biology. Human primary bone precursor cells were seeded in three-dimensional constructs consisting of hydroxyapatite (HA) or poly (lactic-co-glycolic acid) (PLGA). Cells grown on tissue culture polystyrene dishes served as controls. After cultivation for up to 21 days the expression of Ets2 and other important bone-specific genes was assessed by reverse transcription polymerase chain reaction (RT-PCR) and Western Blotting. Ets2 mRNA showed significantly higher expression in controls than in bone-like constructs, and more Ets2 mRNA was expressed in cells grown in HA than in PLGA constructs. At protein level however, Ets2 expression was higher in constructs than in controls after prolonged culture. Our study showed for the first time a differential expression of Ets2 in tissue engineered bone constructs in vitro, demonstrating that scaffold chemistry has an influence on the expression of genes regulating osteogenesis.

Phenothiazine chloride and soft laser light have a biostimulatory effect on human osteoblastic cells.

OBJECTIVE: Low-level laser therapy (LLLT) is a well accepted tool to accelerate wound healing and to reduce inflammation after oral implant insertion. Since there are no in vitro data on a combination of LLLT with prior photosensitization, it was the aim of this study to investigate if photosensitization with phenothiazine chloride results in an alteration of the biostimulatory effect of low-level laser irradiation. BACKGROUND DATA: LLLT and antimicrobial photodynamic therapy are well established for the treatment of peri-implantitis. In vitro studies have shown a biostimulatory effect of LLLT on various cell types, including osteogenic cells. MATERIALS AND METHODS: SaOS-2 cells were treated with the photosensitizer phenothiazine chloride before irradiation with matched laser light. At 24-h intervals the viability and differentiation were analyzed in treated and untreated cells. RESULTS: While the biostimulatory effect of the LLLT could be observed for the lower irradiation dose, the pretreatment with phenothiazine chloride did not significantly affect the growth and differentiation of the SaOS-2 cells. CONCLUSION: It can thus be concluded that combined treatment with phenothiazine chloride and LLLT does not result in a synergistic enhancement of the biostimulatory effect of LLLT, but there was also no evidence for antagonizing effects on growth and differentiation of human osteoblasts.

Potential involvement of MYC- and p53-related pathways in tumorigenesis in human oral squamous cell carcinoma revealed by proteomic analysis.

Oral squamous cellular carcinoma is a malignant tumor with poor prognosis. Discovery of early markers to discriminate between malignant and normal cells is of high importance in clinical diagnosis. Subcellular fractions from 10 oral squamous cell carcinoma and corresponding control samples, enriched in mitochondrial and cytosolic proteins, as well as blood from the tumor were analyzed by proteomics, two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Three-hundred and fifty different gene products were identified. Twenty proteins showed deranged levels in oral squamous cell carcinoma in comparison with the control samples and are potentially involved in tumor growth and metastasis. Of these, 16 proteins were upregulated. By applying pathway analysis, we found 8 of the upregulated gene products to be linked to three main locus genes, p53, MYC, and MYCN, and could be candidate biomarkers for OSCC. The findings of this pilot study show that OSCC gene ontology combined with proteomic analysis is a powerful tool in systems biology for the elucidation of the complexity of expression profiles in cellular processes. Application of such pathway analysis has the potential to generate new insights into complex molecular mechanisms underlying disease related processes and could therefore significantly contribute to the efficient performance of the entire discovery process.

Initial effects of low-level laser therapy on growth and differentiation of human osteoblast-like cells.

Low-level laser therapy is a clinically well established tool for enhancement of wound healing. In vitro studies have also shown that low level laser therapy has a biostimulatory effect on cells of different origin. The aim of this in vitro study was to investigate the initial effect of low-level laser therapy on growth and differentiation of human osteoblast-like cells. SaOS-2 cells were irradiated with laser doses of 1 J/cm2 and 2 J/cm2 using a diode laser with 670 nm wave length and an output power of 400 mW. Untreated cells were used as controls. At 24 h, 48 h and 72 h post irradiation, cells were collected and assayed for viability of attached cells and alkaline phosphatase specific activity. In addition, mRNA expression levels of osteopontin and collagen type I were assessed using semi-quantitative RT-PCR. Over the observation period, cell viability, alkaline phosphatase activity and the expression of osteopontin and collagen type I mRNA were slightly enhanced in cells irradiated with 1 J/cm2 compared with untreated control cells. Increasing the laser dose to 2 J/cm2 reduced cell viability during the first 48 h and resulted in persistently lower alkaline phosphatase activity compared with the other two groups. The expression of osteopontin and collagen type I mRNA slightly decreased with time in untreated controls and cells irradiated with 1 J/cm2, but their expression was increased by treatment with 2 J/cm2 after 72 h. These results indicate that low-level laser therapy has a biostimulatory effect on human osteoblast-like cells during the first 72 h after irradiation. Further studies are needed to determine the potential of low-level laser therapy as new treatment concept in bone regeneration.

Assessing the function of human UNC-93B in Toll-like receptor signaling and major histocompatibility complex II response.

The high sequence identity observed between UNC-93B of mouse and human imply common evolutionary ancestors and a conserved function. A nonconservative point mutation in the mouse Unc93b1 gene has been associated with defective Toll-like receptor (TLR) signaling and impaired major histocompatibility complex (MHC) I and II restricted antigen responses. Like murine UNC-93B, the human homologue is predicted to form 12 transmembrane domains, and it localizes to the endoplasmic reticulum. In human beings its expression is highest in professional antigen-presenting cells such as dendritic cells and macrophages. Interestingly, UNC-93B itself is specifically induced by TLR3 signaling in monocyte-derived dendritic cells and macrophages. To study the effect of UNC-93B deficiency in TLR signaling and antigen-presentation in human beings, UNC-93B message was knocked down in monocyte-derived dendritic cells and a reduced TNFalpha production in response to TLR3 agonists was observed. In the same experiment, the achieved knockdown had no effect on an MHC II-dependent antigen response, suggesting that the reduced quantity of human UNC-93B was still capable of supporting class II antigen presentation or that UNC-93B is not required for class II antigen presentation in human antigen-presenting cells.