A Poroelasticity Theory Approach to Study the Mechanisms Leading to Elevated Interstitial Fluid Pressure in Solid Tumours

Although the mechanisms responsible for elevated interstitial fluid pressure (IFP) in tumours remain obscure, it seems clear that high IFP represents a barrier to drug delivery (since the resulting adverse pressure gradient implies a reduction in the driving force for transvascular exchange of both fluid and macromolecules). R. Jain and co-workers studied this problem, and although the conclusions drawn from their idealized mathematical models offered useful insights into the causes of elevated IFP, they by no means gave a definitive explanation for this phenomenon. In this paper, we use poroelasticity theory to also develop a macroscopic mathematical model to describe the time evolution of a solid tumour, but focus our attention on the mechanisms responsible for the rise of the IFP, from that for a healthy interstitium to that measured in malignant tumours. In particular, we discuss a number of possible time scales suggested by our mathematical model and propose a tumour-dependent time scale that leads to results in agreement with experimental observations. We apply our mathematical model to simulate the effect of “vascular normalization” (as proposed by Jain in Nat Med 7:987–989, 2001) on the IFP profile and discuss and contrast our conclusions with those of previous work in the literature.     

Purification of cell culture‐derived modified vaccinia ankara virus by pseudo‐affinity membrane adsorbers and hydrophobic interaction chromatography

A purification scheme for cell culture‐derived smallpox vaccines based on an orthogonal downstream process of pseudo‐affinity membrane adsorbers (MA) and hydrophobic interaction chromatography (HIC) was investigated. The applied pseudo‐affinity chromatography, based on reinforced sulfated cellulose and heparin‐MA, was optimized in terms of dynamic binding capacities, virus yield and process productivity. HIC was introduced as a subsequent method to further reduce the DNA content. Therefore, two screens were undertaken. First, several HIC ligands were screened for different adsorption behavior between virus particles and DNA. Second, elution from pseudo‐affinity MA and adsorption of virus particles onto the hydrophobic interaction matrix was explored by a series of buffers using different ammonium sulfate concentrations. Eventually, variations between different cultivation batches and buffer conditions were investigated.The most promising combination, a sulfated cellulose membrane adsorber with subsequent phenyl HIC resulted in overall virus particle recoveries ranging from 76% to 55% depending on the product batch and applied conditions. On average, 61% of the recovered virus particles were infective within all tested purification schemes and conditions. Final DNA content varied from 0.01% to 2.5% of the starting material and the level of contaminating protein was below 0.1%. Biotechnol. Bioeng. 2010;107: 312–320. © 2010 Wiley Periodicals, Inc.     

The Role of Glycosaminoglycan Binding of Staphylococci in Attachment to Eukaryotic Host Cells

Attachment of microorganisms to host cells is believed to be a critical early step in microbial pathogenesis. The aim of the study was to determine the role of the known glycosaminoglycan (GAG) binding activity of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) in their attachment to six different eukaryotic cell lines. Three staphylococcal species expressing GAG binding capacity—S. aureus, S. epidermidis, and S. hemolyticus—were chosen for investigation. Six different eukaryotic cell lines, endothelial HUVEC and EA. hy 926 cells, epithelial A549 and HeLa S3 cells, fibroblasts HEL Sp 12 and macrophages J774.A1, were included. A modified ELISA with biotinylated bacteria was used for estimating the adhesion of staphylococci to each of the cell lines. Our results showed that staphylococci adhered to each of the cell lines studied, although the binding of CoNS strains to epithelial cells was lower than to the other cells. The attachment to all cell types could be partially decreased by pretreatment of the bacteria with various polysulfated agents (highest inhibition was 60%), as well as by chlorate and heparitinase treatment of the cells. These observations may suggest that at least one mode of staphylococcal attachment utilizes GAG chains present on the surface of virtually all adherent cells. Received: 6 September 2000 / Accepted: 29 December 2000     

Comparison of early passage, senescent and hTERT immortalized endothelial cells

The need for standardized experimental conditions to gain relevant and reproducible results has increased the demand for well characterized continuously growing cell lines that exhibit the characteristics of their normal counterparts. Immortalization of normal human cells by ectopic expression of the catalytic subunit of human telomerase (hTERT) has shown to result in highly differentiated cell lines. However, the influence of the increased telomerase activity on the protein expression profile was not investigated so far. Therefore, we have immortalized human umbilical vein endothelial cells (HUVECs) by hTERT overexpression and compared them to their normal early passage and senescent counterparts. This study, including a proteomic approach, shows that ectopic hTERT expression leads to a stable growing cell line. Although these cells are highly differentiated, the protein expression profile of the cell line is different to that of normal early passage and senescent cells.     

Genes and enzymes involved in caffeic acid biosynthesis in the actinomycete Saccharothrix espanaensis

The saccharomicins A and B, produced by the actinomycete Saccharothrix espanaensis, are oligosaccharide antibiotics. They consist of 17 monosaccharide units and the unique aglycon N-(m,p-dihydroxycinnamoyl)taurine. To investigate candidate genes responsible for the formation of trans-m,p-dihydroxycinnamic acid (caffeic acid) as part of the saccharomicin aglycon, gene expression experiments were carried out in Streptomyces fradiae XKS. It is shown that the biosynthetic pathway for trans-caffeic acid proceeds from L-tyrosine via trans-p-coumaric acid directly to trans-caffeic acid, since heterologous expression of sam8, encoding a tyrosine ammonia-lyase, led to the production of trans-p-hydroxycinnamic acid (coumaric acid), and coexpression of sam8 and sam5, the latter encoding a 4-coumarate 3-hydroxylase, led to the production of trans-m,p-dihydroxycinnamic acid. This is not in accordance with the general phenylpropanoid pathway in plants, where trans-p-coumaric acid is first activated before the 3-hydroxylation of its ring takes place.     

New strategy for identification of novel Cry-type genes from Bacillus thuringiensis strains

We designed five degenerate primers for detection of novel cry genes from Bacillus thuringiensis strains. An efficient strategy was developed based on a two-step PCR approach with these primers in five pair combinations. In the first step, only one of the primer pairs is used in the PCR, which allows amplification of DNA fragments encoding protein regions that include consensus domains of representative proteins belonging to different Cry groups. A second PCR is performed by using the first-step amplification products as DNA templates and the set of five primer combinations. Cloning and sequencing of the last-step amplicons allow both the identification of known cry genes encoding Cry proteins covering a wide phylogenetic distance and the detection and characterization of cry-related sequences from novel B. thuringiensis isolates.