Tina Zupancic

Inclusion bodies as potential vehicles for recombinant protein delivery into epithelial cells.

BackgroundWe present the potential of inclusion bodies (IBs) as a protein delivery method for polymeric filamentous proteins. We used as cell factory a strain of E. coli, a conventional host organism, and keratin 14 (K14) as an example of a complex protein. Keratins build the intermediate filament cytoskeleton of all epithelial cells. In order to build filaments, monomeric K14 needs first to dimerize with its binding partner (keratin 5, K5), which is then followed by heterodimer assembly into filaments.ResultsK14 IBs were electroporated into SW13 cells grown in culture together with a “reporter” plasmid containing EYFP labeled keratin 5 (K5) cDNA. As SW13 cells do not normally express keratins, and keratin filaments are built exclusively of keratin heterodimers (i.e. K5/K14), the short filamentous structures we obtained in this study can only be the result of: a) if both IBs and plasmid DNA are transfected simultaneously into the cell(s); b) once inside the cells, K14 protein is being released from IBs; c) released K14 is functional, able to form heterodimers with EYFP-K5.ConclusionsSoluble IBs may be also developed for complex cytoskeletal proteins and used as nanoparticles for their delivery into epithelial cells.

Intestinal cell barrier function in vitro is severely compromised by keratin 8 and 18 mutations identified in patients with inflammatory bowel disease

Keratin 8 and 18 (K8/K18) mutations have been implicated in the aetiology of certain pathogenic processes of the liver and pancreas. While some K8 mutations (K8 G62C, K8 K464N) are also presumed susceptibility factors for inflammatory bowel disease (IBD), the only K18 mutation (K18 S230T) discovered so far in an IBD patient is thought to be a polymorphism. The aim of our study was to demonstrate that these mutations might also directly affect intestinal cell barrier function. Cell monolayers of genetically engineered human colonocytes expressing these mutations were tested for permeability, growth rate and resistance to heat-stress. We also calculated the change in dissociation constant (Kd, measure of affinity) each of these mutations introduces into the keratin protein, and present the first model of a keratin dimer L12 region with in silico clues to how the K18 S230T mutation may affect keratin function. Physiologically, these mutations cause up to 30% increase in paracellular permeability in vitro. Heat-stress induces little keratin clumping but instead cell monolayers peel off the surface suggesting a problem with cell junctions. K18 S230T has pronounced pathological effects in vitro marked by high Kd, low growth rate and increased permeability. The latter may be due to the altered distribution of tight junction components claudin-4 and ZO-1. This is the first time intestinal cells have been suggested also functionally impaired by K8/K18 mutations. Although an in vitro colonocyte model system does not completely mimic the epithelial lining of the intestine, nevertheless the data suggest that K8/K18 mutations may be also able to produce a phenotype in vivo.

Archaeosomes can efficiently deliver different types of cargo into epithelial cells grown in vitro

Archaeosomes are a type of liposomes prepared from the polar lipids of various Archaeobacteria. These have unique structural features that increase the lipid bilayers stability even under high temperatures, low or high pH, presence of phospholipases and bile salts. This makes them ideal as basis for the development of new drug, gene and vaccine delivery systems. In this study we prepared large unilamellar archaeosomes (400nm size) from Aeropyrum pernix K1 and demonstrated their potential as base for the development of an efficient and universal system for drug or therapy delivery to epithelial cells. Our archaeosomes may be used to deliver small fluorescent molecules (calcein), smaller proteins (60kDa listeriolysin), large protein aggregates (e.g. keratin 14) and plasmid DNA, into epithelial cells grown in culture. The delivery efficiency for small molecules is already quite high at this initial stage of development, around 40%. Our unilamellar archaeosomes are also not toxic to keratinocytes even at high doses (500μg/ml).

The Caco-2 cell culture model enables sensitive detection of enhanced protein permeability in the presence of N-decyl-β-d-maltopyranoside.

Appropriate assessment of transepithelial permeability in vitro is needed to estimate and model trans-mucosal bioavailability to achieve oral delivery of protein biopharmaceuticals. The Caco-2 cell-based intestinal epithelium model is widely used for this purpose for low molecular mass drugs. The aim of this study was to test the suitability of the Caco-2 model for assessing enhanced transepithelial permeability to proteins. Four unrelated proteins were chosen to test the permeability of Caco-2 monolayers. It was found that proteins could cross the epithelium model, in spite of their size. All tested proteins had similar very low apparent permeability coefficients (Papp) of around 4×10(-10)cm/s. Protein stability over three-hour exposure to Caco-2 was also confirmed. Their crossing rate in a cell-free setup was also measured, to determine the upper limit of permeability to proteins. An epithelium permeability enhancer N-decyl-β-d-maltopyranoside (MP C10) was used to demonstrate accelerated permeability conditions. Papp values could be increased dose dependently up to about 1×10(-7)cm/s, close to the level in the cell-free setup, indicating distinctive potential of the model. This along with enhancing effect of known specific route permeators suggests involvement of the paracellular route in protein transport. Our results thus indicate that the Caco-2 model is a suitable tool for in vitro assessment of enhanced permeability to proteins.

Phosphate Removal During Long Nocturnal Hemodialysis/Hemodiafiltration: A Study With Total Dialysate Collection

The aim of our prospective study was to quantify phosphate removal during long nocturnal high‐flux hemodialysis or hemodiafiltration (HD) with total dialysate collection. Eight patients (two women) were studied for the first‐in‐the‐week HD session that lasted 7–8 h. Total dialysate was collected. Serum and collected dialysate phosphate concentrations were measured every hour. Phosphate removal as assessed from the serum concentration was most important during the first 2 h of HD, and then a plateau was reached. The highest average phosphate concentration in the total dialysate was in the 1st hour, thereafter the concentration decreased but remained stable. The average total removed mass of phosphate quantified from hourly collected dialysate was 5195.7 ± 1898 mg. Phosphate had been removed in a consistent manner during the whole duration of nocturnal HD as assessed through dialysate (despite stable serum phosphate concentration). This could indicate phosphate transfer from intracellular space. The total removed phosphate quantified from the total dialysate collection was higher than previously reported and exceeded the normal phosphate food intake.

Keratinocyte-based cell assays : their potential pitfalls

As an in vitro model system, patient-derived epidermolysis bullosa simplex keratinocytes have had an immense impact on what we know today about keratin filament function and their role in disease development. In the absence of gene therapy, screening compound libraries for new or better drugs is another approach to improve existing treatments for genodermatoses. However in this study, we report of the potential pitfalls when using this type of cell lines as a “reporter” system. When cell lines with different genetic backgrounds are being used in cell-based assays, the greatest obstacle is to determine the most appropriate culture conditions (i.e., the composition of medium, number of cells plated and number of days in culture). We demonstrate how culture conditions can greatly interfere with the cellular response in cell-based assays (cell proliferation, metabolic activity and migration), potentially also giving rise to misleading data.

Phosphate or Phosphorus Removal During Nocturnal Hemodialysis: Are We Using Correct Terms?

Dear Editor, Last year we published an article regarding phosphate removal during nocturnal hemodialysis (1). It has recently been kindly brought to our attention in a personal correspondence from Professor John T. Daugirdas, that our measured results differ significantly from the results of a kineticmodel he has developed (2), and that we might have made a calculation error. We have therefore looked at the results and calculations again and found that while the results “per se” are correct, they can still be misunderstood and misleading, and therefore we wish to publish a correction. The main issue is the way phosphate intake and removal is reported in the literature. While often in the literature on phosphate in chronic kidney disease and dialysis (3,4) the term “phosphate” (PO4 , the form in which “phosphorus”[P] occurs in humans) is used, what is actually reported is the amount of phosphorus. While there is no difference when reported in SI units (mmol), the difference is threefold when phosphorus and phosphate are expressed in conventional units (mg). Although both terms are often considered synonymous in the literature, the usage of the term phosphate, when actually referring to phosphorus, is incorrect from the chemical point of view and misleading (5). This inconsistency is also the reason our data can be misunderstood. In our article we have reported a mean removal of phosphate to be 5195 ± 1898 mg per dialysis procedure (54.7 mmol). This is “per se” correct, as we were referring to the phosphate, meaning literally the PO4 ion. We would kindly ask for a correction, as a more conventional way would be to express it as phosphorus, which is then 1696 ± 575 mg per procedure for the whole group, 1510 ± 507 mg per procedure for on-line HDF and 2253 ± 453 mg per procedure for high-flux HD, P = 0.21. Although the difference is threefold and we have cited amounts expressed as phosphorus in the discussion, the content of the discussion nevertheless remains valid. We would like to thank Professor Daugirdas for bringing this issue to our attention.

Keratin gene mutations influence the keratinocyte response to DNA damage and cytokine induced apoptosis

The keratin filament cytoskeleton is vital to the normal function of epithelial cells. It provides structural support and regulates different aspects of cell metabolism. Mutations in keratins 5 and 14 cause a skin fragility disorder, epidermolysis bullosa simplex (EBS). Patients with severe EBS have an increased cumulative risk for basal cell carcinoma. In this study, we tested how keratin 5 and 14 mutant EBS patient-derived keratinocytes behave in the face of two different types of stressors that are able to induce cell death: ionizing radiation and cytokines TNF-α and TRAIL. The data point out to a substantial difference between how normal and keratin mutant keratinocytes deal with such stresses. When case of DNA damage, the ATM/Chk2-pathway is one of the two main tracks that can prevent the progression of mitosis and so allow repair. This was altered in all investigated keratin mutants with a particular down-regulation of the activated form of checkpoint kinase 2 (pChk2). Keratin mutants also appear less sensitive than normal cells to treatment with TNF-α or TRAIL, and this may be linked to the up-regulation of two pro-survival proteins, Bcl-2 and FLIP. Such changes are likely to have a profound effect on mutant keratinocytes ability to survive and withstand stress, and in theory this may be also a contributing factor to cell transformation.

Death receptor signaling and DNA damage response mechanisms are altered in EBS Dowling-Meara keratinocytes

S | Growth Factors and Signal Transduction