Tailoring secretion of proteoglycan 4 (PRG4) in tissue-engineered cartilage

Articular cartilage provides a low-friction surface for joint articulation, with boundary lubrication facilitated by proteoglycan 4 (PRG4), which is secreted by chondrocytes of the superficial zone. Chondrocytes from different zones are phenotypically distinct, and their phenotypes in vitro are influenced by the system in which they are cultured. We hypothesized that culturing cells from the superficial (S) zone in two-dimensional monolayer or three-dimensional alginate would affect their synthesis of PRG4, and that subsequently seeding them atop alginate-recovered cells from the middle/ deep (M) zone in various proportions would result in tissue-engineered constructs with varying levels of PRG4 secretion and matrix accumulation. During monolayer culture, S cells retained their PRG4-secreting phenotype, whereas in alginate culture the percentage of cells secreting PRG4 decreased with time. Constructs formed with increasing percentages of S cells decreased in thickness and matrix accumulation, depending on both the culture conditions before construct formation and the S-cell density. PRG4-secreting cells were localized to the S-cell seeded construct surface, with secretion rates of 0.1–4 pg/cell/day or 0.1–1 pg/cell/day for constructs formed with monolayer-recovered or alginate-recovered S cells, respectively. Tailoring secretion of PRG4 in cartilage constructs may be useful for enhancing low-friction properties at the articular surface, while maintaining other surfaces free of PRG4 for enhancing integration with surrounding tissues.

Short-term retention of labeled chondrocyte subpopulations in stratified tissue-engineered cartilaginous constructs implanted in vivo in mini-pigs

It is likely that effective application of cell-laden implants for cartilage defects depends on retention of implanted cells and interaction between implanted and host cells. The objectives of this study were to characterize stratified cartilaginous constructs seeded sequentially with superficial (S) and middle (M) chondrocyte subpopulations labelled with fluorescent cell tracking dye PKH26 (*) and determine the degree to which these stratified cartilaginous constructs maintain their architecture in vivo after implantation in mini-pigs for 1 week. Alginate-recovered cells were seeded sequentially to form stratified S*/M (only S cells labelled) and S*/M* (both S and M cells labelled) constructs. Full-thickness defects (4 mm diameter) were created in the patellofemoral groove of adult Yucatan mini-pigs and filled with portions of constructs or left empty. Constructs were characterized biochemically, histologically, and biomechanically, and stratification visualized and quantified, before and after implant. After 1 week, animals were sacrificed and implants retrieved. After 1 week in vivo, glycosaminoglycan and collagen content of constructs remained similar to that at implant, whereas DNA content increased. Histological analyses revealed features of an early repair response, with defects filled with tissues containing little matrix and abundant cells. Some implanted (PKH26-labeled) cells persisted in the defects, although constructs did not maintain a stratified organization. Of the labelled cells, 126 +/- 38% and 32 +/- 8% in S*/M and S*/M* constructs, respectively, were recovered. Distribution of labelled cells indicated interactions between implanted and host cells. Longer-term in vivo studies will be useful in determining whether implanted cells are sufficient to have a positive effect in repair.

Foreword

Ghrelin was first identified in 1999 by Kojima and colleagues (Kojima et al. 1999) as the natural ligand of an orphan G-protein coupled receptor, the Growth Hormone (GH) secretagogue receptor (GHS-R), which had been identified several years earlier through the actions of a growing number of synthetic growth hormone releasing peptides (GHRPs) and non-peptidyl GH secretagogues (Howard et al. 1996). Early studies, therefore, focussed on the actions of ghrelin as an important regulator of GH secretion. As a result Kojima et al (1999) designated this GH-releasing peptide, ghrelin (ghre is the Proto-Indo-European root of the word 'grow'). We now recognise that the functions of ghrelin extend well beyond its GH releasing actions and that it is a multi-functional peptide with both endocrine and autocrine/paracrine modes of action.

Rodent blood-stage Plasmodium survive in dendritic cells that infect naive mice.

Plasmodium spp. parasites cause malaria in 300 to 500 million individuals each year. Disease occurs during the blood-stage of the parasite’s life cycle, where the parasite is thought to replicate exclusively within erythrocytes. Infected individuals can also suffer relapses after several years, from Plasmodium vivax and Plasmodium ovale surviving in hepatocytes. Plasmodium falciparum and Plasmodium malariae can also persist after the original bout of infection has apparently cleared in the blood, suggesting that host cells other than erythrocytes (but not hepatocytes) may harbor these blood-stage parasites, thereby assisting their escape from host immunity. Using blood stage transgenic Plasmodium berghei-expressing GFP (PbGFP) to track parasites in host cells, we found that the parasite had a tropism for CD317+ dendritic cells. Other studies using confocal microscopy, in vitro cultures, and cell transfer studies showed that blood-stage parasites could infect, survive, and replicate within CD317+ dendritic cells, and that small numbers of these cells released parasites infectious for erythrocytes in vivo. These data have identified a unique survival strategy for blood-stage Plasmodium, which has significant implications for understanding the escape of Plasmodium spp. from immune-surveillance and for vaccine development.

A variant of the KLK4 gene is expressed as a cis sense-antisense chimeric transcript in prostate cancer cells

In humans, more than 30,000 chimeric transcripts originating from 23,686 genes have been identified. The mechanisms and association of chimeric transcripts arising from chromosomal rearrangements with cancer are well established, but much remains unknown regarding the biogenesis and importance of other chimeric transcripts that arise from nongenomic alterations. Recently, a SLC45A3–ELK4 chimera has been shown to be androgen-regulated, and is overexpressed in metastatic or high-grade prostate tumors relative to local prostate cancers. Here, we characterize the expression of a KLK4 cis sense–antisense chimeric transcript, and show other examples in prostate cancer. Using non-protein-coding microarray analyses, we initially identified an androgen-regulated antisense transcript within the 3′ untranslated region of the KLK4 gene in LNCaP cells. The KLK4 cis-NAT was validated by strand-specific linker-mediated RT-PCR and Northern blotting. Characterization of the KLK4 cis-NAT by 5′ and 3′ rapid amplification of cDNA ends (RACE) revealed that this transcript forms multiple fusions with the KLK4 sense transcript. Lack of KLK4 antisense promoter activity using reporter assays suggests that these transcripts are unlikely to arise from a trans-splicing mechanism. 5′ RACE and analyses of deep sequencing data from LNCaP cells treated ±androgens revealed six high-confidence sense–antisense chimeras of which three were supported by the cDNA databases. In this study, we have shown complex gene expression at the KLK4 locus that might be a hallmark of cis sense–antisense chimeric transcription.

Expression of PSA-RP2, an alternatively spliced variant from the PSA gene, is increased in prostate cancer tissues but the protein is not secreted from prostate cancer cells

PSA-RP2 is a variant transcript expressed from the PSA gene that is conserved in gorillas, chimpanzees and humans suggesting a particular relevance for this transcript in these primates. We demonstrated by qRT-PCR that PSA-RP2 is upregulated in prostate cancer compared with benign prostatic hyperplasia tissues. The PSA-RP2 protein was not detected in seminal fluid and was cytoplasmically localised but not secreted from LNCaP or transfected PC3 prostate cells, despite secretion from transfected Cos-7 and HEK293 kidney cell lines. PSA-RP2-transfected PC3 cells showed slightly decreased proliferation and increased migration towards PC3-conditioned medium that could suggest a functional role in prostate cancer.