Cell manipulation in autologous chondrocyte implantation: from research to cleanroom.

In the field of orthopaedics, autologous chondrocyte implantation is a technique currently used for the regeneration of damaged articular cartilage. There is evidence of the neo-formation of tissue displaying characteristics similar to hyaline cartilage. In vitro chondrocyte manipulation is a crucial phase of this therapeutic treatment consisting of different steps: cell isolation from a cartilage biopsy, expansion in monolayer culture and growth onto a three-dimensional biomaterial to implant in the damaged area. To minimise the risk of in vitro cell contamination, the manipulation must be performed in a controlled environment such as a cleanroom. Moreover, the choice of reagents and raw material suitable for clinical use in humans and the translation of research protocols into standardised production processes are important. In this study we describe the preliminary results obtained by the development of chondrocyte manipulation protocols (isolation and monolayer expansion) in cleanrooms for the application of autologous implantation.

Loss of heterozygosity at 17p13.3-ter, distal to TP53, correlates with negative hormonal phenotype in sporadic breast cancer.

Genetic alterations on chromosome 17p are frequent in a variety of human malignancies such as sporadic breast carcinomas. The clinico-pathological significance of it remains to be elucidated. The purpose of this study was to explore whether the allelic loss (LOH) in 17p13.3, which is suggested by the presence of tumour suppressor genes, independent of TP53, are related to current clinico-pathological criteria for characterising breast cancer. A group of sporadic breast carcinomas, with no alteration in TP53 locus, were analysed for the presence of LOH in D17S34 and D17S30/5 loci, mapped to the 17p13.3 region, distinct from and telomeric to TP53. LOH by at least one marker was observed in 13 of 47 informative cases (27.6%). Clinicopathological parameters such as age, menopausal status, histological type, tumour size, nodal status, grading, ploidy, labelling index, S-phase fraction and hormonal phenotype, were evaluated. LOH at distal 17p13.3 significantly correlated with the absence of oestrogen receptors (ER) (P=0.018), progesterone receptors (PgR) (P=0.009) and concordant absence of either ER or PgR (P=0.006). This may provide a basis for speculation as to the function of the putative tumour suppressor genes in 17p13-ter in sporadic breast cancer.

Mitochondrial DNA D-loop as a new target of Saporin 6 nuclease activity.

The single-chain ribosome-inactivating proteins (RIPs) from plant origin, including Saporin 6 from the seeds of Saponaria officinalis, are ribotoxins known to act as N-glycosidases which depurinate the conserved alpha sarcin loop of large rRNAs. As a consequence, the eukaryotic ribosomes become inactivated, thereby arresting the protein synthesis at the elongation step. RIPs are currently under study as antiviral and antiproliferative agents. Additional in vitro activities of RIPs against either RNA or DNA have been recently described. A specific nuclease activity on plasmidic DNA was demonstrated by either purified or bacterial-recombinant molecules. We report here that human mitochondrial DNA (mtDNA) is a new specific target of Saporin 6 nuclease activity. A unique site of cleavage has been identified and mapped within the most variable part of the D-loop region of the covalently closed circular mtDNA molecule.

Loss of heterozygosity at pseudoautosomal regions in human breast cancer and association with negative hormonal phenotype.

To determine the possible involvement of X-linked genes in breast cancer, a group of human sporadic breast carcinomas were analyzed for loss of heterozygosity (LOH) at 12 polymorphic loci distributed along the whole chromosome X. LOH by at least one marker was observed in 14 of 46 informative cases and two regions of consistent LOH in 10 of 14 (71.4%) were identified at pseudoautosomal regions (PAR). Allelic losses in these regions significantly correlated with the absence of estrogen receptors (P<0.05) and concordant absence of either estrogen (ER) or progesterone (PgR) receptors (P<0.05). The clinicopathological parameters evaluated, (like age, menopausal status, histological type, tumor size, nodal status, grading, ploidy, labeling index, and S-phase fraction), were independent from the LOH present in the PAR regions of X chromosome. This study suggests a role as a prognostic factor for LOH and ER(-)/PgR(-) when associated and provides some additional support for the existence of candidate tumor suppressor gene on PAR regions, whose alteration may play a role in breast cancer development and progression.