Hematopoietic stem/progenitor cell sources to generate reticulocytes for Plasmodium vivax culture.

The predilection of Plasmodium vivax (P. vivax) for reticulocytes is a major obstacle for its establishment in a long-term culture system, as this requires a continuous supply of large quantities of reticulocytes, representing only 1-2% of circulating red blood cells. We here compared the production of reticulocytes using an established in vitro culture system from three different sources of hematopoietic stem/progenitor cells (HSPC), i.e. umbilical cord blood (UCB), bone marrow (BM) and adult peripheral blood (PB). Compared to CD34+-enriched populations of PB and BM, CD34+-enriched populations of UCB produced the highest amount of reticulocytes that could be invaded by P. vivax. In addition, when CD34+-enriched cells were first expanded, a further extensive increase in reticulocytes was seen for UCB, to a lesser degree BM but not PB. As invasion by P. vivax was significantly better in reticulocytes generated in vitro, we also suggest that P. vivax may have a preference for invading immature reticulocytes, which should be confirmed in future studies

The Usefulness of Toxicogenomics for Predicting Acute Skin Irritation on in Vitro Reconstructed Human Epidermis

In vitro models aiming at replacing the traditional animal test for determining the skin irritation potential of a test substance have been developed, evaluated in prevalidation studies and recently validated by the European Centre for the Validation of Alternative Methods (ECVAM). To investigate the usefulness of toxicogenomic technologies to identify novel mechanistic endpoints for skin irritation responses, the present work challenged the human reconstituted epidermis model validated by ECVAM with 4 irritant chemicals and 4 non classified chemicals tested at subcytotoxic concentrations. Using a specifically designed low density DNA array, about 50 genes out of 240 were found to be significantly and differentially expressed between tissues exposed to irritant and non irritant chemicals for at least one test chemical when compared to the seven others. These genes are involved in cell signalling, stress response, cell cycle, protein metabolism and cell structure. Among them, 16 are expressed in the same way whatever the irritant compound applied. The differential gene expressions might represent new or additional endpoints useful for the mechanistic understanding and perhaps also the hazard assessment of the skin irritation potential of chemicals and products.JRC.I.2-Validation of biomedical testing method

1912-2012: a century of research on Plasmodium vivax in vitro culture

The development of a continuous Plasmodium vivax blood cycle in vitro was first attempted 100 years ago. Since then, and despite the use of different methods, only short-term cultures have been achieved so far. The available literature has been reviewed in order to provide a critical overview of the currently available knowledge on P. vivax blood cycle culture systems and identify some unexplored ways forward. Results show that data accumulated over the past century remain fragmented and often contradictory, making it difficult to draw conclusions. There is the need for an international consortium on P. vivax culture able to collect, update, and share new evidence, including negative results, and thus better coordinate current efforts towards the establishment of a continuous P. vivax culture

Cryopreserved reticulocytes derived from hematopoietic stem cells can be invaded by cryopreserved Plasmodium vivax isolates

The development of a system for the continuous culture of Plasmodium vivax in vitro would benefit from the use of reticulocytes derived from differentiated hematopoietic stem cells (HCS). At present, the need to use both fresh reticulocytes and fresh P. vivax isolates represents a major obstacle towards this goal, particularly for laboratories located in non-endemic countries. Here, we describe a new method for the cryopreservation of HSC-derived reticulocytes to be used for both P. falciparum and P. vivax invasion tests. Cryopreserved P. falciparum and P. vivax isolates could invade both fresh and cryopreserved HSC-derived reticulocytes with similar efficiency. This new technique allows the storage of HSC-derived reticulocytes which can be used for later invasion tests and represents an important step towards the establishment of a continuous P. vivax culture.status: publishe

Hematopoietic stem/progenitor cell sources to generate reticulocytes for Plasmodium vivax culture

The predilection of Plasmodium vivax (P. vivax) for reticulocytes is a major obstacle for its establishment in a long-term culture system, as this requires a continuous supply of large quantities of reticulocytes, representing only 1-2% of circulating red blood cells. We here compared the production of reticulocytes using an established in vitro culture system from three different sources of hematopoietic stem/progenitor cells (HSPC), i.e. umbilical cord blood (UCB), bone marrow (BM) and adult peripheral blood (PB). Compared to CD34+-enriched populations of PB and BM, CD34+-enriched populations of UCB produced the highest amount of reticulocytes that could be invaded by P. vivax. In addition, when CD34+-enriched cells were first expanded, a further extensive increase in reticulocytes was seen for UCB, to a lesser degree BM but not PB. As invasion by P. vivax was significantly better in reticulocytes generated in vitro, we also suggest that P. vivax may have a preference for invading immature reticulocytes, which should be confirmed in future studies.status: publishe