Extremophiles - Platform Strains for Sustainable Production of Polyhydroxyalkanoates

Polyhydroxyalkanoates (PHA) are biodegradable polyesters, which are produced by various bacteria including numerous halophiles. Employment of halophilic strain for PHA production brings numerous benefits such as robustness of the process against contamination by ubiquitous mesophiles or possibility to isolate polymer from bacterial biomass via hypotonic lysis. In this work, we screened three moderate halophiles – Halomonas halophila, Halomonas organivorans and Halomonas salina for the presence of phaC gene encoding for PHA synthase and, subsequently, we have investigated their PHA production potential on various sugars. Among tested strains, H. organivorans demonstrated the extraordinary capacity of PHA production in particular on galactose and mannose since on these saccharides PHA content in dried bacterial cells reached 83 and 90 wt. % on mannose and galactose, respectively. Therefore, H. organivoras can be considered being promising PHA producing strain in particular suitable for the valorization of lignocellulose materials rich in galactomannans such as spent coffee grounds.

IHP-Loaded Red Blood Cells as Preventive Therapy In Sickle Cell Disease

Abstract 1625 Sickle cell disease (SCD) is a genetic disorder characterized by abnormal hemoglobin S (HbS) that polymerizes under hypoxic conditions leading to sickled-shape red blood cells (RBCs). Vaso-occlusive crisis (VOC) is one of the major clinical manifestations of the disease, very painful for patients and causing irreversible organ damages. RBC exchange is commonly used as preventive and curative treatment of the disease. However, the therapeutic action of RBC exchange only relies on the removal of HbS-containing RBCs (SS-RBCs) and their transient replacement by normal RBCs (AA-RBCs). Recent works have shown that sickled reticulocytes, activated platelets and leukocytes play a critical role in the onset of VOC. They aggregate with endothelial cells creating local hypoxia, enhancing sickling and thus capillary blockade. Oxygen deprivation that occurs in venous capillaries may widely contribute to the severity of the occlusion. Therefore, increasing the oxygenation level in capillaries could help to prevent SS-RBCs from sickling and avoid crisis. This may be possible by transfusing patients with AA-RBCs loaded with Inositol HexaPhosphate (IHP), an allosteric effector that binds tightly to hemoglobin. The resulting suspension (IHP-RBCs) has the ability to increase oxygen release by 2 to 3 fold compared to normal AA-RBCs. The objective of the present study was to evaluate in vivo the benefit of using IHP-RBCs treatment in SCD. We used BERK transgenic mouse model that fully mimics human SCD in childhood with specific features of splenomegaly, reticulocytosis and leukocytosis. IHP-RBCs were prepared by loading IHP into murine C57BL6J RBCs using reversible hypotonic lysis method. RBCs subjected to reversible hypotonic lysis but without IHP were used as control suspension. Study was designed with repeated RBC exchanges scheduled every 2 weeks. First RBC exchange using IHP-RBCs or control suspension was performed on 6–7 week-old mice followed by 2 further injections. Mice were sacrificed one week after last RBC exchange and critical hematological parameters (reticulocyte, leukocyte, platelet counts and sickled cells) as well as serum inflammation markers were used as readouts to evaluate the risk of VOC. The first study was performed in normoxic conditions. After the therapy, approximately 42% of mouse RBCs had been replaced by IHP-RBCs or control suspension. Strong reduction of spleen weight (50%) and circulating sickled RBCs was observed in both cases due to the dilution of SS-RBCs. Interestingly, IHP-RBCs treatment enabled to significantly lower reticulocytes (18% vs 31%), leukocytes (5.3 vs 8.4 103/μl) and platelet counts (1057 vs 1518 103/μl) compared to not treated mice. Additionally, Serum Amyeloid Protein (SAP), an inflammation marker analogous of human C-Reactive Protein was also significantly reduced with IHP-RBCs (450 vs 750 μg/ml) indicating lowered severity of inflammation. The analysis of VCAM and HIF-1 factors in both spleen and lungs were very low in both treated and not treated mice. Further experiments demonstrated that hypoxic stress is needed to induce significative inflammation at the organ level. The study will thus be repeated in hypoxic conditions to evaluate the effect of IHP-RBCs treatment on organ damaging. We had in a previous study demonstrated in vitro the ability of IHP-RBCs to reduce sickling of human SS-RBCs (Bourgeaux et al, Transfusion, in press). The present in vivo study brings new evidence of the therapeutic potential of IHP-RBCs with the observation of a significant reduction of VOC risk factors and SAP level in treated mice. These results strongly support the fact that loading IHP into AA-RBCs may improve the effectiveness of conventional transfusion therapy. Disclosures: No relevant conflicts of interest to declare.

A Novel Population of Oct-4+ SSEA-1+ CXCR4+ CD34+ CD133+ Lin− CD45− Very Small Embryonic-Like (VSEL) Stem Cells Identified in Human Cord Blood.

Mononuclear cells (MNC) isolated from bone marrow (BM) or cord blood (CB) contributes to organ/tissue regeneration, however, the identity of the specific cell type(s) involved remains unknown. Recently we identified in murine BM a homogenous population of rare (~0.01% of BM MNC) Sca-1+ lin− CD45− cells that express by RQ-PCR and immunhistochemistry markers of pluripotent stem cells (PSC) such as SSEA-1, Oct-4, Nanog and Rex-1, highly express Rif-1 telomerase protein and display several features typical for primary embryonic stem cells such as a small size (~2–4 um in diameter), a large nuclei surrounded by a narrow rim of cytoplasm, and open-type chromatin (euchromatin) that is typical for embryonic stem cells (Leukemia2006;20,857–869). These cells were named very small embryonic-like (VSELs) stem cells. We will present a new two step isolation procedure to purify a similar population of cells from human CB, which is based on isolation of CB mononuclear cells (CB MNC) by hypotonic lysis and multiparameter FACS sorting. Accordingly, we perform hypotonic lysis of CB to remove erythrocytes and to enrich for CB MNC combined with multiparameter sorting for CXCR4+AC133+CD34+lin−CD45− CB MNC. CB-derived VSELs (CB-VSELs) isolated this way similarly as those isolated from adult murine BM are very small (3–5 um), possess large nuclei containing unorganized euchromatin, express nuclear embryonic transcription factors Oct-4 and Nanog and surface embryonic antigen SSEA-4. In vitro cultures CB-VSELs are able to grow neurospheres that gave rise to neuronal lineages (beta-III tubulin+, nestin+, O4+, MBP+, GFAP+) and cardiomyocytes (beta-myosin heavy chain+, alpha-sarcomeric actin. Based on this we conclude that CB contains VSELs and that the majority of these CB VSELs are lost during routine procedures employed currently for banking of CB MNC. Thus based on our observations, new more efficient methods of CB banking are needed that will enrich/preserve these cells in CB units during preparation before storage. Furthermore, we conclude that CB tissue/organ regenerating potential may be much higher than initially postulated if the proper fraction of CB MNC is employed and we are currently testing this hypothesis in animal models.