Intrabone Injection of T-Cell Depleted Peripheral Blood Stem Cells from HLA-Haploidentical Donors to Reduce the Risk of Graft Rejection in Children

Background. Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is an effective treatment option for patients with malignant and non-malignant hematologic disorders lacking an HLA-compatible donor. Strategies for T-cell depletion (TCD) of the graft, such as positive selection of CD34+ cells, offer the potential to prevent acute and chronic graft-versus-host disease (GVHD). The risk of graft rejection associated with the extensive depletion of both T lymphocytes and accessory cells can be overcome by infusing a very high number (megadose) of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSC) (exceeding 10x106/kg recipient body weight) to overcome the HLA barrier (Aversa F. et al. Blood 1994). Moreover, the infusion of a megadose of CD34+ cells (higher than 20x106/kg and 12.4x106/kg, respectively) has been shown to result in faster immunological recovery and improved leukemia-free survival probability in children (Handgretinger R. et al. Bone Marrow Transplant 2001; Klingebiel T. et al. Blood 2010). Nevertheless, in the case of donors considered “poor mobilizers” (10-30% of cases), the threshold dose of CD34+ cells needed to ensure the inoculum of a megadose of stem cells might not be achieved. In the setting of cord blood (CB) transplantation, one of the strategies aimed at overcoming the problem of low cellularity is represented by the intrabone injection of CB stem cells, with good engraftment rates even in adult patients. We explored the same strategy in the context of T-cell depleted haplo-HSCT and low graft cellularity due to poor donor mobilization, ensuing in inadequate dose of CD34+cells available after positive selection TCD. Patients and methods. From September 2009 to April 2013, 11 pediatric patients affected by malignant or non-malignant hematological disorders (5 acute lymphoblastic leukemias, 1 acute myeloid leukemia, 1 myelodysplastic syndrome, 2 dyskeratosis congenita, 1 Fanconi anemia) received a T-cell depleted CD34+positively selected PBSC allograft from an HLA-haploidentical related donor. Due to the failure to achieve a target cell dose higher than 12x106 purified CD34+ cells/kg, part of the stem cell inoculum was infused as intrabone injection. The procedure was carried out at the patient bedside by multiple intrabone injections in the superior-posterior iliac crests under sedoanalgesia, as previously described (Frassoni F. et al. Lancet Oncol 2008). The median dose of CD34+ cells infused was 9x106/kg (range, 5-12) while the median number of CD3+ lymphocytes was 0.7x104/kg recipient body weight (range, 0.3-11). About one third of the stem cell inoculum, corresponding to a total volume of 20-40 ml, was given intrabone, while the remaining stem cell portion was infused intravenously. Results.No complication occurred during, or immediately after, the intrabone injection. Nine out of the 11 patients achieved a complete donor engraftment, while graft rejection occurred in 2 patients. The median time for neutrophil engraftment was 13.5 days (range, 12-20), while the median time for platelet recovery was 14 days (range, 13-24). One patient developed grade II acute GVHD and only 1 case of limited chronic GVHD was observed. No transplant-related deaths were observed. Conclusions. Our data suggest that, in the haplo-HSCT setting, the intrabone injection of positively selected CD34+ cells, can be safely used in cases of low graft cellularity due to poor donor mobilization, with the aim of minimizing the risk of graft rejection or poor engraftment. Our preliminary data need to be confirmed in larger series of patients and compared with those obtained with conventional intravenous administration of comparable dose of CD34+ cells. Disclosures No relevant conflicts of interest to declare.

Virulence and Karyotype Analyses of rad52Mutants of Candida albicans: Regeneration of a TruncatedChromosome of a Reintegrant Strain (rad52/RAD52) in the Host

ABSTRACT The virulence of Candida albicans mutants lacking one or both copies of RAD52, a gene involved in homologous recombination (HR), was evaluated in a murine model of hematogenously disseminated candidiasis. In this study, the virulence of the rad52Δ mutant was dependent upon the inoculum concentration. Mice survived at a cell inoculum of 1 × 106, but there was a decrease in survival time at dosages of 1.5 × 106 and especially at 3 × 106 cells per animal. The heterozygote RAD52/rad52 behaved like wild type, whereas a reintegrant strain was intermediate in its ability to cause death compared to these strains and to the avirulent rad52/rad52 null at inocula of 1 × 106 and 1.5 × 106 cells. A double mutant, lig4/lig4/rad52/rad52, was avirulent at all inocula used. PCR analysis of the RAD52 and/or LIG4 loci showed that all strains recovered from animals matched the genotype of the inoculated strains. Analysis of the electrophoretical karyotypes indicated that the inoculated, reintegrant strain carried a large deletion in one copy of chromosome 6 (the shortest homologue, or Chr6b). Interestingly, truncated Chr6b was regenerated in all the strains recovered from moribund animals using the homologue as a template. Further, regeneration of Chr6b was paralleled by an increase in virulence that was still lower than that of wild type, likely because of the persistent loss of heterozygosity in the regenerated region. Overall, our results indicate that systemic candidiasis can develop in the absence of HR, but simultaneous elimination of both recombination pathways, HR and nonhomologous end-joining, suppresses virulence even at very high inocula.

Influence of chlorsulfuron on rhizobial growth, nodule formation, and nitrogen fixation with chickpea

Sulfonylurea residues have been found to inhibit the growth of some legume crops and pastures in seasons following application. Negative effects of these herbicides on symbiotic nitrogen fixation by legume crops and pastures have been demonstrated. Reductions in nitrogen fixation may result from a direct effect of the herbicide on rhizobial growth and/or an indirect effect on plant growth. In this study the influence of chlorsulfuron on the growth of chickpea rhizobia [Mesorhizobium ciceri (CC1192)], the growth of chickpea plants, and the extent of nodulation and nitrogen fixation by the chickpea/rhizobia symbiosis were examined. In vitro studies (in yeast mannitol broth and a defined medium) showed that chlorsulfuron applied at double the recommended field application rate did not influence the growth of chickpea rhizobia. An experiment using 14C-labelled chlorsulfuron was conducted to determine if rhizobial cells exposed to chlorsulfuron could deliver the herbicide to the point of root infection and nodule formation. Approximately 1% of the herbicide present in the rhizobial growth medium remained with the cell/inoculum material after rinsing with 1/4 strength Ringer’s solution. This was considered unlikely to affect chickpea growth, nodulation, or nitrogen fixation. A pot experiment was used to define the influence of chlorsulfuron on the growth, nodulation, and nitrogen fixation of chickpeas. The presence of chlorsulfuron in the soil reduced the nodulation and nitrogen fixation of the chickpea plants. Pre-exposing rhizobia to chlorsulfuron before inoculating them into pots with germinating chickpea seeds, reduced the number of nodules formed by 51%. Exposure of chickpeas and chickpea rhizobia to chlorsulfuron can adversely affect the formation and activity of symbiotic nitrogen-fixing nodules, even when only the rhizobial inoculant is exposed briefly to the herbicide.

Production of Cyclic Lipopeptides by Pseudomonas fluorescens Strains in Bulk Soil and in the Sugar Beet Rhizosphere

ABSTRACT The production of cyclic lipopeptides (CLPs) with antifungal and biosurfactant properties by Pseudomonas fluorescens strains was investigated in bulk soil and in the sugar beet rhizosphere. Purified CLPs (viscosinamide, tensin, and amphisin) were first shown to remain highly stable and extractable (90%) when applied (ca. 5 μg g−1) to sterile soil, whereas all three compounds were degraded over 1 to 3 weeks in nonsterile soil. When a whole-cell inoculum of P. fluorescens strain DR54 containing a cell-bound pool of viscosinamide was added to the nonsterile soil, declining CLP concentrations were observed over a week. By comparison, addition of the strains 96.578 and DSS73 without cell-bound CLP pools did not result in detectable tensin or amphisin in the soil. In contrast, when sugar beet seeds were coated with the CLP-producing strains and subsequently germinated in nonsterile soil, strain DR54 maintained a high and constant viscosinamide level in the young rhizosphere for ∼2 days while strains 96.578 and DSS73 exhibited significant production (net accumulation) of tensin or amphisin, reaching a maximum level after 2 days. All three CLPs remained detectable for several days in the rhizosphere. Subsequent tests of five other CLP-producing P. fluorescens strains also demonstrated significant production in the young rhizosphere. The results thus provide evidence that production of different CLPs is a common trait among many P. fluorescens strains in the soil environment, and further, that the production is taking place only in specific habitats like the rhizosphere of germinating sugar beet seeds rather than in the bulk soil.

Cytokine profile of draining lymph node lymphocytes in mice grafted with syngeneic keratinocytes expressing human papillomavirus type 16 E7 protein

Studies on the immune response to human papillomaviruses are compromised by the extreme host and tissue specificity of these viruses. To circumvent this, a mouse model system has been used in which antigen is presented via a differentiated, syngeneic keratinocyte graft expressing human papillomavirus type 16 (HPV-16) E7 protein. Using this model, previous studies have shown that animals grafted with a high cell inoculum (1×107 NEK 16 cells) exhibit a delayed-type hypersensitivity response that is E7-specific and CD4+-mediated, but those receiving a low cell inoculum (5×105 NEK 16 cells) are rendered unresponsive to subsequent and repeated antigen challenge. To investigate the mechanisms underlying this phenomenon, we have analysed the early changes in the cytokine profile of the graft-draining lymph node (GDLN) after high- or low-dose grafts. At 4 days post-grafting, there was a peak secretion of IL-2 associated with a decreased secretion of IL-4 by γδ-TCR+ cells in the group receiving 1×107 NEK 16 cells. At 5 days post-grafting, there was a peak secretion of IL-10 by CD8+ cells in both the high- and low-dose graft groups compared with controls. In contrast, low dose-grafted animals showed an increase in IL-4 production by CD8+ cells at this time-point. Low antigen challenge in this model system is associated with the appearance of a CD8+ population in the GDLN that secretes both IL-4 and IL-10. This population may represent a Tc2 or Ts subset that could induce further unresponsiveness.

Optimization of conditions for growth of wild-type and genetically transformed Trypanosoma cruzi on agarose plates

Growth of Trypanosoma cruzi as colonies on solid medium has not been widely used as an experimental procedure. We therefore sought to establish a reliable and routine plating method. The optimal results were achieved with a matrix of 0·65% low melting point agarose onto which epimasigotes from the mid-to-late logarithmic phase of growth were spread. Colonies could be isolated after incubation for 21 days in a humidified 5% CO2 environment at 28°C. Plating efficiencies in the range of 40% were obtained by this method and clones could be recovered into liquid medium or onto blood-agar slopes with a high success rate. The procedure has also been adapted for the isolation of genetically transformed clones after electroporation of epimastigotes with either plasmid or cosmid vectors. This was best achieved by inclusion of the electroporated cell inoculum in a 0·6% agarose overlay containing G418 as the selective drug, on top of a 0·8% agar base. Transformation efficiencies were as high as 10−5 cells per μg of DNA. A reliable plating method for T. cruzi will have many applications and is a significant step towards the use of ‘shotgun transformation’ to generate libraries of T. cruzi recombinants.