Microsatellite characterization and marker development for the fungus Penicillium digitatum, causal agent of green mold of citrus.

Penicillium digitatum is one of the most important postharvest pathogens of citrus on a global scale causing significant annual losses due to fruit rot. However, little is known about the diversity of P. digitatum populations. The genome of P. digitatum has been sequenced, providing an opportunity to determine the microsatellite distribution within P. digitatum to develop markers that could be valuable tools for studying the population biology of this pathogen. In the analyses, a total of 3,134 microsatellite loci were detected; 66.73%, 23.23%, 8.23%, 1.24%, 0.16%, and 0.77% were detected as mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats, respectively. As consistent with other ascomycete fungi, the genome size of P. digitatum does not seem to correlate with the density of microsatellite loci. However, significantly longer motifs of mono-, di-, and tetranucleotide repeats were identified in P. digitatum compared to 10 other published ascomycete species with repeats of over 800, 300, and 900 motifs found, respectively. One isolate from southern California and five additional isolates from other countries ("global isolates") were used to initially screen microsatellite markers developed in this study. Twelve additional isolates, referred to as the "local isolates," were also collected from citrus at the University of California Riverside agricultural experiment station and were subsequently used to screen the primers that sequenced well and were polymorphic based on the global isolates. Thirty-six primers were screened, and nine trinucleotide loci and one hexanucleotide locus were chosen as robust markers. These loci yielded two to seven alleles and will be useful to study population genetic structure of P. digitatum populations

Absence of CD11a Expression Identifies Embryonic Hematopoietic Stem Cell Precursors via Competitive Neonatal Transplantation Assay.

Hematopoietic stem cells (HSCs) are defined by their self-renewal, multipotency, and bone marrow (BM) engraftment abilities. How HSCs emerge during embryonic development remains unclear, but are thought to arise from hemogenic endothelium through an intermediate precursor called "pre-HSCs." Pre-HSCs have self-renewal and multipotent activity, but lack BM engraftability. They can be identified functionally by transplantation into neonatal recipients, or by in vitro co-culture with cytokines and stroma followed by transplantation into adult recipients. While pre-HSCs express markers such as Kit and CD144, a precise surface marker identity for pre-HSCs has remained elusive due to the fluctuating expression of common HSC markers during embryonic development. We have previously determined that the lack of CD11a expression distinguishes HSCs in adults as well as multipotent progenitors in the embryo. Here, we use a neonatal transplantation assay to identify pre-HSC populations in the mouse embryo. We establish CD11a as a critical marker for the identification and enrichment of pre-HSCs in day 10.5 and 11.5 mouse embryos. Our proposed pre-HSC population, termed "11a- eKLS" (CD11a- Ter119- CD43+ Kit+ Sca1+ CD144+), contains all in vivo long-term engrafting embryonic progenitors. This population also displays a cell-cycle status expected of embryonic HSC precursors. Furthermore, we identify the neonatal liver as the likely source of signals that can mature pre-HSCs into BM-engraftable HSCs

Graft conditioning with fluticasone propionate reduces graft‐versus‐host disease upon allogeneic hematopoietic cell transplantation in mice

Abstract Hematopoietic cell transplantation (HCT) treats many blood conditions but remains underused due to complications such as graft‐versus‐host disease (GvHD). In GvHD, donor immune cells attack the patient, requiring powerful immunosuppressive drugs like glucocorticoids (GCs) to prevent death. In this study, we tested the hypothesis that donor cell conditioning with the glucocorticoid fluticasone propionate (FLU) prior to transplantation could increase hematopoietic stem cell (HSC) engraftment and reduce GvHD. Murine HSCs treated with FLU had increased HSC engraftment and reduced severity and incidence of GvHD after transplantation into allogeneic hosts. While most T cells died upon FLU treatment, donor T cells repopulated in the hosts and appeared less inflammatory and alloreactive. Regulatory T cells (Tregs) are immunomodulatory and survived FLU treatment, resulting in an increased ratio of Tregs to conventional T cells. Our results implicate an important role for Tregs in maintaining allogeneic tolerance in FLU‐treated grafts and suggest a therapeutic strategy of pre‐treating donor cells (and not the patients directly) with GCs to simultaneously enhance engraftment and reduce GvHD upon allogeneic HCT

Graft conditioning with fluticasone propionate reduces graft‐versus‐host disease upon allogeneic hematopoietic cell transplantation in mice

Hematopoietic cell transplantation (HCT) treats many blood conditions but remains underused due to complications such as graft-versus-host disease (GvHD). In GvHD, donor immune cells attack the patient, requiring powerful immunosuppressive drugs like glucocorticoids (GCs) to prevent death. In this study, we tested the hypothesis that donor cell conditioning with the glucocorticoid fluticasone propionate (FLU) prior to transplantation could increase hematopoietic stem cell (HSC) engraftment and reduce GvHD. Murine HSCs treated with FLU had increased HSC engraftment and reduced severity and incidence of GvHD after transplantation into allogeneic hosts. While most T cells died upon FLU treatment, donor T cells repopulated in the hosts and appeared less inflammatory and alloreactive. Regulatory T cells (Tregs) are immunomodulatory and survived FLU treatment, resulting in an increased ratio of Tregs to conventional T cells. Our results implicate an important role for Tregs in maintaining allogeneic tolerance in FLU-treated grafts and suggest a therapeutic strategy of pre-treating donor cells (and not the patients directly) with GCs to simultaneously enhance engraftment and reduce GvHD upon allogeneic HCT