Stacie M. Anderson

Bone marrow cells regenerate infarcted myocardium

Myocardial infarction leads to loss of tissue and impairment of cardiac performance. The remaining myocytes are unable to reconstitute the necrotic tissue, and the post-infarcted heart deteriorates with time. Injury to a target organ is sensed by distant stem cells, which migrate to the site of damage and undergo alternate stem cell differentiation; these events promote structural and functional repair. This high degree of stem cell plasticity prompted us to test whether dead myocardium could be restored by transplanting bone marrow cells in infarcted mice. We sorted lineage-negative (Lin-) bone marrow cells from transgenic mice expressing enhanced green fluorescent protein by fluorescence-activated cell sorting on the basis of c-kit expression. Shortly after coronary ligation, Lin- c-kitPOS cells were injected in the contracting wall bordering the infarct. Here we report that newly formed myocardium occupied 68% of the infarcted portion of the ventricle 9 days after transplanting the bone marrow cells. The developing tissue comprised proliferating myocytes and vascular structures. Our studies indicate that locally delivered bone marrow cells can generate de novo myocardium, ameliorating the outcome of coronary artery disease.

Postnatal NG2 proteoglycan–expressing progenitor cells are intrinsically multipotent and generate functional neurons

Neurogenesis is known to persist in the adult mammalian central nervous system (CNS). The identity of the cells that generate new neurons in the postnatal CNS has become a crucial but elusive issue. Using a transgenic mouse, we show that NG2 proteoglycan–positive progenitor cells that express the 2′,3′-cyclic nucleotide 3′-phosphodiesterase gene display a multipotent phenotype in vitro and generate electrically excitable neurons, as well as astrocytes and oligodendrocytes. The fast kinetics and the high rate of multipotent fate of these NG2+ progenitors in vitro reflect an intrinsic property, rather than reprogramming. We demonstrate in the hippocampus in vivo that a sizeable fraction of postnatal NG2+ progenitor cells are proliferative precursors whose progeny appears to differentiate into GABAergic neurons capable of propagating action potentials and displaying functional synaptic inputs. These data show that at least a subpopulation of postnatal NG2-expressing cells are CNS multipotent precursors that may underlie adult hippocampal neurogenesis.

Resistance to Parvovirus B19 Infection Due to Lack of Virus Receptor (Erythrocyte P Antigen)

BACKGROUND The presence of a specific cellular receptor is thought to be necessary for susceptibility to viral infection. The erythrocyte P antigen is the cellular receptor for parvovirus B19. We hypothesized that the rare persons with the p phenotype, whose erythrocytes do not have this receptor, would be naturally resistant to B19 infection, which causes erythema infectiosum. METHODS Blood samples were collected from two populations in cross-sectional studies. We determined the P antigen phenotype of the red cells and tested plasma for anti-B19-specific antibodies. Bone marrow from donors of known P antigen phenotype was inoculated with parvovirus B19. Infectivity was measured by assays of erythroid progenitor cells, dot blot analysis, and in situ hybridization for B19 DNA, and an immunofluorescence assay for viral-capsid proteins. RESULTS Of the 17 subjects with the p red-cell phenotype, who did not have P antigen on their erythrocytes, none (0 of 11 and 0 of 6) had serologic evidence of previous parvovirus B19 infection. In contrast, the seropositivity rates in the two control groups were 71 percent (53 of 75, P < 0.001) and 47 percent (32 of 68, P = 0.03). In vitro, bone marrow from donors with the p phenotype maintained normal erythropoiesis despite very high concentrations of virus, with no evidence of infection of erythroid progenitor cells by parvovirus B19. CONCLUSIONS People who do not have P antigen, which is the cellular receptor for parvovirus B19, are naturally resistant to infection with this pathogen.

The fusion gene Cbfb-MYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia.

The fusion gene Cbfb - MYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia

Increased expression of Fas antigen on bone marrow CD34+ cells of patients with aplastic anaemia

Summary Fas antigen, a receptor molecule that mediates signals for programmed cell death, is involved in T‐cell‐mediated killing of malignant, virus‐infected or allogeneic target cells. Interferon‐γ (IFN‐γ) and tumour necrosis factored (TNF‐α), potent inhibitors of haemopoiesis, enhance Fas receptor expression on bone marrow (BM) CD34+ cells, and both cytokines render haemopoietic progenitor cells susceptible to Fas‐mediated inhibition of colony formation due to the induction of apoptosis. Haemopoietic suppression in aplastic anaemia (AA) has been associated with aberrant IFN‐γ, increased TNF‐β expression, and elevated numbers of activated cytotoxic T‐cells in marrow. We have now examined Fas antigen expression in fresh AA BM samples. In normal individuals few CD34+ cells expressed Fas antigen and normal marrow cells had low sensitivity to Fas‐mediated inhibition of colony formation. In contrast, in early AA, BM CD34+ cells showed markedly increased percentages of Fas receptor‐expressing CD34+ cells, which correlated with increased sensitivity of AA marrow cells to anti‐Fas antibody‐mediated inhibition of colony formation. The proportion of Fas antigen‐bearing cells was lower in recovered patients’BM. Fas antigen was also detected in the marrow of some patients with myelodysplasia, especially the hypocellular variant. These results are consistent with the hypothesis that AA CD34+ cells, probably including haemopoietic progenitor cells, express high levels of Fas receptor due to in vivo exposure to IFN‐γ and/or TNF‐α and are suitable targets for T‐cell‐mediated killing. Our results suggest that the Fas receptor/Fas ligand system are involved in the pathophysiology of BM failure.

Wnt5a inhibits canonical Wnt signaling in hematopoietic stem cells and enhances repopulation

The mechanisms that regulate hematopoietic stem cell (HSC) fate decisions between proliferation and multilineage differentiation are unclear. Members of the Wnt family of ligands that activate the canonical Wnt signaling pathway, which utilizes β-catenin to relay the signal, have been demonstrated to regulate HSC function. In this study, we examined the role of noncanonical Wnt signaling in regulating HSC fate. We observed that noncanonical Wnt5a inhibited Wnt3a-mediated canonical Wnt signaling in HSCs and suppressed Wnt3a-mediated alterations in gene expression associated with HSC differentiation, such as increased expression of myc. Wnt5a increased short- and long-term HSC repopulation by maintaining HSCs in a quiescent G0 state. From these data, we propose that Wnt5a regulates hematopoiesis by the antagonism of the canonical Wnt pathway, resulting in a pool of quiescent HSCs.

Autoimmune Lymphoproliferative Syndrome with Defective Fas: Genotype Influences Penetrance

Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of lymphocyte homeostasis and immunological tolerance. Most patients have a heterozygous mutation in the APT1 gene, which encodes Fas (CD95, APO-1), mediator of an apoptotic pathway crucial to lymphocyte homeostasis. Of 17 unique APT1 mutations in unrelated ALPS probands, 12 (71%) occurred in exons 7-9, which encode the intracellular portion of Fas. In vitro, activated lymphocytes from all 17 patients showed apoptotic defects when exposed to an anti-Fas agonist monoclonal antibody. Similar defects were found in a Fas-negative cell line transfected with cDNAs bearing each of the mutations. In cotransfection experiments, Fas constructs with either intra- or extracellular mutations caused dominant inhibition of apoptosis mediated by wild-type Fas. Two missense Fas variants, not restricted to patients with ALPS, were identified. Variant A(-1)T at the Fas signal-sequence cleavage site, which mediates apoptosis less well than wild-type Fas and is partially inhibitory, was present in 13% of African American alleles. Among the ALPS-associated Fas mutants, dominant inhibition of apoptosis was much more pronounced in mutants affecting the intracellular, versus extracellular, portion of the Fas receptor. Mutations causing disruption of the intracellular Fas death domain also showed a higher penetrance of ALPS phenotype features in mutation-bearing relatives. Significant ALPS-related morbidity occurred in 44% of relatives with intracellular mutations, versus 0% of relatives with extracellular mutations. Thus, the location of mutations within APT1 strongly influences the development and the severity of ALPS.

Expression of the green fluorescent protein in the oligodendrocyte lineage: a transgenic mouse for developmental and physiological studies.

We generated a transgenic mouse expressing the enhanced green fluorescent protein (EGFP) under the control of the 2′‐3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP) promoter. EGFP+ cells were visualized in live tissue throughout embryonic and postnatal development. Immunohistochemical analysis in brain tissue and in sciatic nerve demonstrated that EGFP expression was restricted to cells of the oligodendrocyte and Schwann cell lineages. EGFP was also strongly expressed in “adult” oligodendrocyte progenitors (OPs) and in gray matter oligodendrocytes. Fluorescence‐activated cell sorting allowed high‐yield purification of EGFP+ oligodendrocyte‐lineage cells from transgenic brains. Electrophysiological patch clamp recordings of EGFP+ cells in situ demonstrated that OP cells displayed large outward tetraethylammonium (TEA)‐sensitive K+ currents and very small inward currents, whereas mature oligodendrocytes were characterized by expression of large inward currents and small outward K+ currents. The proliferation rate of EGFP+ cells in developing white matter decreased with the age of the animals and was strongly inhibited by TEA. Oligodendrocyte development and physiology can be studied in live tissue of CNP‐EGFP transgenic mice, which represent a source of pure EGFP+ oligodendrocyte‐lineage cells throughout development.

Differential Expression of Interleukin-17A and -17F Is Coupled to T Cell Receptor Signaling via Inducible T Cell Kinase

T helper 17 (Th17) cells play major roles in autoimmunity and bacterial infections, yet how T cell receptor (TCR) signaling affects Th17 cell differentiation is relatively unknown. We demonstrate that CD4(+) T cells lacking Itk, a tyrosine kinase required for full TCR-induced phospholipase C-gamma (PLC-gamma1) activation, exhibit decreased interleukin-17A (IL-17A) expression in vitro and in vivo, despite relatively normal expression of retinoic acid receptor-related orphan receptor-gammaT (ROR-gammaT) and IL-17F. IL-17A expression was rescued by pharmacologically induced Ca(2+) influx or constitutively activated nuclear factor of activated T cells (NFAT). Conversely, decreased TCR stimulation or calcineurin inhibition preferentially reduced IL-17A expression. We further found that the promoter of Il17a but not Il17f has a conserved NFAT binding site that bound NFATc1 in wild-type but not Itk-deficient cells, even though both exhibited open chromatin conformations. Finally, Itk(-/-) mice also showed differential regulation of IL-17A and IL-17F in vivo. Our results suggest that Itk specifically couples TCR signaling to Il17a expression and the differential regulation of Th17 cell cytokines through NFATc1.

SAP regulates T cell–mediated help for humoral immunity by a mechanism distinct from cytokine regulation

X-linked lymphoproliferative disease is caused by mutations affecting SH2D1A/SAP, an adaptor that recruits Fyn to signal lymphocyte activation molecule (SLAM)-related receptors. After infection, SLAM-associated protein (SAP)−/− mice show increased T cell activation and impaired humoral responses. Although SAP−/− mice can respond to T-independent immunization, we find impaired primary and secondary T-dependent responses, with defective B cell proliferation, germinal center formation, and antibody production. Nonetheless, transfer of wild-type but not SAP-deficient CD4 cells rescued humoral responses in reconstituted recombination activating gene 2−/− and SAP−/− mice. To investigate these T cell defects, we examined CD4 cell function in vitro and in vivo. Although SAP-deficient CD4 cells have impaired T cell receptor–mediated T helper (Th)2 cytokine production in vitro, we demonstrate that the humoral defects can be uncoupled from cytokine expression defects in vivo. Instead, SAP-deficient T cells exhibit decreased and delayed inducible costimulator (ICOS) induction and heightened CD40L expression. Notably, in contrast to Th2 cytokine defects, humoral responses, ICOS expression, and CD40L down-regulation were rescued by retroviral reconstitution with SAP-R78A, a SAP mutant that impairs Fyn binding. We further demonstrate a role for SLAM/SAP signaling in the regulation of early surface CD40L expression. Thus, SAP affects expression of key molecules required for T–B cell collaboration by mechanisms that are distinct from its role in cytokine regulation.