Bone marrow-derived mesenchymal stromal cells express cardiac-specific markers, retain the stromal phenotype, and do not become functional cardiomyocytes in vitro.

Although bone marrow-derived mesenchymal stromal cells (MSCs) may be beneficial in treating heart disease, their ability to transdifferentiate into functional cardiomyocytes remains unclear. Here, bone marrow-derived MSCs from adult female transgenic mice expressing green fluorescent protein (GFP) under the control of the cardiac-specific alpha-myosin heavy chain promoter were cocultured with male rat embryonic cardiomyocytes (rCMs) for 5-15 days. After 5 days in coculture, 6.3% of MSCs became GFP(+) and stained positively for the sarcomeric proteins troponin I and alpha-actinin. The mRNA expression for selected cardiac-specific genes (atrial natriuretic factor, Nkx2.5, and alpha-cardiac actin) in MSCs peaked after 5 days in coculture and declined thereafter. Despite clear evidence for the expression of cardiac genes, GFP(+) MSCs did not generate action potentials or display ionic currents typical of cardiomyocytes, suggesting retention of a stromal cell phenotype. Detailed immunophenotyping of GFP(+) MSCs demonstrated expression of all antigens used to characterize MSCs, as well as the acquisition of additional markers of cardiomyocytes with the phenotype CD45(-)-CD34(+)-CD73(+)-CD105(+)-CD90(+)-CD44(+)-SDF1(+)-CD134L(+)-collagen type IV(+)-vimentin(+)-troponin T(+)-troponin I(+)-alpha-actinin(+)-connexin 43(+). Although cell fusion between rCMs and MSCs was detectable, the very low frequency (0.7%) could not account for the phenotype of the GFP(+) MSCs. In conclusion, we have identified an MSC population displaying plasticity toward the cardiomyocyte lineage while retaining mesenchymal stromal cell properties, including a nonexcitable electrophysiological phenotype. The demonstration of an MSC population coexpressing cardiac and stromal cell markers may explain conflicting results in the literature and indicates the need to better understand the effects of MSCs on myocardial injury. Disclosure of potential conflicts of interest is found at the end of this article.

Chemical treatment of anti-D results in improved efficacy for the inhibition of Fcgamma receptor-mediated phagocytosis.

BACKGROUND: This study investigated whether treatment of immunoglobulins anti-D or intravenous immune globulin (IVIG) with chemicals previously shown to inhibit phagocytosis could result in an enhancement of Fcgamma receptor (FcgammaR) blockade in vitro. If successful, this approach may provide the possibility of targeting these chemicals to monocyte-macrophages for increased efficacy of immunoglobulin-based therapies in vivo. STUDY DESIGN AND METHODS: For proof-of-concept, the chemical thimerosal, a prototype FcgammaR inhibitor, was combined with RhIG or IVIG. Residual chemical was removed by extensive dialysis. With a monocyte monolayer assay (MMA) and a concentration of immunoglobulin alone that results in 50 percent inhibition of MMA phagocytosis of antibody-coated red blood cells, the effect of thimerosal treatment on the ability of the immunoglobulin to show a significant enhancement of efficacy was determined. RESULTS: It is shown that combining thimerosal with anti-D, either slide and rapid tube or commercially available (WinRho SDF, Cangene), results in a highly significant increase in efficacy over anti-D alone to inhibit phagocytosis in vitro. This effect was not due to residual unbound compound or to cellular toxicity of the chemically treated immunoglobulins. Treatment of IVIG with thimerosal had no significant effect on its ability to inhibit in vitro phagocytosis. CONCLUSION: Our results indicate that it is possible to modify an immunoglobulin by chemical treatment such that the treated immunoglobulin demonstrates significantly enhanced ability to inhibit FcgammaR-mediated phagocytosis. It is also demonstrated that IVIG and anti-D appear to respond differently after chemical treatment. Further examination of this strategy is warranted and has the potential to reduce the dose, cost, and possibly, adverse effects of immunoglobulin-based therapies.

Constitutively polarized granules prime KHYG-1 NK cells.

The major mechanism for NK cell lysis of tumor cells is granule-mediated cytotoxicity. Polarization of granules is a prelude to the release of their cytotoxic contents in response to target-cell binding. We describe the novel observation of constitutive granule polarization in the cytotoxic NK cell line, KHYG-1. Continuous degranulation of KHYG-1 cells, however, does not occur and still requires target-cell contact. Disruption of microtubules with colcemid is sufficient to disperse the granules in KHYG-1 and significantly decreases cytotoxicity. A similar effect is not obtained by inhibiting extracellular signal-related kinase 2 (ERK2), the most distal kinase investigated in the cytolytic pathway. Disruption of microtubules significantly down-regulates activation receptors, NKp44 and NKG2D, implicating them as potential microtubule-trafficking receptors. Such changes in upstream receptor expression may have caused deactivation of ERK2, since NKG2D cross-linking also leads to receptor down-regulation and diminished ERK phosphorylation. Thus, a functional role for NKG2D in KHYG-1 cytotoxicity is demonstrated. Moreover, the novel primed state may contribute to the high cytotoxicity exhibited by KHYG-1.