Mitotically inactivated embryonic stem cells can be used as an in vivo feeder layer to nurse damaged myocardium after acute myocardial infarction: a preclinical study.

RATIONALE: Various types of viable stem cells have been reported to result in modest improvement in cardiac function after acute myocardial infarction. The mechanisms for improvement from different stem cell populations remain unknown. OBJECTIVE: To determine whether irradiated (nonviable) embryonic stem cells (iESCs) improve postischemic cardiac function without adverse consequences. METHODS AND RESULTS: After coronary artery ligation-induced cardiac infarction, either conditioned media or male murine or male human iESCs were injected into the penumbra of ischemic myocardial tissue of female mice or female rhesus macaque monkeys, respectively. Murine and human iESCs, despite irradiation doses that prevented proliferation and induced cell death, significantly improved cardiac function and decreased infarct size compared with untreated or media-treated controls. Fluorescent in situ hybridization of the Y chromosome revealed disappearance of iESCs within the myocardium, whereas 5-bromo-2'-deoxyuridine assays revealed de novo in vivo cardiomyocyte DNA synthesis. Microarray gene expression profiling demonstrated an early increase in metabolism, DNA proliferation, and chromatin remodeling pathways, and a decrease in fibrosis and inflammatory gene expression compared with media-treated controls. CONCLUSIONS: As a result of irradiation before injection, ex vivo and in vivo iESC existence is transient, yet iESCs provide a significant improvement in cardiac function after acute myocardial infarction. The mechanism(s) of action of iESCs seems to be related to cell-cell exchange, paracrine factors, and a scaffolding effect between iESCs and neighboring host cardiomyocytes.

Pharmacokinetic study of oral prednisolone compared with intravenous methylprednisolone in patients with juvenile dermatomyositis.

OBJECTIVE: To determine areas under the curve (AUCs) of oral prednisolone (OP) and intravenous methylprednisolone (IVMP) in patients with juvenile dermatomyositis (DM) and assess the association with nailfold end-row loops (ERLs). Patients with active disease have fewer ERLs that possibly occur in the gastrointestinal tract, impairing absorption of oral medications. METHODS: Six patients with juvenile DM received 50 mg/m(2) of OP (day 1) and IVMP (day 2). Blood was drawn at baseline and at 5, 15, 30, 45, 60, and 90 minutes, and hourly (hours 2-8) after each dose. Samples were analyzed by reverse-phase high-performance liquid chromatography for levels of prednisolone and methylprednisolone. AUCs of OP and IVMP were determined by the trapezoid method; pharmacokinetic parameters were obtained using noncompartmental and compartmental analysis. ERLs were determined from freeze-frame video microscopy and nailfold capillaroscopy. RESULTS: There was a trend toward significance in difference in mean AUC of IVMP (116.72 microg x ml/hour) compared with OP (65.16 microg x ml/hour; P = 0.059). Mean peak concentration was higher for IVMP (34.49 microg/ml) than OP (7.08 microg/ml); mean half-life was shorter for IVMP (1.90 hours) than OP (2.36 hours). There was an inverse association between DeltaAUCs (IVMP AUC - OP AUC) and ERLs (R = -0.68, P = 0.044). CONCLUSION: Patients with juvenile DM and ERL loss may have decreased bioavailability of OP compared with IVMP. This can provide the rationale for greater efficacy of IVMP in patients with active vasculopathy of juvenile DM. Further studies investigating the pharmacokinetics and pharmacodynamics of high-dose IVMP need to be performed in patients with juvenile DM.

A novel approach to the analysis of specificity, clonality, and frequency of HIV-specific T cell responses reveals a potential mechanism for control of viral escape.

Escape from the CD8(+) T cell response through epitope mutations can lead to loss of immune control of HIV replication. Theoretically, escape from CD8(+) T cell recognition is less likely when multiple TCRs target individual MHC/peptide complexes, thereby increasing the chance that amino acid changes in the epitope could be tolerated. We studied the CD8(+) T cell response to six immunodominant epitopes in five HIV-infected subjects using a novel approach combining peptide stimulation, cell surface cytokine capture, flow cytometric sorting, anchored RT-PCR, and real-time quantitative clonotypic TCR tracking. We found marked variability in the number of clonotypes targeting individual epitopes. One subject recognized a single epitope with six clonotypes, most of which were able to recognize and lyse cells expressing a major epitope variant that arose. Additionally, multiple clonotypes remained expanded during the course of infection, irrespective of epitope variant frequency. Thus, CD8(+) T cells comprising multiple TCR clonotypes may expand in vivo in response to individual epitopes, and may increase the ability of the response to recognize virus escape mutants.