John J. Byrnes

Transendocardial Mesenchymal Stem Cells and Mononuclear Bone Marrow Cells for Ischemic Cardiomyopathy: The TAC-HFT Randomized Trial

IMPORTANCE Whether culture-expanded mesenchymal stem cells or whole bone marrow mononuclear cells are safe and effective in chronic ischemic cardiomyopathy is controversial. OBJECTIVE To demonstrate the safety of transendocardial stem cell injection with autologous mesenchymal stem cells (MSCs) and bone marrow mononuclear cells (BMCs) in patients with ischemic cardiomyopathy. DESIGN, SETTING, AND PATIENTS A phase 1 and 2 randomized, blinded, placebo-controlled study involving 65 patients with ischemic cardiomyopathy and left ventricular (LV) ejection fraction less than 50% (September 1, 2009-July 12, 2013). The study compared injection of MSCs (n=19) with placebo (n = 11) and BMCs (n = 19) with placebo (n = 10), with 1 year of follow-up. INTERVENTIONS Injections in 10 LV sites with an infusion catheter. MAIN OUTCOMES AND MEASURES Treatment-emergent 30-day serious adverse event rate defined as a composite of death, myocardial infarction, stroke, hospitalization for worsening heart failure, perforation, tamponade, or sustained ventricular arrhythmias. RESULTS No patient had a treatment-emergent serious adverse events at day 30. The 1-year incidence of serious adverse events was 31.6% (95% CI, 12.6% to 56.6%) for MSCs, 31.6% (95% CI, 12.6%-56.6%) for BMCs, and 38.1% (95% CI, 18.1%-61.6%) for placebo. Over 1 year, the Minnesota Living With Heart Failure score improved with MSCs (-6.3; 95% CI, -15.0 to 2.4; repeated measures of variance, P=.02) and with BMCs (-8.2; 95% CI, -17.4 to 0.97; P=.005) but not with placebo (0.4; 95% CI, -9.45 to 10.25; P=.38). The 6-minute walk distance increased with MSCs only (repeated measures model, P = .03). Infarct size as a percentage of LV mass was reduced by MSCs (-18.9%; 95% CI, -30.4 to -7.4; within-group, P = .004) but not by BMCs (-7.0%; 95% CI, -15.7% to 1.7%; within-group, P = .11) or placebo (-5.2%; 95% CI, -16.8% to 6.5%; within-group, P = .36). Regional myocardial function as peak Eulerian circumferential strain at the site of injection improved with MSCs (-4.9; 95% CI, -13.3 to 3.5; within-group repeated measures, P = .03) but not BMCs (-2.1; 95% CI, -5.5 to 1.3; P = .21) or placebo (-0.03; 95% CI, -1.9 to 1.9; P = .14). Left ventricular chamber volume and ejection fraction did not change. CONCLUSIONS AND RELEVANCE Transendocardial stem cell injection with MSCs or BMCs appeared to be safe for patients with chronic ischemic cardiomyopathy and LV dysfunction. Although the sample size and multiple comparisons preclude a definitive statement about safety and clinical effect, these results provide the basis for larger studies to provide definitive evidence about safety and to assess efficacy of this new therapeutic approach. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00768066.

Autologous Mesenchymal Stem Cells Produce Concordant Improvements in Regional Function, Tissue Perfusion, and Fibrotic Burden When Administered to Patients Undergoing Coronary Artery Bypass Grafting The Prospective Randomized Study of Mesenchymal Stem Cell Therapy in Patients Undergoing Cardiac Surgery (PROMETHEUS) Trial

Rationale: Although accumulating data support the efficacy of intramyocardial cell-based therapy to improve left ventricular (LV) function in patients with chronic ischemic cardiomyopathy undergoing CABG, the underlying mechanism and impact of cell injection site remain controversial. Mesenchymal stem cells (MSCs) improve LV structure and function through several effects including reducing fibrosis, neoangiogenesis, and neomyogenesis. Objective: To test the hypothesis that the impact on cardiac structure and function after intramyocardial injections of autologous MSCs results from a concordance of prorecovery phenotypic effects. Methods and Results: Six patients were injected with autologous MSCs into akinetic/hypokinetic myocardial territories not receiving bypass graft for clinical reasons. MRI was used to measure scar, perfusion, wall thickness, and contractility at baseline, at 3, 6, and 18 months and to compare structural and functional recovery in regions that received MSC injections alone, revascularization alone, or neither. A composite score of MRI variables was used to assess concordance of antifibrotic effects, perfusion, and contraction at different regions. After 18 months, subjects receiving MSCs exhibited increased LV ejection fraction (+9.4±1.7%, P=0.0002) and decreased scar mass (−47.5±8.1%; P<0.0001) compared with baseline. MSC-injected segments had concordant reduction in scar size, perfusion, and contractile improvement (concordant score: 2.93±0.07), whereas revascularized (0.5±0.21) and nontreated segments (−0.07±0.34) demonstrated nonconcordant changes (P<0.0001 versus injected segments). Conclusions: Intramyocardial injection of autologous MSCs into akinetic yet nonrevascularized segments produces comprehensive regional functional restitution, which in turn drives improvement in global LV function. These findings, although inconclusive because of lack of placebo group, have important therapeutic and mechanistic hypothesis-generating implications. Clinical Trial Registration: URL: http://clinicaltrials.gov/show/NCT00587990. Unique identifier: NCT00587990.

Rationale and design of the Transendocardial Injection of Autologous Human Cells (bone marrow or mesenchymal) in Chronic Ischemic Left Ventricular Dysfunction and Heart Failure Secondary to Myocardial Infarction (TAC-HFT) trial: A randomized, double-blind, placebo-controlled study of safety and efficacy

Although there is tremendous interest in stem cell (SC)-based therapies for cardiomyopathy caused by chronic myocardial infarction, many unanswered questions regarding the best approach remain. The TAC-HFT study is a phase I/II randomized, double-blind, placebo-controlled trial designed to address several of these questions, including the optimal cell type, delivery technique, and population. This trial compares autologous mesenchymal SCs (MSCs) and whole bone marrow mononuclear cells (BMCs). In addition, the study will use a novel helical catheter to deliver cells transendocardially. Although most trials have used intracoronary delivery, the optimal method is unknown and data suggest that the transendocardial approach may have important advantages. Several trials support the benefit of SCs in patients with chronic ischemic cardiomyopathy (ICMP), although the sample sizes have been small and the number of trials sparse. After a pilot phase of 8 patients, 60 patients with ICMP (left ventricular ejection fraction 15%-50%) will be randomized to group A (30 patients further randomized to receive MSC injection or placebo in a 2:1 fashion) or group B (30 patients further randomized to BMCs or placebo in a 2:1 fashion). All patients will undergo bone marrow aspiration and transendocardial injection of SCs or placebo. The primary and secondary objectives are, respectively, to demonstrate the safety and efficacy (determined primarily by cardiac magnetic resonance imaging) of BMCs and MSCs administered transendocardially in patients with ICMP.

Green Tea Polyphenols Induce Apoptosis in vitro in Peripheral Blood T Lymphocytes of Adult T-Cell Leukemia Patients

Green tea polyphenols (TEA) are known to exhibit antioxidative activity as well as tumor‐suppressing activity. In order to examine the tumor‐suppressing activity of TEA against adult T‐cell leukemia (ATL), we cultivated peripheral blood T lymphocytes of ATL patients (ATL PBLs), an HTLV‐I‐infected T‐cell line (KODV) and healthy controls (normal PBLs) for 3 days in the presence of TEA and its main constituent, epigallocatechin‐3‐gallate (EGCg), to measure cell proliferation and apoptosis, and to quantitate mRNAs of HTLV‐I pX and β‐actin genes of the cultured cells. Growth of ATL PBLs was significantly inhibited by 9–27 μg/ml of TEA and EGCg, in contrast to minimal growth inhibition of T cells of normal PBLs. Inhibition of KODV was intermediate between ATL PBLs and normal PBLs. The ATL PBLs and KODV treated with 27 μg/ml of either TEA or EGCg induced apoptotic DNA fragmentation, producing terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labeling (TUNEL)‐positive cells, while the normal PBLs treated with the same concentration of TEA or EGCg produced a negligibly small number of TUNEL‐positive cells, in which apoptotic DNA fragmentation was not detectable. Expression of HTLV‐I pX mRNA was suppressed more than 90% in ATL PBLs by treatment with 3–27 μg/ml of either TEA or EGCg, while expression of β‐actin mRNA was much less suppressed by treatment with the same concentration of TEA or EGCg. These results indicate that TEA and EGCg inhibit growth of ATL PBLs, as well as HTLV‐I‐infected T‐cells, by suppressing HTLV‐I pX gene expression and inducing apoptotic cell death.

Sinus histiocytosis with massive lymphadenopathy. A case with unusual skin involvement and a therapeutic response to vinblastine‐loaded platelets

A patient with sinus histiocytosis with massive lymphadenopathy had unusual skin lesions and progressive internal involvement. Both aspects responded dramatically to the administration of vinblastine‐loaded platelets. Vinblastine‐loaded, idiopathic thrombocytopenic purpura antibody‐coated platelets are a rationale therapy for a disease characterized by the presence of actively phagocytosing histiocytes.

Structural and functional properties of DNA polymerase delta from rabbit bone marrow

SummaryDNA polymerase delta, the most recently described class of eukaryotic DNA polymerase, has been purified to apparent homogeneity from rabbit bone marrow. Unlike the previously known eukaryotic DNA polymerases, delta has a 3′ to 5′ exonuclease as an integral component of its 122 000 molecular weight, single polypeptide structure. Similar to the function with prokaryotic DNA polymerases, the 3′ to 5′ exonuclease assists DNA polymerase delta in maintaining the fidelity of DNA synthesis by excising misincorporated nucleotides. DNA polymerase delta and the longer known eukaryotic DNA polymerase alpha are similar in many features. Both are very sensitive to sulfhydryl inhibitors such as N-ethylmaliemide (NEM) and to the antibiotic aphidicolin. Such criteria distinguish alpha and delta from DNA polymerases beta and gamma. This has led to the conclusion that nuclear DNA replication, which is sensitive to NEM and aphidicolin, is carried out by DNA polymerase alpha. However, the similar sensitivity of delta to these reagents requires that the role of alpha and delta in nuclear DNA replication be further defined. In many features DNA polymerase delta is also similar to the viral induced DNA polymerases such as the Herpes simplex virus DNA polymerases which also have associated 3′ to 5′ exonuclease. Understanding of DNA synthesis and the mechanism of DNA replication fidelity in mammalian cells depends upon a further understanding of both DNA polymerases alpha and delta and the nature of the relationship they have to each other.

Protection from apoptotic cell death by interleukin-4 is increased in previously treated chronic lymphocytic leukemia patients

Chronic lymphocytic leukemia (CLL) cells were cultured in a medium supplemented with 0.01-1 ng/ml interleukin-4 (IL-4) for 18 h, fixed and analyzed on a flow cytometer. The percentage of apoptotic (AP) cells with hypodiploid DNA content was determined from DNA histograms. IL-4 at 0.01 ng/ml protected from spontaneous apoptosis of cells from previously treated CLL patients, but had very little effect on apoptotic death in cultures of cells from untreated patients. The number of AP cells in the absence of IL-4 was similar in cultures from treated and untreated patients. The concentration of IL-4 which inhibited spontaneous apoptosis by 50% was less than 0.01 ng/ml for pretreated patients and close to 1 ng/ml for untreated patients. Stage of the disease had no effect on the level of spontaneous apoptosis and its sensitivity to IL-4. Protection from apoptosis by IL-4 was not accompanied by the upregulation of bcl-2 protein. The number of AP cells in methylprednisolone hemisuccinate (MP) treated cultures from previously treated patients was significantly lower than in cultures from untreated patients in the presence of 0.01-1.0 ng/ml IL-4. Treatment with the combination L-phenylalanine mustard (L-PAM)+ fludarabine induced synergistic apoptotic response. Apoptosis induced by this combination was relatively resistant to IL-4 in patients treated with chlorambucil and prednisone, but not in patients previously treated with fludarabine. Protection from cytotoxicity by IL-4 may be one of the mechanisms of acquired drug resistance in CLL.

Clinical spectrum of HTLV-I in South Florida

A total of 113 patients with infection due to human T-cell leukemia virus type 1 (HTLV-I) were evaluated at the University of Miami from January 1988 to March 1993. Forty patients were identified with adult T-cell leukemia/lymphoma (ATLL) and 63 with HTLV-I-associated myelopathy (HAM). Three had concomitant ATLL and HAM. Two HAM patients co-infected with human immunodeficiency virus type 1 (HIV-I) developed clonal lymphoproliferative disease during the study period. Patients with ATLL have a poor prognosis; multiple chemotherapy regimens including high-dose cytotoxic agents have been utilized with a small impact on survival. Most of our patients are currently treated with experimental regimens. Rheumatologic or autoimmune illnesses were identified, mostly in HAM patients, and a small number developed immunodeficiencies in the absence of other definable etiologic factors. Most of the patients were immigrants from areas of endemicity in the Caribbean basin, although many Americans were also recognized. HTLV-I/II infection was diagnosed serologically and typed as HTLV-I by polymerase chain reaction (PCR) or a modified Western blot when a DNA sample was not available. In 24 of 40 patients with ATLL, Southern blot hybridization performed on DNA extracted from peripheral blood lymphocytes or tumor tissue demonstrated clonal HTLV-I integration. In South Florida, ATLL and HAM are now seen frequently. Since HTLV-I infection is associated with a 4% lifetime risk of developing ATLL and an additional 0.25% lifetime risk for developing HAM, a large pool of asymptomatically infected individuals must exist here.(ABSTRACT TRUNCATED AT 250 WORDS)

Dose Comparison Study of Allogeneic Mesenchymal Stem Cells in Patients With Ischemic Cardiomyopathy (The TRIDENT Study)

Rationale: Cell dose and concentration play crucial roles in phenotypic responses to cell-based therapy for heart failure. Objective: To compare the safety and efficacy of 2 doses of allogeneic bone marrow–derived human mesenchymal stem cells identically delivered in patients with ischemic cardiomyopathy. Methods and Results: Thirty patients with ischemic cardiomyopathy received in a blinded manner either 20 million (n=15) or 100 million (n=15) allogeneic human mesenchymal stem cells via transendocardial injection (0.5 cc per injection × 10 injections per patient). Patients were followed for 12 months for safety and efficacy end points. There were no treatment-emergent serious adverse events at 30 days or treatment-related serious adverse events at 12 months. The Major Adverse Cardiac Event rate was 20.0% (95% confidence interval [CI], 6.9% to 50.0%) in 20 million and 13.3% (95% CI, 3.5% to 43.6%) in 100 million (P=0.58). Worsening heart failure rehospitalization was 20.0% (95% CI, 6.9% to 50.0%) in 20 million and 7.1% (95% CI, 1.0% to 40.9%) in 100 million (P=0.27). Whereas scar size reduced to a similar degree in both groups: 20 million by −6.4 g (interquartile range, −13.5 to −3.4 g; P=0.001) and 100 million by −6.1 g (interquartile range, −8.1 to −4.6 g; P=0.0002), the ejection fraction improved only with 100 million by 3.7 U (interquartile range, 1.1 to 6.1; P=0.04). New York Heart Association class improved at 12 months in 35.7% (95% CI, 12.7% to 64.9%) in 20 million and 42.9% (95% CI, 17.7% to 71.1%) in 100 million. Importantly, proBNP (pro-brain natriuretic peptide) increased at 12 months in 20 million by 0.32 log pg/mL (95% CI, 0.02 to 0.62; P=0.039), but not in 100 million (−0.07 log pg/mL; 95% CI, −0.36 to 0.23; P=0.65; between group P=0.07). Conclusions: Although both cell doses reduced scar size, only the 100 million dose increased ejection fraction. This study highlights the crucial role of cell dose in the responses to cell therapy. Determining optimal dose and delivery is essential to advance the field, decipher mechanism(s) of action and enhance planning of pivotal Phase III trials. Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02013674.

Treatment of idiopathic thrombocytopenic purpura

ITP is a common disease that is sorely in need of better management. Treatment strategy requires consideration of both long-term benefits and long-term hazards of each available therapeutic option. This discussion reviews conventional therapy as well as newer approaches to refractory ITP, including immunosuppressants, vinca alkaloids, colchicine, androgens, tamoxifen, and plasmapheresis.