Differentiation of Human Cardiac Atrial Appendage Stem Cells into Adult Cardiomyocytes: A Role for the Wnt Pathway?

Human cardiac stem cells isolated from atrial appendages based on aldehyde dehydrogenase activity (CASCs) can be expanded in vitro and differentiate into mature cardiomyocytes. In this study, we assess whether Wnt activation stimulates human CASC proliferation, whereas Wnt inhibition induces cardiac maturation. CASCs were cultured as described before. Conventional PCR confirmed the presence of the Frizzled receptors. Small-molecule inhibitors (IWP2, C59, XAV939, and IWR1-endo) and activator (CHIR99021) of the Wnt/ß -catenin signaling pathway were applied, and the effect on ß-catenin and target genes for proliferation and differentiation was assessed by Western blot and RT-qPCR. CASCs express multiple early cardiac differentiation markers and are committed toward myocardial differentiation. They express several Frizzled receptors, suggesting a role for Wnt signaling in clonogenicity, proliferation, and differentiation. Wnt activation increases total and active ß-catenin levels. However, this does not affect CASC proliferation or clonogenicity. Wnt inhibition upregulated early cardiac markers but could not induce mature myocardial differentiation. When CASCs are committed toward myocardial differentiation, the Wnt pathway is active and can be modulated. However, despite its role in cardiogenesis and myocardial differentiation of pluripotent stem-cell populations, our data indicate that Wnt signaling has limited effects on CASC clonogenicity, proliferation, and differentiation.

Cardiac atrial appendage stem cells promote angiogenesis in vitro and in vivo.

Cardiac atrial appendage stem cells (CASCs) show extraordinary myocardial differentiation properties, making them ideal candidates for myocardial regeneration. However, since the myocardium is a highly vascularized tissue, revascularization of the ischemic infarct area is essential for functional repair. Therefore, this study assessed if CASCs contribute to cardiac angiogenesis via paracrine mechanisms. First, it was demonstrated that CASCs produce and secrete high levels of numerous angiogenic growth factors, including vascular endothelial growth factor (VEGF), endothelin-1 (ET-1) and insulin-like growth factor binding protein 3 (IGFBP-3). Functional in vitro assays with a human microvascular endothelial cell line (HMEC-1) and CASC CM showed that CASCs promote endothelial cell proliferation, migration and tube formation, the most important steps of the angiogenesis process. Addition of inhibitory antibodies against identified growth factors could significantly reduce these effects, indicating their importance in CASC-induced neovascularization. The angiogenic potential of CASCs and CASC CM was also confirmed in a chorioallantoic membrane assay, demonstrating that CASCs promote blood vessel formation in vivo. In conclusion, this study shows that CASCs not only induce myocardial repair by cardiomyogenic differentiation, but also stimulate blood vessel formation by paracrine mechanisms. The angiogenic properties of CASCs further strengthen their therapeutic potential and make them an optimal stem cell source for the treatment of ischemic heart disease.

From Bone Marrow to Cardiac Atrial Appendage Stem Cells for Cardiac Repair: A Review.

Traditionally the heart is considered a terminally differentiated organ. However, at the beginning of this century increased mitotic activity was reported in ischemic and idiopathic dilated cardiomyopathy hearts, compared to healthy controls, underscoring the potential of regeneration after injury. Due to the presence of adult stem cells in bone marrow and their purported ability to differentiate into other cell lineages, this cell population was soon estimated to be the most suited candidate for cardiac regeneration. Clinical trials with autologous bone marrow-derived mononuclear cells, using either an intracoronary or direct intramyocardial injection approach consistently showed only minor improvement in global left ventricular ejection fraction. This was explained by their limited cardiomyogenic differentiation potential. To obtain more convincing improvement in cardiac function, based on true myocardial regeneration, the focus of research has shifted towards resident cardiac progenitor cells. Several isolation procedures have been described: the c-kit surface marker was the first to be used, however experimental research has clearly shown that c-kit+ cells only marginally contribute to regeneration post myocardial infarction. Sphere formation was used to isolate the so-called cardiosphere derived cells (CDC), and also in this cell population cardiomyogenic differentiation is a rare event. Recently a new type of stem cells derived from atrial tissue (cardiac atrial stem cells - CASCs) was identified, based on the presence of the enzyme aldehyde dehydrogenase (ALDH). Those cells significantly improve both regional and global LV ejection fraction, based on substantial engraftment and consistent differentiation into mature cardiomyocytes (98%).

Cardiac atrial appendage stem cells engraft and differentiate into cardiomyocytes in vivo: A new tool for cardiac repair after MI.

BACKGROUND: This study assessed whether autologous transplantation of cardiac atrial appendage stem cells (CASCs) preserves cardiac function after myocardial infarction (MI) in a minipig model. METHODS AND RESULTS: CASCs were isolated from right atrial appendages of Göttingen minipigs based on high aldehyde dehydrogenase activity and expanded. MI was induced by a 2h snare ligation of the left anterior descending coronary artery. Upon reperfusion, CASCs were intramyocardially injected under NOGA guidance (MI-CASC, n=10). Non-transplanted pigs (MI, n=8) received sham treatment. 3D electromechanical mapping (EMM) and cardiac MRI were performed to assess left ventricular (LV) function. MI pigs developed LV dilatation at 2 months (2M), while in the MI-CASC group volumes remained stable. Global LV ejection fraction decreased by 16 ± 8% in MI animals vs 3 ± 10% in MI-CASC animals and regional wall thickening in border areas was better preserved in the MI-CASC group. EMM showed decreased viability and wall motion in the LV for both groups POST-MI, whereas at 2M these parameters only improved in the MI-CASC. Substantial cell retention was accompanied by cardiomyogenic differentiation in 98±1% of the transplanted CASCs, which functionally integrated. Second harmonic generation microscopy confirmed the formation of mature sarcomeres in transplanted CASCs. Absence of cardiac arrhythmias indicated the safety of CASC transplantation. CONCLUSION: CASCs preserve cardiac function by extensive engraftment and cardiomyogenic differentiation. Our data indicate the enormous potential of CASCs in myocardial repair.

Mesenchymal stem cell secreted platelet derived growth factor exerts a pro-migratory effect on resident Cardiac Atrial appendage Stem Cells.

Mesenchymal stem cells (MSCs) modulate cardiac healing after myocardial injury through the release of paracrine factors, but the exact mechanisms are still unknown. One possible mechanism is through mobilization of endogenous cardiac stem cells (CSCs). This study aimed to test the pro-migratory effect of MSC conditioned medium (MSC-CM) on endogenous CSCs from human cardiac tissue. By using a three-dimensional collagen assay, we found that MSC-CM improved migration of cells from human cardiac tissue. Cell counts, perimeter and area measurements were utilized to quantify migration effects. To examine whether resident stem cells were among the migrating cells, specific stem cell properties were investigated. The migrating cells displayed strong similarities with resident Cardiac Atrial appendage Stem Cells (CASCs), including a clonogenic potential of ~21.5% and expression of pluripotency associated genes like Oct-4, Nanog, c-Myc and Klf-4. Similar to CASCs, migrating cells demonstrated high aldehyde dehydrogenase activity and were able to differentiate towards cardiomyocytes. Receptor tyrosine kinase analysis and collagen assays performed with recombinant platelet derived growth factor (PDGF)-AA and Imatinib Mesylate, a PDGF receptor inhibitor, suggested a role for the PDGF-AA/PDGF receptor α axis in enhancing the migration process of CASCs. In conclusion, our findings demonstrate that factors present in MSC-CM improve migration of resident stem cells from human cardiac tissue. These data open doors towards future therapies in which MSC secreted factors, like PDGF-AA, can be utilized to enhance the recruitment of CASCs towards the site of myocardial injury.

Bone marrow-derived mesenchymal stem cells promote colorectal cancer progression through paracrine neuregulin 1/HER3 signalling.

OBJECTIVE: Bone marrow-derived mesenchymal stem cells (BM-MSC) migrate to primary tumours and drive tumour progression. This study aimed to identify the molecular mechanisms associated with these heterotypic cellular interactions and analyse their relevance in colorectal cancer (CRC). DESIGN: Paracrine interactions of BM-MSC with CRC cells were studied using collagen invasion assays, cell counts, flow cytometric cell-cycle analysis and tumour xenograft models. The role of neuregulin 1 (NRG1) and the human epidermal growth factor receptor (HER) family pathways were investigated using tyrosine kinase assays, mass spectrometry, pharmacological inhibition, antibody-mediated neutralisation and RNA interference. Transmembrane neuregulin 1 (tNRG1), HER2 and HER3 expression was analysed in primary CRC (n=54), adjacent normal colorectal tissues (n=4), liver metastases (n=3) and adjacent normal liver tissues (n=3) by immunohistochemistry. RESULTS: BM-MSC stimulate invasion, survival and tumorigenesis of CRC through the release of soluble NRG1, activating the HER2/HER3-dependent PI3K/AKT signalling cascade in CRC cells. Similarly, tumour-associated mesenchymal cells (T-MC) in CRC demonstrate high tNRG1 expression, which is significantly associated with advanced Union for International Cancer Control stage (p=0.005) and invasion depth (p=0.04) and decreased 5-year progression-free survival (p=0.01). HER2 and HER3 show membrane localisation in cancer cells of CRC tissue. CONCLUSION: Paracrine NRG1/HER3 signals initiated by BM-MSC and T-MC promote CRC cell progression, and high tNRG1 expression is associated with poor prognosis in CRC.

The cardiac atrial appendage stem cell: a new and promising candidate for myocardial repair.

AIMS: Considerable shortcomings in the treatment of myocardial infarction (MI) still exist and therefore mortality remains high. Cardiac stem cell (CSC) therapy is a promising approach for myocardial repair. However, identification and isolation of candidate CSCs is mainly based on the presence or absence of certain cell surface markers, which suffers from some drawbacks. In order to find a more specific and reliable identification and isolation method, we investigated whether CSCs can be isolated based on the high expression of aldehyde dehydrogenase (ALDH). METHODS AND RESULTS: An ALDH(+) stem cell population, the cardiac atrial appendage stem cells (CASCs), was isolated from human atrial appendages. CASCs possess a unique phenotype that is clearly different from c-kit(+) CSCs but that seems more related to the recently described cardiac colony-forming-unit fibroblasts. Based on immunophenotype and in vitro differentiation studies, we suggest that CASCs are an intrinsic stem cell population and are not mobilized from bone marrow or peripheral blood. Indeed, they possess a clonogenicity of 16% and express pluripotency-associated genes. Furthermore, compared with cardiosphere-derived cells, CASCs possess an enhanced cardiac differentiation capacity. Indeed, differentiated cells express the most important cardiac-specific genes, produce troponin T proteins, and have an electrophysiological behaviour similar to that of adult cardiomyocytes (CMs). Transplanting CASCs in the minipig MI model resulted in extensive cardiomyogenic differentiation without teratoma formation. CONCLUSION: We have identified a new human CSC population able to differentiate into functional CMs. This opens interesting perspectives for cell therapy in patients with ischaemic heart disease.

Clinical benefits of the addition of lower extremity low-intensity resistance muscle training to early aerobic endurance training intervention in patients with coronary artery disease: a randomized controlled trial.

OBJECTIVE: Muscle resistance training is often combined with aerobic endurance training during rehabilitation of patients with coronary artery disease. However, the clinical effects of additional lower-extremity low-intensity muscle resistance training during early rehabilitation (within the first month after coronary revascularization) in patients with coronary artery disease remain unclear. DESIGN: Prospective randomized controlled trial. SUBJECTS: Sixty patients with coronary artery disease. METHODS: Subjects were randomly assigned to early aerobic endurance training (n = 30) or combined aerobic endurance and resistance muscle training (n = 30). Subjects performed 18 (standard deviation 2) exercise sessions (at 65% VO(2peak), for 40 mins/session). In resistance muscle training, additional low-intensity (12-20 repetition maximum) resistance muscle exercises were performed. The following parameters were evaluated: exercise capacity, body composition, blood lipid profile, glycaemic control, blood endothelial progenitor cell and cytokine content, and muscle performance. RESULTS: A total of 47 patients with coronary artery disease completed the intervention. Total body lean tissue mass tended to increase with greater magnitude (p = 0.07), and blood high-density lipid cholesterol content increased with significantly greater magnitude in resistance muscle training (p < 0.05), compared with aerobic endurance training. Maximal exercise capacity, ventilatory threshold, and muscle performance increased, and steady-state exercise respiratory exchange ratio, and adipose tissue mass reduced significantly (p < 0.05), without differences between groups (p < 0.05). CONCLUSION: In early aerobic endurance training intervention in patients with coronary artery disease, additional low-intensity resistance muscle training contributes to a greater increase in blood high-density lipid cholesterol content, and tends to affect lean tissue mass.

Mesenchymal stem cells or cardiac progenitors for cardiac repair? A comparative study.

In the past, clinical trials transplanting bone marrow-derived mononuclear cells reported a limited improvement in cardiac function. Therefore, the search for stem cells leading to more successful stem cell therapies continues. Good candidates are the so-called cardiac stem cells (CSCs). To date, there is no clear evidence to show if these cells are intrinsic stem cells from the heart or mobilized cells from bone marrow. In this study we performed a comparative study between human mesenchymal stem cells (hMSCs), purified c-kit(+) CSCs, and cardiosphere-derived cells (CDCs). Our results showed that hMSCs can be discriminated from CSCs by their differentiation capacity towards adipocytes and osteocytes and the expression of CD140b. On the other hand, cardiac progenitors display a greater cardiomyogenic differentiation capacity. Despite a different isolation protocol, no distinction could be made between c-kit(+) CSCs and CDCs, indicating that they probably derive from the same precursor or even are the same cells.

Memory CD4+CD127high T cells from patients with multiple sclerosis produce IL-17 in response to myelin antigens.

Myelin-reactive T helper-17 cells are implicated in the pathogenesis of multiple sclerosis (MS). Here, we analyzed the reactivity of peripheral naive and memory conventional CD4(+)CD127(high) T cells (Tconv) of MS patients and healthy controls (HC) towards myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG) and tetanus toxoid (TT). Proliferative responses of Tconv cells towards MBP, MOG and TT were not significantly different between MS patients and HC. However, MBP and MOG but not TT reactive memory Tconv cells from MS patients, in contrast to HC, produced IL-17. These results suggest that myelin antigen reactive Th-17 cells are enriched in MS patients.

The use of human tissue in epidemiological research; ethical and legal considerations in two biobanks in Belgium.

This paper discusses the legal implications of setting up two new biobanks in Belgium. The first is hospital-based and will archive tissue from patients with haematologic cancer, whereas the second is linked to a general practice based morbidity registry and will involve storage of blood samples. To date, Belgium has no specific legislation that regulates storage of human tissue and related databases. Several issues concerning the protection of individuals with regard to the processing of personal medical data are discussed from the existing privacy legislation. We will address the principle of consent (broad versus specific) and the type of data recorded (anonymous, encoded and identifiable) for both biobanks.

Human bone marrow stem cells co-cultured with neonatal rat cardiomyocytes display limited cardiomyogenic plasticity.

BACKGROUND AIMS: This study investigated whether neonatal rat cardiomyocytes (NRCM), when co-cultured, can induce transdifferentiation of either human mesenchymal stromal cells (MSC) or hematopoietic stem cells (HSC) into cardiomyocytes. Stem cells were obtained from patients with ischemic heart disease. METHODS: Ex vivo-expanded MSC or freshly isolated HSC were used to set-up a co-culture system between NRCM and MSC or HSC. 5-azacytidin (5-aza) or dimethylsulfoxide (DMSO) was used as differentiation-inducing factor. Co-cultured stem cells were separated from NRCM by flow sorting, and cardiac gene expression was analyzed by reverse transcriptase-polymerase chain reaction. Cellular morphology was analyzed by immunofluorescence and transmission electron microscopy (TEM). RESULTS: Co-culturing MSC induced expression of troponin T and GATA-4. However, no expression of alpha-actinin, myosin heavy chain or troponin I was detected. In the case of HSC, only expression of troponin T could be induced. Immunofluorescence and TEM confirmed the absence of sarcomeric organization in co-cultured MSC and HSC. Adding 5-aza or DMSO to the co-cultures did not influence differentiation. CONCLUSIONS: This in vitro co-culture study obtained no convincing evidence of transdifferentiation of either MSC or HSC into functional cardiomyocytes. Nevertheless, induction of troponin T was observed in MSC and HSC, and GATA-4 in MSC. However, no morphologic changes could be detected by immunofluorescence or by TEM. These data could explain why only limited functional improvement was reported in clinical stem cell trials.

Natural naive CD4+CD25+CD127low regulatory T cell (Treg) development and function are disturbed in multiple sclerosis patients: recovery of memory Treg homeostasis during disease progression.

Patients with relapsing-remitting multiple sclerosis (RR-MS) show a suboptimal CD4(+)CD25(+) regulatory T cell (Treg) function, whereas no Treg alterations are observed in secondary progressive MS (SP-MS) patients. To clarify the difference in Treg activity between early and chronic disease stages in MS, we analyzed the functional capacity and homeostatic parameters of naive CD4(+)CD25(+)CD127(low)CD45RA(+) Tregs (nTregs) and their memory counterparts CD4(+)CD25(+)CD127(low)CD45RO(+) Tregs (mTregs) in untreated MS patients and healthy controls. Interestingly, whereas the suppressive capacity of FACS-sorted nTregs was impaired in both early and chronic MS patients, only the latter group showed a restored mTreg function. Consistent with this observation, chronic MS patients had increased numbers of mTregs as compared with age-matched early MS patients, whereas nTreg frequencies did not differ significantly. TCR excision circle numbers were reduced in nTregs of early MS patients, suggestive of a diminished nTreg thymic output. Moreover, a decreased number of CD31(+) mTregs were observed in early vs chronic MS patients, indicating that inflammatory processes drive the homeostatic turnover of mTregs during the early disease stage. Additionally, early MS patients showed a more restricted nTreg and mTreg TCR BV gene profile as compared with healthy controls and chronic MS patients. Finally, analysis of IFN-beta and glatiramer acetate-treated MS patients showed that these immunomodulatory drugs modify nTreg homeostasis. Taken together, this study provides strong evidence for a disturbed thymic nTreg development and function in MS patients. Moreover, memory Treg but not naive Treg homeostasis recovers during disease progression.

Compromised CD4+ CD25(high) regulatory T-cell function in patients with relapsing-remitting multiple sclerosis is correlated with a reduced frequency of FOXP3-positive cells and reduced FOXP3 expression at the single-cell level.

CD4+ CD25(high) regulatory T cells (Tregs) of patients with relapsing-remitting (RR) multiple sclerosis (MS), in contrast to those of patients with secondary progressive (SP) MS, show a reduced suppressive function. In this study, we analysed forkhead box P3 (FOXP3) at the single-cell level in MS patients and controls (healthy individuals and patients with other neurological diseases) by means of intracellular flow cytometry. Our data revealed a reduced number of peripheral blood CD4+ CD25(high) FOXP3+ T cells and lower FOXP3 protein expression per cell in RR-MS patients than in SP-MS patients and control individuals, which was correlated with the suppressive capacity of Tregs in these patients. Interestingly, interferon (IFN)-beta-treated RR-MS patients showed restored numbers of FOXP3+ Tregs. Furthermore, a higher percentage of CD4+ CD25(high) FOXP3+ Tregs in RR-MS patients, as compared with controls and SP-MS patients, expressed CD103 and CD49d, adhesion molecules involved in T-cell recruitment towards inflamed tissues. This was consistent with a significantly increased number of CD27+ CD25(high) CD4+ T cells in the cerebrospinal fluid (CSF), as compared with peripheral blood, in RR-MS patients. Taken together, these data show aberrant FOXP3 expression at the single-cell level correlated with Treg dysfunction in RR-MS patients. Our results also suggest that Tregs accumulate in the CSF of RR-MS patients, in an attempt to down-regulate local inflammation in the central nervous system.

Effect of prebiotic galacto-oligosaccharide, long-chain fructo-oligosaccharide infant formula on serum cholesterol and triacylglycerol levels.

OBJECTIVE: Cholesterol is a nutrient of essential importance in infant feeding because it is necessary in membrane development. In adults with high lipid levels, high doses of inulin (oligofructose) inconsistently decreased levels of serum cholesterol. The aim of the present study was to evaluate cholesterol and triacylglycerol levels in infants receiving a formula with a specific mixture of 0.6 g/100 mL of galacto-oligosaccharides (GOS) and long-chain fructo-oligosaccharides (lcFOS) in a ratio of 9/1, a control formula, or breast milk. Because the level of lcFOS in the infant milk is low, we hypothesized that there would be no differences between the formula groups. METHODS: Two hundred fifteen infants were included in a prospective, randomized, double-blinded, placebo-controlled trial during the first 6 mo of life. Formula-fed infants were randomized to receive a standard infant formula with a specific mixture of 0.6 g/100 mL of GOS/lcFOS, in a ratio of 9/1, or a control formula. Breast-fed infants were randomized to receive one of these two formulas after the mother had decided to discontinue breastfeeding. Serum levels of cholesterol, high-density lipoprotein, low-density lipoprotein (LDL), and triacylglycerol were determined at 8 and 26 wk of age and were provided for infants who received the GOS/lcFOS formula or control formula from birth or after cessation of breastfeeding and for the subgroups that were fully fed with breast milk and formula. RESULTS: One hundred eighty-seven infants completed the study. Total cholesterol and LDL levels at 8 and 26 wk were significantly lower in the formula-fed groups than in the breast-fed infants. There were no significant differences between the formula-fed groups. Levels of triacylglycerols and high-density lipoprotein did not differ between groups. CONCLUSION: Our study demonstrated no differences in total cholesterol and LDL cholesterol in infants receiving an infant formula with GOS/lcFOS from infants receiving a control infant formula. Furthermore, total cholesterol and LDL cholesterol levels were higher in breast-fed infants than in formula-fed infants.

A CFSE based assay for measuring CD4+CD25+ regulatory T cell mediated suppression of auto-antigen specific and polyclonal T cell responses.

CD4(+)CD25(+) regulatory T cells (Tregs) are considered to play a key role as suppressors of immune mediated reactions. The analysis of Treg function in patients with autoimmune, allergic or oncogenic diseases has emerged over the past years. In the present study we describe a CFSE based protocol to measure Treg mediated suppression of CD4(+) T cells. Measuring Treg suppressive capacity towards proliferation of anti-CD3 Ab stimulated CD4(+)CD25(-) T cells in coculture experiments by means of a CFSE based and a classical [(3)H]thymidine incorporation assay gave similar results, provided that CD4(+)CD25(+) T cells were anergic. However, when CD4(+)CD25(+) T cells proliferated upon mitogenic stimulation, data obtained by the CFSE assay allowed the detection of a significant Treg suppression whereas this was clearly underestimated using the [(3)H]thymidine assay. In addition, an indirect CFSE based method was developed to analyze antigen specific responses of total CD4(+) T cells and Treg depleted CD4(+) T cells (i.e. CD4(+)CD25(-) T cells). Our results indicate that, in healthy individuals, CD4(+) T cell responses against the multiple sclerosis (MS) auto-antigens, myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG), were increased in Treg depleted CD4(+) T cells as compared to total CD4(+) T cells. Our initial data suggest that Tregs in MS patients show an impaired suppression of myelin reactive T cells when compared to healthy controls. Moreover, this experimental setup permits the measurement of cytokine production of the antigen proliferated CFSE(low) T cells by additional flow cytometric analyses. In conclusion, the described CFSE based Treg suppression assay is a valuable tool to study suppressor T cells in (auto)immune disorders.

Recovery of regional but not global contractile function by the direct intramyocardial autologous bone marrow transplantation: results from a randomized controlled clinical trial.

BACKGROUND: Recent trials have shown that intracoronary infusion of bone marrow cells (BMCs) improves functional recovery after acute myocardial infarction. However, whether this treatment is effective in heart failure as a consequence of remodeling after organized infarcts remains unclear. In this randomized trial, we assessed the hypothesis that direct intramyocardial injection of autologous mononuclear bone marrow cells during coronary artery bypass graft (CABG) could improve global and regional left ventricular ejection fraction (LVEF) at 4-month follow-up. METHODS AND RESULTS: Twenty patients (age 64.8+/-8.7; 17 male, 3 female) with a postinfarction nonviable scar, as assessed by thallium (Tl) scintigraphy and cardiac magnetic resonance imaging (MRI), scheduled for elective CABG, were included. They were randomized to a control group (n =10, CABG only) or a BMC group (CABG and injection of 60.10(6)+/-31.10(6) BMC). Primary end points were global LVEF change and wall thickening changes in the infarct area from baseline to 4-month follow-up, as measured by MRI. Changes in metabolic activity were measured by Tl scintigraphy and expressed as a score with a range from 0 to 4, corresponding to percent of maximal myocardial Tl uptake (4 indicates <50%, nonviable scar; 3, 50% to 60%; 2, 60% to 70%; 1, 70% to 80%; 0>80%). Global LVEF at baseline was 39.5+/-5.5% in controls and 42.9+/-10.3% in the BMC group (P=0.38). At 4 months, LVEF had increased to 43.1+/-10.9% in the control group and to 48.9+/-9.5% in the BMC group (P=0.23). Systolic thickening had improved from -0.6+/-1.3 mm at baseline to 1.8+/-2.6 mm at 4 months in the cell-implanted scars, whereas nontreated scars remained largely akinetic (-0.5+/-2.0 mm at baseline compared with 0.4+/-1.7 mm at 4 months, P=0.007 control versus BMC-treated group at 4 months). Defect score decreased from 4 to 3.3+/-0.9 in the BMC group and to 3.7+/-0.4 in the control group (P=0.18). CONCLUSIONS: At 4 months, there was no significant difference in global LVEF between both groups, but a recovery of regional contractile function in previously nonviable scar was observed in the BMC group.

Secondary progressive in contrast to relapsing-remitting multiple sclerosis patients show a normal CD4+CD25+ regulatory T-cell function and FOXP3 expression.

Accumulating evidence indicates an immunosuppressive role for CD4(+)CD25(+) regulatory T cells (Tregs) in autoimmune diseases. Although an impaired Treg function in patients with relapsing-remitting multiple sclerosis (RR-MS) has been reported recently, no information is available so far about Treg function in the progressive stage of the disease. In the present study, the phenotypic and functional characteristics of CD4(+)CD25(+) T cells isolated from the peripheral blood of patients with RR-MS and secondary progressive multiple sclerosis (SP-MS) were investigated. No significant quantitative or phenotypic abnormalities in CD4(+)CD25(+) T cells from RR- and SP-MS patients were detected. However, whereas a reduced suppressor function of CD4(+)CD25(+) T cells toward proliferation and interferon-gamma production of CD4(+)CD25(-) responder T cells was found in RR-MS patients, SP-MS patients showed a normal Treg function. The suppressive capacity of MS-derived CD4(+)CD25(+) T cells was correlated with disease duration but not with age, indicating that Treg function is more affected in the early phase of the disease process. Consistently with the suppressive capacity, CD4(+)CD25(+) T cells from SP-MS patients showed normal levels of FOXP3 mRNA in contrast to RR-MS patients that had a reduced FOXP3 expression. These data are the first to demonstrate differences in function and FOXP3 expression of CD4(+)CD25(+) T cells from patients with RR- and SP-MS.