Hematopoietic stem and progenitor cell mobilization in mice and humans by a first-in-class mirror-image oligonucleotide inhibitor of CXCL12.

NOX-A12 is a PEGylated mirror-image oligonucleotide (a so-called Spiegelmer) that binds to CXCL12 (stromal cell-derived factor-1, SDF-1) with high affinity thereby inhibiting CXCL12 signaling on both its receptors, CXCR4 and CXCR7. In animals, NOX-A12 mobilized white blood cells (WBCs) and hematopoietic stem and progenitor cells (HSCs) into peripheral blood (PB). In healthy volunteers, single doses of NOX-A12 had a benign safety profile and also dose-dependently mobilized WBCs and HSCs into PB. HSC peak mobilization reached a plateau at five times the baseline level at an i.v. dose of 5.4 mg/kg. In accordance with the plasma half-life of 38 h, the duration of the WBC and HSC mobilization was long lasting and increased dose-dependently to more than 4 days at the highest dose (10.8 mg/kg). In conclusion, NOX-A12 may be appropriate for therapeutic use in and beyond mobilization of HSCs, e.g., in long-lasting mobilization and chemosensitization of hematological cancer cells.

Influence of mannose-binding lectin genotypes and serostatus in allo-SCT: analysis of 131 recipients and donors.

Mannan-binding lectin (MBL) deficiency is determined by MBL gene polymorphisms and is associated with an increased infection risk. To clarify the role of MBL in Allo-SCT, 131 recipients-donors were analysed. MBL genotypes were determined by PCR and heteroduplex analyses, MBL serum levels by ELISA, and MBL oligomers by western blotting. MBL levels <400 ng/ml were associated with increased susceptibility to fungal pneumonia (7/12 vs 35/111; P=0.04, adjusted P=0.002), HSV/VZV (7/12 vs 26/111; P=0.03), CMV reactivation and acute GVHD. Donor genotypes had no influence. Pre-SCT MBL levels corresponded to recipients' genotypes (P<0.001), changed significantly post-SCT, but were not influenced by donors' genotypes. MBL oligomer profiles were similar pre-/post-SCT. Cultured CD34+ cells were found not to synthesise MBL. In conclusion, low MBL levels pre-transplant predisposed patients to sepsis, fungal and viral infection. Donors' MBL genotypes did not influence infection rates. Prospective studies should clarify the importance of MBL as a prelude for MBL replacement after SCT.

Lenalidomide as salvage therapy after allo-SCT for multiple myeloma is effective and leads to an increase of activated NK (NKp44(+)) and T (HLA-DR(+)) cells.

We investigated efficacy and toxicity of lenalidomide in 24 heavily pretreated myeloma patients with a median age of 59 years (range: 37-70) and relapse after allo-SCT. Lenalidomide was given at a dose of 15 mg (n=4), or 25 mg (n=20), orally once daily on day 1 to day 1 every 28 days, with (n=20) or without (n=4) DHAP. The median number of lenalidomide cycles was five (range: 2-17). Major side effects were leukopenia (grade 4: 4%, grade 3: 21% and grade 2: 17%) and thrombocytopenia (grade 3: 17% and grade 2: 29%); infectious complications were observed in 50%. Non-hematological toxicity consisted of muscle cramps (n=9), fatigue (n=5) and constipation (n=2). Mild grade I-II GVHD was seen in three patients. Response was achieved in 66%: CR in 8%, VGPR in 8%, PR in 50% and SD in 13%. The median time to progression was 9.7 months (95% confidence interval (CI): 7.5-11.9), and median OS was 19.9 months (95% CI: 17.3-22.5). Immunomonitoring after lenalidomide showed significant increase of activated NK (NKp44(+)) and T (HLA-DR(+)) cells, as well as regulatory T cells (CD4(+), CD25(+), CD127(lo)), supporting an immunomodulating anti-myeloma effect of lenalidomide.

Percutaneous intramyocardial stem cell injection in patients with acute myocardial infarction: first-in-man study.

BACKGROUND: Clinical studies on intracoronary stem cell infusion in patients with acute myocardial infarction (AMI) have shown promising results for left ventricular ejection fraction (LVEF). However, preclinical studies have shown that intramyocardial cell injection is better than the intracoronary approach. OBJECTIVE: To test safety and feasibility of intramyocardial cell injection and left ventricular electromechanical mapping (EMM) early after AMI. DESIGN: On day 10.5 (5) (mean (SD)) after AMI and percutaneous coronary intervention with stent implantation (culprit lesion: 15 LAD, 3 circumflex and 2 right coronary arteries) 20 patients (mean (SD) 60.4 (11.4) years) received bone marrow derived mononuclear cells in the low-voltage area using EMM-guided percutaneous intramyocardial injection. EMM and coronary angiography were performed in 15 patients at 6-months' follow-up. Echocardiography, recording of laboratory data and clinical assessment (6-month and 12-month follow-up) were carried out in all 20 patients. RESULTS: None of the patients showed periprocedural complications. Three patients received an implantable cardioverter-defibrillator for primary prevention of sudden cardiac death and 6 (30%) patients showed in-stent restenosis. One patient underwent bypass surgery owing to chronic stent occlusion after 6 months. 2.0 (0.6)x10(8) cells, including 1.0 (0.3)x10(6) CD45(dim)/CD34(hi) stem cells, were injected in each patient. EMM showed a mean (SD) improvement from a baseline unipolar voltage of 45.5 (14.3)% to 59.3 (19.8)% of normal voltage (p = 0.002) and reduction of the low-voltage area from 28.7 (12.1)% to 20.3 (13.5)%; (p = 0.016). During the 12-month follow-up, the left ventricular ejection fraction (LVEF) improved from 40.8 (6.9)% to 47.1 (10.6)%; (p = 0.037). CONCLUSION: Left ventricular EMM and percutaneous intramyocardial cell injection in patients with AMI was shown to be a safe procedure. It is associated with improved LVEF and electromechanical parameters after 12-months' follow-up. TRIAL REGISTRATION NUMBER: Eudra-CT-No 2005-003629-19.

Transportation and cryopreservation may impair haematopoietic stem cell function and engraftment of allogeneic PBSCs, but not BM.

Recent data suggest that the practice of using frozen allogeneic grafts is becoming increasingly common among transplant centres. Therefore, we retrospectively analysed 31 frozen allogeneic PBSC and 8 BM grafts by flow cytometry with regard to their CD34+ content, membrane integrity (7-AAD) and stem cell-specific enzyme activity (aldehyde dehydrogenase, ALDH) in relation to individual transplantation results. Membrane integrity of CD34+ cells was significantly impaired in cryopreserved PBSC but not in BM compared to unfrozen allografts. In 9 out of 31 frozen PBSC (but none of the BM) grafts numbers of SSC(lo)ALDH(br) cells per kg body weight (BW) were significantly reduced while in the same grafts the numbers of CD34+ cells per kg BW were close to normal. Overall, 9 out of 33 patients (27%) who received unrelated PBSC allografts cryopreserved after transportation did not achieve engraftment. For comparison, primary graft failure was observed in our centre in only 7 out of 493 recipients (1.4%) of fresh allogeneic PBSC grafts. Moreover, we did not see any graft failure in patients receiving frozen/thawed BM or autologous PBSC transplants. We, therefore, conclude that PBSC grafts become much more sensitive to cryopreservation after transport and/or storage. Importantly, the engraftment potential of frozen HSC grafts may reliably be predicted by measuring ALDH activity.

Hepatocytic gene expression in cultured rat mesenchymal stem cells.

The origin of liver cells from distinct bone marrow stem cells, eg, hematopoietic stem cells or multipotent adult progenitor cells has been recently described using in vitro studies. Cell culture experiments revealed the key role of growth factors and the organ-specific environment for the induction of liver-specific genes. We investigated the in vitro potential of rat mesenchymal stem cells to differentiate into hepatocytic cells in cocultures with isolated rat liver cells. Rat mesenchymal stem cells (MSCs) propagated in culture, and transduced with green fluorescent protein (GFP) were cloned. Cells from selected clones were either cultured under liver-stimulating conditions, using serum free medium supplemented with HGF, EGF, SCF, and FGF-4 alone on fibronectin-coated surfaces, or cocultured with freshly isolated rat liver cells. Cocultured cells were harvested after two weeks and sorted into GFP-positive (GFP+) and GFP-negative (GFP-) cells. RT-PCR for liver specific markers CK-18 and albumin were performed on the different cell populations. After 2 weeks, the specified culture conditions led to the expression of albumin and CK-18 RNA in GFP-positive sorted MSCs from the cocultures, whereas MSCs cultured without liver cells did not express the studied genes. The results indicate, that when cocultured with liver cells MSCs from the bone marrow have the potential to differentiate toward hepatocytic cells in vitro. We conclude that MSC may possess an enhanced capacity to differentiate into functional liver cells. Additionally, environmental factors seem to be crucial for specific and directed differentiation.

Aldehyde dehydrogenase activity as a marker for the quality of hematopoietic stem cell transplants.

Taking advantage of fluorescent substrates for their metabolic marker aldehyde dehydrogenase (ALDH), hematopoietic stem cells (HSC) were defined as SSC(lo)ALDH(br) - reflecting their low orthogonal light scattering and bright fluorescence intensity in flow cytometry. Based thereon, we investigated the usefulness of ALDH activity for characterizing HSC graft quality, particularly under stress conditions. We first compared the expression of ALDH vs CD34 in bone marrow and peripheral blood stem cell (PBSC) samples over 7 days. We noted that (i) only ALDH activity but not CD34 expression strongly reflected colony-forming ability over time, and that (ii) PBSC grafts stored at room temperature lost most of their progenitor cells within just 48 h. We then retrospectively related ALDH and CD34 expression as well as granulocyte-macrophage colony-forming units (CFU-GM) potential for 19 cryopreserved allogeneic PBSC grafts to engraftment data. Strikingly, in all six patients who received markedly decreased numbers of SSC(lo)ALDH(br) cells, this was associated not only with almost complete loss of CFU-GM potential but also with delayed establishment/permanent absence of full hematopoietic donor cell chimerism, whereas all other patients showed early complete donor chimerism. In conclusion, we suggest to measure ALDH activity as a surrogate marker for HSC activity, and to transport and store PBSC under controlled cooling conditions.

[The nature of the changes in the systemic hemodynamic parameters under the combined action of vasoactive substances]. / Kharakter izmenenii sistemnykh gemodinamicheskikh parametrov pri sochetannom deistvii vazoaktivnykh veshchestv.

The direction and obviousness of changes in systemic arterial and venous vascular beds occurring in combined action of two similarly acting pressor and depressor vasoactive drugs, were studied in acute experiments in cats. The changes of last and general peripheral resistance of vessels were greater under the action of the two drugs as compared to the effect of a single drug, and yet less obvious than their algebraic sum. The shifts in cardiac output and venous return were not greater than the most obvious response to a single drug. The same was true for the blood flow changes in v. v. cavae. Irrespective of the mechanism of action of vasoactive drugs and the direction of shifts of systemic AP, single and combined drugs increased the blood flow in the v. cava anterior whereas the v. cava posterior's blood flow changed in different directions.

[The dependence of the hemodynamic shifts in the systemic arterial and venous vascular beds on the degree of change in the arterial pressure under the action of vasoactive substances]. / Zavisimost' gemodinamicheskikh sdvigov v sistemnom arterial'nom i venoznom sosudistom rusle ot stepeni izmeneniia arterial'nogo davleniia pri deistvii vazoaktivnykh veshchestv.

The participation of hemodynamic parameters in formation of systemic pressor displacement due to action of norepinephrine and angiotensin was shown in acute experiments in cats. An arterial pressure rise by 15 +/- 2% and 25 +/- 3% depended on total peripheral resistance and cardiac output enhancement. The maximal arterial pressure rise only depended on total peripheral resistance increase. The reduction of venous return resulted from a decrease of the blood flow in v. cava posterior whereas the blood stream in v. cava anterior increased. Depressor actions of acetylcholine and histamine were enhanced owing to a fall in the vessels resistance.

[Character of arterial pressure changes under combined action of pressor and depressor agents]. / Kharakter izmenenii arterial'nogo davleniia pri sochetannom deistvii pressornykh i depressornykh veshchestv.

It is shown in acute experiments in cats that byphasic arterial pressure changes occurred due to action of two heterodirectional and equivalent humoral stimuli. Magnitudes of both initial depressor and following pressor phases were authentically less than the ones due to separate action of those stimuli. It is noticed that the character of sum reaction depends neither on action mechanisms of vasoactive agents in the blood circulation system nor differences in the latent period of the effect of those drugs, but connected with intensiveness of stimuli. Predominance of depressor phase magnitude was shown to observe due to a rise of stimuli intensiveness down to complete disappearance of pressor reaction.