Cryopreserved human heart valve allografts: a ten-year single centre experience.

This study provides an overview of tissue banking activities at the Croatian Cardiovascular Tissue Bank (CTB) during past ten years and presents the outcomes of cryopreserved heart valve allografts (CHAs) use in different patient groups. From June 2011 until December 2021, 75 heart donations were referred to CTB: 41 recipient of heart transplant (RHT), 32 donors after brain death (DBD) and 2 donors after circulatory death (DCD) donations. Processing resulted in 103 valves of which 65 met quality requirements for clinical use. Overall tissue discard rate was 37%. The most frequent reasons for discard were inadequate morphology (12%) in RHT donations and microbiological contamination (19%) in DBD donations. Altogether, 38 CHAs were transplanted to 36 patients. Recipients were divided in three groups; infective endocarditis (IE), non-infectious heart disease and congenital heart disease group. In the IE group, the 30-day, 1-year and 3-year survival was 71%, 53% and 47%, respectively. Freedom from re-operation due to all graft-related causes was 76% and due to structural valve deterioration 88%. There were no cases of graft reinfection. In the congenital heart disease group CHAs were predominantly (94%) used for right ventricular outflow tract reconstruction and 88% of patients recovered without graft-related complications. At present, the number of demands for CHAs at CTB considerably outweighs their availability.

Recurrent Aortic Infections Due to Unrecognized Aorto-Enteric Fistula.

We report a case of an infrarenal abdominal aortic aneurysm (AAA) with unrecognized primary aortoduodenal fistula (ADF), treated by endovascular aortic repair (EVAR). Endograft infection was diagnosed 12 months thereafter. The associated ADF was uncovered during open surgery, which included endograft extraction, in situ aortic reconstruction with a cryopreserved homograft (CHG) and duodenal repair. The patient was urgently reoperated in the early postoperative course, due to CHG rupture and subsequent hemorrhagic shock. After establishing control of hemorrhage, CHG was explanted, followed by aortic ligation and extraanatomical reconstruction with axillofemoral bypass. The importance of timely diagnosis of primary ADF prior to AAA repair, as well as treatment options and optimal materials for simultaneous aortic and bowel reconstruction in the setting of primary or secondary ADF, are discussed.

Comparative Analysis of Biological and Functional Properties of Bone Marrow Mesenchymal Stromal Cells Expanded in Media with Different Platelet Lysate Content.

Due to their ability to induce immunological tolerance in the recipient, mesenchymal stromal cells (MSCs) have been utilized in the treatment of various hematological and immune- and inflammation-mediated diseases. The clinical application of MSCs implies prior in vitro expansion that usually includes the use of fetal bovine serum (FBS). The present study evaluated the effect of different platelet lysate (PL) media content on the biological properties of MSCs. MSCs were isolated from the bone marrow of 13 healthy individuals and subsequently expanded in three different culture conditions (10% PL, 5% PL, 10% FBS) during 4 passages. The cells cultured in different conditions had comparable immunophenotype, clonogenic potential, and differentiation capacity. However, MSC growth was significantly enhanced in the presence of PL. Cultures supplemented with 10% PL had a higher number of cumulative population doublings in all passages when compared to the 5% PL condition (p < 0.03). Such a difference was also observed when 10% PL and 10% FBS conditions were compared (p < 0.005). A statistically significant difference in population doubling time was determined only between the 10% PL and 10% FBS conditions (p < 0.005). Furthermore, MSCs cultured in 10% PL were able to cause a 66.9% reduction of mitogen-induced lymphocyte proliferation. Three chromosome aberrations were detected in PL conditions. Since two changes occurred in the same do nor, it is possible they were donor dependent rather than caused by the culture condition. These findings demonstrate that a 10% PL condition enables a higher yield of MSCs within a shorter time without altering MSC properties, and should be favored over the 5% PL condition.

The organic arsenic derivative GMZ27 induces PML-RARα-independent apoptosis in myeloid leukemia cells.

Arsenic trioxide (ATO) is an inorganic arsenic derivative that is very effective against acute promyelocytic leukemia. However, organic arsenic derivatives (OAD) have a more favorable toxicity profile than ATO. We herein characterized dipropil-S-glycerol arsenic (GMZ27), a novel OAD. GMZ27 had potent antiproliferative activity against human acute myeloid leukemia (AML) cell lines that was higher than that of ATO. In contrast to ATO, GMZ27 only marginally induced maturation of leukemia cells and had no effect on the cell cycle. The anti-leukemia activity of GMZ27 against AML cells was independent of the presence of the PML-RARα fusion protein. GMZ27 dissipates mitochondrial transmembrane potential, and induces cleavage of caspase 9 and activation of caspase 3 without altering the expression levels of (BCL-2), BAX and BCL-xl. GMZ27 induces the formation of intracellular superoxide, a reactive oxygen species (ROS) which plays a major role in the antileukemia activity of this OAD. In addition to ROS generation, GMZ27 concomitantly reduces intracellular glutathione which markedly weakens the cellular antioxidant capacity, thus enhancing the detrimental intracellular effects of ROS production. These results indicate that GMZ27 induces apoptosis in AML cells in a PML-RARα-independent fashion, through the induction of ROS production. This activity provides the rationale for the testing of GMZ27 in patients with AML.

[Mesenchymal stem cells: immunomodulatory properties and clinical application]. / Mezenhimske maticne stanice: imunomodulatorne znacajke i klinicka primjena.

Mesenchymal stem cells (MSCs) are multipotent nonhematopoietic cells that were first identified in bone marrow. Clinical interest for MSCs was initiated by the observation that MSCs are immunoprivileged cells that display immunomodulatory properties in vitro. Ex vivo expanded MSCs have therefore become a new type of cellular therapy in development with a wide range of potential clinical applications. So far many clinical studies confirmed safety of their use and showed that infused MSCs suppress graft versus host disease, support engraftment of transplanted allogeneic hematopoietic stem cells and stimulate growth in patients with osteogenesis imperfecta. Although underlying immunomodulatory mechanisms of action are not completely understood, potential benefit of MSC therapy justifies its clinical use in a broad range of disorders. In this report we give historical overview of MSC discovery and current scientific and clinical achievements in this field. Better insight into their biological properties and mechanisms of action are needed.

MER1, a novel organic arsenic derivative, has potent PML-RARalpha-independent cytotoxic activity against leukemia cells.

Arsenic trioxide (ATO) is an inorganic arsenic derivative that is highly effective against PML-RARalpha-positive leukemia but much less against other hematological malignancies. We synthesized an organic arsenic derivative (OAD), S-dimethylarsino-thiosuccinic acid (MER1), which offers a superior toxicity profile and comparable in vitro activity relative to ATO. In Swiss Webster mice, maximally-tolerated cumulative dose of MER1 when given i.v. for 5 days was 100 mg/kg/d. We demonstrated that MER1 induced apoptosis and dose- and time-dependent inhibition of survival and growth in a panel of myeloid leukemia cell lines. Unlike ATO, this activity was independent of PML-RARalpha status and was not associated with induction of myeloid maturation. In NB4 and HL60 cells, MER1 and ATO induced caspase activation and dissipation of mitochondrial transmembrane potential. At the same time, MER1 induced generation of reactive oxygen species (ROS) and cell cycle arrest in G2/M phase and proved to be more potent than ATO at inducing apoptosis. ROS generation and intracellular glutathione levels were key modulators of MER1-induced cytotoxicity as evidenced by abrogation of apoptosis in myeloid leukemia cell lines pretreated with the disulfide bond-reducing agent dithiothreitol or the radical scavenger N-acetyl-L-cysteine. Collectively, these data indicate that MER1 induces apoptosis in PML-RARalpha-positive and -negative myeloid leukemia cells by enhancing oxidative stress. This agent, therefore, combines low in vivo toxicity with formidable in vitro pro-apoptotic ROS-mediated activity, and may represent a novel OAD suitable for clinical development against a variety of hematological malignancies.

Adaphostin has significant and selective activity against chronic and acute myeloid leukemia cells.

Adaphostin is a tyrphostin that was designed to inhibit Bcr/Abl tyrosine kinase by altering the binding site of peptide substrates rather than that of adenosine triphosphate, a known mechanism of imatinib mesylate (IM). However, it has been shown that adaphostin-mediated cytotoxicity is dependent on oxidant production and does not require Bcr/Abl. We have tested adaphostin against both Philadelphia chromosome (Ph)-positive (K562, KBM5, KBM5-R [IM resistant KBM5], KBM7, and KBM7-R [IM-resistant KBM7]) and Ph-negative (OCI/AML2 and OCI/AML3) cells, and against cells from patients with chronic myeloid leukemia (CML) and acute myeloid leukemia (AML). Adaphostin significantly inhibited growth of all cell lines (50% inhibition of cell proliferation [IC50] 0.5-1 microM) except K562 (IC50 13 microM). Ph-positive IM-resistant cell lines showed significant cross resistance to adaphostin. Simultaneous or sequential treatment with adaphostin and IM did not exert a synergistic effect in any KBM line. Adaphostin induced superoxide and apoptosis in a dose-dependent and time-dependent fashion in both Ph-positive and Ph-negative cells. Adaphostin selectively inhibited colony growth of cells from CML (IM-sensitive and IM-resistant) and AML patients. Analysis of tyrosine phosphorylated proteins after treatment with adaphostin revealed alternate effects in different cells consistent with the modulation of multiple targets. In conclusion, adaphostin showed significant and selective activity against CML and AML cells and its development for clinical testing is warranted.

AMN107, a novel aminopyrimidine inhibitor of p190 Bcr-Abl activation and of in vitro proliferation of Philadelphia-positive acute lymphoblastic leukemia cells.

BACKGROUND: Previous studies have shown that patients with Bcr-Abl-positive acute lymphoblastic leukemia (ALL) either have primary disease that is refractory to imatinib mesylate or develop disease recurrence after an initial response. METHODS: The authors investigated the effects of a newly designed Bcr-Abl inhibitor, AMN107, by comparing its in vitro inhibitory potency on p190 Bcr-Abl ALL cell lines with that of imatinib. RESULTS: In two Philadelphia (Ph)-positive ALL cell lines, AMN107 was found to be 30-40 times more potent than imatinib in inhibiting cellular proliferation. AMN107 was also more effective than imatinib in inhibiting phosphorylation of p190 Bcr-Abl tyrosine kinase in cell lines and primary ALL cells. The inhibition of cellular proliferation was associated with the induction of apoptosis in only one of the cell lines. No activity was observed in cell lines lacking the BCR-ABL genotype. CONCLUSIONS: The results of the current study suggest the superior potency of AMN107 compared with imatinib in Ph-positive ALL and support clinical trials of AMN107 in patients with Ph-positive ALL.

AMN107, a novel aminopyrimidine inhibitor of Bcr-Abl, has in vitro activity against imatinib-resistant chronic myeloid leukemia.

Resistance to or intolerance of imatinib in patients with Philadelphia chromosome-positive chronic myelogenous leukemia (CML) has encouraged the development of more potent Bcr-Abl inhibitors. AMN107 is a novel, orally bioavailable ATP-competitive inhibitor of Bcr-Abl. The effects of AMN107 were compared with those of imatinib on imatinib-sensitive (KBM5 and KBM7) and imatinib-resistant CML cell lines (KBM5-STI571R1.0 and KBM7-STI571R1.0). Compared with the antiproliferative activity of imatinib, AMN107 was 43 times more potent in KBM5 (IC50 of 11.3 versus 480.5 nmol/L) and 60 times more potent in KBM7 (IC50 of 4.3 versus 259.0 nmol/L) cells. IC50 for AMN107 and imatinib were 2,418.3 and 6,361.4 nmol/L, respectively, in KBM5-STI571R1.0, and 97.2 and 2,497.3 nmol/L, respectively, in KBM7-STI571R1.0 cells. AMN107 inhibited autophosphorylation of Bcr-Abl kinase more effectively than imatinib in all cell lines. They had similar effects on cell cycle progression and apoptotic response in these cell lines. Among severe combined immunodeficient mice bearing KBM5 cells, mean survival times of groups treated with 10, 20, and 30 mg/kg/d of AMN107, starting day 20 after leukemic cell grafting and continuing for 20 days, were 144%, 159%, and 182%, respectively, compared with controls. These results strongly support investigation of the clinical efficacy of AMN107 in patients with CML.

Troxacitabine and imatinib mesylate combination therapy of chronic myeloid leukaemia: preclinical evaluation.

The in vitro and in vivo activity of a deoxycytidine analogue, troxacitabine, alone or in combination with imatinib mesylate (IM), was evaluated against human chronic myeloid leukaemia (CML) cell lines both sensitive (KBM5 and KBM7) and resistant (KBM5-R and KBM7-R) to IM. These cell lines differ in their sensitivity to IM but all showed similar sensitivity to treatment with troxacitabine (IC50 = 0.5-1 micromol/l). Combined treatment with troxacitabine and IM revealed additive or synergistic effects. Greater apoptotic response was seen with combined treatment than with either agent alone in KBM7-R cells. In clonogenic assays, troxacitabine showed activity against mononuclear cells from CML patients (IC50 = 0.01 micromol/l) with either IM-sensitive or resistant disease. In vivo efficacy studies were carried out in severe combined immunodeficient mice bearing KBM5 or KBM5-R cells. Troxacitabine was administered i.p. daily for 5 d starting on day 20, at doses of 5, 10, 20, or 25 mg/kg. IM was administered i.p. twice a day for 10 d at a dose of 50 mg/kg starting on day 25. In this setting of late stage disease, troxacitabine led to a significant increase in life span, while IM did not. When IM was combined with troxacitabine at 10 and 25 mg/kg in the KBM5 xenograft model, a further increase in life span was observed and some mice achieved long-term survival. These data indicate that the combination of troxacitabine and IM has significant preclinical activity in advanced CML and that clinical evaluation of this combination is warranted.

In vitro activity of dimethylarsinic acid against human leukemia and multiple myeloma cell lines.

PURPOSE: Arsenic trioxide (As(2)O(3)), an inorganic arsenic compound, has recently been approved for the treatment of relapsed or refractory acute promyelocytic leukemia. However, systemic toxicity associated with As(2)O(3) treatment remains a problem. Inorganic arsenic is detoxified in vivo by methylation reactions into organic arsenic compounds that are less toxic. METHODS AND RESULTS: We investigated the antiproliferative and cytotoxic activity of dimethylarsinic acid (DMAA), an organic arsenic derivative and major metabolic by-product of As(2)O(3), against a panel of eight leukemia and multiple myeloma cell lines. As(2)O(3) was tested in comparison. In clonogenic assay, the average concentration of DMAA that suppressed cell colony growth by 50% was 0.5-1 m M, while for As(2)O(3) it was on average 1-2 microM. At those concentrations DMAA and As(2)O(3) had significantly less effect on colony growth of normal progenitor cells. Cytotoxic doses of DMAA and As(2)O(3) in 3-day trypan blue dye exclusion assay experiments were similar to doses effective in clonogenic assay. Assessment of apoptosis by annexin V assay revealed a high rate of apoptosis in all cell lines treated with DMAA and As(2)O(3), but significantly less effect on normal progenitor cells. DMAA, unlike As(2)O(3), had no effect on the maturation of leukemic cells. CONCLUSIONS: DMAA exerts differential antiproliferative and cytotoxic activity against leukemia and multiple myeloma cells, with no significant effect on normal progenitor cells. However, concentrations of DMAA needed to achieve such efficacy are up to 1000 times those of As(2)O(3). Evaluation of novel organic arsenic that would combine the high efficacy of As(2)O(3) and the low toxicity of DMAA is warranted.

[Origin and development of haematopoietic cells]. / Podrijetlo i razvoj krvotvornih stanica.

The development of new treatment strategies ofhaematopoietic stem cell (HSC) transplantation for a large number of non-malignant and malignant diseases has again led to an increasing interest in investigation of HSC biology, especially of molecular mechanisms responsible for the formation and development of haematopoietic tissue. During the last decade a number of experimental models have been introduced to investigate the nature of the HSC. One of the most popular models is in vitro differentiation of embryonic stem (ES) cells. The analysis of ES cells with targeted gene disruption is a new approach in the identification of genes that play a key role in the establishment of hematopoiesis. The most important advantage of this strategy is the possibility to investigate the function of the genes whose mutations cause death of embryo. Applied in vivo, the technology of mutated ES forms a basis for creation and investigation of "knock-out gene" animals. In combination with transgenic animals, this model has revealed a group of genes encoding transcription factors that play the key role in the development and differentiation of haematopoietic tissue. The purpose of the first part of this review is to make the reader familiar with recent results of haematopoietic stem cell studies in vivo and in vitro. In the second part attention of the reader is focused on molecular mechanisms that control hematopoiesis, especially transcription factors.