The cytotoxic effects of bendamustine in combination with cytarabine in mantle cell lymphoma cell lines.

Bendamustine is clinically useful in mantle-cell lymphoma (MCL). Its favorable toxicity profile in-vivo favors its combination with other cytotoxic drugs. Cytarabine is a key drug in the treatment of younger patients with MCL. The current study investigated the in-vitro cytotoxic effect of bendamustine and cytarabine, alone or combined, on two MCL cell lines representing the classic and blastoid variant of the lymphoma subtype (JEKO-1 and GRANTA-519). Cell lines were exposed to each drug alone, or simultaneously and consecutively to both drugs, for different time schedules. Apoptosis was measured by flow cytometry. Mitochondrial damage, cell proliferation/metabolic activity, and cell cycle analysis were also assessed. The synergistic, additive, or antagonistic effects of the drugs were calculated with the combination index (CI) method. Bendamustine and cytarabine alone exhibited relevant cytotoxic activity on both cell lines. Both drugs induced cell cycle arrest in S phase. Drug combinations were associated with significantly higher cytotoxic effects than each drug alone. Among the combination schedules, the consecutive incubation of bendamustine followed by cytarabine was associated with the lower CI, being 10-100-fold lower than with simultaneous incubations. The cytotoxic effect of the consecutive combination was prominent on both cell lines, indicating a very strong and highly significant synergy in inducing apoptosis. Similar results were obtained measuring mitochondrial damage or the decline of the metabolic activity in all cell lines. The strong synergistic effect of bendamustine and cytarabine on MCL cells provides a rationale for developing schedules combining these agents in the treatment of MCL.

A study on mutual interaction between cytokine induced killer cells and umbilical cord-derived mesenchymal cells: Implication for their in-vivo use.

Recently a number of cellular therapy based-clinical trials have been carried out using mesenchymal stromal cells (MSC) or cytokine-induced-killer (CIK) cells aiming to improve outcome of allogeneic hematopoietic stem cell transplantation. We have isolated MSC from umbilical cord (UC) exploring the interaction between CIK cells and UC-MSC. We found that UC-MSC could suppress CIK cells activity, when co-cultured in a cell-to-cell system. In addition, CIK cells could potentially lyse UC-MSC in a time and ratio dependent manner that could have implications for their in vivo use. Here we provide experimental data on the mutual interaction of CIK cells and UC-MSC, suggesting a negative interference when the two cell types are used in combination. In the light of our observations, when CIK and UC-MSC will be used in clinical trials, timing and sequencing of their infusion should be considered.

The cytotoxic action of the CD56+ fraction of cytokine-induced killer cells against a K562 cell line is mainly restricted to the natural killer cell subset.

BACKGROUND: Cytokine-induced killer cells are polyclonal T cells generated ex vivo and comprise two main subsets: the CD56- fraction, possessing an alloreactive potential caused by T cells (CD3+CD56-), and the CD56+ fraction, characterised by a strong antitumour capacity induced by natural killer-like T cells (NK-like T, CD3+CD56+) and natural killer cells (NK, CD3-CD56+ bright). MATERIALS AND METHODS: We investigated the cytotoxic action of selected CD56+ cell subpopulations against a human chronic myeloid leukaemia (K562) cell line. RESULTS: After immunomagnetic selection of the CD56+ cell fraction, NK bright cells (CD3-CD56+ bright) and two subsets of NK-like T cells (CD3+CD56+), called NK-like T CD56 dim and NK-like T CD56 bright, could be identified. The cytotoxic effect against K562 cells was mainly exerted by the NK bright subpopulation and resulted to be inversely correlated with the percentage of NK-like T CD56 dim cells in the culture. The lytic action appeared to be independent of cell degranulation as suggested by the lack of change in the expression of CD107a. DISCUSSION: We conclude that the cytotoxic action of CD56+ cells against a K562 cell line is mainly due to the NK cells.

[A double blind randomised clinical trial to assess the efficacy of the treatments of the superficial pressure sores]. / Studio clinico randomizzato controllato in doppio cieco sull'efficacia dei trattamenti delle lesioni da decubito.

In spite of the progresses of knowledge and care, pressure sores continue to be a clinically relevant problem. A double blind randomised controlled trial was organised to assess the efficacy of triticum vulgaris (Fitostimoline) vs placebo in the re-epithelisation of superficial pressure sores. Patients with stage NPUAP II or superficial pressure sores, with an expected survival of more than 3 months and eligible for a follow-up up to 8 weeks were included, over a period of 2 years in 46 clinical sites. The protocol was approved by local ethical committees and informed consent was obtained before randomisation. Medications were performed by nurses if the patient was hospitalised and by nurses or properly instructed caregivers at home. Weekly follow-up controls were assumed by nurses. Out of the 294 randomised patients 270 were included in the analyses. The two groups are comparable for the main characteristics except for Norton Scale mean values, less severe in the group assigned to active treatment (10.1+/-3.7 vs 8.9+/-3.2). The mean follow-up was of 3.8 and 4.2 weeks with a mean duration of 26+/-18 and 29+/-18 days for the experimental group and controls respectively. Seventy-six patients in the treatment group and controls (58.0 and 54.7) had their lesions re-epithelized. Adjusting results for age, initial Norton and Push scores there are no differences between treated and controls (OR 0.99 95% IC 0.60-1.67). This multicentre study, sponsored by a research group of nurses, failed to support the hypothesis that triticum vulgaris, the active component of the product Fitostimoline, given on top of recommended treatment, provides a specific therapeutic advantage in terms of frequency and timing of re-epithelization in superficial pressure sores.

Multivariate statistical data analysis as a tool to analyze ex vivo expansion dynamics of cytokine-induced killer cells.

BACKGROUND: Cytokine-induced killer (CIK) cells, obtained after mononucleated cell stimulation with interferon-γ, interleukin-2, and anti-CD3 antibody, are constituted by CD3(+) CD56(+) (CIK) cells and a minority of natural killer (NK; CD3(-) CD56(+) ) cells and T-lymphocytes (CD3(+) CD56(-) ) with antitumor effect against hematological malignancies, thus representing a promising immunotherapy strategy. To ensure in vivo antitumor activity it is mandatory to maximize the percentage of CD3(+) 56(+) effector cells, which is highly variable depending on the starting sample and the harvesting day. Based on cytofluorimetric data, we have retrospectively applied multivariate statistical data analysis (MVDA) to 30 expansions building mathematical models able to predict the expansion fate and the optimal CIK harvesting day. METHODS: Cell phenotype was monitored during culture; multivariate batch statistical process control was applied to monitor cell expansion and orthogonal projections to latent structures to predict CIK percentage. RESULTS: Ten expansions had CD3(+) CD56(+) cells ≥ 40% (good batches) and 20 had CD3(+) CD56(+) cells ≤ 40%. In 36.7%, CD3(+) CD56(+) cells reached the highest concentration at day 17 and the others at day 21. We built a highly predictive regression model for estimating CD3(+) CD56(+) cells during culture. Three variables resulted highly informative: NK % at day 0, cytotoxic T-lymphocytes % (CTLs, CD3(+) CD8(+) ) at day 4, and CIK % at day 7. "Good batches" are characterized by a high percentage of CTLs and CD3(+) CD56(+) cells at day 4 and day 7, respectively. CONCLUSION: By applying MVDA it is possible to optimize CIK expansion, deciding the optimal cell harvesting day. A predictive role for CTL and CIK was evidenced.

Cytosine arabinoside potentiates the apoptotic effect of bendamustine on several B- and T-cell leukemia/lymphoma cells and cell lines.

Bendamustine and cytosine arabinoside (ara-c) are commonly used cytotoxic agents with unique mechanisms of action. We have previously reported a striking additive cytotoxic effect of the consecutive combination of bendamustine and ara-c in mantle cell lymphoma (MCL) cell lines. In the present study, cell lines of follicular lymphoma (DOHH-2), chronic lymphocytic leukemia/lymphoma (EHEB), diffuse large B-cell lymphoma (SU-DHL-4), T-cell leukemia/lymphoma (JURKAT and KARPAS-299) and MCL (JEKO-1 and GRANTA-519) were exposed to the two single drugs or the drugs combined, given simultaneously and consecutively. Peripheral blood chronic lymphocytic leukemia (CLL) B-cells from five patients were also analyzed. Apoptosis, cell proliferation/metabolic activity and mitochondrial damage were evaluated. The combination index (CI) was used to assess synergy between the drugs. Bendamustine exhibited a relevant cytotoxic effect that was dose- and time-dependent, except for SU-DHL-4 and T-cell lymphoma cells. The addition of ara-c after bendamustine significantly potentiated the single-drug cytotoxic effect of bendamustine on all cell lines, including 17p - CLL B-cells, JURKAT and SU-DHL-4, the latter presenting the highest synergism (CI < 0.01). Bendamustine and ara-c are highly synergistic on T- and B-cell lymphoma cells and cell lines, similar to MCL, overcoming resistance to the single agents.