Eryptosis Is Altered in Peritoneal Dialysis Patients.

BACKGROUND: Red blood cells (RBCs) undergo programmed cell death known as eryptosis. Triggers of eryptosis include increased cytosolic Ca(2+) concentration, oxidative stress, osmotic shock, energy depletion and several uremic toxins. Little is known about the pathogenesis of eryptosis in peritoneal dialysis (PD) patients; furthermore, its relevance in worsening clinical conditions in these patients is still not completely defined. OBJECTIVES: We investigated eryptosis levels in PD patients and its association with inflammatory and clinical parameters. MATERIAL AND METHODS: A total of 46 PD patients and 17 healthy subjects (CTR) were enrolled. All eryptosis measurements were made in freshly isolated RBCs using the flow cytometer. RESULTS: Eryptosis was significantly higher in PD patients than that in CTR (p < 0.001). Eryptosis levels did not differ significantly between PD patients with and without diabetes, with and without hypertension, and with and without cardiovascular disease. Eryptosis showed no significant differences between patients treated with continuous ambulatory PD/automated PD, with Kt/Vurea value ≤1.7 and >1.7, with a negative or positive history of peritonitis. On the contrary, eryptosis showed significantly lower levels in PD patients with weekly creatinine clearance ≥45 L/week/1.73 m2 (2.8%, 1.7-4.9 vs. 5.6%, 5.0-13.5; p= 0.049). Eryptosis showed significantly lower levels in PD patients with residual diuresis (n = 23) than that in patients without (3.7%, 2.6-5.6 vs. 5%, 3.1-16; p = 0.03). In these 23 patients, significant negative correlations between percentage of eryptosis and residual glomerular filtration rate (rGFR; Spearman's rho = -0.51, p = 0.01) and diuresis volume (Spearman's rho = -0.43, p = 0.05) were found. CONCLUSIONS: The present study demonstrated higher eryptosis levels in PD patients compared to corresponding levels in CTR. Furthermore, important PD comorbidity and main PD parameters do not influence eryptosis. Importantly, our data have reported an increase in eryptosis levels with progressive residual diuresis and rGFR loss, probably due to decreased uremic toxins clearance.

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.

Enumeration of residual white blood cells in leukoreduced blood products: Comparing flow cytometry with a portable microscopic cell counter.

Transfusion of blood components is potentially associated to the risk of cell-mediated adverse events and current guidelines require a reduction of residual white blood cells (rWBC) below 1 × 10(6) WBC/unit. The reference method to enumerate rare events is the flow cytometry (FCM). The ADAM-rWBC microscopic cell counter has been proposed as an alternative: it measures leukocytes after their staining with propidium iodide. We have tested the Adam-rWBC for the ability to enumerate rWBC in red blood cells and concentrates. We have validated the flow cytometry (FCM) for linearity, precision accuracy and robustness and then the ADAM-rWBC results have been compared with the FCM. Our data confirm the linearity, accuracy, precision and robustness of the FCM. The ADAM-rWBC has revealed an adequate precision and accuracy. Even if the Bland-Altman analysis of the paired data has indicated that the two systems are comparable, it should be noted that the rWBC values obtained by the ADAM-rWBC were significantly higher compared to FCM. In conclusion, the Adam-rWBC cell counter could represent an alternative where FCM technology expertise is not available, even if the risk that borderline products could be misclassified exists.

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.

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.

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.

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.

Epidermal growth factor, basic fibroblast growth factor and platelet-derived growth factor-bb can substitute for fetal bovine serum and compete with human platelet-rich plasma in the ex vivo expansion of mesenchymal stromal cells derived from adipose tissue.

BACKGROUND AIMS: Human mesenchymal stromal cells (MSC) are multipotent cells possessing self-renewal capacity, long-term viability and multilineage potential. We analyzed the effect of four different medium supplements on the expansion and differentiation of adipose tissue-derived MSC (ADSC) in order to avoid the use of xenogeneic serum. METHODS: We compared fetal bovine serum (FBS) with 10% human platelet-rich plasma (hPRP), 3% human platelet-poor plasma (hPPP) and with a cytokine cocktail composed of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and platelet-derived growth factor-bb (PDGFbb) added to 3% hPPP. This mixture was developed testing EGF, bFGF, granulocyte-colony-stimulating factor (G-CSF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF-I), PDGFbb and transforming growth factor (TGF)-ß1 added alone or in combination with hPPP. RESULTS: Our data demonstrate that the addition of EGF, bFGF and PDGFbb, in a medium supplemented with hPPP, obtainable from 150-200 mL whole autologous blood, supports ADSC expansion better than FBS, as confirmed by cumulative population doublings (cPD; 15.0 ± 0.5 versus 9.4 ± 2.8). The addition of human platelet-rich plasma (hPRP) further improved ADSC proliferation (cPD 20.0 ± 1.2), but the achievement of hPRP presented a major drawback, requiring 1000-1200 mL autologous or donor whole blood. The medium supplements did not influence ADSC phenotype: they expressed CD105, CD90 and CD44 lacking hematopoietic antigens. The exposure to the proposed cocktail or to hPRP increased adipogenic and osteogenic differentiation. CONCLUSIONS: The addition of EGF, bFGF and PDGFbb to hPPP could ensure a sufficient number of ADSC for clinical applications, avoiding the use of animal serum and representing a novel approach in regenerative medicine.

Effect of platelet lysate on the functional and molecular characteristics of mesenchymal stem cells isolated from adipose tissue.

BACKGROUND: Mesenchymal stem cells (MSCs) are non-hematopoietic, adult, fibroblast-like, multipotent cells that are plastic adherent in standard culture conditions. They can be isolated from several tissues, but it is always necessary to expand them for clinical practice. AIM: We investigated the effect of human platelet lysate (hPL) on the expansion of human MSCs isolated from adipose tissue (AT), comparing it with fetal bovine serum (FBS) and human platelet-poor plasma (hPPP). MATERIALS AND METHODS: Human AT-MSCs, hPL and hPPP were obtained from 7 healthy subjects. AT-MSCs were seeded at 1500 cells/cm(2) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 10% hPPP or 10% hPL. Cells were harvested, counted and analyzed by flow cytometry every 7 days for 5 passages (P). The differentiation assays, RNA isolation and co-culture with allogeneic lymphocytes were performed at the end of P2. RESULTS: AT-MSCs achieved a better proliferation rate when cultured with hPL than with hPPP or FBS (20 ±2 versus 8 ±3 and 6 ±3, respectively, at the end of P5 [p<0.01]). hPL preserved the differentiation capacity and typical expression of surface antigens, avoiding the risks associated with the use of animal derivatives. AT-MSCs demonstrated a stronger inhibitory effect on lymphocyte proliferation with hPL than with other culture conditions, even at a AT-MSCs:T cells ratio of 1:10. The transcriptional level of matrix metalloproteinase 2, used to evaluate stemness, was very high in all conditions tested. CONCLUSIONS: hPL represents an effective and safe supplement for MSC expansion to be used in the clinical setting.