A quantitative study on the growth variability of tumour cell clones in vitro

Objectives: In this study, we quantify the growth variability of tumour cell clones from a human leukemia cell line. Materials and methods: We have used microplate spectrophotometry to measure the growth kinetics of hundreds of individual cell clones from the Molt3 cell line. The growth rate of each clonal population has been estimated by fitting experimental data with the logistic equation. Results: The growth rates were observed to vary among different clones. Up to six clones with a growth rate above or below the mean growth rate of the parent population were further cloned and the growth rates of their offsprings were measured. The distribution of the growth rates of the subclones did not significantly differ from that of the parent population thus suggesting that growth variability has an epigenetic origin. To explain the observed distributions of clonal growth rates we have developed a probabilistic model assuming that the fluctuations in the number of mitochondria through successive cell cycles are the leading cause of growth variability. For fitting purposes, we have estimated experimentally by flow cytometry the maximum average number of mitochondria in Molt3 cells. The model fits nicely the observed distributions of growth rates, however, cells in which the mitochondria were rendered non functional (rho-0 cells) showed only a 30% reduction in the clonal growth variability with respect to normal cells. Conclusions: A tumor cell population is a dynamic ensemble of clones with highly variable growth rate. At least part of this variability is due to fluctuations in the number of mitochondria.Comment: 31 pages, 5 figure

Epithelial-to-mesenchymal transition (EMT) induced by inflammatory priming elicits mesenchymal stromal cell-like immune-modulatory properties in cancer cells

Background: Epithelial-to-mesenchymal transition (EMT) has a central role in cancer progression and metastatic dissemination and may be induced by local inflammation. We asked whether the inflammation-induced acquisition of mesenchymal phenotype by neoplastic epithelial cells is associated with the onset of mesenchymal stromal cell-like immune-regulatory properties that may enhance tumour immune escape. Methods: Cell lines of lung adenocarcinoma (A549), breast cancer (MCF7) and hepatocellular carcinoma (HepG2) were co-cultured with T, B and NK cells before and after EMT induction by either the supernatant of mixed-lymphocyte reactions or inflammatory cytokines. Results: EMT occurrence following inflammatory priming elicited multiple immune-regulatory effects in cancer cells resulting in NK and T-cell apoptosis, inhibition of lymphocyte proliferation and stimulation of regulatory T and B cells. Indoleamine 2,3-dioxygenase, but not Fas ligand pathway, was involved at least in part in these effects, as shown by the use of specific inhibitors. Conclusions: EMT induced by inflammatory stimuli confers to cancer cells some mesenchymal stromal cell-like immune-modulatory properties, which could be a cue for cancer progression and metastatic dissemination by favouring immune escape

Association of kidney disease measures with risk of renal function worsening in patients with type 1 diabetes

Background: Albuminuria has been classically considered a marker of kidney damage progression in diabetic patients and it is routinely assessed to monitor kidney function. However, the role of a mild GFR reduction on the development of stage 653 CKD has been less explored in type 1 diabetes mellitus (T1DM) patients. Aim of the present study was to evaluate the prognostic role of kidney disease measures, namely albuminuria and reduced GFR, on the development of stage 653 CKD in a large cohort of patients affected by T1DM. Methods: A total of 4284 patients affected by T1DM followed-up at 76 diabetes centers participating to the Italian Association of Clinical Diabetologists (Associazione Medici Diabetologi, AMD) initiative constitutes the study population. Urinary albumin excretion (ACR) and estimated GFR (eGFR) were retrieved and analyzed. The incidence of stage 653 CKD (eGFR < 60 mL/min/1.73 m2) or eGFR reduction > 30% from baseline was evaluated. Results: The mean estimated GFR was 98 \ub1 17 mL/min/1.73m2 and the proportion of patients with albuminuria was 15.3% (n = 654) at baseline. About 8% (n = 337) of patients developed one of the two renal endpoints during the 4-year follow-up period. Age, albuminuria (micro or macro) and baseline eGFR < 90 ml/min/m2 were independent risk factors for stage 653 CKD and renal function worsening. When compared to patients with eGFR > 90 ml/min/1.73m2 and normoalbuminuria, those with albuminuria at baseline had a 1.69 greater risk of reaching stage 3 CKD, while patients with mild eGFR reduction (i.e. eGFR between 90 and 60 mL/min/1.73 m2) show a 3.81 greater risk that rose to 8.24 for those patients with albuminuria and mild eGFR reduction at baseline. Conclusions: Albuminuria and eGFR reduction represent independent risk factors for incident stage 653 CKD in T1DM patients. The simultaneous occurrence of reduced eGFR and albuminuria have a synergistic effect on renal function worsening

Acetone-induced changes in the toxicokinetics of 2,5-hexanedione in rabbits

Male rabbits were intravenously injected with 2,5-hexanedione or 2,5-hexanedione plus acetone. A toxicokinetic analysis showed that a two-compartment model satisfactorily described the kinetics of 2,5-hexanedione in rabbits. Simultaneous dosing with acetone altered the toxicokinetic model which best described the plasma concentration versus time data. The model-independent parameter body clearance, calculated according to the trapezoidal rule, showed a decrease in the body clearance of 2,5-hexanedione in rabbits simultaneously injected with acetone. The results suggest that toxicokinetic interference may partly explain the neurotoxic potentiation of that which occurs in 2,5-hexanedione-induced axonopathy as a response to simultaneous exposure to acetone

Adult T-cell acute lymphoblastic leukemia: prognostic impact of myeloid associated antigens.

valuation of: Khabori M, Samiee S, Fung S et al. Adult precursor T-lymphoblastic leukemia/lymphoma with myeloid-associated antigen expression is associated with a lower complete remission rate following induction chemotherapy. Acta Haematol. 120, 5\u201310 (2008). Despite the recent improvement in the treatment of the disease, the prognosis of adult T-cell acute lymphoblastic leukemia (T-ALL) remains poor. In the last several decades, many reports analyzed clinical and/or biological factors in order to identify prognostic parameters suitable for risk stratification of T-ALL patients. The article under review analyzed the prognostic impact of myeloid-associated antigen expression in a monocentric cohort of adult T-ALL patients

Controlled comparison of ketanserin and nifedipine in Raynaud's phenomenon

Twenty-eight patients suffering from either primary or secondary Raynaud's phenomenon were treated with nifedipine and ketanserin. Each patient was treated with one of the two drugs administered after an adequate washout period. Furthermore each patient was submitted before and after treatment with each drug to computerized digital thermometry to evaluate the therapeutic response. The data obtained during the intake of the two drugs at zero, five, and twenty-three minutes were compared with thermometry-relevant baseline data at the same periods. Ketanserin proved to be useful in the treatment of Raynaud's phenomenon and statistically significantly superior (alpha less than 0.05) with respect to nifedipine in the thermometric controls and also in the subjective evaluation of the patients (p less than 0.02). In this study nifedipine did not show particular efficacy. Furthermore only 2 patients had to discontinue treatment with ketanserin, whereas 8 had to discontinue treatment with nifedipine (p less than 0.001)

Cross-talks within the innate immune system: the role of neutrophils

Neutrophils represent a key component of the inflammatory response and act as a powerful defensive system against invading bacteria. Following activation by various stimuli, neutrophils have the capacity to release lytic enzymes with potent antimicrobial potential and to generate reactive oxygen intermediates, both essential for pathogen killing. Neutrophils can also produce, upon appropriate stimulation, a variety of proteins in vitro and in vivo, including cytokines and chemokines, that are involved in their effector functions, as well as in the recruitment, activation and programming of other immune cells. In this latter context, recent studies have shown that neutrophils, through secreted products and/or cell-cell contact, might potentially interact with monocytes, dendritic cells and NK cells, T and B lymphocytes, in turn suggesting that neutrophils play an important role in the development and/or modulation of the immune response. In the context of the innate immune system, for instance, it has been demonstrated that neutrophils, under specific settings: i) induce dendritic cell maturation and activation; ii) release chemokines strongly chemotactic for immature dendritic cells; iii) deliver antigen molecules to dendritic cells, thus eliciting antigen-specific T lymphocytes responses. On the other hand, the interaction of neutrophils with either NK cells or T cell subsets is less documented and somewhat contradictory. Nonetheless, the concept that cooperation between cells of the innate immune system is important in sculpting the ensuing inflammatory response and that neutrophils actively participate in this process is continuously better supported. It is thus our purpose to provide additional experimental evidence which extends the current knowledge of neutrophils as cells involved in the cross-talks with other leukocyte types

On the co-purification of 6-sulfo LacNAc(+) dendritic cells (slanDC) with NK cells enriched from human blood.

The ability of NK cells to directly recognize pathogens and be activated via Toll-like receptors (TLR) is increasingly recognized. Nevertheless, controversial results on the NK cell ability to be directly activated by lipopolysaccharide (LPS), the ligand of TLR4, have been recently reported. To start elucidating the reasons explaining the contrasting observations of the literature, we focused on the potential role of currently used NK cell purification procedures to condition putative NK cell responsiveness to LPS. To do so, human NK cells were isolated by negative selection, using three different commercial kits, to be comparatively evaluated for the production of IFN\u3b3 in response to ultra-pure LPS and/or IL-2. Despite the lack of surface TLR4, we found that two out of the three NK cell-enriched populations released IFN\u3b3 (and one of the two, IL-12p70 as well) in response to the LPS plus IL-2 combination, whereas the last one did not. However, the two LPS plus IL-2-responsive NK cell populations were found variably contaminated with 6-sulfo LacNAc+ dendritic cells (slanDC), demonstrated responsible for triggering, via the production of IL-12p70 in response to LPS, the release of IFN\u3b3 by IL-2-stimulated NK cells. Accordingly, slanDC depletion completely abrogated the capacity to produce both IL-12p70 and IFN\u3b3 in response to LPS plus IL-2 by slanDC-containing NK cells. Taken together, our data uncover that two commercially available kits, specifically designed to isolate NK cells by negative selection, also co-purify variable amounts of slanDC. The latter cells may dramatically affect the outcome of experiments carried on to evaluate NK cell responsiveness to TLR agonists such as LPS

Analysis of B-cell receptor signal transduction in B-cell chronic lymphocytic leukemia by phosphospecific flow-cytometry

B-cell chronic lymphocytic leukemia (B-CLL) is a clonal lymphoproliferative disease characterized by the expansion of mature CD5+ B-cells. Recent studies indicated that variability in clinical outcomes observed in CLL patients is related to differences in the ability to transduce B-cell receptor (BCR)-mediate signals. The BCR signal transduction routes include, in the initial phases, Syk tyrosin kinase and BCR prolonged stimulation has been associated to activation of kinases including mitogen activated protein kinases (MAPK). In this study we used phosphospecific flow cytometry to study single-cell signaling triggered by BCR in peripheral blood B-cells from 21 B-CLL patients. The following proteins were analyzed: Syk, MAPK (Erk, p38, JNK) and NF-kappaB, either in basal condition or after BCR cross-linking achieved by treatment with anti-IgM F(ab\u2019)2. Most cases showed constitutive phosphorylation of Syk and NF-kappaB (86% and 93%, respectively). Erk was constitutively phosphorylated in 81% cases, JNK in 5%, and p38 in 0% (Table 1). The BCR cross-linking increased the constitutive phosphorylation of Syk in 50% cases, of Erk in 27%, and of NF-kappaB in 21%. No cases showed p38 or JNK increased phosphorylation (Table 1). Recent works shows that efficient activation of BCR signaling depends on inactivation of phosphatase proteins (PTPs), and that endogenously generated H2O2 is an important transiently PTPs inhibitor. Therefore, we cross-linked the BCR in the presence of H2O2. In this condition we observed an increase of phosphorylation of all proteins with respect to treatment with anti-IgM alone (Table 1). Furthermore, the treatment of B-CLL cells with H2O2 alone induced increased phosphorylation of signaling proteins with respect to the constitutive level (Syk in 21%, p38 in 56%, Erk in 38%, JNK in 9%, and NF-kappaB in 43% cases, Table1). Conversely, PTP inhibition in normal B cells did not significantly modify the phosphorylation of these proteins. Taken together, these results revealed a clear-cut remodeling of BCR-mediated signaling in B-CLL cells with respect to normal B cells, thus confirming and extending previous results obtained by Western blot analysis. Furthermore, this study provides the first analysis of BCR signaling by phosphospecific flow cytometry in B-CLL cells and indicate that single-cells measurement of phosphoproteins in response to BCR engagement enables to define cell network phenotypes in B-CLL by multidimensional molecular profiles of signaling