Clonal composition of human multipotent mesenchymal stromal cells

Multipotent mesenchymal stromal cells (MMSCs) are a heterogeneous population consisting of cells with a distinct proliferative potential. The aim of this study was to define clonal composition in MMSCs and trace the dynamics of individual clones in MMSC subpopulations with different proliferative potentials during the process of cultivation. The investigation was performed at single-cell level using genetically marked cells. Specifically, human bone marrow MMSCs were infected with a lentiviral vector-bearing marker gene. Integration site analysis was performed for clones at each passage by ligation-mediated polymerase chain reaction and Southern blot hybridization. Sibling connections between clones and clonal composition of MMSC culture at each passage were revealed. The MMSC population contained multiple, different, mainly small, clones. It was found that large long-living clones with a high, but limited proliferative potential could be detected rarely in MMSCs population. These data suggest that the human MMSC population does not fit the "stem cell" criteria, however, MMSCs may contain a subpopulation of large clones with a high proliferative potential.     

Differential expression of cytokines in breast cancer patients receiving different chemotherapies: implications for cognitive impairment research

Purpose

Altered levels of cytokines and chemokines may play a role in cancer- and cancer treatment-related cognitive difficulties. In many neurodegenerative diseases, abnormal concentrations of cytokines and chemokines affect neuronal integrity leading to cognitive impairments, but the role of cytokines in chemotherapy-related cognitive difficulties in cancer patients is not well understood. Patients receiving doxorubicin-based (with cyclophosphamide, or cyclophosphamide plus fluorouracil; AC/CAF) chemotherapy or cyclophosphamide, methotrexate, and fluorouracil (CMF) chemotherapy report experiencing cognitive difficulties; because these regimens work by different modes of action, it is possible that they differentially affect cytokine levels.

Methods

This study examined the relationships between cytokine levels (i.e., IL-6, IL-8, and MCP-1) and type of chemotherapy among 54 early-stage breast cancer patients receiving AC/CAF or CMF. Cytokine levels were assessed at two time-points: prior to on-study chemotherapy cycle?2 (cycle?2) and after two consecutive chemotherapy cycles (prior to on-study cycle?4; cycle?4).

Main results

Analyses of variance using cycle?2 levels as a covariate (ANCOVA) were used to determine differences between chemotherapy groups. Levels of IL-6, IL-8, and MCP-1 increased in the AC/CAF group and decreased in the CMF group; the only significant between-group change was in IL-6 (p?<?0.05).

Conclusions

These results, although preliminary based on the small sample size, suggest that AC/CAF chemotherapy is more cytokine inducing than CMF. Future studies should confirm these results and explore the distinct inflammatory responses elicited by different chemotherapy regimens when assessing cognitive function in cancer patients.     

Does better access to FPs decrease the likelihood of emergency department use?: Results from the Primary Care Access Survey

Objective

To determine whether better access to FP services decreases the likelihood of emergency department (ED) use among the Ontario population.

Design

Population-based telephone survey.

Setting

Ontario.

Participants

A total of 8502 Ontario residents aged 16 years and older.

Main outcome measures

Emergency department use in the 12 months before the survey.

Results

Among the general population, having a regular FP was associated with having better access to FPs for immediate care (P < .001) but was not associated with a decreased likelihood of ED visits (odds ratio [OR] = 1.49, P = .03). Better actual access to FP services for immediate care was associated with a decreased likelihood of ED use (OR = 0.62, P < .001) among the general population. Among those with chronic diseases, having a regular FP was associated with a decreased likelihood of ED use (OR = 0.47, P = .01). Of the Ontario population, 39.3% wanted to see FPs for immediate care at least once a year; 63.1% of them had seen FPs without difficulties and were significantly less likely to use EDs than those who did not see FPs or had difficulties accessing physicians when needed (OR = 0.62, P < .001). Having a chronic health condition, recent immigrant status, residence in rural and northern parts of Ontario, and lower educational and income levels were significant predictors of a higher likelihood of ED use, independent of access to FPs (P < .05).

Conclusion

A decreased likelihood of ED use is strongly associated with having a regular FP among those with chronic diseases and with having access to FPs for immediate care among the general population. Further research is needed to understand what accounts for a higher likelihood of ED use among those with regular FPs, new immigrants, residents of northern and rural areas of Ontario, and people with low socioeconomic status when actual access and sociodemographic characteristics have been taken into consideration. More important, this study demonstrates a need of distinguishing between potential and actual access to care, as having a regular FP and having timely and effective access to FP care might mean different things and have different effects on ED use.     

Inhibition of NADPH oxidases prevents chronic ethanol-induced bone loss in female rats

Previous in vitro data suggest that ethanol (EtOH) activates NADPH oxidase (Nox) in osteoblasts leading to accumulation of reactive oxygen species (ROS). This might be a mechanism underlying inhibition of bone formation and increased bone resorption observed in vivo after EtOH exposure. In a rat model in which cycling females were infused intragastrically with EtOH-containing liquid diets, EtOH significantly decreased bone formation and stimulated osteoblast-dependent osteoclast differentiation. These effects were reversed by exogenous 17-β-estradiol coadministration. Moreover, coadministration of N-acetyl cysteine (NAC), an antioxidant, or diphenylene iodonium (DPI), a specific Nox inhibitor, also abolished chronic EtOH-associated bone loss. EtOH treatment up-regulated mRNA levels of Nox1, 2, 4, and the receptor activator of nuclear factor-κB ligand (RANKL), an essential factor for differentiation of osteoclasts in bone. Protein levels of Nox4, a major Nox isoform expressed in nonphagocytic cells, was also up-regulated by EtOH in bone. 17-β-Estradiol, NAC, and DPI were able to normalize EtOH-induced up-regulation of Nox and RANKL. In vitro experiments demonstrated that EtOH directly up-regulated Nox expression in osteoblasts. Pretreatment of osteoblasts with DPI eliminated EtOH-induced RANKL promoter activity. Furthermore, EtOH induced RANKL gene expression, and RANKL promoter activation in osteoblasts was ROS-dependent. These data suggest that inhibition of Nox expression and activity may be critical for prevention of chronic EtOH-induced osteoblast-dependent bone loss.     

Clinical pharmacokinetics of tyrosine kinase inhibitors: focus on 4-anilinoquinazolines

The 4-anilinoquinazolines (gefitinib, erlotinib and lapatinib) are members of a class of potent and selective inhibitors of the human epidermal growth factor receptor (HER) family of tyrosine kinases that have been developed to treat patients with tumours with defined genetic alterations of the HER tyrosine kinase domain. They are characterized by a moderate rate of absorption after oral administration with peak plasma concentrations at several hours post-dose. Absolute bioavailability of gefitinib and erlotinib is about 60%. Low bioavailability is assumed for lapatinib. The drugs are extensively distributed in human tissues, including tumour tissues, have a large volume of distribution at least 3-fold exceeding the volume of body water and are extensively (about 95%) protein bound to α(1)-acid glycoprotein and albumin. Existing human data for gefitinib and erlotinib indicate that these substances penetrate into the central nervous system and accumulate in brain tumours, possibly due to leaks in the blood-brain barrier. Gefitinib, erlotinib and the absorbed fraction of lapatinib undergo extensive metabolism - mainly via hepatic and intestinal cytochrome P450 (CYP) 3A4 and also via CYP2D6 (gefitinib) and CYP1A2 (erlotinib) - and are primarily eliminated by biotransformation. The excretion of unchanged gefitinib, erlotinib, lapatinib and their metabolites occurs predominantly in the faeces and only a minor fraction is excreted in the urine. No relevant effects of age, sex, bodyweight or race on their pharmacokinetics have been reported to date. Limited available data indicate that genetic polymorphisms in enzymes and transporters involved in the pharmacokinetics of gefitinib (CYP2D6) and erlotinib (CYP3A4, CYP3A5 and ABCG2 [breast cancer resistance protein]) alter the exposure to these drugs. Modification of drug dose should be considered in patients with severe hepatic impairment receiving these tyrosine kinase inhibitors and in current smokers receiving erlotinib. Existing recommendations for dose adjustment (i.e. a dose decrement or increment for gefitinib, erlotinib and lapatinib in the presence of CYP3A4 inhibitors or inducers, respectively; a dose increase for erlotinib in smoking patients) need to be validated in clinical studies. Further investigations are required to explain the large interindividual variability in the pharmacokinetics of these drugs and to assess the clinical relevance of interaction potential and inhibitory effects on the metabolizing enzymes and transporters.     

Ethanol Impairs Estrogen Receptor Signaling Resulting in Accelerated Activation of Senescence Pathways,Whereas Estradiol Attenuates the Effects of Ethanol in Osteoblasts

Epidemiological and animal studies have suggested that chronic alcohol consumption is a major risk factor for osteoporosis. Using bone from cycling female rats infused chronically with ethanol (EtOH) in vivo and osteoblastic cells in vitro, we found that EtOH significantly increased estrogen receptor α (ERα) and β (ERβ) mRNA and ERα protein levels. Treatment with 17β‐estradiol (E2) in vivo and in vitro interfered with these effects of EtOH on bone and osteoblastic cells. ERα agonist propylpyrazoletriol (PPT) and ERβ agonist diarylpropionitrile (DPN) attenuated EtOH‐induced ERα and ERβ gene overexpression, respectively. Similar to the ER antagonist ICI 182780, EtOH blocked nuclear translocation of ERα‐ECFP in the presence of E2 in UMR‐106 osteoblastic cells. EtOH also downregulated ERE‐luc reporter activity. On the other hand, EtOH by itself upregulated some common ERα‐ and ERβ‐mediated genes apparently by an ER‐independent pathway. EtOH also transactivated the luciferase activity of the p21 promoter region independent of additional exogenous ERα, activated p21 and p53, and stimulated senescence‐associated β‐galactosidase activity in rat stromal osteoblasts. E2 treatment attenuated these EtOH actions. We conclude that inhibitory cross‐talk between EtOH and E2 in osteoblasts on ERs, p53/p21, and cell senescence provides a pathophysiologic mechanism underlying bone loss and the protective effects of estrogens in alcohol‐exposed females.     

Phosphorus dendrimers affect Alzheimer's (Aβ1-28) peptide and MAP-Tau protein aggregation

Alzheimer's disease (AD) is characterized by pathological aggregation of β-amyloid peptides and MAP-Tau protein. β-Amyloid (Aβ) is a peptide responsible for extracellular Alzheimer's plaque formation. Intracellular MAP-Tau aggregates appear as a result of hyperphosphorylation of this cytoskeletal protein. Small, oligomeric forms of Aβ are intermediate products that appear before the amyloid plaques are formed. These forms are believed to be most neurotoxic. Dendrimers are highly branched polymers, which may find an application in regulation of amyloid fibril formation. Several biophysical and biochemical methods, like circular dichroism (CD), fluorescence intensity of thioflavin T and thioflavin S, transmission electron microscopy, spectrofluorimetry (measuring quenching of intrinsic peptide fluorescence) and MTT-cytotoxicity assay, were applied to characterize interactions of cationic phosphorus-containing dendrimers of generation 3 and generation 4 (CPDG3, CPDG4) with the fragment of amyloid peptide (Aβ(1-28)) and MAP-Tau protein. We have demonstrated that CPDs are able to affect β-amyloid and MAP-Tau aggregation processes. A neuro-2a cell line (N2a) was used to test cytotoxicity of formed fibrils and intermediate products during the Aβ(1-28) aggregation. It has been shown that CPDs might have a beneficial effect by reducing the system toxicity. Presented results suggest that phosphorus dendrimers may be used in the future as agents regulating the fibrilization processes in Alzheimer's disease.     

Synthesis of new potential anticancer agents based on 4-thiazolidinone and oleanane scaffolds

The synthesis and evaluation of the anticancer activity of new acylated oximes derivatives of oleanolic acid with 4-thiazolidinone-3(5)-carboxylic acid moieties were described. Newly synthesized compounds were elucidated on the basis of elemental analyses and spectral data (IR, ¹H, and ¹³C NMR). Anticancer activity of the tested compounds has been evaluated in vitro at National Cancer Institute (NCI) in which some structure activity relationships (SARs) were discussed. Among the tested compounds, 3-[(2,4-thiazolidinedione-5-ylidene)-carboxyimino]olean-12-en-28-oic acid methyl ester (IVm) was superior to other related compounds with mean values of pGI₅₀?=?5.51/5.57, pTGI?=?5.09/5.13, and pLC₅₀?=?4.62/4.64, low toxicity and moderate activity level in vivo hollow fiber assay.