BN Cells Research Articles

Experimental and theoretical study of 90Sr/90Y-n-Si/ZnO betavoltaic battery and theoretical prediction of homojunction betavoltaic cells performance

Today, betavoltaic batteries has been considered due to their high energy density and long life for operating electrical systems in inaccessible and hostile environments. Conventional electrochemical batteries, despite their widespread use in electronic devices, have a limited lifetime and tend to degrade in extreme environmental conditions. The current paper pursues three goals. The first goal is to the experimental and theoretical investigation of a n-Si/ZnO heterojunction betavoltaic battery based on 90Sr/90Y source. The second goal is to optimize ZnO, SnO2, BN, and diamond homojunction betavoltaic cells in two planar and cubical models by Monte Carlo simulation (MCNP code). The third goal is to present a new approach for estimating the standard error in calculating the parameters of betavoltaic batteries. In order to fabrication of a n-Si/ZnO heterojunction, the ZnO nanospheres were placed on the n-Si (100) substrate using chemical bath deposition (CBD) technology. The Al and Au electrodes were deposited on the formed sample. Then this sample was exposed to the radiation of an external 90Sr/90Y source with an activity of 12.8 mCi. The experimental values obtained for the short circuit current (Isc), open circuit voltage (Voc), efficiency (η), and maximum output power (Pmax) were 0.047 μA, 0.015 V, 3.4 × 10−4 percent, and 0.141 nW, respectively. To compare with the experiment, we investigated the n-Si/ZnO betavoltaic cell by MCNP code. In the simulation, the beta spectrum of the 90Sr/90Y source was considered. The calculated theoretical values for Isc, Voc, η, and Pmax were 0.063 μA, 0.020 V, 6.4 × 10−4 percent, and 0.265 nW, respectively. The experimental results show that the simulation results can be valid. In the optimization of ZnO, SnO2, BN, and diamond homojunction betavoltaic cells in two planar and cubical models by MCNP code, it was found that the Isc, Voc, η, and Pmax of the cubical model are better compared to the planar model. In the cubical model, Pmax of ZnO, SnO2, BN, and diamond betavoltaic batteries is 2633.34 nW ± 0.16 %, 1670.49 nW ± 0.15 %, 198.20 nW ± 0.17 %, and 1315.24 nW ± 0.15 %, respectively. In other words, Pmax of the ZnO betavoltaic battery is about 58 %, 1229 %, and 100 % more than Pmax of SnO2, BN, and diamond betavoltaic batteries, respectively. Pmax of the SnO2 betavoltaic battery is about 743 % and 27 % more than Pmax of BN and diamond betavoltaic batteries, respectively. The results show that ZnO and SnO2 betavoltaic batteries can perform better compared to BN and diamond betavoltaic batteries. Also, their growth process is less expensive compared to BN and diamond.

Observations on a Novel Bacterial Pathogen of Root-Knot Nematodes (Meloidogyne spp.).

A novel Gram-negative pathogenic bacterium (BN) was discovered in second-stage juveniles (J2) of root-knot nematodes (RKN, Meloidogyne spp.). Mature bacteria showed a peculiar rod morphology characterized by four cells sequentially joined at septa. Mature rods measured 4–5 × 0.5–0.6 μm and were characterized by the emptying and tapering of both apical cells. The data showed an electron-dense external matrix forming a coating capsule involved in host attachment. The rods were not motile and packed in parallel inside the J2 body. After J2 penetration by adhering, germinating cells, the bacterium proliferated until the host body content was completely digested, producing a lethal disease. Parasitized hosts were recognized using light microscopy by a pale creamy-brown color assumed at parasitism completion. At death, the whole nematode body was filled with cells and only a few sclerotized esophageal structures (i.e., stylet, median bulb) remained visible. The BN cells were quickly released at host body rupture, suggesting that J2 infection occurs through passive adhesion of cells dispersed in soil. The bacterium appeared fastidious, as attempts to obtain pure cultures on common nutritive media failed.

Mechanical behaviour of 2D hybrid structure fabricated by doping graphene with triangular h-BN cells

A two dimensional (2D) hybrid structure has been fabricated by doping graphene with hexagonal boron nitride (h-BN) cells. Substantial changes are expected as carbon-carbon (C) bonds are substituted by B–N bonds and C–B and C–N bonds are formed between the BN cells and graphene. In this paper, molecular dynamics simulations are performed to explore the tensile behaviour and fracture mechanisms of the 2D HS, and reveal how and to what extent the BN cells impact its overall tensile properties. A comprehensive study of free boundary effects is also conducted for the first time for 2D materials. Herein, the 2D nonlinear elastic constitutive relation has been formulated in terms of the equivalent elastic modulus Ye that decreases almost linearly with BN content α due to the distinct elastic properties between B–N bonds and C–C bonds. The downward trend of the fracture stress with growing α is also achieved due to the α− dependence of Ye and the limit on the fracture strain imposed by the newly formed C–B bonds. In addition, evidence has been accumulated showing that the free boundaries can significantly decrease the elastic moduli and fracture stress but largely enhance the fracture toughness of the 2D nanosheets.

Adipose-derived stromal cell therapy combined with a short course nonmyeloablative conditioning promotes long-term graft tolerance in vascularized composite allotransplantation.

The risks of chronic immunosuppression limit the utility of vascularized composite allotransplantation (VCA) as a reconstructive option in complex tissue defects. We evaluated a novel, clinically translatable, radiation-free conditioning protocol that combines anti-lymphocyte serum (ALS), tacrolimus, and cytotoxic T-lymphocyte-associated protein 4 immunoglobulin (CTLA4-Ig) with adipose-derived stromal cells (ASCs) to allow VCA survival without long-term systemic immunosuppression. Full-mismatched rat hind-limb-transplant recipients received tacrolimus (0.5mg/kg) for 14days and were assigned to 4 groups: controls (CTRL) received no conditioning; ASC-group received CTLA4-Ig (10mg/kg body weight i.p. postoperative day [POD] 2, 4, 7) and donor ASCs (1×106 iv, POD 2, 4, 7, 15, 28); the ASC-cyclophosphamide (CYP)-group received CTLA4-Ig, ASC plus cyclophosphamide (50mg/kg ip, POD 3); the ASC-ALS-group received CTLA4-Ig, ASCs plus ALS (500µL ip, POD 1, 5). Banff grade III or 120days were endpoints. ASCs suppressed alloresponse in vitro. Median rejection-free VCA survival was 28days in CTRL (n=7), 34 in ASC (n=6), and 27.5 in ASC-CYP (n=4). In contrast, ASC-ALS achieved significantly longer, rejection-free VCA survival in 6/7 animals (86%), with persistent mixed donor-cell chimerism, and elevated systemic and allograft skin Tregs , with no signs of acute cellular rejection. Taken together, a regimen comprised of short-course tacrolimus, repeated CTLA4-Ig and ASC administration, combined with ALS, promotes long-term VCA survival without chronic immunosuppression.

A role for ATP Citrate Lyase in cell cycle regulation during myeloid differentiation

Differentiation of myeloid progenitor cells into macrophages is accompanied by increased PU.1 concentration and increasing cell cycle length, culminating in cell cycle arrest. Induction of PU.1 expression in a cultured myeloid cell line expressing low PU.1 concentration results in decreased levels of mRNA encoding ATP-Citrate Lyase (ACL) and cell cycle arrest. ACL is an essential enzyme for generating acetyl-CoA, a key metabolite for the first step in fatty acid synthesis and for histone acetylation. We hypothesized that ACL may play a role in cell cycle regulation in the myeloid lineage. In this study, we found that acetyl-CoA or acetate supplementation was sufficient to rescue cell cycle progression in cultured BN cells treated with an ACL inhibitor or induced for PU.1 expression. Acetyl-CoA supplementation was also sufficient to rescue cell cycle progression in BN cells treated with a fatty acid synthase (FASN) inhibitor. We demonstrated that acetyl-CoA was utilized in both fatty acid synthesis and histone acetylation pathways to promote proliferation. Finally, we found that Acly mRNA transcript levels decrease during normal macrophage differentiation from bone marrow precursors. Our results suggest that regulation of ACL activity is a potentially important point of control for cell cycle regulation in the myeloid lineage.

Absence of genotoxic impact assessed by micronucleus frequency in circulating lymphocytes of workers exposed to cadmium

PurposeCd is considered as a genotoxic carcinogen for which a threshold can be identified. This threshold has, however, not been established and the shape of the relationship between Cd exposure and genotoxic effects is unknown. The aim of the present study was to analyse the shape of the dose-response relationship for the genotoxic effects of Cd in occupational settings. MethodsThe study has a cross-sectional design and includes 60 healthy male and female workers with known Cd exposure selected from two plants manufacturing or recycling nickel-Cd batteries. The frequency of MN was measured in circulating lymphocytes, and related to internal Cd doses (Cd-B, Cd-U). Determinants of MN frequency were traced by multivariate regression analysis. ResultsCd exposure covered a wide range as measured by Cd-B (0.02–1.26 μg/dL), Cd-U (0.26–15.80 μg/g creat) and seniority in the plant (1–42 years). Gender was the only parameter significantly associated with MN frequency, women having on average 8.5 additional MN/1000 BN cells compared to men. Cd-B, Cd-U or Ni-U did not influence MN frequency when adjusted for gender and other potential confounders. ConclusionThis finding is consistent with the existing knowledge on the mechanisms governing the genotoxic activity of Cd, which are all non-stochastic and thresholded. The threshold for systemic genotoxic effects of Cd is thus beyond the range of internal exposure considered in the present investigation.

Buccal micronucleus cytome biomarkers in Algerian couples with idiopathic infertility

The buccal micronucleus cytome (BMCyt) assay is a useful and a minimally invasive cytogenetic method for measuring genomic damage. The aim of the present study is to evaluate the extent of chromosomal damage in couples with idiopathic infertility using a BMCyt.This study included 54 patients (27 couples) with idiopathic infertility and 30 fertile subjects (15 couples). When evaluated by individual (each subject from the couple is considered separately), the frequencies of micronucleated cells (MNC), total micronuclei (TMN), nuclear buds (NBUD), and binucleated cells (BN) were significantly higher in the infertile individuals than in the fertile ones (p = 0.009, p = 0.009, p = 0.003 and p < 0.0001, respectively). Among the cells reflecting cell death events, condensed chromatin (CC), karyorrhectic (KHC) and pyknotic (PYK) cells were significantly higher in the infertile individuals (p = 0.0001, p = 0.003, p = 0.001, respectively). Identical results were obtained when data were analysed by couple (female + male). The frequencies of MNC, TMN, NBUD, and BN cells were significantly higher in the infertile couples (p = 0.019, p = 0.021, p = 0.013, and p < 0.0001, respectively). Likewise, CC, KHC and PYK cells were significantly higher in the infertile couples (p = 0.002, p = 0.034, p = 0.008, respectively). BN cells showed the most pronounced difference between the fertile and infertile groups. The basal (BAS) and karyolitic (KYL) cells did not show a significant difference. In conclusion, this study showed that, in comparison to controls, couples with idiopathic infertility had significantly higher frequencies of DNA damage biomarkers (MN and NBUD), biomarkers of cytokinesis-failure or arrest (BN cells) and cell death biomarkers (CC, KHC and PYK cells). These results suggest a possible role of chromosomal damage in idiopathic infertility that may be due to an imbalance between DNA damage rates and DNA repair mechanisms.

Characterization of primary normal and malignant breast cancer cell and their response to chemotherapy and immunostimulatory agents

BackgroundThe phenomenon of chemotherapy-resistant cancers has necessitated the development of new therapeutics as well as the identification of specific prognostic markers to predict the response to novel drugs. Primary cancer cells provide a model to study the multiplicity of tumourigenic transformation, to investigate alterations of the cellular response to various molecular stimuli, and to test therapeutics for cancer treatment.MethodsHere, we developed primary cultures of human breast tissue – normal cells (BN1), cancer cells (BC5), and cells from a chemotherapy-treated tumour (BrCCh1) to compare their response to conventional chemotherapeutics and to innate immunity stimulators with that of the immortalized breast cells MCF7, MDA-MB-231, and MCF10A. Expression of the progesterone receptor (PGR), oestrogen receptor (ER) α and β, human epidermal growth factor receptor (HER) 2 and 3 and aromatase CYP19, as well as expression of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) mRNA in human breast cells were characterized.ResultsWe revealed that BC5 carcinoma cells were PGRlow/ERbhigh/ERa−/Cyp19+, the BrCCh1 cells that originated from the recurrent tumour were PGR−/ERb+/ERa−/Cyp19+, and normal BN cells were PGR−/ERb+/ERa−/Cyp19high. The treatment of primary culture cells with antitumour therapeutics revealed that BrCCh1 cells were doxorubicine-resistant and sensitive to cisplatin. BC5 cells exhibited low sensitivity to tamoxifen and cisplatin. The innate immunity activators interferon-α and an artificial small nucleolar RNA analogue increased expression of IFIT3 at different levels in primary cells and in the immortalized breast cells MCF7, MDA-MB-231, and MCF10A. The relative level of activation of IFIT3 expression was inversely correlated with the baseline level of IFIT3 mRNA expression in breast cell lines.ConclusionOur data demonstrated that primary cancer cells are a useful model for the development of novel cancer treatments. Our findings suggest that expression of IFIT3 mRNA can be used as a prognostic marker of breast cancer cell sensitivity to immunostimulating therapeutics.

Calendula officinalis Extracts Protect against H2O2 Induced Chromosome Damage on HacaT Human Skin Cells

Background: Calendula officinalis extracts contain antioxidant compounds. The extracts tested in the current study protect against reactive oxygen species (ROS) induced cytotoxic activity towards human cells. Objective: To determine the ability of Calendula officinalis extracts to protect against H2O2 induced chromosome damage on HaCaT human skin cells. Methodology: The chromosome damage protection by four Calendula officinalis extracts was investigated in vitro using a dose-response (0.125, 0.5 and 1.0% (v/v)) on HaCaT human skin cells. Two water/ethanol based proprietary calendula extracts A and B and two proprietary aqueous calendula extracts C and D from Biodynamically grown plants were examined. Extracts were characterised using Folin-Ciocalteau and DPPH assays. Protection of extracts against chromosome damage was measured via micronuclei induction using the cytokinesis-blocked micronucleus assay. No genotoxicity was induced by the extracts themselves. Cells were also exposed to the Calendula extracts for 47 h before being exposed to an oxidative stress (300-μM hydrogen peroxide for 1 h). Results: The oxidative stress induced micronuclei (MNi) at a frequency significantly higher (P<0.05; range=20-25 MNi/1000 binucleated cells, n=3) than the background frequency (media alone control; MNi range=7-13 MNi/1000 binucleated cells, n=3). When the cells were pre-treated with extracts before the oxidative stress, the level of protection against chromosomal damage by all Calendula extracts was significant (P<0.05). The frequency of MNi in the presence of extracts was reduced by an average increment of 20 MNi/1000 BN cells to 2-9 MNi/1000 BN cells at all doses tested. Conclusion: The Calendula extracts protected against the chromosomal damage induced by oxidative stress in vitro. Such form of genetic damage has been linked with carcinogenesis.

PU.1 promotes cell cycle exit in the murine myeloid lineage associated with downregulation of E2F1

Acute myeloid leukemia (AML) is characterized by increased proliferation and reduced differentiation of myeloid lineage cells. AML is frequently associated with mutations or chromosomal rearrangements involving transcription factors. PU.1 (encoded by Sfpi1) is an E26 transformation-specific family transcription factor that is required for myeloid differentiation. Reduced PU.1 levels, caused by either mutation or repression, are associated with human AML and are sufficient to cause AML in mice. The objective of this study was to determine whether reduced PU.1 expression induces deregulation of the cell cycle in the myeloid lineage. Our results showed that immature myeloid cells expressing reduced PU.1 levels (Sfpi1(BN/BN) myeloid cells) proliferated indefinitely in cell culture and expanded invivo. Transplantation of Sfpi1(BN/BN) cells induced AML in recipient mice. Cultured Sfpi1(BN/BN) cells expressed elevated messenger RNA transcript and protein levels of E2F1, an important regulator of cell cycle entry. Restoration of PU.1 expression in Sfpi1(BN/BN) myeloid cells blocked proliferation, induced differentiation, and reduced E2F1 expression. Taken together, these data show that PU.1 controls cell cycle exit in the myeloid lineage associated with downregulation of E2F1 expression.

Inter-individual variance in micronuclei frequency in lymphocytes of patients with cervical insufficiency

The aim of this work was to explain the inter-individual variance in baseline and induced micronuclei (MN) frequency in peripheral blood lymphocytes (PBLs) of females with uterine cervical insufficiency. The baseline and induced MN response of the PBLs in 32 patients were investigated using the cytokinesis-block micronucleus (CBMN) method. The obtained mean values of induced MN frequency (11.31 ? 4.44/1000 BN cells), were significantly higher (p &lt; 0.001) in comparison to their baseline MN frequencies (7.81 ? 3.33 MN/1000 BN cells). The analysis of variance (ANOVA) showed statistically significant difference between-group vs. within-group variance in baseline (p &lt; 0.05) and induced MN frequency (p &lt; 0.001), in professionally exposed patients when compared to the non-exposed. Multivariate logistic regression analysis confirmed that patients with high baseline MN frequency (cut-off 8.5 MN/1000 MN), had significantly increased (odds ratio OR = 5.875; CI = 1.28 - 26.795, p &lt; 0.05) induced MN frequency (&gt;12 MN/1000 BN cells). Our results suggest that the baseline MN frequency in PBLs directly influences the scope of the induced MN response.

Chromosomal instability in peripheral blood lymphocytes of patients with reproductive failure assessed by micronucleus assay

We investigated chromosomal instability in peripheral blood lymphocytes (PBL) of patients with reproductive failure in respect to age, smoking habits, gender, miscarriages, and semen parameters. The study involved 36 individual cases of reproductive failure (18 men and 18 women) attended at the Clinical Centre of Kragujevac, Serbia, and 30 healthy subjects (15 men and 15 women). Micronuclei (MN) frequency was estimated in PBL using the cytokinesis-block micronucleus (CBMN) assay. The baseline MN frequencies were significantly higher (p=0.031; p<0.001) in male [(9.22 ± 4.70) MN per 1000 BN cells] and female patients [(13.50 ± 2.5) MN per 1000 BN cells] than in male and female healthy controls [(6.27 ± 2.66) MN per 1000 BN cells; (6.80 ± 2.98) MN per 1000 BN cells]. The mean baseline MN frequency did not significantly differ between miscarriage groups and between patients with and without normal values of semen parameters. The correlations between poor sperm concentration (<20x106 mL-1), rapid progressive motility (<25 %), normal morphology (<30 %), and MN frequencies were negative, but not statistically significant. We found that only gender significantly influenced the MN rates in analysed patients. There were no significant differences between age groups and between smokers and non-smokers in patients and control samples. We conclude that the increase in baseline MN frequency in PBL of patients with reproductive failure corresponds to the increase in chromosomal damage, which occurs as a result of complex events that cause reproductive disorders.

High Micronucleus Frequency in Peripheral Blood Lymphocytes of Untreated Cancer Patients Irrespective of Gender, Smoking and Cancer Sites

Chromosomal instability could be one of primary causes for malignant cell transformation. The objective of the present study was to evaluate the spontaneous genetic damages in circulated lymphocytes of newly diagnosed cancer patients by using cytokinesis-block micronucleus (CBMN) assay, with respect to the factors that might affect micronucleus frequency (i.e. age, gender, smoking habits and cancer sites). Micronuclei (MN) are small nuclei that are originated from chromosome fragments or whole chromosomes. The analyzed samples included 44 untreated cancer patients (19 females and 25 males with mean age of 60.89 years) with different cancer sites (12 patients with breast cancer, 5 with uterine cancer and 27 with cancer of pharynx). Control group included 40 healthy donors (28 females and 12 males with mean age of 43.95 years). The mean baseline MN frequency was significantly higher (p < 0.001) in cancer patients (15.18 +/- 5.05 MN/1000 BN cells ranging from 4 to 27) than the baseline frequency in healthy controls (6.45 +/- 2.75 MN/1000 BN cells, ranging from 1 to 11). There was no gender difference in baseline MN frequency in cancer patients and healthy controls. Moreover, the MN frequency did not significantly differ among cancer sites, and between smokers and non-smokers in both patient and control samples. In conclusion, untreated cancer patients may be associated with an increase of chromosomal instability in peripheral blood lymphocytes, irrespective of gender, cigarette smoking and cancer sites.

Micronuclei frequency in peripheral blood lymphocytes of thyroid cancer patients after radioiodine therapy and its relationship with metastasis

In most cancers peripheral blood lymphocytes exhibit DNA damage. In the case of thyroid cancer the micronucleus (MN) assay has been used to assess DNA damage before and after exposure to iodine-131 ( 131I). The aim of our study was to use this method to assess DNA damage in peripheral blood lymphocytes of thyroid cancer patients and search for its relationship with metastasis as well as 131I exposure. A significant increase in micronuclei frequency was observed in peripheral blood lymphocytes of 54 thyroid cancer patients in comparison to 38 controls ( p = 0.000). Further analysis revealed significant elevation in micronuclei index from 48.5 MN/1000 BN cells (range: 25.1–111.2, n = 25) in patients without metastasis to 68.1 MN/1000 BN cells (range: 26.2–135.5, n = 29, p = 0.001) in group of patients with metastasis to one or more sites. There was no clear correlation between the micronuclei frequency and the therapeutic 131I dose ranging from 0.41 to 31.5 GBq with the exposure interval of <1 to 126 months. In addition, age and sex did not show any influence on micronuclei frequency in either patients or control population. These findings are indicative of increased basal DNA damage in thyroid cancer patients before treatment. Radioiodine treatment did not increase DNA damage measured by the micronuclei frequency for the interval between the last radioiodine dose administered and analysis of blood sample. However a significant increase of peripheral blood lymphocytes micronuclei was observed in thyroid cancer patients with metastasis.

Treatment of Pregnant Women with a Betamimetic and Verapamil Increases the Micronuclei Frequency in Umbilical Cord Blood Lymphocytes

In prevention of preterm labor, betamimetics are used in gynecological practice mostly combined with antiarrhythmic verapamil because of their therapeutic cardiovascular side effects. The aim of this study was to investigate the influence of a betamimetic (ritodrine hydrochloride, fenoterol or hexoprenaline) and verapamil (administered to mothers) on the frequency of micronuclei (MN) in umbilical cord blood lymphocytes of neonates, using cytokinesis-block micronucleus test. The analyzed sample included 23 babies whose mothers received the therapy and 30 control babies whose mothers received no therapy. The average MN frequency was significantly higher in the neonates whose mothers received the therapy (8.13 +/- 2.69 MN/1000 BN cells), in comparison with the baseline frequency in untreated controls (3.30 +/- 2.63 MN/1000 BN cells), with probability p < 0.05. The highest MN frequency was found in neonates whose mothers received fenoterol and verapamil (2.8-fold i.e. 9.10 +/- 3.00 MN/1000 BN cells), while ritodrine hydrochloride and hexoprenaline combined with verapamil induced 2.3-fold and 2.2-fold higher MN values than in controls (7.50 +/- 3.33 MN/1000 BN cells and 7.29 +/- 0.95 MN/1000 BN cells). Multiple linear regression analysis showed that MN frequency was affected only by the maternal therapeutic treatment, while the neonates' sex, maternal age, cigarette smoking, and therapeutic doses did not affect the MN frequency in umbilical lymphocytes of neonates. We conclude that the treatment of pregnant women with a betamimetic and verapamil significantly increases the MN frequency in umbilical cord blood lymphocytes of neonates, regardless to therapeutic doses.

Exploitation of Novel Hyperdiploid and Nonhyperdiploid Myeloma Cell Lines for Studying Innovative Interventions for Myeloma and Its Associated Bone Disease.

Establishment of clinically relevant human myeloma (MM) cell lines is central for our understanding of MM pathogenesis and development of novel therapies for the disease. Unfortunately, most available lines were generated from extramedullary sites and categorized as nonhyperdiploid. In contrast, hyperdiploid MM cell lines are rare. We established procedures for establishment of stroma-dependent MM lines by passaging primary MM in SCID-hu or SCID-rab mice followed by maintenance in co-culture with stromal cells. We described the establishment and characterization of 2 hyperdiploid (LD, and CF) and 2 nonhyperdiploid (JB and BN) cell lines. LD, CF, JB and BN cells have approximately 51, 57, 44 and 43 chromosomes, respectively. Using our animal models, we also established bortezomib-sensitive and -resistant BN lines. These cell lines were cellularly, phenotypically and molecularly characterized using flow cytometry immunophenotyping, DNA content, G-band and multicolor spectral karyotyping (SKY), and microarray. The cell lines were infected with lentiviral-expressing luciferase for detection of tumor cells at high sensitivity level and for monitoring MM growth in co-cultures and in vivo by live animal imaging. We have reported that hyperdiploid MM cells express high level of the Wnt signaling inhibitor, DKK1, and that DKK1 expression is correlated with level of bone disease. The CF cells which typically represent hyperdiploid patients based on pattern of trisomies, DKK1 expression and induction of severe osteolysis, were engrafted in SCID-rab mice and upon establishment of MM were locally treated with control and DKK1 neutralizing antibodies (R&D Systems, 100 μg/day, S.C.) for 4 weeks. Anti-DKK1 had no significant effect on CF cell growth however; in contrast to control group, bone mineral density (BMD) of the myelomatous bone was preserved by anti-DKK. Prevention of bone loss by anti-DKK1 antibody was associated with increased number of osteocalcin-expressing osteoblasts (27±4 vs. 8±3 in control bones, p<0.003) and reduced numbers of TRAP-expressing multinucleated osteoclasts in myelomatous bones (59±6 vs. 32±3 in control bones, p<0.006). Recent clinical observations indicate bortezomib increases osteoblast activity in MM patients. For studying the effect of bortezomib on MM bone disease, SCID-rab mice engrafted with luciferase-expressing BN cells (n=5 per group) were treated with saline, bortezomib (S.C. 0.5 mg/kg) and melphalan (2.5 mg/kg) twice a week for 4 weeks. Both agents effectively inhibited MM growth as shown by live imaging and hIg ELISA, however, while in bortezomib-treated hosts myelomatous BMD increased by 17±12%, it decreased in saline- and melphalan-treated hosts by 27±9% and 20±9%, respectively (p<0.003 bortezomib vs. saline), indicating that the bone anabolic effect was unique to bortezomib. Ex vivo growth of luciferase-expressing BN cells in co-culture with osteoblasts was lowered by 53±3% (p<0.0005) than their growth rate in co-culture with the supporting mesenchymal stem cells, suggesting that increased bone formation and osteoblast activity negatively impact certain myelomas and that anti-MM mechanism of action of bortezomib involves effect on bone metabolism. These MM cell lines and the procedures used for their establishment provide essential tools for studying myeloma biology and therapy.

Validation of micronuclei frequency in peripheral blood lymphocytes as early cancer risk biomarker in a nested case–control study

Aim of this work was to assess the predictive value of micronuclei (MN) frequency in peripheral blood lymphocytes (PBL) for the risk of cancer death in disease-free individuals. Blood samples from 1650 subjects selected from the general population of Pisa, Italy, were collected between June 1991 and November 1993. The follow-up until January 2005 recorded a total of 111 deaths (52 for cancer). MN frequency was assessed for 49 cancer cases and 101 matched controls. A significantly higher MN frequency was found in cancer cases (4.7 ± 3.4 MN/1000 BN cells) versus controls (1.5 ± 1.7; p < 0.0001). Donors were stratified in two classes and multivariate logistic regression analysis confirmed that individuals with high MN frequency (>2.5 MN/1000 BN cells) had a significantly increased risk of cancer death (OR = 10.7; 95% CI = 4.6–24.9; p < 0.0001) when compared to individuals with low MN frequency (≤2.5 MN/1000 BN cells). Ageing was associated with a 6% increased risk per year ( p = 0.03). No influence of other potential confounders (gender, occupation, smoking and drinking habits) was observed. Finally, subjects with a higher MN frequency showed a higher MR for CVD (Logrank test, p = 0.001). These findings provide strong evidence that MN frequency assessed in PBL of disease-free subjects is a good predictor of cancer death risk, evaluated by a nested case–control study performed 14 years after the original recruitment.

Proteolytic Disassembly Is a Critical Determinant for Reovirus Oncolysis

See page 1406Mammalian ortheoreoviruses are currently being investigated as novel cancer therapeutics, but the cellular mechanisms that regulate susceptibility to reovirus oncolysis remain poorly understood. In this study, we present evidence that virion disassembly is a key determinant of reovirus oncolysis. To penetrate cell membranes and initiate infection, the outermost capsid proteins of reovirus must be proteolyzed to generate a disassembled particle called an infectious subviral particle (ISVP). In fibroblasts, this process is mediated by the endo/lysosomal proteases cathepsins B and L. We have analyzed the early events of infection in reovirus-susceptible and -resistant cells. We find that, in contrast to susceptible glioma cells and Ras-transformed NIH3T3 cells, reovirus-resistant cancer cells and untransformed NIH3T3 cells restrict virion uncoating and subsequent gene expression. Disassembly-restrictive cells support reovirus infection, as in vitro-generated ISVPs establish productive infection, and pretreatment with poly(I:C) does not prevent infection in cancer cells. We find that the level of active cathepsin B and L is increased in tumors and that disassembly-restrictive glioma cells support reovirus oncolysis when grown as a tumor in vivo. Together, these results provide a model in which proteolytic disassembly of reovirus is a critical determinant of susceptibility to reovirus oncolysis.

Cytogenetic damage in female Chilean agricultural workers exposed to mixtures of pesticides

The VIII Region of Bio-Bio is a major fruit-growing area of Chile that makes intensive use of agricultural pesticides. The cytogenetic damage associated with exposure to mixtures of pesticides was evaluated by comparing peripheral blood lymphocyte micronucleus (MN) frequencies in a group of 64 female agricultural workers and 30 female controls. The exposed subjects worked during the spring and summer in thinning and pruning fruit trees and in harvesting and packing different fruits, such as raspberries, grapes, apples, and kiwis. They did not use any protective measures during their work activities. A significant increase in the frequency of binucleated cells with micronuclei (BNMN) was found in the exposed women as compared with the controls (36.94 +/- 14.47 vs. 9.93 +/- 6.17 BNMN/1000 BN cells; P < 0.001). The frequency of BNMN varied as a function of age in both the exposed and control groups, but no correlation was found between BNMN frequency and the duration of exposure. Also, smoking and other habits had no effect on MN frequency. Our study confirms that occupational exposure to pesticide mixtures results in cytogenetic damage.

Enhancement of gene delivery by an analogue of α-MSH in a receptor-independent fashion

In order to transfect melanoma specifically by receptor-mediated endocytosis we prepared dioctadecyl aminoglycylspermine (lipospermine)–DNA complexes with [Nle4,D-Phe7]-α-MSH(4–10), a pseudo-peptide analogue of α-melanocyte stimulating hormone (α-MSH) linked to a thiol-reactive phospholipid. With these complexes we obtained an up to 70-fold increase of transfection with B16-F1 melanoma cells. However when B16-G4F, an α-MSH receptor negative melanoma cell line was transfected, an up to 700-fold increased transfection efficiency was observed. The peptide hormone analogue was equally efficient when it was only mixed with lipospermine–DNA complexes without covalent coupling. In addition to melanoma cells we also obtained up to 30-fold increased transfection with BN cells (embryonic liver cells). Our data show that an α-MSH analogue increased transfection independently of the MSH receptor expression but reaches efficiencies approaching those obtained with peptides derived from viral fusion proteins. The absence of targeting of constructs containing [Nle4,D-Phe7]-α-MSH(4–10) can probably be attributed due to the relatively modest number of MSH receptors at the surface of melanoma. We suggest, however, that the peptide hormone analogue used in this study has membrane-active properties and could be of interest as helper agent to enhance non-viral gene delivery presumably by endosomal-destabilizing properties.