A new flow cytometry assay identifies recipient IgG subtype antibodies binding donor cells: increasing donor availability for highly sensitised patients.

Objectives: There are four immunoglobulin (IgG) subtypes that have varying complement-activating ability: strong (IgG3 and IgG1) and weak (IgG2 and IgG4). The standard flow cytometric crossmatch (FCM) assay does not distinguish between the various subtypes of the IgG molecule. This study outlines the development and use of a novel cell-based IgG subtype-specific FCM assay that is able to detect the presence of and quantitate the IgG subtypes bound to donor cells. Methods: A six-colour lyophilised reagent was designed that specifically detects the four IgG subtypes, as well as distinguishes between T cells and B cells in the lymphocyte population. To test the efficacy of this reagent, a retrospective evaluation of a group of highly sensitised patients awaiting heart and kidney transplant was carried out, who, because of positive standard FCM results, had been deemed incompatible with numerous prior potential donors. Results: Observations in this study demonstrate that the positive standard FCM results were mainly because of the presence of noncomplement-activating IgG2 or IgG4 antibodies. The results were supported by the absence of C3d-binding donor-specific antibodies (DSA) and a negative complement-dependent cytotoxicity crossmatch (CDC). Conclusion: Preliminary data presented in this study demonstrate the reliability of the novel IgG subtype assay to detect the presence of pretransplant, complement-activating antibodies bound to donor cells. The knowledge gained from the IgG subtype assay and the C3d-binding specificities of DSAs provides improved identification of donor suitability in pretransplant patients, potentially increasing the number of transplants.

Structural and Functional Characterization of Deceased Donor Stem Cells: A Viable Alternative to Living Donor Stem Cells.

Stem cells can be isolated from various human tissues including bone marrow (BM) and adipose tissue (AT). Our study outlines a process to isolate adult stem cells from deceased donors. We have shown that cell counts obtained from deceased donor BM were within established living donor parameters. Evaluation of demographic information exhibited a higher percentage of hematopoietic stem cells (HSC) in males versus females, as well as a higher percentage of HSC in the age bracket of 25 years and under. For the first time, we show that deceased donor femur BM grew cell colonies. Our introduction of new technology for nonenzymatic AT processing significantly increased cell recovery over the traditional enzymatic processing method. Cell counts from the deceased donor AT exceeded living donor parameters. Furthermore, our data illustrated that AT from female donors yielded a much higher number of total nucleated cells (TNC) than males. Together, our data demonstrates that our approach to isolate stem cells from deceased donors could be a routine practice to provide a viable alternative to living donor stem cells. This will offer increased accessibility for patients awaiting stem cell therapies.

Simultaneous inhibition of cell-cycle, proliferation, survival, metastatic pathways and induction of apoptosis in breast cancer cells by a phytochemical super-cocktail: genes that underpin its mode of action.

Traditional chemotherapy and radiotherapy for cancer treatment face serious challenges such as drug resistance and toxic side effects. Complementary / Alternative medicine is increasingly being practiced worldwide due to its safety beneficial therapeutic effects. We hypothesized that a super combination (SC) of known phytochemicals used at bioavailable levels could induce 100% killing of breast cancer (BC) cells without toxic effects on normal cells and that microarray analysis would identify potential genes for targeted therapy of BC. Mesenchymal Stems cells (MSC, control) and two BC cell lines were treated with six well established pro-apoptotic phytochemicals individually and in combination (super cocktail), at bioavailable levels. The compounds were ineffective individually. In combination, they significantly suppressed BC cell proliferation (>80%), inhibited migration and invasion, caused cell cycle arrest and induced apoptosis resulting in 100% cell death. However, there were no deleterious effects on MSC cells used as control. Furthermore, the SC down-regulated the expression of PCNA, Rb, CDK4, BcL-2, SVV, and CD44 (metastasis inducing stem cell factor) in the BC cell lines. Microarray analysis revealed several differentially expressed key genes (PCNA, Rb, CDK4, Bcl-2, SVV, P53 and CD44) underpinning SC-promoted BC cell death and motility. Four unique genes were highly up-regulated (ARC, GADD45B, MYLIP and CDKN1C). This investigation indicates the potential for development of a highly effective phytochemical combination for breast cancer chemoprevention / chemotherapy. The novel over-expressed genes hold the potential for development as markers to follow efficacy of therapy.

Characterization of coordinated immediate responses by p16INK4A and p53 pathways in UVB-irradiated human skin cells.

While the precise mechanisms of melanoma development are unknown, recent in vivo studies have revealed that the p16(Ink4a)/Rb pathway is disrupted in melanomagenesis. Here, we characterize the role of p16/Rb in coordinating the early events in UVB-irradiated skin. Foreskins and melanoma cell cultures were irradiated with low and high acute UVB doses and examined for cell-cycle- and apoptosis-associated genes. In melanoma cells, low UVB dose upregulated p16, p53, and p21 expression levels in Malme-3M, and high UVB dose accentuated the expression of p53 and p21(Cip1/Waf1), in particular; however, in SkMel-28 cells only p16 expression was upregulated in response to UV irradiation. In HaCaT cells, high UVB dose caused dramatic increase in p53 expression followed by upregulation of p21(Cip1/Waf1) and Bax, and downregulation of Bcl-2 leading to apoptosis. In HaCaT cells, reinstatement of p16 pathway restored cell-cycle arrest in response to low dose. Foreskin organ culture experiments confirmed our in vitro cell results. These data indicate that the p53 and p16 pathways respond independently to UVB insult. The p16 pathway is favored at low doses and results in cell-cycle arrest; the p53 pathway is more responsive to higher doses and induces apoptosis depending on p53 mutation status.

Synergistic action of dietary phyto-antioxidants on survival and proliferation of ovarian cancer cells.

OBJECTIVES: A number of herbal dietary antioxidant supplements containing Indole-3 Carbinol (I3C) and Resveratrol (RE) have been established as anti-proliferative agents in cancer. These compounds have both similar as well as unique molecular targeting profiles. The purpose of this study is to analyze their mechanism of action when used individually and in combination in ovarian cancer. METHODS: SK-OV-3 ovarian cancer cells were treated with various doses of I3C, RE or I3C+RE. AlamarBlue dye assay was used to examine cell growth and proliferation. Cell cycle and apoptosis were analyzed by flow cytometry. Western blot was performed to determine the expression of the genes associated with cell cycle and apoptosis. CA-125, a functional marker of ovarian cancer, and nitric oxide, were analyzed by ELISA. RESULTS: I3C or RE inhibited cell proliferation, and caused cell contraction and apoptosis. Analysis of apoptosis-associated genes revealed an inhibition of Retinoblastoma protein (Rb) and Survivin (SVV) gene expression. This was accompanied by elevation of p21, a tumor suppressor. Cell cycle was inhibited at both G1 and G2/M by individual treatments, and accentuated by a combination. AlamarBlue assay revealed a clear synergistic action of I3C+RE. CA125 was inhibited by either I3C or RE treatments. In contrast, basal nitric oxide production was inhibited by I3C and I3C+RE but not RE alone. CONCLUSIONS: This is the first evidence demonstrating the effects of I3C on ovarian cancer cells and its synergism with RE. Based on this model, our data indicate that combinations of compounds with different targeting properties will be more effective in chemoprevention and/or chemotherapy of ovarian and possibly other cancers.

Differential effects of leptin on the invasive potential of androgen-dependent and -independent prostate carcinoma cells.

Obesity has been linked with an increased risk of prostate cancer. The formation of toxic free oxygen radicals has been implicated in obesity mediated disease processes. Leptin is one of the major cytokines produced by adipocytes and controls body weight homeostasis through food intake and energy expenditure. The rationale of the study was to determine the impact of leptin on the metastatic potential of androgen-sensitive (LNCaP) cells as well as androgen-insensitive (PC-3 and DU-145) cells. At a concentration of 200 nm, LNCaP cells showed a significant increase (20% above control; P < .0001) in cellular proliferation without any effect on androgen-insensitive cells. Furthermore, exposure to leptin caused a significant (P < .01 to P < .0001) dose-dependent decrease in migration and invasion of PC3 and Du-145 prostate carcinoma cell lines. At the molecular level, exposure of androgen-independent prostate cancer cells to leptin stimulates the phosphorylation of MAPK at early time point as well as the transcription factor STAT3, suggesting the activation of the intracellular signaling cascade upon leptin binding to its cognate receptor. Taken together, these results suggest that leptin mediates the invasive potential of prostate carcinoma cells, and that this effect is dependent on their androgen sensitivity.

Elevation of serum riboflavin carrier protein in hepatocellular carcinoma.

Early detection of hepatocellular cancer (HCC), makes it surgically resectable with a potential for cure. The test most commonly used to detect HCC is the measurement of serum alpha-fetoprotein (AFP) levels. However, the AFP test is negative in HCC detection in more than 30% of the cases. Riboflavin carrier protein (RCP) is a growth and developmental protein, synthesized and secreted by the liver and hence was of interest to measure its levels in HCC. A prospective double blind evaluation of RCP levels in serum from 93 subjects was undertaken. These included 22 proven cases of HCC, 25 normal controls, 20 cases of alcoholic hepatitis, 20 cirrhotics, and 6 cases of primary biliary cirrhosis (PBC). RCP was measured by a sensitive and specific radioimmunoassay (RIA). RCP was immunohistochemically localized in paraffin sections of liver specimens using standard methods. Mean serum RCP levels in HCC were 21.75+/-14.66ng/ml and were significantly higher (p<0.0001) than those in normal controls (0.73+/-0.25ng/ml), alcoholic hepatitis (1.92+/-0.82ng/ml), PBC (2.16+/-0.74ng/ml), or cirrhosis (5.02+/-1.52ng/ml). Serum RCP levels were elevated in all 22 HCC cases. In contrast serum AFP levels were elevated in 11 of 22 HCC cases. Immunohistochemical analyses revealed positive staining for RCP in liver tumors. We have previously demonstrated elevation of serum RCP levels in breast adenocarcinoma. Our results suggest that serum RCP levels are significantly elevated in HCC also and could potentially serve as a marker for HCC detection under conditions where breast cancer is ruled out. In combination with AFP, serum RCP levels have the potential of serving as a panel of markers for better detection of HCC.