Cytotoxic drug sensitivity of Epstein-Barr virus transformed lymphoblastoid B-cells.

BACKGROUND: Epstein-Barr virus (EBV) is the causative agent of immunosuppression associated lymphoproliferations such as post-transplant lymphoproliferative disorder (PTLD), AIDS related immunoblastic lymphomas (ARL) and immunoblastic lymphomas in X-linked lymphoproliferative syndrome (XLP). The reported overall mortality for PTLD often exceeds 50%. Reducing the immunosuppression in recipients of solid organ transplants (SOT) or using highly active antiretroviral therapy in AIDS patients leads to complete remission in 23-50% of the PTLD/ARL cases but will not suffice for recipients of bone marrow grafts. An additional therapeutic alternative is the treatment with anti-CD20 antibodies (Rituximab) or EBV-specific cytotoxic T-cells. Chemotherapy is used for the non-responding cases only as the second or third line of treatment. The most frequently used chemotherapy regimens originate from the non-Hodgkin lymphoma protocols and there are no cytotoxic drugs that have been specifically selected against EBV induced lymphoproliferative disorders. METHODS: As lymphoblastoid cell lines (LCLs) are well established in vitro models for PTLD, we have assessed 17 LCLs for cytotoxic drug sensitivity. After three days of incubation, live and dead cells were differentially stained using fluorescent dyes. The precise numbers of live and dead cells were determined using a custom designed automated laser confocal fluorescent microscope. RESULTS: Independently of their origin, LCLs showed very similar drug sensitivity patterns against 29 frequently used cytostatic drugs. LCLs were highly sensitive for vincristine, methotrexate, epirubicin and paclitaxel. CONCLUSION: Our data shows that the inclusion of epirubicin and paclitaxel into chemotherapy protocols against PTLD may be justified

Extended Field Laser Confocal Microscopy (EFLCM): Combining automated Gigapixel image capture with in silico virtual microscopy

<p>Abstract</p> <p>Background</p> <p>Confocal laser scanning microscopy has revolutionized cell biology. However, the technique has major limitations in speed and sensitivity due to the fact that a single laser beam scans the sample, allowing only a few microseconds signal collection for each pixel. This limitation has been overcome by the introduction of parallel beam illumination techniques in combination with cold CCD camera based image capture.</p> <p>Methods</p> <p>Using the combination of microlens enhanced Nipkow spinning disc confocal illumination together with fully automated image capture and large scale <it>in silico </it>image processing we have developed a system allowing the acquisition, presentation and analysis of maximum resolution confocal panorama images of several Gigapixel size. We call the method Extended Field Laser Confocal Microscopy (EFLCM).</p> <p>Results</p> <p>We show using the EFLCM technique that it is possible to create a continuous confocal multi-colour mosaic from thousands of individually captured images. EFLCM can digitize and analyze histological slides, sections of entire rodent organ and full size embryos. It can also record hundreds of thousands cultured cells at multiple wavelength in single event or time-lapse fashion on fixed slides, in live cell imaging chambers or microtiter plates.</p> <p>Conclusion</p> <p>The observer independent image capture of EFLCM allows quantitative measurements of fluorescence intensities and morphological parameters on a large number of cells. EFLCM therefore bridges the gap between the mainly illustrative fluorescence microscopy and purely quantitative flow cytometry. EFLCM can also be used as high content analysis (HCA) instrument for automated screening processes.</p

Fibroblasts—a key host cell type in tumor initiation, progression, and metastasis

Tumor initiation, growth, invasion, and metastasis occur as a consequence of a complex interplay between the host environment and cancer cells. Fibroblasts are now recognized as a key host cell type involved in host–cancer signaling. This review discusses some recent studies that highlight the roles of fibroblasts in tumor initiation, early progression, invasion, and metastasis. Some clinical studies describing the prognostic significance of fibroblast-derived markers and signatures are also discussed

Confrontation of fibroblasts with cancer cells in vitro: Gene network analysis of transcriptome changes and differential capacity to inhibit tumor growth

Background: There is growing evidence that emerging malignancies in solid tissues might be kept under control by physical intercellular contacts with normal fibroblasts. Methods: Here we characterize transcriptional landscapes of fibroblasts that confronted cancer cells. We studied four pairs of in vitro and ex vivo fibroblast lines which, within each pair, differed in their capacity to inhibit cancer cells. The natural process was modeled in vitro by confronting the fibroblasts with PC-3 cancer cells. Fibroblast transcriptomes were recorded by Affymetrix microarrays and then investigated using network analysis. Results: The network enrichment analysis allowed us to separate confrontation- and inhibition-specific components of the fibroblast transcriptional response. Confrontation-specific differences were stronger and were characterized by changes in a number of pathways, including Rho, the YAP/TAZ cascade, NF-kB, and TGF-beta signaling, as well as the transcription factor RELA. Inhibition-specific differences were more subtle and characterized by involvement of Rho signaling at the pathway level and by potential individual regulators such as IL6, MAPK8, MAP2K4, PRKCA, JUN, STAT3, and STAT5A. Conclusions: We investigated the interaction between cancer cells and fibroblasts in order to shed light on the potential mechanisms and explain the differential inhibitory capacity of the latter, which enabled both a holistic view on the process and details at the gene/protein level. The combination of our methods pointed to proteins, such as members of the Rho pathway, pro-inflammatory signature and the YAP1/TAZ cascade, that warrant further investigation via tools of experimental perturbation. We also demonstrated functional congruence between the in vitro and ex vivo models. © 2015 Alexeyenko et al

Symbionts as Major Modulators of Insect Health: Lactic Acid Bacteria and Honeybees

Lactic acid bacteria (LAB) are well recognized beneficial host-associated members of the microbiota of humans and animals. Yet LAB-associations of invertebrates have been poorly characterized and their functions remain obscure. Here we show that honeybees possess an abundant, diverse and ancient LAB microbiota in their honey crop with beneficial effects for bee health, defending them against microbial threats. Our studies of LAB in all extant honeybee species plus related apid bees reveal one of the largest collections of novel species from the genera Lactobacillus and Bifidobacterium ever discovered within a single insect and suggest a long (>80 mya) history of association. Bee associated microbiotas highlight Lactobacillus kunkeei as the dominant LAB member. Those showing potent antimicrobial properties are acquired by callow honey bee workers from nestmates and maintained within the crop in biofilms, though beekeeping management practices can negatively impact this microbiota. Prophylactic practices that enhance LAB, or supplementary feeding of LAB, may serve in integrated approaches to sustainable pollinator service provision. We anticipate this microbiota will become central to studies on honeybee health, including colony collapse disorder, and act as an exemplar case of insect-microbe symbiosis

Cytotoxic drug sensitivity of Epstein-Barr virus transformed lymphoblastoid B-cells

BACKGROUND: Epstein-Barr virus (EBV) is the causative agent of immunosuppression associated lymphoproliferations such as post-transplant lymphoproliferative disorder (PTLD), AIDS related immunoblastic lymphomas (ARL) and immunoblastic lymphomas in X-linked lymphoproliferative syndrome (XLP). The reported overall mortality for PTLD often exceeds 50%. Reducing the immunosuppression in recipients of solid organ transplants (SOT) or using highly active antiretroviral therapy in AIDS patients leads to complete remission in 23–50% of the PTLD/ARL cases but will not suffice for recipients of bone marrow grafts. An additional therapeutic alternative is the treatment with anti-CD20 antibodies (Rituximab) or EBV-specific cytotoxic T-cells. Chemotherapy is used for the non-responding cases only as the second or third line of treatment. The most frequently used chemotherapy regimens originate from the non-Hodgkin lymphoma protocols and there are no cytotoxic drugs that have been specifically selected against EBV induced lymphoproliferative disorders. METHODS: As lymphoblastoid cell lines (LCLs) are well established in vitro models for PTLD, we have assessed 17 LCLs for cytotoxic drug sensitivity. After three days of incubation, live and dead cells were differentially stained using fluorescent dyes. The precise numbers of live and dead cells were determined using a custom designed automated laser confocal fluorescent microscope. RESULTS: Independently of their origin, LCLs showed very similar drug sensitivity patterns against 29 frequently used cytostatic drugs. LCLs were highly sensitive for vincristine, methotrexate, epirubicin and paclitaxel. CONCLUSION: Our data shows that the inclusion of epirubicin and paclitaxel into chemotherapy protocols against PTLD may be justified

Differential Trafficking of Oxidized LDL and Oxidized LDL Immune Complexes in Macrophages: Impact on Oxidative Stress

Oxidized low-density lipoproteins (oxLDL) and oxLDL-containing immune complexes (oxLDL-IC) contribute to formation of lipid-laden macrophages (foam cells). It has been shown that oxLDL-IC are considerably more efficient than oxLDL in induction of foam cell formation, inflammatory cytokines secretion, and cell survival promotion. Whereas oxLDL is taken up by several scavenger receptors, oxLDL-IC are predominantly internalized through the FCgamma receptor I (FCgamma RI). This study examined differences in intracellular trafficking of lipid and apolipoprotein moieties of oxLDL and oxLDL-IC and the impact on oxidative stress.Fluorescently labeled lipid and protein moieties of oxLDL co-localized within endosomal and lysosomal compartments in U937 human monocytic cells. In contrast, the lipid moiety of oxLDL-IC was detected in the endosomal compartment, whereas its apolipoprotein moiety advanced to the lysosomal compartment. Cells treated with oxLDL-IC prior to oxLDL demonstrated co-localization of internalized lipid moieties from both oxLDL and oxLDL-IC in the endosomal compartment. This sequential treatment likely inhibited oxLDL lipid moieties from trafficking to the lysosomal compartment. In RAW 264.7 macrophages, oxLDL-IC but not oxLDL induced GFP-tagged heat shock protein 70 (HSP70) and HSP70B', which co-localized with the lipid moiety of oxLDL-IC in the endosomal compartment. This suggests that HSP70 family members might prevent the degradation of the internalized lipid moiety of oxLDL-IC by delaying its advancement to the lysosome. The data also showed that mitochondrial membrane potential was decreased and generation of reactive oxygen and nitrogen species was increased in U937 cell treated with oxLDL compared to oxLDL-IC.Findings suggest that lipid and apolipoprotein moieties of oxLDL-IC traffic to separate cellular compartments, and that HSP70/70B' might sequester the lipid moiety of oxLDL-IC in the endosomal compartment. This mechanism could ultimately influence macrophage function and survival. Furthermore, oxLDL-IC might regulate the intracellular trafficking of free oxLDL possibly through the induction of HSP70/70B'

Effect of frequently used chemotherapeutic drugs on the cytotoxic activity of human natural killer cells.

Tumors are considered to be possible targets of immunotherapy using stimulated and expanded autologous or allogeneic natural killer (NK) cells mismatched for MHC class I molecules and inhibitory NK receptors. NK cell-based immunoadjuvant therapies are carried out in combination with standard chemotherapeutic protocols. In the presented study, we characterized the effect of 28 frequently used chemotherapeutic agents on the capacity of NK cells to kill target cells. We found that treatment of NK cells with the drugs vinblastine, paclitaxel, docetaxel, cladribine, chlorambucil, bortezomib, and MG-132 effectively inhibited NK cell-mediated killing without affecting the viability of NK cells. On the other hand, the following drugs permitted efficient NK cell-mediated killing even at concentrations comparable with or higher than the maximally achieved therapeutic concentration in vivo in humans: asparaginase, bevacizumab, bleomycin, doxorubicin, epirubicin, etoposide, 5-fluorouracil, hydroxyurea, streptozocin, and 6-mercaptopurine

Effect of frequently used chemotherapeutic drugs on cytotoxic activity of human cytotoxic T-lymphocytes.

Tumors are considered to be possible targets of immunotherapy using stimulated and expanded cytotoxic T-lymphocytes (CTL). It is important to consider the drug-induced effects when chemotherapeutic regimens and CTL-mediated immunotherapy is planned to be used in parallel. In this study, we characterized the effect of 29 frequently used chemotherapeutic agents on the cytotoxic activity of autologous and allogeneic CTLs. We found that treatment of CTLs with the following drugs: docetaxel, vincristine, chlorambucil, mitomycin C, oxaliplatin, doxorubicin, and bleomycin effectively inhibited CTL-mediated killing, without affecting their viability. On the other hand, the following drugs enhanced or permitted efficient CTL-mediated killing in vitro at concentrations comparable with the maximally achieved therapeutic concentration in vivo in humans: daunorubicin, prednisolone, vinorelbine, cisplatin, methotrexate, hydroxyurea, cytarabine, cyclophosphamide, topotecan, epirubicin, fluorouracil, carboplatin, asparaginase, 6-mercaptopurine, and bortezomib. Our results could potentially be used in the future to design new CTL-based adjuvant immunotherapy protocols