Rec Cells Research Articles

G-Protein-Coupled Receptors Mediate Modulations of Cell Viability and Drug Sensitivity by Aberrantly Expressed Recoverin 3 within A549 Cells

To elucidate the currently unknown molecular mechanisms responsible for the aberrant expression of recoverin (Rec) within cancerous cells, we examined two-dimensional (2D) and three-dimensional (3D) cultures of Rec-negative lung adenocarcinoma A549 cells which had been transfected with a plasmid containing human recoverin cDNA (A549 Rec) or an empty plasmid as a mock control (A549 MOCK). Using these cells, we measured cytotoxicity by several anti-tumor agents (2D), cellular metabolism including mitochondrial and glycolytic functions by a Seahorse bio-analyzer (2D), the physical properties, size and stiffness of the 3D spheroids, trypsin sensitivities (2D and 3D), and RNA sequencing analysis (2D). Compared with the A549 MOCK, the A549 Rec cells showed (1) more sensitivity toward anti-tumor agents (2D) and a 0.25% solution of trypsin (3D); (2) a metabolic shift from glycolysis to oxidative phosphorylation; and (3) the formation of larger and stiffer 3D spheroids. RNA sequencing analysis and bioinformatic analyses of the differentially expressed genes (DEGs) using Gene Ontology (GO) enrichment analysis suggested that aberrantly expressed Rec is most likely associated with several canonical pathways including G-protein-coupled receptor (GPCR)-mediated signaling and signaling by the cAMP response element binding protein (CREB). The findings reported here indicate that the aberrantly expressed Rec-induced modulation of the cell viability and drug sensitivity may be GPCR mediated.

MiR-122 promotes diabetic retinopathy through targeting TIMP3

ABSTRACT Diabetic retinopathy (DR) is a primary complication of diabetes mellitus. DR can cause severe vision loss for patients. miR-122 is elevated in DR patients, while its role in DR is unclear. Hence, the purpose of this study was to analyze the effect of miR-122 on the function of high glucose-induced REC cells and the underlying molecular mechanisms. In this study, our results revealed that miR-122 was up-regulated in high glucose-induced human retinal pigment epithelial cells (ARPE-19). High glucose decreased the cell viability of ARPE-19 cells, which was then restored by miR-122 knockdown. In addition, miR-122 knockdown suppressed apoptosis of high glucose-induced ARPE-19 cells. High glucose also inhibited B-cell lymphoma-2 (Bcl-2) level and increased cleaved caspase-3 level in ARPE-19 cells, which were reversed by miR-122 knockdown. Tissue inhibitor of metalloproteinases-3 (TIMP3) was a direct target of miR-122. TIMP3 was decreased in high glucose-induced ARPE-19 cells, and the decrease was abrogated by miR-122 knockdown. In addition, the effects of miR-122 overexpression in cell viability and apoptosis of high glucose-induced ARPE-19 were abolished by overexpression of TIMP3. In conclusion, the effect and mechanism of miR-122 on high glucose-induced ARPE-19 cells were demonstrated for the first time. miR-122 promoted diabetic retinopathy through targeting TIMP3, making miR-122 a promising target for diabetic retinopathy therapy.

Abstract 1757: The novel BTK inhibitor CC-292 exerts in vitro and in vivo antitumor activity, interferes with adhesion, cell migration, and synergizes with lenalidomide in MCL models

Abstract Background B-cell receptor (BCR) signaling contributes to the pathogenesis of B cell malignancies and has emerged as a new target for therapy with special relevance for tumor cell-microenvironment crosstalk. Recently, blockade of the BCR-related kinase Btk (Bruton tyrosin kinase) with the first-in-class inhibitor Ibrutinib, has shown impressive clinical responses in Mantle cell lymphoma (MCL) and Chronic Lymphocytic Leukemia. However, resistances to treatment have already appeared, opening the door to new BTK inhibitors. In this study, we have evaluated the antitumor profile of a novel and highly selective BTK inhibitor CC-292(Celgene)in MCL. Methods Representative MCL lines (MINO, JEKO-1, REC-1, UPN1, Z138, HBL2, GRANTA, JVM-2) were used for in vitro and in vivo studies. The effects of CC-292 on lymphoma cell growth and apoptosis were analyzed by MTT and Annexin V/PI staining, respectively. BTK phosphorylation at Y223 residue was used as a read-out of BTK activity. Microenvironment-derived BTK activation was mimicked by IgM crosslinking or SDF1α/CXCL12 stimulation. Actin polymerization experiments were performed using Phalloidin-TRITC staining, and adhesion assays were done on EIA/RIA 96-well plates coated with Fibronectin or VCAM-1. In vivo activity was assessed in a subcutaneous mouse xenograft model, where daily oral dosing was started when tumors were palpable and extended for a total of 18 days. Results BTK activation (pBtkY(223)) was detected in all MCL cell lines analyzed. CC-292 (10-1000nM) exerted cytostatic effect in part of the cell lines, where REC-1, MINO, JVM2 and UPN1 were the most sensitive cell lines. No correlation between constitutive Btk activation and response to CC-292 was found. Noteworthy, CC-292 potently inhibited constitutive and IgM/CXCL12-induced Btk activation and interfered with tumor cell-microenvironment interactions, by blocking SDF1α/CXCL12-induced actin polymerization, as well as IgM-induced adhesion to VCAM and FN. In CC-292 sensitive cell lines, the combination with the immunomodulatory drug lenalidomide was synergistic and was accompanied with IRF4 decrease in MINO and REC cell lines. Finally, CC-292 (30mg/kg) showed antitumor activity in vivo in a sc mouse xenograft (UPN-1 cell line), reducing tumor outgrowth by 52% Conclusions In summary, these results warrant further investigation of CC-292 for MCL therapy, both alone and in combination with the immunomodulatory agent lenalidomide. Citation Format: Anna Vidal-Crespo, Vanina Rodríguez, Alba Matas-Céspedes, Elías Campos, Armando López-Guillermo, Gael Roué, Dolors Colomer, Patricia Pérez-Galán. The novel BTK inhibitor CC-292 exerts in vitro and in vivo antitumor activity, interferes with adhesion, cell migration, and synergizes with lenalidomide in MCL models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1757. doi:10.1158/1538-7445.AM2014-1757

Daratumumab, a Novel Human Anti-CD38 Monoclonal antibody shows Anti-Tumor Activity In Mouse Models Of MCL, FL and CLL

Daratumumab (DARA) is a human CD38 antibody with broad-spectrum killing activity. DARA induces killing of tumor cells, mainly via complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) (de Weers M, J Immunol 2011). DARA is currently being evaluated in phase I/II clinical trials in patients with multiple myeloma. In these clinical studies the adverse events have been manageable and marked reductions in paraprotein and bone marrow plasma cells have been observed. We have previously reported (Blood (ASH annual meeting abstracts). Nov 2012, 120 (21): 3935) that DARA induces cytotoxic activity in vitro via ADCC in primary cells and cell lines from Mantle Cell Lymphoma (MCL), Follicular Lymphoma (FL) and Chronic Lymphoctic Leukemia (CLL). CDC induction was low, which is associated to high expression of the complement inhibitors and reduced number of CD38 molecules per cell in these indications. This suggests a threshold for CD38-targeted CDC lysis. In addition, based on known interactions between CD38-CXCR4, we also demonstrated that in the CLL subtype, with high CD38 and more migratory capacity, DARA (10-30 µg/mL) inhibited in vitro CXCL12/SDF1α-mediated migration up to 70%.Here, we present the first preclinical in vivo results of DARA in mouse models of MCL, transformed FL(tFL) and CLL. We generated heterotopic and systemic mouse models of these entities by subcutaneous or intravenous inoculation of tumor cell lines in SCID mice, that retain both NK cells and macrophages as potential effector cells.In MCL (REC cell line) and tFL (RL cell line) subcutaneous mouse models, we tested DARA activity in both prophylactic (treatment initiation simultaneous to lymphoma cell inoculation) and therapeutic settings(treatment initiation one week after lymphoma cell inoculation, when tumors were about 100 mm3 in size). In the prophylactic setting, mice received 3 doses of DARA or control IgG bi-weekly (20/10/10 mg/kg). In the therapeutic setting, treatment was intensified to 4 doses of DARA or control IgG weekly (20/10/10/10 mg/kg). In both schedules, mice were sacrificed one week after the last dose. DARA completely abrogated tumor growth of REC or RL cells in the prophylactic setting. Moreover, in the therapeutic setting, DARA induced total tumor regression of REC tumors in 4 out of 6 mice, and prevented the splenomegaly observed in control IgG treated mice. In the case of tFL and therapeutic setting, DARA reduced 60% of tumor growth compared to control IgG treated mice at day 32, when experiment was finished. In CLL, we analyzed the effect of DARA on cell homing to lymphoid organs together with its therapeutic properties in a systemic CLL mouse model. Using NOD/SCID/gamma null mice (lacking NK cells and effective macrophages), we analyzed the effect of DARA on primary CLL cell migration from Peripheral Blood (PB) to bone marrow (BM) and Spleen. In this system, NSG mice were pretreated (day 0) with DARA, control IgG or anti-CXCR4 as positive control for inhibition of cell homing, followed by fresh CLL cell inoculation (50×106 cells/per mice) on day 1. PB, BM and spleen cells were isolated on day 2 and CLL cells were identified by staining for CD45/CD19/CD5 and counted using a flow cytometer. Cell counting showed that CLL cells mainly migrate to the spleen, and that DARA significantly reduced this migration (55% inhibition on average, p<0.05). Migration of CLL cells to BM was limited and was not affected by pretreatment of mice with DARA.Finally, we tested DARA therapeutic activity in a systemic CLL mouse model (MEC2 cell line), following the schedule described before (4 doses of control IgG/ DARA weekly (20/10/10/10 mg/kg)), and assessed efficacy on mice overall survival. Mice treated with control IgG started to lose weight and showed signs of disease between days 30-40 and were sacrificed for ethical reasons. In the DARA treated group, in 7 out of 8 mice survival was extended up to day 90, when the experiment was stopped. In conclusion, DARA demonstrated strong in vivo activity in immunocompromised mouse models of MCL, tFL and CLL cell lines and interfered with homing of primary CLL cells to the spleen. These results warrant further investigation of DARA in clinical trials for these indications. Disclosures:Lopez-Guillermo:Roche: Membership on an entity's Board of Directors or advisory committees. Lammerts van Bueren:Genmab: Employment, Stocks Other. Bakker:Genmab: Employment, Stocks Other. Parren:Genmab: Employment, Stocks Other. Perez-Galan:Genmab: Research Funding.

Comparison of Pyrometric Co-C and Re-C Eutectic-Point Cells Between VNIIM and LNE-Cnam

VNIIM and LNE-Cnam have collaborated for several years in the field of metal-carbon eutectic points. The first action was the construction of a Pt-C cell at VNIIM using the LNE-Cnam technique and cell design. The Pt-C cells constructed in each of the laboratories were studied and compared in the past. The two laboratories have followed their collaboration work by studying and comparing Co-C and Re-C cells. Different designs and filling techniques were applied. The melting and freezing temperatures observed on the Re-C cells from the two laboratories were measured at VNIIM. The Re-C and Co-C cells were compared at LNE-Cnam in the high-temperature blackbody furnace HTBB 3200pg which was thermally optimized before the measurements. The results of the comparison showed that the Co-C cells were comparable at the level of 0.03 K while the Re-C cells showed a large difference of melting temperatures of about 0.7 K. In this article, the cells used and the methodology of the comparison will be described. The temperature differences that were obtained at the highest temperature will be examined to propose an explanation for this temperature difference.

New Method of Filling of High-Temperature Fixed-Point Cells Based on Metal-Carbon Eutectics/Peritectics

A new method of filling of high-temperature fixed-point cells based on metal-carbon eutectics and peritectics is suggested and tested. In this method a metal and carbon powder mixture is introduced not directly into the crucible, but into an additional container located just above the crucible. The mixture melts inside the container, and the already molten eutectic drops through a small hole in the bottom of the container and fills the crucible drop by drop. The method can be used to obtain a uniform ingot without porous or foundry cavities, to minimize the risk of contamination, and to avoid some other disadvantages. The method was applied to fabricate Re-C and WC-C cells using 5N purity materials. The cells demonstrated a good plateau shape with melting ranges of 0.2 K and 80 mK for Re-C and WC-C, respectively. The Re-C cell was compared with a cell built at NMIJ and showed good agreement with a difference of melting temperatures of only 45 mK.

Assessment of Proliferation Induced in Fibroblasts and Rabbit Corneal Epithelial Cells by a Platelet Lysate Formulation: A Stability Study

Introduction. Platelets contain a mixture of growth factors (GF). These can be released from platelet lysate (PL) whose application to wounds is supposed to favour the healing process. The application of PL in a suitable formulation can improve therapeutic efficacy and patient compliance. A formulation of PL with a selected biocompatible vehicle has been developed for application in buccal mucosal damages (mucositis), that are the most common complications of the nonsurgical therapy of cancer. The same vehicle can be proposed also for ophthalmic formulations as several studies have shown that topical application of the growth factors to the injured cornea facilitates wound repair by rapid regrowth of the epithelial cells. Aim of the present work was to develop in vitro tests for a fast evaluation of PL activity in the formulation to evidence excipient compatibility, and the effect of formulative parameters on PL stability.The proliferative effect on two different models, fibroblasts and a corneal epithelial cell line, was evaluated. The stability of PL in the formulation has been assessed after two weeks at 4–8 °C storage.Methods. PL was mixed in a 1:1 ratio to the selected vehicle and stored at 4–8 °C. Immediately after preparation (time zero, T0) and after 2, 7, 10 and 15 days (T2–T15), aliquots were used to assess proliferative effect on cell cultures. Both fibroblast (primary human cell lines) and RCE (Rabbit corneal epithelial cell lines, ECACC) were seeded in 96-well plates with area of 0.34 cm2 at the concentrations of either 10000 or 5000 cells/well. After 24 hours the cells were added with either complete growth medium (Reference), minimal medium not supplemented with foetal calf serum and for REC cells without also EGF (Control), PL formulation diluted 1/20 (containing PL at 5%) and PL formulation diluted 1/40 (containing PL at 2.5%). After 24 hours, neutral red (NR) test was performed (Tox Kit 4, Sigma-Aldrich, Milano I). The NR solution absorbance was determined by means of a plate reader (Perkin Elmer, Milan, I) at wavelength of 490 nm. The absorbance read for each sample was compared with that of Reference, whose proliferation was assumed as 100%.Results. At time zero, the percentage of proliferation showed no significant differences with respect to the cells in complete growth medium (Reference) in the case of fibroblasts, both at 5000 and 10000 seeding level. The two cell models gave comparable results although slightly higher values of proliferation were obtained in fibroblasts. This can be attributed to the epithelial nature of the RCE and to the possible different percentage in the platelet derived mixture of FGF and EGF. In both cases it can be argued that the vehicle is compatible with cell growth. Both for fibroblasts and for RCE no decrease of activity with time was observed, and no statistical differences with respect to the control have been obtained until 15 days storage. (See figure 1).The results obtained suggest that the two cell culture models evaluated can be suitable to assess the activity of PL in easy and fast way. This will be useful to develop formulations intended for the treatment of mucositis and of damages of corneal epithelia. In particular, the formulation tested seems to be stable and to allow the release of GF from PL until 15 days of storage. [Display omitted]

In vitro activity of cyclin-dependent kinase inhibitor CYC202 (Seliciclib, R-roscovitine) in mantle cell lymphomas

Background:: Mantle cell lymphoma (MCL) has the worst prognosis of all B-cell lymphomas and has poor response to conventional therapy. It is characterized by the presence of a chromosomal translocation t(11:14) (q13;q32) which results in deregulated cyclin D1 expression. Since defects in cell cycle regulation and apoptosis are primary events in MCL, small-molecule inhibitors of cdks–cyclins may play an important role in the therapy of this disorder. CYC202 (Seliciclib, R-roscovitine; Cyclacel Ltd., Dundee, UK) is a purine analogue and a selective inhibitor of the cdk2–cyclin E as well as cdk7–cyclin H and cdk9–cyclin T.Materials and methods:: The activity of CYC202 was tested in four human MCL cell lines: REC, Granta-519, JeKo-1 and NCEB-1. The effect of CYC202 on the cell cycle and on apoptosis-, cell-cycle- and transcription-regulation-related proteins was assessed.Results:: The IC50 was 25μm for REC, Granta-519 and JeKo-1 cells and 50μm for NCEB-1 cells. CYC202 caused an accumulation of cells in the G2–M phase of the cell cycle and apoptosis. CYC202 caused down-regulation of cyclin D1 and Mcl-1 protein levels, possibly because of the inhibition of transcription elongation.Conclusions:: Our data suggest that CYC202 is an active agent in MCL. The concomitant decrease of the phosphorylated and total forms of RNA polymerase II suggests that this could be the main mechanism mediating the biological effects of CYC202 in MCL cells. The drug might represent a new therapeutic agent in this lymphoma subtype.

767. Intact Microtubules Are Required for Ad-Vector Induction of Chemokine Genes in Epithelial Cells

Upon entry in epithelial cells, adenovirus vectors (Ad-vectors) activate a cascade of signaling pathways including p38 MAPK (p38) and extracellular-signal regulated kinase (ERK). These signaling events occur within minutes and are triggered directly by the viral capsid. The activation of signaling by Ad-vectors leads to the expression of chemokine inflammatory genes such as IP-10. Shortly following internalization, Ad-vectors interact with microtubules that are required to mediate viral translocation to the nucleus. To better understand the role of microtubules in Ad vector induced signaling and chemokine gene expression, epithelial cells (REC) were treated with the microtubule depolymerizing reagent nocodazole (20mM). Nocodazole substantially reduced transduction of REC cells by a first generation Ad-vector, Adβgal, as determined by β-galactosidase expression 24 hours post-transduction. Vector internalization into REC however was unaffected as measured by flow cytometry confirming that microtubules were required to mediate nuclear translocation of Ad-vectors. To determine the effect of microtubule depolymerization on the induction of chemokine mRNA expression, RANTES and IP-10 gene expression was determined at 6 hours following transduction with Adβgal. Compared to untreated cells, nocodazole blocked the Adβgal induction of RANTES and IP-10 expression. Phosphorylated ERK or p38 was increased by nocodazole in the absence of Ad-vector transduction, a response that was not changed following the addition of Adβgal. Although indirect, this finding suggests that p38 and ERK signaling is linked to microtubules and is required but not sufficient to induce the expression of IP-10 and RANTES. In contrast, experiments with the microtubule-stabilizing agent, paclitaxel, did not affect Adβgal transduction of REC cells. Furthermore, Adβgal induction of p38/ERK phosphorylation and IP-10/RANTES gene expression was unchanged following paclitaxel suggesting that microtubule treadmilling or dynamic instability was not required for Ad-vector induction of host immune responses. These preliminary data suggest that intact microtubules are required for the Ad-vector induction of host inflammatory genes in epithelial cells. Understanding the biology of Ad-vector induced intracellular signaling will contribute significantly to the development of novel recombinant vectors for gene therapy.

Differential Activation of Innate Immune Responses by Adenovirus and Adeno-Associated Virus Vectors

Adenovirus vectors induce acute inflammation of infected tissues due to activation of the innate immune system and expression of numerous chemokines and cytokines in transduced target cells. In contrast, adeno-associated virus (AAV) vectors are not associated with significant inflammation experimentally or clinically. We tested the ability of AAV vectors to induce the expression of chemokines in vitro and to activate the innate immune system in vivo. In human HeLa cells and murine renal epithelium-derived cells (REC cells) the adenovirus vector AdlacZ induced the expression of multiple inflammatory chemokines including RANTES, interferon-inducible protein 10 (IP-10), interleukin-8 (IL-8), MIP-1beta, and MIP-2 in a dose-dependent manner. The use of AAVlacZ did not induce the expression of these chemokines above baseline levels despite 40-fold-greater titers than AdlacZ and greater amounts of intracellular AAVlacZ genomes according to Southern and slot blot analysis. This finding confirmed that the lack of AAVlacZ induction of chemokine was not due to reduced transduction. In DBA/2 mice, the intravenous administration of 2.5 x 10(11) particles of AAVlacZ resulted in the rapid induction of liver tumor necrosis factor alpha (TNF-alpha), RANTES, IP-10, MIP-1beta, MCP-1, and MIP-2 mRNAs. However, 6 h following injection, chemokine mRNA levels returned to baseline. As expected, administration of 10-fold less AdlacZ caused an induction of liver TNF-alpha and chemokine mRNAs that persisted for more than 24 h posttransduction. Whereas intravenous administration of 2.5 x 10(11) particles of AAVlacZ triggered a transient infiltration of neutrophils and CD11b(+) cells into liver, this response stood in contrast to widespread inflammation and toxicity induced by AdlacZ. Kupffer cell depletion abolished AAVlacZ but not AdlacZ-induced chemokine expression and neutrophil infiltration. In summary, these results show that AAV vectors activate the innate immune system to a lesser extent than do adenovirus vectors and offer a possible explanation for the reduced inflammatory properties of AAV compared to adenovirus vectors.

Activation of p38 and ERK signaling during adenovirus vector cell entry lead to expression of the C-X-C chemokine IP-10.

The use of adenovirus vectors for human gene therapy is limited by potent inflammatory responses that result in significant morbidity. In kidney-derived epithelial cells (REC), activation of extracellular signal-regulated kinase 1/2 (ERK) and p38 kinase (p38) pathways occurred within 20 min of transduction with the serotype 5 adenovirus vector AdCMV beta gal. Inhibition of ERK and p38 with U0126 and SB203580, respectively, reduced the expression of IP-10 mRNA following transduction with AdCMV beta gal. To determine the role of the coxsackievirus-adenovirus receptor (CAR) or alpha(v) integrins in the activation of ERK and p38 and the expression of IP-10, REC cells were transduced with the fiber-modified and RGD-deleted adenovirus vectors AdL.F(RAEK-HA) and AdL.PB(HA), respectively. Compared with the wild-type capsid vector Ad5Luc, transduction with AdL.F(RAEK-HA) and AdL.PB(HA) resulted in reduced ERK-p38 activation and less IP-10 mRNA expression. The decreased IP-10 expression induced by the tropism-modified vectors was due to diminished transduction, since increasing multiplicity of infection resulted in increased IP-10 expression. Inhibition of adenovirus penetration with bafilomycin A1 or ammonium chloride attenuated the activation of ERK-p38 and IP-10 mRNA expression following infection, suggesting that endosomal escape was required to trigger these pathways. In vivo, direct inhibition of ERK and p38 signaling pathways inhibited adenovirus vector-induced IP-10 expression in mouse liver 1 h following transduction. These results demonstrate the importance of signaling via ERK and p38 in the early host response to adenovirus vectors and will permit the development of novel strategies to improve the safety and efficacy of these agents in human gene therapy.

Adenovirus vector-induced inflammation: capsid-dependent induction of the C-C chemokine RANTES requires NF-kappa B.

Adenovirus vectors for gene therapy activate responses in the host that result in acute inflammation of transduced tissues. Our previous studies in vivo demonstrate that chemokines, including the C-C chemokine RANTES (regulated on activation, normal T cell expressed and secreted), contribute to the acute inflammation induced by adenovirus vectors. Various first-generation adenovirus vectors, including adCMV beta gal, were equally capable of inducing the expression of RANTES 3 hr after transduction in epithelial HeLa and REC cells. Deletional analysis of the human RANTES promoter revealed that induction by adCMV beta gal required the elements spanning base pairs -90 to -25 of the gene. Electrophoretic mobility shift assays demonstrated that nuclear extracts from adCMV beta gal-transduced HeLa cells bound to an NF-kappa B site at position -54. Overexpression of I-kappa B alpha suppressed adCMV beta gal induction of RANTES, confirming that this process was dependent on the nuclear translocation of NF-kappa B. The coxsackievirus-adenovirus receptor (CAR)-independent, serotype 3 adenovirus was equally capable of inducing the expression of RANTES in HeLa cells. This observation suggested that binding to CAR was not specifically required in adenovirus vector-induced RANTES expression. The use of RGD peptides to block adCMV beta gal interactions with alpha(v)-integrins reduced RANTES expression but also transduction efficiency. In CAR-deficient P815 cells, binding of adCMV beta gal to alpha(v)-integrins without efficient cell transduction did not result in increased RANTES expression. Expression of human CAR in P815 cells increased the binding and transduction efficiency of adCMV beta gal and resulted in RANTES expression in these cells. These results suggest that the induction of RANTES by adenovirus vectors is dependent on efficient interaction with its cell surface receptors and vector internalization. Understanding the biology of the host response to adenovirus vectors will impact the design of future generations of these agents aimed at reducing their immunogenicity and improving their safety.

P22phox-containing microparticles from plasma of septic patients are related to reactive oxygen species production and apoptosis of vascular cells in culture

Reactive oxygen species (ROS) are related to vascular dysfunction in several pathologies and may be related to the pathophysiology of sepsis and multiple organ dysfunction. To investigate their role and sources we evaluated the effects of plasma of septic patients (SPP) on reactive ROS production and apoptosis of rabbit endothelial (REC) and aortic smooth muscle (RASM) cells in culture through NAD(P)H (0.3 mM)-driven lucigenin (5 μM) luminescence and annexin V assay respectively. SPP alone showed intrinsic luminescence when compared to plasma from healthy controls (HCP) (51.4 ± 9.5 cpm/min/mg protein vs 7.0 ± 1.2, n = 5, P < 0.05). After incubation with REC and RASM cells SPP induced a two-fold increase in ROS production (n = 5, P < 0.05) when compared to incubation with HCP. This enhancement of ROS production by REC and RASM cells were paralleled by increase in apoptosis rates induced by SPP. The fraction responsible for ROS production and apoptosis induction was determined through filtration and ultracentrifugation to be over 50 kD and to contain microparticles (MP), similar to those from activated platelets. MP showed similar luminescence (60.6 ± 4.4 vs 32.9 ± 3.1, n = 11), blocked by DPI (60%), a flavoenzyme inhibitor, and by SOD (80%). Addition of MP from SPP to REC and RASM also induced increased luminescence when compared to the effect of MP from HCP; These signals were also inhibitable by SOD and DPI. Incubation of REC and RASM with MP from SPP doubled apoptosis rates when compared to incubation with MP from HCP. Apoptosis was also inhibited by addition of SOD or DPI. After lipid extraction, western-blot analysis of the MP was positive for the p22phox and gp91 subunits of the NAD(P)H oxidase. Thus, SPP has intrinsic and enhances REC and RASM ROS production, due to NAD(P)H oxidase activity. It causes apoptosis of REC and RASM, also inhibited by SOD and DPI. These effects may be attributable to MP containing the p22phox and gp91 subunits of NAD(P)H oxidase. ROS derived from microparticles may represent a new signaling pathway involved in the pathophysiology of severe sepsis.

Photodynamic efficiency of liposome-administered tetramethyl hematoporphyrin in two human bladder cancer cell lines.

The main problems presented by superficial bladder carcinoma, its high recurrence rate and multifocal appearance, require treatment of the bladder as a whole. Photodynamic therapy (PDT) is one such experimental treatment for superficial bladder carcinoma, involving the administration of a photosensitizer that accumulates in the tumor tissue, and subsequent irradiation of the tumor with light. Since the photosensitizers used in PDT suffer from several drawbacks, new photosensitizers are being sought. Drug delivery systems are also being investigated for the administration of hydrophobic photosensitizers and enhancement of photodynamic efficiency and tumor selectivity. In this study we examined a new photosensitizer, tetramethyl hematoporphyrin (TMHP), in two human bladder cancer cell lines. In the first pair of the experiments, TMHP was bound to unilamellar liposomes. Cellular uptake, dark toxicity and photodynamic efficiency were then studied. Fluorescence microscopy showed TMHP localization in the cytoplasm in a perinuclear region, sparing the nucleus. Dark toxicity occurred after incubation of cells with TMHP above a concentration of 20 micrograms/ml. Irradiation was carried out using an argon-pumped dye laser emitting a wavelength of 630 nm at a fluence of 3.6 and 7.2 J/cm2. Before irradiation, cells were incubated with TMHP at concentrations of 2.5 and 5 micrograms/ml for 1 h. Cell survival rates after incubation with 5 micrograms/ml TMHP and irradiation at 7.2 J/cm2 were 15.7% of control cells for Rec and 4.5% for Waf cells. Uptake studies showed a higher intracellular TMHP concentration in Waf than in Rec cells. This correlates with the higher PDT efficiency seen in Waf cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of Escherichia coli plasmid and chromosomal recombination by the Ref function of bacteriophage P1.

The Ref activity of phage P1 enhances recombination between two defective lacZ genes in the Escherichia coli chromosome (lac- x lac- recombination). Plasmid recombination, both lac- x lac- and tet- x tet-, was measured by transformation of recA strains, and was also assayed by measurement of beta-galactosidase. The intracellular presence of recombinant plasmids was verified directly by Southern blotting. Ref stimulated recombination of plasmids in rec+ and rec(BCD) cells by 3-6-fold, and also the low level plasmid recombination in recF cells. RecA-independent plasmid recombination, either very low level (recA cells) or high level (recB recC sbcA recA cells), was not stimulated. Ref stimulated both intramolecular and intermolecular plasmid recombination. Both normal and Ref-stimulated lac- x lac- chromosomal recombination, expected to be mostly RecBC-dependent in wild-type bacteria, were affected very little by a recF mutation. We have previously reported Ref stimulation of lac- x lac- recombination in recBC sbcB bacteria, a process known to be RecF-dependent. Chromosomal recombination processes thought to involve activated recombination substrates, e.g., Hfr conjugation, P1 transduction, were not elevated by Ref activity. We hypothesize that Ref acts by unknown mechanisms to activate plasmid and chromosomal DNA for RecA-mediated recombination, and that the structures formed are substrates for both RecF-dependent (plasmid, chromosomal) and Rec(BCD)-dependent (chromosomal) recombination pathways.

Inviability of dam recA and dam recB cells of Escherichia coli is correlated with their inability to repair DNA double-strand breaks produced by mismatch repair.

The molecular basis for the inviability of dam-3 recA200(Ts) and dam-3 recB270(Ts) cells was studied. The dam-3 recA200(Ts) cells were inviable in yeast extract-nutrient broth or in minimal medium at 42 degrees C. Although the dam-3 recB270(Ts) cells were inviable in yeast extract-nutrient broth at 42 degrees C, they were viable at 42 degrees C in minimal medium, in which the high salt content suppresses the mutant phenotype caused by the recB270(Ts) mutation at 42 degrees C. Under the growth conditions rendering dam rec cells inviable, the cells accumulated double-strand breaks in their DNA. Introduction of a mutL or mutS mutation restored the viability of dam-3 recB270(Ts) cells grown in yeast extract-nutrient broth at 42 degrees C and eliminated the formation of DNA double-strand breaks in these cells. We conclude that the inability to repair DNA double-strand breaks produced by the mismatch repair process accounts for the inviability of the dam recA and dam recB cells.

Effect of chromosome homology of plasmid transformation and plasmid conjugal transfer in Haemophilus influenzae.

Homology between a resident plasmid in H. influenzae and a plasmid entering the cell has been shown to be very important for the acquisition by the cell of genetic markers from the incoming plasmid. For example, the frequency of transformation of recombination-proficient H. influenzae by two 51-kbp plasmids increases by more than 3 orders of magnitude when the recipient cells contain a partially homologous plasmid (1). This increase is not simply the result of increased uptake of plasmid DNA into the cell, since no transformation could be observed with Rec− recipients which take up DNA as well as the wild type. Furthermore, one of the plasmids has been shown to contain the DNA uptake sites necessary for efficient uptake into competent H. influenzae cells (Setlow and Albritton, unpublished experiments), and the data suggest that the other plasmid also contains such sites. Thus, the plasmids are able to transform Rec cells containing homologous DNA at a higher frequency because of events occurring following DNA uptake.

DYSMORPHIC SYNDROME, SPHEROCYTOSIS AND PARTIAL DELETION OF THE SHORT ARM OF CHROMOSOME 8

Congenital spherocytic anemia is a relatively common disorder but the genetic defect has not yet been definitively mapped to a chromosome. We studied a family in which 2 of 3 children were dysmorphic, affected by severe spherocytosis requiring intermittant transfusions, and showed deletion of a portion of the short arm of chromosome 8 (8p-). Both parents and the sibling were normal and showed no evidence of hemolysis or the deletion. Assays of carbonic anhydrase II, factor VII, thryroglobulin, and glutathione reductase, genes previously assigned to 8p were unremarkable. In order to elucidate the membrane defect, red cell membranes were electrophoresed using 1-D Fairbanks SDS polyacrylamide gels and a modified O'Farrell 2-D technique. Lipids were examined using thin-layer chromatography. Coomasie blue stained 1-D and silver stained 2-D electrophoretograms were normal; in particular members of the ternary binding complex, spectrin (α and β), band 4.1, and actin were normal as were membrane cholesterol and phospholipids. The association between the gene deletion and spherocytosis in the affected family members suggests that a gene for this defect lies on 8p, but does not affect the quantities of membrane proteins conventionally associated with rec cell membrane stability.

Homology and repair of UV-irradiated plasmid DNA in haemophilus influenzae.

UV-irradiated plasmid pNov1 containing a cloned fragment of chromosomal DNA could be repaired by excision, but plasmid p2265 without homology to the chromosome could not. Establishment of pNov1 was more UV resistant in Rec(-) than in Rec(+) cells.

Contraction and organization of collagen gels by cells cultured from periodontal ligament, gingiva and bone suggest functional differences between cell types.

Monkey periodontal ligament fibroblasts (MPLF cells), human gingival fibroblasts (HGF cells), rat embryonic calvaria cells (REC cells), porcine periodontal ligament epithelial cells (PPLE cells) and rat osteosarcoma 17/2 cells (ROS cells) were incorporated into 3-dimensional collagen gels plated in 60 mm Petri dishes in order: first, to measure the capacity of these cell types to contract; second, to investigate cell-collagen and intercellular relationships during contraction; and third, to define the cellular contribution to tissue contraction in an in vitro system. Measurements at times up to 72 h on 3 ml gels containing 5 x 10(5) cells and with a collagen concentration of 1.20 mg/ml showed that MPLF cells contracted the gels at a significantly greater rate (P less than 0.001) than did the other cell types. In addition, contraction started sooner and was of greater extent than with the other cells. HGF cells contracted the gels more rapidly than REC and PPLE cells, while ROS cells caused no contraction. Several stages of gel compaction could be defined: (1) the attachment of cells to collagen; (2) cellular spreading within the collagen fibre matrix; (3) organization and alignment of collagen fibres by cell processes; (4) cell migration; (5) establishment of intercellular contacts; and (6) the development of a cellular reticular arrangement within the gel and the extension of this arrangement into a 3-dimensional, tissue-like, honeycomb network. Electron microscopic observations on 0.1 ml gels containing MPLF cells showed that, in the early contractile phase, numerous cell processes attached to or enclosed collagen fibrils. These processes contained microfilamentous material and few organelles. In compacted gels, the cells contained an increased amount of distended rough endoplasmic reticulum and Golgi membranes. Since MPLF cells have the capacity for vigorous contraction of the collagen gels and since they develop a reticular, 3-dimensional structure in compacted gels that is reminiscent of the relationship of periodontal ligament fibroblasts to collagen fibres in vivo, it is suggested that they could provide the major force necessary for tooth eruption in vivo. This system also provides a well-defined in vitro model to study the sequential stages that occur during contraction processes.