Mouse Ascites Research Articles

Peroxisome Proliferator-Activated Receptor (PPAR)γ Can Inhibit Chronic Renal Allograft Damage

Chronic inflammation and fibrosis are the leading causes of chronic allograft failure. The nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma is a transcription factor known to have antidiabetogenic and immune effects, and PPARgamma forms obligate heterodimers with the retinoid X receptor (RXR). We have reported that a retinoic acid (RAR)/RXR-agonist can potently influence the course of renal chronic allograft dysfunction. In this study, in a Fischer to Lewis rat renal transplantation model, administration of the PPARgamma-agonist, rosiglitazone, independent of dose (3 or 30 mg/kgBW/day), lowered serum creatinine, albuminuria, and chronic allograft damage with a chronic vascular damage score as follows: 35.0 +/- 5.8 (controls) vs. 8.1 +/- 2.4 (low dose-Rosi; P < 0.05); chronic tubulointerstitial damage score: 13.6 +/- 1.8 (controls) vs. 2.6 +/- 0.4 (low dose-Rosi; P < 0.01). The deposition of extracellular matrix proteins (collagen, fibronectin, decorin) was strikingly lower. The expression of transforming growth factor-beta1 was inhibited, whereas that of bone morphogenic protein-7 (BMP-7) was increased. Intragraft mononuclear cells and activated fibroblast numbers were reduced by 50%. In addition, the migratory and proliferative activity of these cells was significantly inhibited in vitro. PPARgamma activation diminished the number of cells expressing the proinflammatory and fibrogenic proteoglycan biglycan. In macrophages its secretion was blocked by rosiglitazone in a predominantly PPARgamma-dependent manner. The combination of PPARgamma- and RAR/RXR-agonists resulted in additive effects in the inhibition of fibrosis. In summary, PPARgamma activation was potently immunosuppressive and antifibrotic in kidney allografts, and these effects were enhanced by a RAR/RXR-agonist.

Abstract LB-256: Catena: A novel cellular entity composed of ovarian cancer stem cells

Abstract Metastasis of cancer cells into the peritoneal cavity is a clinical manifestation of advanced-stage ovarian cancer and is associated with poor survival. In an orthotopic NOD/SCIDIL2R −/− (NSG) model of advanced ovarian serous adenocarcinoma, we have identified a novel cellular structure consisting of a chain of cells coated with an extensive pericellular glycocalyx, free-floating in the ascites of mice following intra-peritoneal engraftment with ovarian cancer cell lines (Ovcar3, Ovcar5, A2780) or primary ovarian cancer cells. We have termed these chains “catenae“ (Latin for “chains”). Ovcar3 has epithelial characteristics and expresses E-cadherin. In the inflammatory ascites of NSG mice undergoes an “epithelial to mesenchymal transition (EMT)” with E-cadherin loss, followed by a “mesenchymal to catena transition (MCT)”. Ovcar5 and A2780 are mesenchymal and do not require in vivo passage to undergo MCT and catena formation was readily obtained under our selective culture conditions. By transmission and scanning electron microscopy catenae are undifferentiated with high nuclear to cytoplasmic ratio and have amoeboid features with extensive membrane blebbing and abundant microvilli, yet retain epithelial-type ZO-1+ tight junctional connections. Time-lapse microscopy showed that a catena develops from a single cell by up to 7 rapid (16 hrs) symmetric divisions. We can expand catenae in vitro (∼10,000 fold in 7-9 days) for prolonged periods with the majority of cells retaining CSC function as determined by single cell recloning and limiting dilution engraftment in NSG mice. As few as 2 cells when injected with matrigel produce subcutaneous tumors within 1-2 weeks with the morphology of serous papillary ovarian adenocarcinoma. To our knowledge, this is the first report to show isolation of a homogeneous population of CSC, that can be expanded extensively in vitro without differentiation, yet retain the capacity to fully differentiate to all cellular components of the original tumor. At high cell density, catenae develop into spheroids by a process of “rolling up”. Spheroids contain ∼10% CSC while epithelial tumor monolayers have &amp;lt;1% CSC. Time-lapse studies showed that spheroids extrude fresh catenae. Microarray and massive parallel RNA-sequencing revealed a CSC gene signature with hyaluronan synthase-2 (HAS2), hyaluronan and proteoglycan link protein 1 (HAPLN1) and PDGFRA the top 3 genes differentially upregulated genes. Catenae produce PDGFC and respond to autocrine signaling via PDGFRA. HAS2 and HAPLN1 are responsible for generating the hyaluronan component of the protective glycocalyx. Treatment of catena with hyaluronidase removes the glycocalyx resulting in reversal of the MCT and EMT and loss of CSC capacity. We have adapted the catena and spheroid system for high-throughput screening for identification of compounds that specifically inhibit CSC by inducing apoptosis, proliferation inhibition or differentiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-256.

Abstract 2926: The synthetic triterpenoid CDDO-methyl ester and the rexinoid LG100268 extend survival in the KPC transgenic mouse model of pancreatic cancer

Abstract Pancreatic cancer is the fourth leading cause of cancer-related deaths in the U.S. and is almost always fatal. While prevention offers the most promising approach for reducing the mortality of this disease, there is still a need to develop effective drugs for the prevention and treatment of pancreatic cancer. We have previously reported that the triterpenoid CDDO-methyl ester (CDDO-Me) and the rexinoid LG100268 (268) can prevent and treat experimental lung and breast cancer in rodents. These drugs also inhibit proliferation and induce apoptosis in human pancreatic cancer cells but have not been tested in vivo against pancreatic cancer. Hingorani et al have developed the autochthonous LSL-KrasG12D/+;LSL-Trp53R127H/+;Pdx-1-Cre (KPC) mouse model of pancreatic cancer. These KPC transgenic mice closely replicate the genetic mutations, clinical symptoms, and histopathology found in human pancreatic cancer. We have generated pancreatic cancer cell lines from a solid pancreatic tumor and from ascites of KPC mice. The triterpenoid CDDO-Me directly interacts with both STAT3 and IKK in these cells. As a result, CDDO-Me (0.3-1 μM) decreases constitutive IL-6 secretion, inhibits constitutive and IL-6 inducible STAT3 phosphorylation, and blocks the degradation of IKBα in cells challenged with TNFα. CDDO-Me also inhibits proliferation and induces apoptosis of these pancreatic cancer cell lines. Although the rexinoid 268 has little effect on epithelial cells in vitro, it regulates macrophages and other immune cells that drive tumorigenesis in the KPC mice. In order to test these drugs in vivo, KPC mice were fed powdered control diet or diet containing CDDO-Me (50 mg/kg diet), 268 (30 mg/kg diet) or the combination, beginning at 4 weeks of age until the mice displayed overt symptoms (cachexia, abdmonial distension) of pancreatic cancer. CDDO-Me, 268, and the combination (n = 25 per group) all significantly (P &amp;lt; 0.05) increased survival compared to the controls (n = 81), although only by 3-4 weeks (control 20.1 wks, CDDO-Me 23.5 wks, 268 23 wks, and CDDO-Me + 268 24.1 wks). Notably, Olive et al have recently shown that gemcitabine, the current standard of care for human pancreatic cancer even though it is only marginally effective, does not extend survival in KPC mice. These results suggest that drugs with promising in vitro effects on human pancreatic cell lines or in xenograft models should be evaluated in the challenging, yet highly relevant, KPC model before entering clinical trials and that additional drugs and drug combinations need to be developed for pancreatic cancer. Supported by the Sidney Kimmel Foundation for Cancer Research, NIH grant RO1 CA101207 and Reata Pharmaceuticals, Inc. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2926.

Characterization of metal chelate methacrylate monolithic disk for purification of polyclonal and monoclonal immunoglobulin G

Dynamic binding capacity (DBC) of commercial metal-chelate methacrylate monolith-convective interaction media (CIM) was performed with commercial human immunoglobulin G (IgG) (Cohn fraction II, III). Monoliths are an attractive stationary phase for purification of large biomolecules because they exhibit very low back pressure even at high flow rates and flow-unaffected binding properties. Adsorption of IgG onto CIM-IDA disk immobilized with Cu 2+, Ni 2+ and Zn 2+ were studied with Tris-acetate (TA), phosphate-acetate (PA) and MMA (MES, MOPS and acetate) buffer systems at different flow rates. Adsorption and elution of IgG varied with different buffers and adsorption of IgG was maximum with MMA buffer. Adsorption of human IgG from Cohn fractions (II, III) was high when Cu 2+ was used as ligand. CIM-IDA disk showed dynamic binding capacity in the range of 14–16 mg/ml with Cu 2+ and 7–9 mg/ml with Ni 2+ for human IgG with MMA buffer. In the case of CIM-IDA-Zn 2+ column, the binding capacity was only about 0.5 mg/ml of support. Different desorption strategies like lowering of pH and increasing of competitive agent were also studied to achieve maximum recovery. Chromatographic runs with human serum and mouse ascites fluid were also carried out with metal chelate methacrylate monolithic disk and the results indicate the potential of this technique for polyclonal human IgG and monoclonal IgG purification from complex biological samples.

Cancer-induced Immunostimulatory CD11b+Gr-1+ cells (100.30)

Abstract Both innate and adaptive immune systems are considered equally important for cancer prevention, immunosurveillance, and control of cancer progression. It is known that while both systems initially eliminate emerging tumor cells efficiently, tumors eventually escape immune attack by a variety of mechanisms including differentiation and recruitment of immunosuppressive CD11b+Gr-1+ myeloid suppressor cells into the tumor microenvironment. However, we show here that CD11b+Gr-1+ cells found in ascites of epithelial ovarian cancer-bearing mice at advanced stages of disease are immunostimulatory rather than being immunosuppressive. These cells consist of a homogenous population of cells that morphologically resemble neutrophils. Moreover, like dendritic cells, immunostimulatory CD11b+Gr-1+ cells can strongly cross-prime, augmenting the proliferation of functional CTLs via signaling through the expression of costimulatory molecule CD80. Adoptive transfer of these immunostimulatory CD11b+Gr-1+ cells from ascites of ovarian cancer-bearing mice results in the significant regression of subcutaneous tumors even without being pulsed with exogenous tumor antigen prior to adoptive transfer. We now show for the first time that adaptive immune responses against cancer can be augmented by these cancer-induced granulocyte-like immunostimulatory myeloid (CD11b+Gr-1+) cells, thereby mediating very effective anti-tumor immunity.

Inhibitory effect of Ad-hTERTp-HSV-TK/GCV system on mice ascites production planted by liver cancer cell line

Objective To observe the effect of Ad-bTERTp-HSV-TK/GCV system on malignant ascites of mice and probe into its mechanism of action.Methods The SX1 inbred strain mice were injected with H22 cell line of liver cancer and were divided into 4 groups at random.The mice in each group were given corresponding treatment after 48 hours.The production of ascites and survival period were evaluated. The apoptosis rates of tumor cells were detected by FCM.Morphological changes of tumor cells were studied by electromicroscope.Results Compared with other groups.Ad-hTERTp-HSV-TK/GCV Can obviously inhibit the production of ascites(P<0.01),prolong the survival period (P<0.01),and apoptosis rate in this group (27.12±2.12)% was significantly higher than that in other groups.No obvious side effect Was found during the treatment.Conclusion Ad-hTERTp-HSV-TK/GCV system Can inhibit production of ascites and prolong the survical period of mice by inducing apoptosis of hepatoma cells,which is a safe and feasible treatment for hepatoma therapy. Key words: Liver neoplasms,experimental; Gene therapy; Kinase adenosine; Adenoviruses.human; Ascites

Hospicells (ascites‐derived stromal cells) promote tumorigenicity and angiogenesis

The microenvironment is known to play a dominant role in cancer progression. Cells closely associated with tumoral cells, named hospicells, have been recently isolated from the ascites of ovarian cancer patients. Whilst these cells present no specific markers from known cell lineages, they do share some homology with bone marrow-derived or adipose tissue-derived human mesenchymal stem cells (CD9, CD10, CD29, CD146, CD166, HLA-1). We studied the role of hospicells in ovarian carcinoma progression. In vitro, these cells had no effect on the growth of human ovarian carcinoma cell lines OVCAR-3, SKOV-1 and IGROV-1. In vivo, their co-injection with adenocarcinoma cells enhanced tumor growth whatever the tumor model used (subcutaneous and intraperitoneally established xenografts in athymic mice). In addition, their injection increased the development of ascites in tumor-bearing mice. Fluorescent macroscopy revealed an association between hospicells and ovarian adenocarcinoma cells within the tumor mass. Tumors obtained by coinjection of hospicells and human ovarian adenocarcinoma cells presented an increased microvascularization indicating that the hospicells could promote tumorigenicity of ovarian tumor cells in vivovia their action on angiogenesis. This effect on angiogenesis could be attributed to the increased HIF1alpha and VEGF expression associated with the presence of the hospicells. Collectively, these data indicate a role for these ascite-derived stromal cells in promoting tumor growth by increasing angiogenesis.

Ovarian Tumor-Induced T Cell Suppression Is Alleviated by Vascular Leukocyte Depletion

The ovarian cancer microenvironment recruits an array of immune cells to the site of tumor growth. Within the peritoneal ascites of both humans and mice, the predominant population of tumor-infiltrating leukocytes is a CD11c+CD11b+ population variably referred to as vascular leukocytes (VLCs), tumor-associated macrophages, and immature dendritic cells. We have previously shown that these cells are critical for tumor growth because their selective elimination from the peritoneal tumor microenvironment inhibited tumor progression. However, the underlying mechanism by which this therapy was efficacious is poorly understood. Here, we use the murine ID8 ovarian tumor model to demonstrate that the tumor microenvironment induces in vivo immunosuppression of T cells and that the SR-A+ VLCs mediate this suppression. Importantly, the elimination of SR-A+ VLCs from the peritoneum of tumor-bearing mice relieves the T cell suppression. Moreover, the profound changes that VLC elimination has on the immune system are T cell-dependent because the protective antitumor effect of VLC elimination does not occur when CD8 T cells are concomitantly depleted. These results were confirmed and extended with the use of a genetic model for VLC depletion, which demonstrated that short-term therapeutic depletion of VLCs alleviates immunosuppression and allows for efficacious vaccination against model tumor antigens in tumor-bearing mice. These studies provide a mechanistic explanation for how leukocytes contribute to ovarian tumor progression and, correspondingly, how leukocyte depletion inhibits tumor growth.

Small interfering RNA targeting MDR1 inhibits ovarian cancer growth and increases efficacy of chemotherapy in vivo

To further validate a knockdown approach for circumventing the multidrug resistance gene (MDR1), we used small interfering RNA(siRNA) targeting MDR1 gene to inhibit the expression of MDR1 gene and P-glycoprotein(P-gp) in vivo. Ascite tumor xenografts were established by implanting human ovarian carcinoma cells SKOV3/AR intraperitoneally into the nude mice. The mice were randomized into the following three treatment groups with each group six mice respectively: Taxol, Taxol with lipofectamine and Taxol with siRNA/MDR1-lipofectamine intraperitoneal injection. The tumor growth rate and the ascite growth rate of mice were investigated. The expressions of MDR1 gene and P-gp in mice were determined by reverse transcription-polymerase chain reaction(RT-PCR) and immunohistochemistry respctively. The growth of tumors and ascites in mice treated with Taxol and siRNA/MDR1-lipofectamine was significantly inhibited compared with those in mice of other groups. After 28 days’ treatment, the average tumor weight and ascite volume decreased by 43.6% and 29.7% in the group treated with Taxol and siRNA/MDR1-lipofectamine compared with these treated with Taxol alone (P<0.001). The expressions of MDR1 gene and P-gp in the group treated with Taxol and siRNA/MDR1-lipofectamine were also decreased compared with those in the group treated with Taxol alone (P<0.001). Small interfering RNA targeting-MDR1 can effectively and specifically suppress the expression of MDR1(P-glycoprotein) and inhibit ovarian cancer growth in vivo.

Antitumour Activity of Methanolic Extract of Plumeria alba L. Leaves Against Dalton Lymphoma Ascites in Mice

Purpose: To evaluate Plumeria alba leaves for antitumor activity against Ehrlich ascites carcinoma (EAC) and Dalton lymphoma ascites (DLA) bearing Swiss albino mice. Method: The antitumour activity of the methanolic extract of Plumeria alba leaves (MPA) was evaluated against EAC and DLA using in-vitro cytotoxic and mean survival time, a decrease in the tumour volume and viable cell count in the DLA tumour hosts. The animal was observed for improvement in the haematological parameters (e.g., heamoglobin content, red and white blood cells count, and differential cell count) following MPA treatment of the tumour bearing mice. Results: MPA was found to be cytotoxic in the invitro model. Intraperitoneal administration of MPA increased the survival time, dead cell count haematological parameters and solid tumour mass was also significantly reduced. Conclusion: MPA possesses significant antitumour activity. Keywords: Antitumour, cytotoxicity Plumeria alba L.

Targeting the mammalian target of Rapamycin to inhibit VEGF and cytokines for the treatment of primary effusion lymphoma

Primary effusion lymphoma (PEL) is a fatal malignancy, which typically presents as a lymphomatous effusion that later disseminates. Rapamycin (Rapa), which targets mTOR (mammalian target of Rapa), is currently evaluated as a treatment for PEL, but the recent development of PEL in Rapa-treated post-transplant recipients questions the drug's use in PEL. Here, we used a murine model of PEL effusion that mimics the human disease to investigate the anti-PEL activity of Rapa. We found that Rapa reduces ascites accumulation and extends mouse survival. Initially, Rapa reduced PEL load compared to control mice, but most mice rapidly showed PEL progression. Levels of VEGF, which promotes vascular permeability contributing to effusion formation, were significantly reduced in ascites of Rapa-treated mice compared to controls. Expression of IL-10, the principal autocrine growth factor for PEL, was initially reduced in PEL from Rapa-treated mice but rapidly increased despite treatment. We found that the hypoxic environment of ascites and Rapa cooperate in stimulating IL-10 expression in PEL, which likely contributes to the emergence of drug resistance. These results identify Rapa an effective drug to reduce PEL effusions but illustrate the rapid development of drug resistance, which likely limits the efficacy of Rapa in PEL.

Rapamycin reduces primary effusion lymphoma progression by targeting VEGF production and VEGF responses

Kaposi's sarcoma herpesvirus (KSHV)-infected primary effusion lymphoma (PEL) typically presents as a malignant effusion in body cavities that disseminates to distant sites. There is a need for new therapies for PEL as most patients rapidly succumb in spite of high-dose chemotherapy. We examined the potential utility of Rapamycin for PEL treatment. We found that the downstream effectors of the mammalian target of Rapamycin (mTOR) p70S6k and S6 are constitutively phosphorylated in PEL cells, and that Rapamycin inhibits this constitutive p70S6k and S6 phosphorylation. Rapamycin reduces PEL proliferation but is not cytotoxic for PEL cells. Intraperitoneal injection of PEL cells in NOD/SCID mice causes experimental effusion lymphoma. Rapamycin delayed PEL development in this model, markedly reduced accumulation of ascites (p = 0.009), prevented formation of solid tumor masses, and a significantly extended mouse survival (P < 0.001). However, Rapamycin did not eradicate PEL in mice. We examined the mechanisms by which Rapamycin reduces PEL progression in this mouse model. Levels of VEGF, which promotes vascular permeability and is critical to the accumulation of body cavity fluids, were significantly reduced in ascites of Rapamycin-treated mice compared to controls (p = 0.009). Rapamycin inhibited VEGF-induced phosphorylation of VEGF receptor (R) 2 in endothelial cells and activation of the downstream effectors of VEGFR2 phosphorylation src and enos (endothelial nitric oxide synthase). Rapamycin did not alter KSHV genes transcription in PEL cells, and only insignificantly reduced levels of IL-10, the principal growth factor for PEL, in ascites of PEL-bearing mice. Reduction of VEGF secretion by PEL and impairment of endothelial cell responses to residual VEGF likely explain reduced accumulation of ascites in Rapamycin-treated mice. The failure of Rapamycin to significantly reduce IL-10 levels in PEL-bearing mice and to promote PEL cell death likely explain PEL persistence in mice treated with Rapamycin. The successful use of Rapamycin to reduce PEL effusion and disease progression by reducing VEGF secretion and endothelial cell responses to VEGF illustrates a novel application of mTOR inhibition that targets the tumor microenvironment rather than the tumor cells, and is applicable to the treatment of PEL and other malignancies characterized by ascites accumulation and increased vascular permeability.

Experimental elimination and recovery of double minute chromosomes in malignant cell populations.

The influence of the environment on the frequency of cells with double minute chromosomes (dms) was studied in the SEWA mouse ascites tumor. Under normal in vivo conditions, this tumor contained about 90%., cells with one or more dms. Explantation in vitro resulted in a decrease of the dms carrying cells, which after 100 days only amounted to 5% of the population. Following reimplantation in vivo. the original proportion of these cells was restored after an interval of 170 days. A mathematical model is presented describing these changes as selective phenomena. In the stock tumor almost all minute chromosomes were C-band negative. In the experimental populations. both in vivo and in vitro, another type of minute chromosomes appeared which were C-band positive. Also by other morphologic and functional criteria they were shown to be small chromosomes and not dms. In the present paper they have been referred to as microchromosomes. The fact that dms have been found only in malignant cells, and the correlation established in the present experiments between presence of dms and in vivo environment, led to speculations concerning their function. One possibility is that they involve amplification of genic material of significance to tumor development.

Have double minutes functioning centromeres?

Normally, double minute (DM) chromosomes are extremely difficult to observe at other mitotic stages than prophase and metaphase. In a certain clonal subline of the mouse ascites tumor SEWA, however, the DMs were unusually distinct and could be followed through all mitotic stages. Their behavior was extraordinary: During metaphase-anaphase they exhibited no response whatever towards the spindle forces, and it was concluded that the DMs were without functioning centromere. Instead, at metaphase they were attached to or enclosed by the nucleolar matter persisting around the chromosome ends, and at anaphase they were transported to the poles together with the nucleolar matter, sticking to the daughter chromosomes. The daughter halves of each DM were usually carried to the same pole. Thus most of the DMs, in spite of their lack of detectable centromeric activity, were included into the telophase nuclei. Some micronuclei were found, however, probably leading to the loss of a proportion of the DMs. The content of DMs in a certain tumor cell population therefore must depend on the balance between this loss and the gain due to a positive selection value of cells containing DMs.

A hexaploid Ehrlich-Lettré mouse ascites tumor line with low sensitivity to colchicine.

A hexaploid mouse ascites tumor, ELH, derivative of the hyperdiploid Ehrlich-Lettre tumor (ELD), has been studied from different view-points. It originated as an in vivo implant of the hyper-triploid in vitro strain ELDA, which in its turn developed from an in vivo strain of the original in vitro explant in 1964 of the ELD Lund strain. Chromosome number distribution and behavior of different marker chromosomes were followed during the prehistory of ELH and through serial in vivo and in vitro transplantation. Its C- and G-band patterns were analyzed in passages 264 in vivo and 170 in vitro, and its response to colchicine, in passages 105–115, in vivo. ELH was demonstrated to be about 10 times more resistant than ordinary hyperdiploid ELD strains both to the c-mitotic action and to the general toxicity of colchicine.

Chromosomal evolution in the Ehrlich-Lettré complex of hyperdiploid mouse ascites tumors: results from seven laboratory strains.

The chromosomes of 7 strains of the hyperdiploid Ehrlich-Lettre mouse ascites tumor (ELD) were analyzed by conventional stainings and by G- and C-bandings. A tendency to slow decrease in chromosome number during a 20-year period was revealed in the 7 strains and in the ELD complex in general. A G-band idiogram of the normal mouse chromosomes was elaborated with numbering of regions and bands. By G-band analysis the following average composition of the ELD karyotypes was indicated: unchanged mouse chromosomes (30%,), marA, marB, minutes (11%), mar1—mar12, telocentric markers (10%), anonymous telocentric markers, often strongly rearranged (50%). Great differences were revealed among normal chromosomes and markers in their capacity to persist in the tumor stemline as structurally stable units. The presence of some unchanged chromosomes in most cells of all 7 strains indicated that they involved selective advantage. Evidently chromosomal evolution has operated by preferably maintaining certain elements unchanged and by submitting other elements to heavy rearrangement. The C-band analysis showed that, in addition to the centromeric C-heterochromatin present in all chromosomes, some had interstitial C-bands, as marA in all strains, mar5 and mar7 in specific single strains, and others in numbers varying among the strains.

Large double minutes with ring-shape and rod-shape

High incidence of large double minutes (DM) was characteristic of one subline of the SEWA mouse ascites tumor. This subline, SEWA1R, was the one in which the new C-bandless chromosomes (CM) appeared. The incidence of large DM was associated with low incidence of DM per cell, low incidence of DM-carrying cells, and high incidence of CM-carrying cells. The large DM were either round or elongated (rod-shaped). Many of the former kind were suggestive of ring-shape and a few of the largest obviously constituted pairs of rings, free or interlocked. One configuration of 4 interlocked rings was evidently in its second mitotic generation; in the same cell another configuration of 2 pairs of interlocked rings had developed from 1 pair of free rings in the preceding generation. Possible relations between CM and large DM are considered.

Phosphorylation of the Consensus Sites of Protein Kinase A on α1D L-type Calcium Channel

The novel alpha(1D) L-type Ca(2+) channel is expressed in supraventricular tissue and has been implicated in the pacemaker activity of the heart and in atrial fibrillation. We recently demonstrated that PKA activation led to increased alpha(1D) Ca(2+) channel activity in tsA201 cells by phosphorylation of the channel protein. Here we sought to identify the phosphorylated PKA consensus sites on the alpha(1) subunit of the alpha(1D) Ca(2+) channel by generating GST fusion proteins of the intracellular loops, N terminus, proximal and distal C termini of the alpha(1) subunit of alpha(1D) Ca(2+) channel. An in vitro PKA kinase assay was performed for the GST fusion proteins, and their phosphorylation was assessed by Western blotting using either anti-PKA substrate or anti-phosphoserine antibodies. Western blotting showed that the N terminus and C terminus were phosphorylated. Serines 1743 and 1816, two PKA consensus sites, were phosphorylated by PKA and identified by mass spectrometry. Site directed mutagenesis and patch clamp studies revealed that serines 1743 and 1816 were major functional PKA consensus sites. Altogether, biochemical and functional data revealed that serines 1743 and 1816 are major functional PKA consensus sites on the alpha(1) subunit of alpha(1D) Ca(2+) channel. These novel findings provide new insights into the autonomic regulation of the alpha(1D) Ca(2+) channel in the heart.

Depletion of CD8+ T cells enhances airway remodelling in a rodent model of asthma.

Airway remodelling is induced by persistent airway inflammation and may lead to severe asthma. T cells play a pivotal role in asthmatic airway inflammation but their role in remodelling is poorly understood. Although previous studies have revealed that CD8(+) T cells inhibit the late airway response and airway inflammation in a rat model of asthma, their effects on airway remodelling have not been evaluated. The aim of this study was to examine the role of CD8(+) T cells in airway remodelling. Brown Norway rats were sensitized with ovalbumin (OVA) on day 0. CD8(+) T cells in rats were depleted during the repeated challenges by treating them with a CD8alpha monoclonal antibody (OX-8). Control rats were treated with mouse ascites. Sensitized rats were challenged with OVA on days 14, 19 and 24 or were sham challenged with phosphate-buffered saline. On day 29, bronchoalveolar lavage and lung tissues were harvested. Repeated OVA inhalations evoked significant increases in the numbers of periodic acid-Schiff-positive epithelial cells and proliferating cell nuclear antigen-positive epithelial cells, and in airway smooth muscle mass compared to the control group. CD8-depleted rats had significant enhancement of these changes, principally affecting the large airways. These results suggest that endogenous CD8(+) T cells have inhibitory effects on airway remodelling in this model of asthma.

Myxoma viral serpin, Serp-1, inhibits human monocyte adhesion through regulation of actin-binding protein filamin B

Serp-1 is a secreted myxoma viral serine protease inhibitor (serpin) with proven, highly effective, anti-inflammatory defensive activity during host cell infection, as well as potent immunomodulatory activity in a wide range of animal disease models. Serp-1 binds urokinase-type plasminogen activator (uPA) and the tissue-type PA, plasmin, and factor Xa, requiring uPA receptor (uPAR) for anti-inflammatory activity. To define Serp-1-mediated effects on inflammatory cell activation, we examined the association of Serp-1 with monocytes and T cells, effects on cellular migration, and the role of uPAR-linked integrins and actin-binding proteins in Serp-1 cellular responses. Our results show that Serp-1 associates directly with activated monocytes and T lymphocytes, in part through interaction with uPAR (P<0.001). Serp-1, but not mammalian serpin PA inhibitor-1 (PAI-1), attenuated cellular adhesion to the extracellular matrix. Serp-1 and PAI-1 reduced human monocyte and T cell adhesion (P<0.001) and migration across endothelial monolayers in vitro (P<0.001) and into mouse ascites in vivo (P<0.001). Serp-1 and an inactive Serp-1 mutant Serp-1(SAA) bound equally to human monocytes and T cells, but a highly proinflammatory mutant, Serp-1(Ala(6)), bound less well to monocytes. Serp-1 treatment of monocytes increased expression of filamin B actin-binding protein and reduced CD18 (beta-integrin) expression (P<0.001) in a uPAR-dependent response. Filamin colocalized and co-immunoprecipitated with uPAR, and short interference RNA knock-down of filamin blocked Serp-1 inhibition of monocyte adhesion. We report here that the highly potent, anti-inflammatory activity of Serp-1 is mediated through modification of uPAR-linked beta-integrin and filamin in monocytes, identifying this interaction as a central regulatory axis for inflammation.