Unique histological changes in lung metastases of osteosarcoma patients following therapy with liposomal muramyl tripeptide (CGP 19835A lipid)

Summary We have recently begun a phase II trial in patients with osteosarcoma who developed pulmonary metastases during adjuvant chemotherapy or who presented with pulmonary metastases that persisted despite chemotherapy. Eligible patients were rendered free of visible disease by surgery. Liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (MTP-PE, CGP 19835A lipid) (2 mg/m²) was infused twice weekly for 3 months. In five patients, a single tumor nodule recurred within 6 weeks after completion of therapy. These lesions were resected and submitted for pathological examination. Tissue specimens obtained after therapy were compared to those obtained before therapy. All the patients showed a histological change in the characteristics of the pulmonary tumors. In three patients, peripheral fibrosis surrounded the tumor and inflammatory cell infiltration and neovascularization were present. This is in contrast to central necrosis, with viable peripheral tumor cells and no inflammatory response observed in lesions resected following chemotherapy. In a fourth case, evidence of early fibrotic changes was found. This and the fifth case showed a change in malignant characteristics, from high grade before liposomal therapy to low grade after therapy. The present study provides evidence for a biological effect of liposomal MTP-PE.     

Regulation of lipopolysaccharide-induced tumor necrosis factor production by cyclosporin A in mice primed with muramyl dipeptide

Abstract The effect of cyclosporin A (CsA) on tumor necrosis factor (TNF) or interleukin-6 (IL-6) production was evaluated in vivo in primed or unprimed mice challenged with lipopolysaccharide (LPS). Both pretreatment with BCG infection or with muramyl dipeptide (MDP) prior to LPS challenge resulted in an increase in the cytokine bioactivity level in the blood. CsA administration inhibited the TNF production. In unprimed mice, either normal or sensitized to LPS lethality by galactosamine treatment, a marked decrease in the cytokine level was observed after injection of CsA. After adrenalectomy, the yield of both TNF and IL-6 following LPS injection was markedly elevated but decreased by CsA administration. Ex vivo experiments have shown that the inhibitory effect of CsA could be demonstrated at the level of macrophages from mice previously given the drug. If mice had received MDP, in vitro responses of cells to LPS were enhanced but again CsA decreased the mRNA expression and protein secretion.     

Uptake of hyaluronan in hepatic metastases after blocking of liver endothelial cell receptors

To follow the biodistribution of exogenous hyaluronan in tumor-bearing animals, a total of seventeen inbred rats with hepatic metastases from a colonic adenocarcinoma received 125I-labelled hyaluronan by intravenous injections. Group I received only labeled hyaluronan (25 g), whereas group II received 2.5 mg chondroitin sulphate prior to labeled hyaluronan, to block receptor uptake in normal liver endothelial cells. Animals in group III received intravenous, as well as intraperitoneal chondroitin sulphate (2.5 mg), to see if a better and prolonged blocking could be achieved. Radioactivity was visualized by whole body autoradiography, using phosphorimaging and the average radioactivity determined as phosphoimaging density units of the total area of hepatic metastases, normal liver, and skeletal muscle by computer-based image analysis. At 5 h, tumors in groups II and III showed higher uptake (4.8 ±1.8, P = .01 and 3.6 ±1.1, P = .01, respectively), in comparison to group I (1.8 ±0.6), and the mean normal liver/tumor concentration ratio was reduced from 21.4 ±10.1 in group I to 5.7 ±2.7 in group II and 3.5 ±1.1 in group III (P = .008 and P = .01, respectively).Our study shows that hyaluronan targets liver metastases of a colon adenocarcinoma. Furthermore, chondroitin sulphate pretreatment increases tumor uptake, while uptake at normal receptor sites is significantly reduced. The results also suggest that after blocking of normal hyaluronan/chondroitin sulphate receptors in healthy tissue, hyaluronan may be used to deliver drugs to specific hyaluronan receptor-positive sites of pathology.     

The Italian multicentre dosimetric study for lesion dosimetry in 223Ra therapy of bone metastases: Calibration protocol of gamma cameras and patient eligibility criteria

PurposeThe aims of this work were to explore patient eligibility criteria for dosimetric studies in ²²³Ra therapy and evaluate the effects of differences in gamma camera calibration procedures into activity quantification.MethodsCalibrations with ²²³Ra were performed with four gamma cameras (3/8-inch crystal) acquiring planar static images with double-peak (82 and 154 keV, 20% wide) and MEGP collimator. The sensitivity was measured in air by varying activity, source-detector distance, and source diameter. Transmission curves were measured for attenuation/scatter correction with the pseudo-extrapolation number method, varying the experimental setup. ²²³Ra images of twenty-five patients (69 lesions) were acquired to study the lesions visibility. Univariate ROC analysis was performed considering visible/non visible lesions on ²²³Ra images as true positive/true negative group, and using as score value the lesion/soft tissue contrast ratio (CR) derived from ^99mTc-MDP WB scan.ResultsSensitivity was nearly constant varying activity and distance (maximum s.d. = 2%). Partial volume effects were negligible for object area ⩾960 mm². Transmission curve measurements are affected by experimental setup and source size, leading to activity quantification errors up to 20%. The ROC analysis yielded an AUC of 0.972 and an optimal threshold of CR of 10, corresponding to an accuracy of 92%.ConclusionThe minimum calibration protocol requires sensitivity and transmission curve measurements varying the object size, performing a careful procedure standardisation. Lesions with ^99mTc-MDP CR higher than 10, not overlapping the GI tract, are generally visible on ²²³Ra images acquired at 24 h after the administration, and possibly eligible for dosimetric studies.     

Radiation therapy for bone-only metastases in breast cancer patients: A GOCO survey of current clinical practice

IntroductionThe role of radiation therapy (RT) for patients with bone-only metastatic (BOM) breast cancer has not been investigated sufficiently. The aim of this survey was to evaluate current clinical practice in treating breast cancer patients with BOM in Radiation Therapy Departments in Catalonia and Occitania within the scope of the GOCO group.Materials and methodsAn electronic questionnaire was completed by experienced radiation oncologists from fourteen RT centers. The items surveyed the professional experience, therapeutic approach, technique, dose stereotactic body RT (SBRT) availability.ResultsAll Radiation Oncology Departments (ROD) in Catalonia (12) and Occitania (2) responded to the survey. Eleven (78.5%) of the RODs advise RT for BOM as initial treatment in the oligometastatic setting. RT to asymptomatic bone oligometastases is more often restricted for “risky lesions”. The most inconsistent approaches were the treatment for asymptomatic lesions, when to treat bone metastases with respect to systemic treatment (ST) and the indication for RT after a complete response to ST.ConclusionWhile BOM breast cancer patients have a relatively good prognosis, there is a lack of consistency in their approach with RT. This can be explained by the absence of evidence-based guidelines and an incomplete availability of SBRT.     

Peritoneal M2 macrophage transplantation as a potential cell therapy for enhancing renal repair in acute kidney injury

Acute kidney injury (AKI) is a clinical condition that is associated with high morbidity and mortality. Inflammation is reported to play a key role in AKI. Although the M2 macrophages exhibit antimicrobial and anti-inflammatory activities, their therapeutic potential has not been evaluated for AKI. This study aimed to investigate the protective effect of peritoneal M2 macrophage transplantation on AKI in mice. The macrophages were isolated from peritoneal dialysates of mice. The macrophages were induced to undergo M2 polarization using interleukin (IL)-4/IL-13. AKI was induced in mice by restoring the blood supply after bilateral renal artery occlusion for 30 minutes. The macrophages were injected into the renal cortex of mice. The changes in renal function, inflammation and tubular proliferation were measured. The M2 macrophages were co-cultured with the mouse primary proximal tubular epithelial cells (PTECs) under hypoxia/reoxygenation conditions in vitro. The PTEC apoptosis and proliferation were analysed. The peritoneal M2 macrophages effectively alleviated the renal injury and inflammatory response in mice with ischaemia-reperfusion injury (IRI) and promoted the PTEC proliferation in vivo and in vitro. These results indicated that the peritoneal M2 macrophages ameliorated AKI by decreasing inflammatory response and promoting PTEC proliferation. Hence, the peritoneal M2 macrophage transplantation can serve as a potential cell therapy for renal diseases.     

肝纤维化基因治疗的进展

肝硬化是慢性肝病晚期的组织学改变 ,以纤维组织大量增生和肝小叶结构无序化为特征 ,因此又称肝纤维化。近年来随着分子生物学的发展 ,肝纤维化的分子机制逐渐得以阐明 ,从而使肝纤维化的基因治疗成为可能。肝纤维化的基因治疗主要起到阻止纤维化发展、刺激肝细胞分裂和肝组织结构重建三方面的作用。目前 ,常用的方法一般是通过缺陷病毒 (如腺病毒 )转入特定的细胞因子和酶 (如肝细胞生长因子、转化生长因子β1受体、基质金属蛋白酶等 )的基因 ,通过靶细胞表达这些因子作用于受损的肝脏 ,达到延缓和治愈肝纤维化的目的     

Kupffer cells play important roles in the metabolic degradation of a soluble anti-tumor (1 → 3)-β-d-glucan, SSG, in mice

Abstract Metabolic degradation of a soluble highly branched (1 → 3)-β- d -glucan, SSG, was examined in mice using a macrophage blocker, gadolinium chloride (GdCl₃). Intraperitoneally administered SSG distributed in the liver was slowly degraded, and after 5 weeks about 30% of the SSG became anionic. In addition, it is suggested that the metabolites would contain fewer branching points as assessed by the reactivity to limulus factor G. On the other hand, in the spleen, the molecular weight and the degree of branching of SSG were not changed for at least 5 weeks. Blockade of Kupffer cells by GdCl₃ did not significantly change the distribution ratio of SSG in the liver. However, the treatment significantly delayed the degradation of SSG. These results suggested that Kupffer cells play important roles, not in the distribution, but in the oxidative degradation of SSG in the liver. In addition, splenic macrophages did not significantly contribute to the metabolic degradation of SSG.     

Clonal mesenchymal stem cells derived from human bone marrow can differentiate into hepatocyte‐like cells in injured livers of SCID mice

There is increasing evidence that human mesenchymal stem cells (hMSCs) can be a valuable, transplantable source of hepatocytes. Most of the hMSCs preparations used in these studies were likely heterogeneous cell populations, isolated by adherence to plastic surfaces or by density gradient centrifugation. Therefore, the participation of other unknown trace cell populations cannot be rigorously discounted. Here we report the isolation and establishment of a cloned human MSC line (chMSC) from human bone marrow primary culture, through which we confirmed the hepatic differentiation capability of authentic hMSCs. chMSCs expressed markers of mesenchymal cells, but not markers of hematopoietic stem cells. In vitro, chMSCs can differentiate into either mesenchymal cells or cells exhibiting hepatocyte‐like phenotypes. When transplanted intrasplentically into carbon tetrachloride‐injured livers of SCID mice, EGFP‐tagged chMSCs engrafted into the host liver parenchyma, exhibited typical hepatocyte morphology, form a three‐dimensional architecture, and differentiate into hepatocyte‐like cells expressing human albumin and α‐1‐anti‐trypsin. By confocal microscopy, ultrafine intercellular nanotubular structures were visible between adjacent transplanted and host hepatocytes. We postulate that these structures may assist in the phenotype conversion of chMSCs, possibly by exchange of cytoplasmic components between native hepatocytes and transplanted cells. Thus, a clonal pure population of hMSCs, which can be expanded in culture, may have potential as a cellular source for substitution damaged cells in hepatic injury. J. Cell. Biochem. 108: 693–704, 2009. © 2009 Wiley‐Liss, Inc.     

Ribozymes against TGFbeta1 reverse character of activated hepatic stellate cells in vitro and inhibit liver fibrosis in rats

BACKGROUND/AIMS: Transforming growth factor beta (TGFbeta1) is considered the key mediator in the process of liver fibrosis. The purpose of this investigation was to evaluate the activity of ribozymes against TGFbeta1 in a cell-free system and activated hepatic stellate cells (HSCs), and antifibrotic effect in activated HSCs in vitro and in rats. METHODS: Three ribozymes targeting against TGFbeta1 mRNA were designed, and then cloned into the U1 snRNA expression cassette. The chimeric ribozymes were selected for the analysis of their performances in activated HSCs through the detection of their cleavage activities in a cell-free system. After ribozyme-encoding plasmids had been transfected into HSC-T6 cells, the effects of ribozymes on activated HSCs were evaluated through the analysis of proliferation, activation and collagen deposition of HSC-T6. The adenoviral vector expressing the ribozymes was constructed, and then delivered into rat models of hepatic fibrosis induced by carbon tetrachloride. RESULTS: TGFbeta1 expression was efficiently down-regulated in activated HSCs by U1 snRNA chimeric ribozymes which possessed perfect cleavage activity in a cell-free system. Further studies demonstrated that U1 snRNA chimeric ribozymes inhibited the synthesis of collagen I, reduced deposition of collagen I, suppressed BrdU incorporation, but had no effect on desmin and alpha-SMA expression in transfected HSC-T6 cells. Histological analysis demonstrated that the adenoviral vector expressing ribozyme (Rz803) could alleviate fibrotic pathology in rats treated with carbon tetrachloride. CONCLUSIONS: The anti-TGFbeta1 ribozymes could reverse the character of activated HSCs in vitro and improve fibrotic pathology in vivo. It indicated that TGFbeta1 could be considered as a novel candidate for a therapeutic agent against hepatic fibrosis.     

Identification of ribonucleotide reductase M2 as a potential target for pro-senescence therapy in epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is the leading cause of gynecological-related cancer deaths in the United States. There is, therefore, an urgent need to develop novel therapeutic strategies for this devastating disease. Cellular senescence is a state of stable cell growth arrest that acts as an important tumor suppression mechanism. Ribonucleotide reductase M2 (RRM2) plays a key role in regulating the senescence-associated cell growth arrest by controlling biogenesis of 2'-deoxyribonucleoside 5′-triphosphates (dNTPs). The role of RRM2 in EOC remains poorly understood. Here we show that RRM2 is expressed at higher levels in EOCs compared with either normal ovarian surface epithelium (P < 0.001) or fallopian tube epithelium (P < 0.001). RRM2 expression significantly correlates with the expression of Ki67, a marker of cell proliferation (P < 0.001). Moreover, RRM2 expression positively correlates with tumor grade and stage, and high RRM2 expression independently predicts a shorter overall survival in EOC patients (P < 0.001). To delineate the functional role of RRM2 in EOC, we knocked down RRM2 expression in a panel of EOC cell lines. Knockdown of RRM2 expression inhibits the growth of human EOC cells. Mechanistically, RRM2 knockdown triggers cellular senescence in these cells. Notably, this correlates with the induction of the DNA damage response, a known mediator of cellular senescence. These data suggest that targeting RRM2 in EOCs by suppressing its activity is a novel pro-senescence therapeutic strategy that has the potential to improve survival of EOC patients.     

Augmenter of liver regeneration (ALR) gene therapy attenuates CCl4-induced liver injury and fibrosis in rats

Liver fibrosis represents a process of healing and scarring in response to chronic liver injury. Augmenter of liver regeneration (ALR) has been shown to protect hepatocytes from various toxins. The aim of this study was to investigate the effects of ALR gene therapy on liver injury and fibrosis induced by CCl₄ in rats and further explore the underlying mechanisms. Human ALR expression plasmid was delivered via the tail vein. ALR gene therapy might protect the liver from CCl₄-induced injury and fibrogenesis by attenuating the mitochondrial dysfunction, suppressing oxidative stress, and inhibiting activation of HSCs. This report demonstrated that ALR gene therapy protected against the ATP loss, increased the activity of ATPase, decreased intrahepatic reactive oxygen species level, and down-regulated transforming growth factor-β1, platelet-derived growth factor-BB, and α-smooth muscle actin expression. Following gene transfer liver function tests were significantly improved. In brief, ALR gene therapy might be an effective therapeutic reagent for liver fibrosis with potential clinical applications.     

Administration of fibroblast growth factor 2 in combination with bone marrow transplantation synergistically improves carbon-tetrachloride-induced liver fibrosis in mice

We previously reported that fibroblast growth factor 2 (FGF2) facilitated the differentiation of transplanted bone marrow cells (BMCs) into hepatocytes. Our earlier study also demonstrated that administration of FGF2 in combination with bone marrow transplantation (BMT) synergistically activated tumor necrosis factor-alpha signaling and significantly improved liver function and prognosis more than BMT alone. However, the way that it affected the extracellular matrix remained unclear. Here, we investigated the effect of FGF2 treatment together with BMT on liver fibrosis in mice treated with carbon tetrachloride (CCl₄). Transplantation of BMCs and concurrent treatment with FGF2 caused a statistically significant reduction in CCl₄-induced liver fibrosis that was accompanied by strong expression of matrix metalloproteinase 9 as compared with FGF2-only treatment or BMT alone. Moreover, in this process, the proliferation of bone-marrow-derived cells was accelerated without causing apoptosis. Thus, the administration of FGF2 in combination with BMT synergistically improves CCl₄-induced liver fibrosis in mice. This treatment has the potential of being an effective therapy for patients with liver cirrhosis. This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (nos. 16390211 and 16590597) and for translational research from the Ministry of Health, Labor and Welfare (H-trans-5 and H17-Special-015).     

Biological and biochemical characterization of M2B cells: Classical BSE prion is conserved in transgenic mice overexpressing bovine prion protein gene

M2B cells with persistent classical bovine spongiform encephalopathy (C-BSE) have been established previously. In this study, we performed strain characterization of the M2B cell line in bovine PrP^C overexpressing mice (Tg 1896). Mice intracranially inoculated with M2B cells and C-BSE survived for 451 ± 7 and 465 ± 31 d post inoculation, respectively. Although biochemical properties, including deglycosylation and conformational stability, differed between M2B cells and C-BSE, inoculation with M2B cell lysate and C-BSE resulted in comparable phenotypes. Comparable vacuolation scores and PrP^Sc depositions were observed in the brain of Tg 1896 inoculated with both M2B cell lysate and C-BSE. Our results show that biochemical and biological characteristics of M2B cells and C-BSE are classifiable in the same strain.     

Correlation of micro vessel density and c-Myc expression in breast tumor of mice following mesenchymal stem cell therapy

Stem cell therapy for degenerative diseases has been established; however there are controversies over the treatment of solid tumors with stem cell transplantation. In the present study, the anti-tumor action of mesenchymal stem cells (MSCs) has been examined in a mouse model of breast cancer with emphasize on tumor growth, angiogenesis and c-Myc expression in breast tumors. For this purpose, MSCs were isolated from bone marrow of Balb/c mice and characterized. A Balb/c mouse model of breast cancer was developed and subjected to cell therapy intra venous (I.V) or intra tumor (I.T) with MSCs. Tumor growth was measured using a digital caliber for until the end of experiment (30 days). Then the mice were sacrificed and their tumors were removed and processed for histopathological examination, immunohistochemical assay of CD31 and measuring of c-Myc expression using quantitative PCR. Detection of the labeled-MSCs in tumors following injection of the cells (I.V or I.T) clearly showed the homing of MSCs into tumors. Tumor growth in case of MSC-treated mice by I.V and I.T routes was inhibited by approximately 28% and 34% respectively compared to controls. The suppression of angiogenesis was reflected in Micro Vessel Density (MVD) following I.V or I.T delivery of the MSCs. c-Myc gene expression in tumor tissues of mice treated I.V or IT with MSCs was down-regulated to 28.0% and 16.0% respectively compare to control groups. In conclusion, growth inhibition of breast tumors in mice due to MSC therapy is associated with modulation of c-Myc activation and angiogenesis markers.     

Importance of methodology in the evaluation of renal mononuclear phagocytes and analysis of a model of experimental nephritis with Shp1 conditional knockout mice

Tissue resident mononuclear phagocytes (Mophs), comprising monocytes, macrophages, and dendritic cells (DCs), play important roles under physiological and pathological conditions. The presence of these cells in the kidney has been known for decades, and studies of renal Mophs (rMophs) are currently underway. Since no unified procedure has been identified to isolate rMophs, results of flow cytometric analysis of rMophs have been inconsistent among studies. We therefore first evaluated a preparative method for rMophs using collagenous digestion. The yield of rMophs greatly increased after the collagenase digestion. In particular, F4/80^high rMophs, which were positive for CD11c, a specific marker of DCs, dramatically increased. In addition, since neutrophils are sometimes mixed among rMophs in the analysis of flow cytometry, we established a gating strategy for eliminating neutrophils. To determine the contribution of rMophs to the development of autoimmune nephritis, we analyzed an experimental model of autoimmune nephritis that was applied to Shp1 conditional knockout mice (Shp1 CKO). This knockout strain is generated by crossing a mouse line carrying floxed Shp1 allele to mice expressing Cre recombinase under the control of the CD11c promoter. Shp1 CKO therefore specifically lack Shp1 in cells expressing CD11c. As a result, Shp1 CKO were susceptible to that experimental glomerulonephritis and F4/80^high rMophs of Shp1 CKO increased dramatically. In conclusion, our preparative methods for collagenase digestion and gating strategy for neutrophils are necessary for the analysis of rMophs, and Shp1 suppresses the development of autoimmune nephritis through the control of rMophs.