Pathological grade is a useful prognostic factor for stratifying breast cancer patients into favorable  and less favorable  outcome groups. Under the current system of tumor grading, however, a large proportion of tumors are characterized as intermediate-grade, making determination of optimal treatments difficult. In an effort to increase objectivity in the pathological assessment of tumor grade, differences in chromosomal alterations and gene expression patterns have been characterized in low-grade, intermediate-grade, and high-grade disease. In this review, we outline molecular data supporting a linear model of progression from low-grade to high-grade carcinomas, as well as contradicting genetic data suggesting that low-grade and high-grade tumors develop independently. While debate regarding specific pathways of development continues, molecular data suggest that intermediate-grade tumors do not comprise an independent disease subtype, but represent clinical and molecular hybrids between low-grade and high-grade tumors. Finally, we discuss the clinical implications associated with different pathways of development, including a new clinical test to assign grade and guide treatment options. Breast cancer is the most common cancer in women worldwide, and in the United States (US) is estimated to account for ~26% of all new female cancer cases and 15% of all cancer deaths among women.® likely reflect heterogeneity in pathological factors such as estrogen receptor (ER) and HER2 status. Breast carcinomas are heterogeneous at the molecular level, with at least five disease categories identified through differential patterns of gene expression.Pathological assessment of breast cancer is currently based on criteria such as tumor size, lymph node and hormone receptor status, and epidermal growth factor receptor 2 (HER2) expression, but pathology alone does not accurately predict outcomes, even for patients with similar tumor characteristics. Recent studies suggest that, despite use of identical treatment modalities in patients with similar pathological characteristics, clinical outcomes can be highly variable.In this review, we examine use of the Nottingham histological score in assigning grade to breast carcinomas and the clinical utility of the Nottingham score in determining patient risk and outcome. We outline our understanding of how genomic alterations contribute to histological characteristics that define tumor grade and the importance of molecular changes in shaping tumor growth and differentiation in patients with breast cancer. An important focus of this review is the ongoing debate over development of high-grade and low-grade breast disease, specifically on whether low-grade and high-grade breast carcinomas represent separate and distinct diseases. We present molecular evidence supporting a linear model of progression from low-grade to high-grade carcinomas, as well as contradicting genetic data suggesting that low-grade and high-grade tumors develop independently. While debate regarding specific pathways of development continues, molecular data suggest that intermediate-grade tumors do not comprise an independent disease subtype, but represent clinical and molecular hybrids between low-grade and high-grade tumors. Finally, we discuss the clinical implications of different pathways of development, including a new clinical test to assign grade and guide treatment options.The Nottingham combined histological grading system, based on classification parameters developed by Bloom and RichardsonThe Nottingham grading system has clinical utility in determining patient risk and outcome—patients with low-grade carcinomas have ~95% five-year survival compared to just 50% in patients with high-grade disease.14in situ carcinoma, and finally invasive malignancy.Breast cancer progression can be defined by a non-obligatory sequence of histological changes from normal epithelium through atypical hyperplasia, in situ hybridization (FISH) demonstrated that low-grade and high-grade disease were associated with different types of chromosomal alterations in the 16q region.Since the initial models of disease progression were published, a number of studies examining levels and patterns of genomic variation in breast carcinomas have supported the hypothesis that low-grade and high-grade tumors represent separate genetic diseases, based largely on observations that the frequency of alterations at chromosome 16q was significantly higher in low-grade tumors. An allelic imbalance (AI) analysis using three microsatellite markers on chromosome 16q detected a significantly higher frequency of AI events in low-grade (grade 1) compared to high-grade (grade 3) tumors for two of the three markers.in situ (DCIS) lesions further supports a model of two distinct pathways of breast disease development. Higher levels of chromosomal alterations have been detected via CGH in high-grade compared to low-grade DCIS, with loss of 16q found almost exclusively in low-grade lesions.in situ and invasive disease suggest that low-grade and high-grade invasive breast tumors evolve directly from low-grade and high-grade DCIS, respectively  and unfavorable (= 3) scores for each component, significantly higher levels of AI were observed in samples with unfavorable (high) scores for all components.in situ from invasive disease. In contrast, high-grade (poorly-differentiated) invasive tumors did not show significantly higher levels of AI than grade 3 DCIS, but AI events at specific chromosomal regions (1p36 and 11q23) were significantly more frequent in high-grade invasive tumors compared to high-grade DCIS.in situ lesions compared to low-grade invasive carcinomas may reflect the protracted time-to-progression associated with low-grade DCIS. Likewise, increased levels of AI at 1p36 and 11q23 in high-grade carcinomas suggest that these chromosomal regions may harbor genes associated with invasiveness. Therefore, consideration of histological grade when analyzing genetic data has the potential to identify molecular changes associated with invasion and to define molecular signatures of aggressive behavior for low-grade and high-grade disease.The identification of genomic signatures for low-grade and high-grade breast disease provides new insights into the heterogeneity of breast cancer. Under current models of disease progression, low-grade and high-grade breast carcinomas develop independently along different genetic pathways, thus consideration of breast disease without regard to tumor grade may mask molecular  factors specific to one grade.Stratification of low-grade and high-grade breast carcinomas into separate molecular diseases is based on the high frequency of alterations observed for chromosome 16 in low-grade tumors and a low frequency of 16q alterations in high-grade tumors. To localize genes involved in low-grade IDCA, and to refine regions of chromosome 16 that may be important to the development of high-grade disease, Roylance et al characterized 40 low-grade (grade 1) and 17 high-grade (grade 3) IDCA using CGH with nearly contiguous coverage of chromosome 16q.in situ lesions by random accumulation of genetic mutations. These studies hypothesize that low-grade and high-grade breast carcinomas are not necessarily unique genetic diseases. Under this model, cells with the most aggressive/poorly-differentiated characteristics tend to become the dominant cell type during progression from low-grade to high-grade carcinomas.The Nottingham grading system, used to assign histological grade to invasive carcinomas, does not adequately describe internal variation in the degree of differentiation within tumors. Although most pathologists rely on nuclear grade, either alone or in combination with central necrosis, to classify DCIS, one recent attempt to quantify histological diversity in 120 pure DCIS lesions found that ~46% of cases showed localized variability in histological grade. Nearly one-third of lesions with internal grade differences demonstrated further diversity for a panel of immunohistochemistry markers including ER, GATA-binding protein 3 (GATA3), and HER2.Molecular and pathological changes have been associated with low-grade and high-grade breast carcinomas, which represent the extremes of histological differentiation. Conversely, development of a model for intermediate-grade breast tumors presents a particular challenge because intermediate-grade carcinomas contain a blend of histological features common to both low-grade (well-differentiated) and high-grade (poorly-differentiated) tumors. Because carcinomas with intermediate-grade histology represent 30%–60% of all invasive breast cancers, improved understanding of the genetics of these tumors is critical in determining the optimum course of treatment for the large group of patients with intermediate pathological features.32Patterns of genetic changes usually do not differ significantly between intermediate-grade and high-grade carcinomas, supporting the idea that intermediate-grade invasive breast tumors develop from either grade 2 or grade 3 DCIS. Further studies of genomic alterations in breast tumors of different histological grades have shown that although genetic changes were more frequent in grade 3 tumors, alterations of one specific chromosomal region (16q) were significantly lower (P < 0.01) in high-grade (26%) compared to intermediate-grade (54%) tumors.In our ongoing studies of intermediate-grade breast carcinomas, we observed that clinicopathological characteristics and overall levels of genomic alterations in grade 2 tumors were generally intermediate compared to low-grade and high-grade disease.Gene expression analysis has been widely used to identify genetic profiles associated with different stages of breast cancer development. Using laser microdissection to isolate pure populations of tumor cells and prevent cross-contamination from stroma or co-occurring lesions, histological grade rather than pathological stage, was found to correlate with significantly different patterns of gene expression.35Recognizing the inherent subjectivity in assigning histological grade and the need to better characterize intermediate-grade tumors, researchers have begun to analyze combined gene expression data sets from primary breast tumor samples derived from multiple sources. These approaches have led to the development of a gene expression grade index (GGI), based on 97 genes, which summarizes molecular differences between low-grade and high-grade breast tumors.Molecular data (DNA and RNA) suggest that intermediate-grade invasive breast cancer is not a discrete disease, but represents a blend between low-grade and high-grade tumors. However, whether poorly-differentiated tumors arise from well-differentiated carcinomas, or whether low-grade and high-grade tumors develop along independent genetic pathways remains unclear. Although multiple studies have identified significant differences in gene expression between low-grade and high-grade disease,Determining relationships among tumors of different histological grades has important clinical implications for estimating risk and defining treatment options in patients with breast disease. For example, atypical ductal hyperplasia (ADH) specimens typically share a gene expression profile with grade 1 disease and tend to cluster with low-grade DCIS and well-differentiated invasive cancer.Development of molecular signatures that closely correlate with histological differentiation may improve the assessment of tumor grade. At present, debate continues within the pathology community over the best way to assign histological grade. Some studies suggest that a two-tiered grading system comprised of nuclear pleomorphism and mitotic counts is superior to the current tripartite system that includes tubule formation.typeDX™  and MammaPrint®  are now being used more frequently as clinical tools to determine treatment for certain groups of patients. For example, the OncotypeDX™ can be used to make decisions about chemotherapy after surgery for women with node-negative, ER-positive breast cancer. In 2008, Ipsogen (http://www.ipsogen.com/) developed the MapQuant Dx™ Genomic Grade test based on the GGI discussed above.32Finally, molecular profiles such as the OncoMolecular characterization of breast tumors at both the DNA and RNA levels suggests that intermediate-grade carcinomas do not represent an independent disease subtype, but instead share clinical and molecular features of low-grade and high-grade tumors. In contrast, debate continues as to whether poorly-differentiated (high-grade) tumors evolve from well-differentiated (low-grade) tumors or whether low-grade and high-grade carcinomas represent discrete diseases that develop along separate genetic pathways. While efforts continue to improve our understanding of biological factors influencing the development of low-, intermediate-, and high-grade tumors, clinical uses of molecular assays are providing new ways to assign histological grade and guide treatments for patients with breast cancer.


Dietary fat has been posited as another potential risk factor for breast cancer, possibly acting through the estrogen pathway. A new animal study suggests a high-fat diet may alter estrogen metabolism, thereby modifying the effects of maternal exposure to TCDD and increasing mammary cancer risk in the next generation .Some human and animal studies have linked early-life exposure to the endocrine-disrupting chemical 2,3,7,8-tetrachlorodibenzo-a]anthracene (DMBA) at days 35, 49, and 63 after birth in order to initiate mammary tumors. A second cohort of female offspring was treated identically until either day 35 or 49, when morphologic and molecular analyses of their mammary glands were performed.One group of pregnant female FVB/NJ mice (a TCDD-responsive mouse strain) received an olive oil/toluene blend with TCDD; another received an equivalent volume of olive oil/toluene without TCDD. Their female offspring were randomly assigned to either a low-fat or high-fat diet and exposed to the carcinogen 7,12-dimethyl-benz[in utero and fed the low-fat diet. Whereas one-third of TCDD-unexposed litters fed a high-fat diet had DMBA-induced mammary lesions, every litter exposed to both TCDD and a high-fat diet developed mammary lesions.Maternal TCDD exposure was associated with a doubling of mammary tumor incidence only in offspring fed the high-fat diet. In contrast, no mammary tumors arose in mice exposed to TCDD Previous animal studies had shown that prenatal exposure to TCDD alters mammary gland differentiation and increases susceptibility to mammary cancer. However, this is the first to show that TCDD may interact with a high-fat diet during pregnancy and early life. The researchers propose that a high-fat diet may boost sensitivity to maternal TCDD exposure by altering estrogen metabolism.in utero combined with a high-fat diet was also associated with increased expression of Cyp1b1 and decreased expression of Comt in mammary tissue. Human studies have suggested that diminished COMT expression and increased CYP1B1 expression are associated with increased risk of breast cancer and other estrogen-responsive cancers, perhaps by increasing levels of estrogen metabolites that can contribute to carcinogenesis by damaging DNA. They also note that obesity may affect breast cancer risk because TCDD persists in adipose tissue, including that in the breast.The new findings highlight a possible mechanism that may explain epidemiologic data separately linking early-life TCDD exposure and high-fat diets to increased breast cancer risk in humans. In the present study, TCDD exposure


Thallium-201 single-photon emission computerized tomography (SPECT) was used to clarify the relationship between 201Tl uptake and the response in chemotherapy to platinum compounds in 21 patients with small-cell lung cancer. 201Tl-SPECT scans were obtained twice: at 15 min (early scan) and 120 min (delayed scan) after an intravenous injection of 111 MBq (3 mCi) of thallium-201 chloride. We obtained the uptake ratio from each scan and calculated the retention index:uptake ratio = region of interest uptake/contralateral normal lung uptake; retention index = (delayed ratio - early ratio)/early ratio. After 201Tl scintigraphy, 12 patients received chemotherapy consisting of platinum compounds and nine were treated with chemoradiation. Among patients receiving only chemotherapy, the retention index correlated with the responses to chemotherapy. In an in vitro study, ouabain, an inhibitor of the Na,K-ATPase pump, reduced sensitivity to cisplatin and inhibited intracellular thallium uptake in the small-cell lung cancer cell line. These studies suggest that 201Tl-SPECT is a useful indicator of response to chemotherapy with platinum compounds in small-cell lung cancer, and that Na,K-ATPase is commonly involved in transporting both thallium and platinum compounds into cancer cells.


To test the hypothesis that OxPAPC may attenuate lung inflammation and barrier disruption caused by pathologic lung distension, we used a rodent model of ventilator-induced lung injury and an in vitro model of pulmonary endothelial cells exposed to pathologic mechanochemical stimulation.Mechanical ventilation at high tidal volume (HTV) may cause pulmonary capillary leakage and acute lung inflammation resulting in ventilator-induced lung injury. Besides blunting the Toll-like receptor-4-induced inflammatory cascade and lung dysfunction in a model of lipopolysaccharide-induced lung injury, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) exerts direct barrier-protective effects on pulmonary endothelial cells In vitro, the effects of OxPAPC on endothelial barrier dysfunction and GTPase activation were assessed in cells exposed to thrombin and pathologic (18%) cyclic stretch.Rats received a single intravenous injection of OxPAPC (1.5 mg/kg) followed by mechanical ventilation at low tidal volume (LTV) (7 mL/kg) or HTV (20 mL/kg). Bronchoalveolar lavage was performed and lung tissue was stained for histological analysis. In vitro, high-magnitude stretch enhanced thrombin-induced endothelial paracellular gap formation associated with Rho activation. These effects were dramatically attenuated by OxPAPC and were associated with OxPAPC-induced activation of Rac.HTV induced profound increases in bronchoalveolar lavage and tissue neutrophils and in lavage protein. Intravenous OxPAPC markedly attenuated HTV-induced protein and inflammatory cell accumulation in bronchoalveolar lavage fluid and lung tissue. OxPAPC exhibits protective effects in these models of ventilator-induced lung injury. Mechag injury . However studies . Thus, Ain vivo protective effect of OxPAPC may be associated, in part, with its direct effects on the vascular endothelial barrier.Cell-membrane phospholipids and phospholipids present in circulating lipoproteins may undergo oxidation by lipoxygenases or reactive oxygen and nitrogen species as a result of VILI, trauma, or septic inflammation -13. One Previously, we described potent Rac-dependent barrier-protective effects of oxidized phospholipids on cultured pulmonary endothelial cells (ECs) and identified the critical role of cyclopentenone-containing oxidized modifications of arachidonoyl moiety and polar head groups (choline and serine) in the mediation of the OxPAPC effects ,22. Our In this study, we used rodent models of VILI and pulmonary ECs exposed to physiologic and pathologic levels of cyclic stretch (CS) and thrombin stimulation to test the hypotheses that vascular leak caused by mechanical ventilation at HTVs involves the Rho pathway of endothelial barrier dysfunction and that OxPAPC may attenuate Rho activation induced by VILI-associated pathologic mechanochemical stimulation via Rac-dependent mechanisms. Selected parts of this study were presented at the American Thoracic Society International Conference in San Diego, California, 20 to 25 May 2006.-7 mol/mouse) followed by HTV or LTV . Control animals were anesthetized and allowed to breathe spontaneously. At sacrifice, BAL of both lungs was performed with 1 mL of sterile Hanks' balanced salt solution for measurement of inflammatory cells and protein. All animal experiments were approved by the Institutional Animal Care and Use Committee at Johns Hopkins University and the University of Chicago. The animals were housed in pathogen-free conditions in the Johns Hopkins Asthma and Allergy Center and the University of Chicago Animal Care Facilities, where they were cared for in accordance with institutional and National Institutes of Health  guidelines.Adult male Brown Norway rats (250 to 350 g)  or adult male C57BL/6J mice (8 to 10 weeks old with an average weight of 20 to 25 g)  were anesthetized with an intraperitoneal injection of ketamine (75 mg/kg) and acepromazine (1.5 mg/kg). All rat studies were performed using 2-hour mechanical ventilation. Tracheotomy was performed and the trachea was cannulated with a 14-guage intravenous catheter, which was tied into place to prevent air leak. Rats were assigned to either HTV (20 mL/kg) or LTV (7 mL/kg) mechanical ventilation at 85 breaths per minute and 0 positive end-expiratory pressure (PEEP) for 2 hours. Arterial blood pressure and pH were monitored via a carotid artery catheter at 30-minute intervals. External dead space in the HTV group allowed the maintenance of blood pH of 7.30 to 7.44. Intravenous fluid boluses of phosphate-buffered saline (PBS) were given to maintain a mean arterial pressure of greater than 65 mm Hg. Rats were randomly assigned to concurrently receive an intravenous bolus of sterile PBS or OxPAPC (1.5 mg/kg) via the jugular vein at the initiation of mechanical ventilation. At the end of each experiment, rats were killed by exsanguination under anesthesia, and BAL was performed on the left lung using 3 mL of sterile PBS. BAL inflammatory cell counting was performed using a standard hemacytometer technique. Differential cell counts were performed on Diff-Quick-stained  slides with a minimum of 300 cells per slide. The BAL protein concentration was determined by a modified Lowry colorimetric assay using a Bio-Rad DC protein assay kit . In subsequent experiments, mechanical ventilation of mice was performed for 4 hours as we  have preAt sacrifice, the lungs were harvested without lavage collection and fixed in 4% paraformaldehyde. After fixation, the lungs were embedded in paraffin, cut into 4-μm sections, and stained with hematoxylin and eosin. Sections were evaluated at × 400 magnification.Measurement of Evans blue accumulation in the lung tissue was performed by spectrofluorimetric analysis of lung tissue lysates according to the protocol described previously .PAPC was obtained from Sigma-Aldrich  and oxidized by exposure of dry lipid to air for 72 hours. The extent of oxidation was monitored by positive-ion electrospray mass spectrometry as described previously ,21,28. H5 cells per well) onto collagen I-coated flexible-bottom BioFlex plates. After 48 hours of culture, each plate received fresh medium, was mounted onto the Flexercell system, and was exposed for 2 hours to either low-magnitude (5% elongation) or high-magnitude (18% elongation) CS to recapitulate the mechanical stresses experienced by the alveolar endothelium during normal respiration and HTV mechanical ventilation, respectively [All CS experiments were performed using an FX-4000T Flexercell Tension Plus system  equipped with a 25-mm BioFlex Loading Station as previously described ,30. Expeectively ,31,32. Aectively ,33.Rho and Rac activation assays were performed using commercially available assay kits purchased from Upstate Biotechnology  as we have previously described ,33.The cellular barrier properties were analyzed by measurement of transendothelial electrical resistance across confluent human pulmonary artery and human lung microvascular endothelial monolayers using an electrical cell-substrate impedance sensing system  as previously described ,33,34.After exposure to CS and agonist stimulation, ECs were subjected to immunofluorescence staining to visualize actin filaments as previously described ,33.in vivo data are presented as mean ± standard deviation. Group comparisons were evaluated by the analysis of variance test with post hoc Newman-Keuls multiple comparison test. P values of less than 0.05 were considered statistically significant.All in vivo and demonstrated that at these doses OxPAPC alone did not change total cell count, neutrophil count, or protein content in the BAL of uninjured control animals [4 ± 1.79 versus 5.83 × 104 ± 0.72 cells per milliliter in LTV controls)  and BAL PMNs (1.57 ± 0.32 × 104 versus 3.15 ± 0.86 × 103 cells per milliliter in HTV). Statistical analysis of BAL macrophages . Because PAPC is subject to in vivo oxidation to OxPAPC, analyses of BAL protein and cell count were performed in an additional series of experiments in a mouse model of VILI using the oxidation-resistant PAPC analog, DMPC. DMPC had no effect on either BAL cell count or protein in control animals and did not protect against HTV-induced cell and protein accumulation in the BAL  compared with control rats exposed to 'protective' LTV mechanical ventilation (7 mL/kg) -37. In o mL/kg co) Figure . This ef) Figure , bottom,s Figure , top shos Figure . T test Histological analysis of paraffin-embedded rat lung sections stained with hematoxylin and eosin revealed parenchymal inflammatory cell recruitment (neutrophils noted with arrows) and areas of alveolar hemorrhage indicative of vascular disruption with HTV ventilation that was attenuated with OxPAPC Figure . QuantitThe protective effects of OxPAPC against vascular leak were further assessed by measurement of Evans blue leakage into the lung tissue. HTV induced noticeable Evans blue leakage from the vascular space into the lung parenchyma, which was significantly decreased by OxPAPC pretreatment Figure . ImportaIn the following experiments, we investigated a role of Rho-dependent signaling in lung injury induced by mechanical ventilation. Pharmacologic inhibition of Rho-associated kinase by Y-27632 markedly attenuated HTV-induced increases in lung BAL cell count and protein content in our murine VILI model Figure , suggestin vivo and in vitro, increased thrombin levels may become a considerable factor contributing to the Rho-mediated vascular endothelial barrier dysfunction caused by HTV mechanical ventilation. Because the in vivo use of thrombin is limited due to significant intravascular thrombosis, we performed additional experiments using thrombin-derived non-thrombogenic PAR-1 (protease-activated receptor-1) receptor ligand TRAP-6 in our murine VILI model. Mice were given a single dose of intravenous TRAP-6 (3 × 10-7 mol/mouse) followed by 4 hours of HTV mechanical ventilation. Measurements of BAL protein concentration and cell count revealed that TRAP-6 exacerbated HTV-induced lung dysfunction, inducing a 36% ± 6.7% increase in BAL inflammatory cells and a 62% ± 9.2% increase in BAL protein compared with animals treated only with HTV. Notably, OxPAPC, but not its oxidation-resistant analog DMPC (data not shown), significantly reduced these parameters of lung injury in TRAP-6-treated animals  known to activate Rho signaling [Thrombin stimulation of pulmonary ECs exposed to pathologically relevant levels of CS ignaling ,39-42. Uignaling . Human pignaling . Consistignaling , thrombiBecause the site of transvascular flux in the lungs is the microvasculature, and phenotypic differences between macro- and microvascular endothelium are well recognized, we next used human lung microvascular ECs to further characterize the effects of oxidized phospholipids. Our results demonstrate that, as with human pulmonary artery ECs (HPAECs), OxPAPC increased baseline transendothelial electrical resistance in human lung microvascular ECs in a dose-dependent manner Figure . Furtherin vitro at time points corresponding to the acute phase of thrombin-induced barrier disruption (5 to 15 minutes) and the recovery phase (50 minutes) based on our previous studies [The small GTPases Rho and Rac play opposing roles in the regulation of EC permeability ,43,44. T studies . PulmonaWe have previously shown that thrombin stimulation causes a decrease in basal Rac activity, which correlates with reciprocal Rho activation and Rho-dependent paracellular gap formation during the acute phase (15 minutes) of thrombin-induced EC barrier dysfunction. Subsequent increases in Rac activation were seen at later time points and corresponded to EC barrier recovery ,23. In tin vivo model of VILI, we show here for the first time that a single intravenous dose of OxPAPC significantly attenuates the early vascular barrier disruption and acute inflammation induced by mechanical ventilation at HTV. Intravenous OxPAPC significantly reduced alveolar and tissue inflammatory cell recruitment and protein accumulation in the BAL after 2 hours of mechanical ventilation at HTV.Vascular barrier dysfunction and acute lung inflammation are fundamental features that contribute to the significant mortality associated with VILI and ARDS. Despite advances in protective LTV ventilation strategies, effective pharmacotherapy for this devastating syndrome is lacking. Using an aseptic ETF, E2F, Nrf1, CREB, and HIF1) is not found in the list of genes upregulated by LPS [ISRE, cREL, IRF, NF-κ B, ICSBP, and PU.1) detected in the lung tissue are not upregulated by mechanical ventilation [In our previous study, we described similar protective effects of OxPAPC in an animal model of LPS-induced lung injury . In thatd by LPS . In turntilation ,50.in vitro model of pulmonary ECs preconditioned at a pathologically relevant level of CS (18% CS) [in vitro.Clinical studies suggest that the vascular leak observed in VILI patients is caused by a combination of lung mechanical strain and increased levels of edemagenic and inflammatory mediators such as thrombin, histamine, tumor necrosis factor-alpha, and interleukin (IL)-8 and IL-1 -54. Seve(18% CS) ,31,32 anin vitro model of VILI, we demonstrated the protective effects of OxPAPC on barrier disruption induced by high-magnitude (18%) CS and thrombin and linked them with OxPAPC-induced reduction in Rho activity and modest increases in Rac activity. Furthermore, the combination of physiologically relevant CS (5% CS) and OxPAPC dramatically attenuated thrombin-induced Rho activation in the acute phase of EC barrier disruption and further promoted Rac activation associated with EC recovery after thrombin challenge.Previous studies have shown that the EC barrier dysfunction induced by pathologic CS and thrombin is mediated by Rho-dependent mechanisms, whereas restoration of monolayer integrity is dependent on Rac activation . Furtherin vitro and in vivo results presented here, as well as our previously published results [in vivo model of VILI induced by HTV and TRAP-6 and data from pulmonary ECs exposed to pathologic CS and thrombin in vitro show potent barrier-protective effects of OxPAPC in the two-hit models of ALI. Taken together, these results strongly suggest that OxPAPC does not act as a specific anti-thrombin but rather promotes endothelial barrier function via regulation of Rac/Rho signaling leading to acceleration of EC monolayer recovery and enhancement of peripheral cytoskeleton and cell-cell junctions)[in vitro and in vivo studies, we believe that intravascular OxPAPC can act directly on the vascular endothelial layer and cause cytoskeletal changes that serve to counteract a variety of injurious insults that lead to barrier disruption.The  results -22, strounctions)-58. BaseStudies by several groups clearly suggest reciprocal relations between Rho and Rac activation -62 and oSeveral potential mechanisms may be involved in the barrier-protective effects of and suppression of Rho signaling by OxPAPC. OxPAPC stimulates protein kinase A ,66, whicin vivo oxidant stress, and their plasma levels inversely correlate with outcome in patients with ARDS [sn-2-oxygenated, but not sn-2-fragmented, phospholipids (PEIPC and PECPC) are responsible for the OxPAPC-mediated Rac/Cdc42 activation, cytoskeletal remodeling, and induction of barrier-protective effects in the vascular endothelium [sn-2-oxygenated products by precisely monitored oxidation of synthetic phospholipids or direct synthesis of lead compounds will be a promising but challenging task in generating a novel group of phospholipid-derived compounds combining anti-inflammatory and barrier-protective properties. In conclusion, this study demonstrates for the first time the protective effect of OxPAPC in the in vivo and in vitro models of VILI. Although further studies are needed to clarify molecular mechanisms of OxPAPC barrier-protective effects, these findings suggest that oxidized phospholipids and OxPAPC, in particular, may be considered as a new group of therapeutic candidates in ALI/VILI, combining anti-inflammatory and barrier-enhancing properties in the treatment of this devastating disease.There is an apparent controversy between the protective effects of OxPAPC described in this study and the role of oxidant stress, lipid peroxidation, and tissue damage in patients with ARDS and ALI. Isoprostanes, prostanoid compounds primarily formed by non-enzymatic lipid peroxidation, have been used as markers of ith ARDS . Patholoith ARDS ,72. Analith ARDS ,73-75. Iothelium . Thus, sin vivo and in vitro models of VILI, we demonstrated for the first time the protective effect of oxidized phospholipids against early vascular barrier disruption and acute inflammation induced by mechanical ventilation at HTV. In contrast to the direct effects of OxPAPC on inhibition of the TLR-4-mediated inflammatory cascade in the model of LPS-induced lung inflammation and vascular dysfunction, we attribute the protective effects of OxPAPC on the lung vascular endothelium in the aseptic VILI model to the OxPAPC-induced activation of Rac signaling and reduction of Rho-induced endothelial hyperpermeability. These findings suggest that oxidized phospholipids may be considered as a new group of therapeutic candidates in ALI/VILI, combining anti-inflammatory and barrier-enhancing properties in the treatment of this devastating syndrome.The results presented here, as well as our previously published data -22,58, s• Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) significantly attenuates vascular leak, neutrophil accumulation, and Evans blue extravasation into the lung parenchyma caused by mechanical ventilation at high tidal volume and thrombin-derived signaling peptide TRAP-6.• Protective effects of OxPAPC against VILI (ventilator-induced lung injury)-associated lung vascular leak have been reproduced in the pulmonary endothelial cell monolayers exposed to high-magnitude cyclic stretch and thrombin stimulation.• Protection of endothelial monolayer integrity was due to OxPAPC-mediated attenuation of Rho pathway of endothelial barrier dysfunction.ALI = acute lung injury; ARDS = acute respiratory distress syndrome; BAL = bronchoalveolar lavage; CS = cyclic stretch; DMPC = di-myristoyl-sn-glycero-3-phosphorylcholine; EC = endothelial cell; GEF = guanosine nucleotide exchange factor; HPAEC = human pulmonary artery endothelial cell; HPF = high power microscopic field; HTV = high tidal volume; IL = interleukin; LPS = lipopolysaccharide; LTV = low tidal volume; NF-κB = nuclear factor-kappa-B; OxPAPC = oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine; PAPC = 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine; PBS = phosphate-buffered saline; PEEP = positive end-expiratory pressure; PMN = polymorphonuclear leukocyte; RhoGDI = Rho GDP dissociation inhibitor; TLR = Toll-like receptor; TRAP-6 = thrombin receptor activating peptide-6; VILI = ventilator-induced lung injury.The authors declare that they have no competing interests.in vivo studies. KGB initiated, reviewed, and coordinated the study and funded in vivo and in vitro experiments. All authors read and approved the final manuscript.SN performed animal experiments, analyzed results, and was involved in manuscript preparation. PF and JX performed animal experiments. AAB performed immunofluorescence analysis and was involved in the discussion of the results. SC performed biochemical studies. VNB and NL synthesized the OxPAPC and participated in the manuscript discussion. JGNG funded


Non-malignant dermal fibroblast strains, cultured from affected members of a Li-Fraumeni syndrome (LFS) family with diverse neoplasms associated with radiation exposure, display a unique increased resistance to the lethal effects of gamma-radiation. In the studies reported here, this radioresistance (RR) trait has been found to correlate strongly with an abnormal pattern of post-gamma-ray DNA replicative synthesis, as monitored by radiolabelled thymidine incorporation and S-phase cell autoradiography. In particular, the time interval between the gamma-ray-induced shutdown of DNA synthesis and its subsequent recovery was greater in all four RR strains examined and the post-recovery replication rate was much higher and was maintained longer than in normal and spousal controls. Alkaline sucrose sedimentation profiles of pulse-labelled cellular DNA indicated that the unusual pattern of DNA replication in irradiated RR strains may be ascribed to anomalies in both replicon initiation and DNA chain elongation processes. Moreover, the RR strain which had previously displayed the highest post-gamma-ray clonogenic survival was found to harbour a somatic (codon 234) mutation (presumably acquired during culture in vitro) in the same conserved region of the p53 tumour-suppressor gene as the germline (codon 245) mutation in the remaining three RR strains from other family members, thus coupling the RR phenotype and abnormal post-gamma-ray DNA synthesis pattern with faulty p53 expression. Significantly, these two aberrant radioresponse end points, along with documented anomalies in c-myc and c-raf-1 proto-oncogenes, are unprecedented among other LFS families carrying p53 germline mutations. We thus speculate that this peculiar cancer-prone family may possess in its germ line a second, as yet unidentified, genetic defect in addition to the p53 mutation.


Recent studies have shown that bafilomycin A1-sensitive vacuolar-type H(+)-ATPase (V-ATPase) plays important roles in cell growth and differentiation. However, there is no published study that has focused on the expression of V-ATPase in human tumour tissues. This study was designed to examine the mRNA and protein levels for the 16 kilodalton (kDa) proteolipid of V-ATPase in human pancreatic carcinoma tissues. We first investigated the mRNA level for V-ATPase in six cases of invasive pancreatic cancers and two normal pancreases, using reverse transcription-polymerase chain reaction technique. Then, we examined immunohistochemically the level of V-ATPase protein in 49 pancreatic cancers and ten benign cystic neoplasms of the pancreas, using antisera raised against the 16 kDa proteolipid. There was a notable difference in the level of V-ATPase mRNA between normal and pancreatic carcinoma tissues, with no evident difference in the expression of the beta-actin gene. Immunohistochemically, 42 out of 46 invasive ductal cancers (92%) displayed a mild to marked immunoreactivity for V-ATPase in the cytoplasm, whereas neither non-invasive ductal cancers nor benign cystic neoplasms expressed detectable immunoreactive proteins. These findings suggest that the overexpression of V-ATPase protein is characteristic of invasive pancreatic tumours. V-ATPase may play some crucial roles in tumour progression.


Three weeks after DEN treatment, Fas-mediated liver cell apoptosis induced by anti-Fas antibody resulted in a biphasic effect on induction of liver cell tumours, depending on dosage and time of antibody administration. Single or multiple treatment with high dose anti-Fas antibody , induced gross liver cell damage and decreased the incidence of liver cell tumours in DEN-treated mice. In contrast, five treatments with low dose anti-Fas antibody , induced dispersed localized liver cell damage and promoted the number of large-sized liver cell adenomas and hepatocellular carcinomas. These findings suggest that high dose anti-Fas antibody has a marked effect on the clearance of DEN-initiated liver cells, whereas repeated administration of low dose anti-Fas antibody promotes hepatocarcinogenesis. It is concluded that Fas-mediated liver cell apoptosis has a biphasic effect on hepatocarcinogenesis. © 2000 Cancer Research CampaignThe present study was designed to investigate the effect of Fas-mediated liver cell apoptosis, induced by a hamster monoclonal antibody against mouse Fas antigen, on diethylnitrosamine (DEN)-induced hepatocarcinogenesis in mice. DEN (10 μg g


Drosophila melanogaster, a pre-mRNA splicing hierarchy controls sexual identity and ultimately leads to sex-specific Doublesex (DSX) transcription factor isoforms. The male-specific DSXM represses genes involved in female development and activates genes involved in male development. Spatial and temporal control of dsx during embryogenesis is not well documented.In M is specifically expressed in subsets of male somatic gonad cells during embryogenesis. Following testis formation, germ cells remain in contact with DSXM-expressing cells, including hub cells and premeiotic somatic cyst cells that surround germ cells during spermatogenesis in larval and adult testes.Here we show that DSXdsx is transcriptionally regulated in addition to being regulated at the pre-mRNA splicing level by the sex determination hierarchy. The dsx locus is spatially controlled by somatic gonad identity. The continuous expression of DSXM in cells contacting the germline suggests an ongoing short-range influence of the somatic sex determination pathway on germ cell development.We show that  Drosophila, including somatic gonad formation [Sxl and transformer (tra) pre-mRNA such that functional SXL and TRA proteins are produced only in females. Presence of TRA and the constitutive product of transformer-2 (tra2) in females lead to female-specific splicing of the doublesex (dsx) pre-mRNA, which then gives rise to DSXF protein. In X:AA flies, the absence of SXL, and thus TRA, results in male-specific splicing of dsx pre-mRNA. Male-specific dsx mRNA encodes DSXM protein. Both DSXF and DSXM are zinc-finger transcription factors of the DMRT family. Members of this family play important roles in sex determination in most animals that have been examined to date [A regulatory cascade directs all aspects of somatic sexual differentiation in ormation -3. This  to date .Drosophila DSX proteins possess identical N-terminal DNA-binding domains but differ in their C-termini [M is thought to repress genes that are involved in female development and activate male differentiation genes while DSXF is thought to do the opposite [The -termini -7. DSXM opposite -11. Althopposite ,12,13.dsx locus plays a critical role in both somatic gonad development [M have testes but very few germ cells. These germ cells can show evidence of either male or female development. However, dsx is not required within the germline cells, suggesting that the role of dsx in germline development is non-autonomous [dsx transcripts in larvae and adults [dsx in both somatic gonad and germline development, very little is known about when and where DSX is expressed during gonadogenesis.The elopment  and specelopment ,15. Flietonomous . Thus, Dd adults . DespiteDrosophila is initiated in the embryo [Gonad development in e embryo . Germ cee embryo -20. Addie embryo . Followie embryo , whereasdsx mRNA is expressed in the embryo. We have developed an antibody that detected the DSXM isoform and show that, in contrast to SXL, which is expressed uniformly throughout the embryo [M expression was restricted to the initial somatic cells that form the somatic gonad in male embryos. Furthermore, DSXM is expressed in the male-specific somatic gonadal precursors that are later recruited to the gonad and are maintained in male embryos. DSXM is not detected outside the gonad, in the germ cells, or in the late arriving somatic cells that surround the embryonic testis. Male germ cells are in direct contact with DSXM-expressing somatic cells through adulthood, as DSXM is specifically expressed in the two somatic cyst cells surrounding developing germline cysts during pre-meiotic spermatogenesis. Only differentiating sperm appear to be unaccompanied by DSXM-positive somatic cells.In this report we show that the male-specific isoform of e embryo ,24, DSXMdsx transcripts are not maternally deposited and are specifically expressed in the somatic precursors of the gonads just prior to gonad coalescence [dsx mRNA isoform. We sorted male and female embryos bearing a female-specific Sxl early promoter [dsxm mRNA is expressed in embryos. dsxm mRNA was not readily detected in 3–10 hour old embryos of either sex, but was easily detected in 10–16 hour and 16–22 hour old male embryos. Sequencing of the RT-PCR products verified that they derived from predicted dsxm transcripts. Thus, RT-PCR experiments showed that dsxmis expressed solely in male embryos.A systematic survey of embryonic expression patterns has shown that lescence . Howeverpromoter  attachedM expression pattern, we raised polyclonal antisera against a peptide from the male-specific C-terminus of DSXM. To determine antibody specificity we performed immuno-labeling experiments focusing on whether: 1) the cell-staining pattern matched the in situ hybridization pattern, 2) the signal was male-specific and 3) the signal was absent from male embryos mutant for dsx.To further dissect the DSXM during embryonic stage 13 dsx15/In(3R)dsx23)  Fig .M-expressing cells, we performed co-immunofluorescence staining experiments with anti-DSXM and antibodies against several somatic gonad precursor markers. We focused on mid- to late embryogenesis when DSXM is strongly expressed and when gonad formation occurs  to detect the somatic gonad precursors [M indicated that DSXM was not expressed in the germline. Anti-TJ and anti-DSXM staining indicated that DSXM was expressed in somatic cells intermingled with the germline in stage 13 embryos . EYA is a transcription factor, which is also expressed in the somatic gonad [M revealed co-expression at the cellular level throughout the entire somatic gonad of stage 13 and older embryos  . In antiing Fig. . Taken tM expression. However, if sex determination or maintenance of sexual identity is an ongoing process, then germline sexual identity might require continued expression of DSX. We readily detected dsxm transcripts by RT-PCR with RNA from adult testes (not shown). To explore the cellular expression pattern of DSXM in testes of larvae and adult flies, we performed whole-mount antibody immunofluorescence staining experiments.In contrast to many other cell types, the germline stem cells continue to divide in adults in order to produce gametes. If germline sexual identity is irreversibly determined during embryogenesis, there may be no need for post-embryonic somatic DSXM expression in the somatic hub cells of larval . These data are consistent with the idea that the determination or maintenance of germ cell sexual identity requires ongoing DSXM expression until the end of spermatogenic transcription at meiosis, but is no required once germline transcription ceases.Germline cells leaving the niche divide four times to produce 16-cell cysts. Germline cysts are enveloped by two somatic cyst cells, which become flattened as the germline cysts enlarge 20-fold in volume in preparation for meiosis. These engorged cells are highly transcriptionally active. Sperm differentiation occurs postmeiotically under translational control . The tralls Fig. . Anti-DSEYA Fig.  and 7E–HM expression should not be dependent on a germline. To investigate whether germ cells were necessary for DSXM expression in somatic gonadal precursor cells, we examined embryos lacking germ cells due to the maternal grandchildless mutation gs(l)N26 [M staining was readily detectable in germlineless embryonic testes N26 . Unsurprtes Fig.  indicatieya gene was a candidate regulator of dsx expression as EYA was always expressed in DSXM-positive gonad cells, and preceded DSXM expression temporally in the embryo . We therefore determined whether DSXM expression required EYA function. This experiment was complicated by the fact that EYA is essential for gonadogenesis. In late embryonic stages, eya mutant embryos have germ cells that are scattered throughout posterior regions and only a few somatic gonad precursor cells develop. The few somatic gonad precursors do not coalesce into a gonad, rather they form clumps with associated germ cells [eya mutant male embryos, we were able to detect DSXM expression in the sporadically formed somatic gonad precursor cells  was used for sexing embryos [gs(l)N26 [Df(3R)dsx15 and In(3R)dsx23 [; eyacli-IIEand eyacli-DI [tjeo2 [All flies were raised and maintained on standard cornmeal media  at 25°C. A fly strain bearing a construct containing the early , SxlPE,  upstream embryos . Only fegs(l)N26 ; Df(3R)d3R)dsx23 ; eyacli-yacli-DI  and tjeo+-mRNA from all samples was done using the Oligotex mRNA Mini Kit . RT-PCR was performed using the OneStep RT-PCR Kit . A pair of primers  flanking two introns was used to amplify an 801 bp cDNA fragment from the dsxm transcript. The amplicons were sequenced for verification. For loading control a separate RT-PCR of each mRNA sample was carried out in parallel using a primer pair  for the ubiquitously expressed gene β3-tubulin [Total RNA was prepared using TRIzol reagent  from 3–10, 10–16, 16–22 hour old, sex sorted embryos as well as from testes of male adult flies. Preparation of polyA-tubulin , which aM antibody was made using standard methods . A peptide containing the C-terminal amino acids SSNGAYHHGHHL was used for immunization of two rats, both of which gave useful antibodies of good titer and were used here.The polyclonal DSXFor cell staining experiments, pre-stage 17 embryos were fixed, devitellinized, and immunostained as described . BecauseM at 1:500 in general and the rabbit anti-SOX100B at 1:1000  at 1:50, rabbit anti-SOX100B  at 1:1000 in Fig  the TSA The following secondary antibodies were used, all at 1:500: Cy5 goat anti-rabbit, biotin-coupled goat anti-rat, and biotin-coupled goat anti-rabbit , Alexa 647 goat anti-guinea pig, Alexa 488 goat anti-guinea pig, Alexa 488 goat anti-rabbit, Alexa 647 goat anti-mouse, Alexa 488 goat anti-mouse, Alexa 647 goat anti-chicken . Images were acquired using a Zeiss LSM 510 Meta Confocal Microscope  and processed using Photoshop 7.0 . All figures were annotated using Illustrator 10.0 . All images in figures are single optical sections as indicated.DAPI  staining was used to visualize the nuclei.M localizes to the nucleus. (A-D) Stage 13 testis immunofluorescence using (A) anti- DSXM, (B) anti-VASA, and (C) DAPI. (D) Merged images A-C. (E-H) Stage 15 male testis immunofluorescence using (E) anti- DSXM, (F) anti-VASA and (G) DAPI. (H) Merged images E-G. The scale bars = 10 mm. Anterior is to the left.DSXClick here for file


Mathematical models based on kinetics of HIV-1 plasma viremia after initiation of combination antiretroviral therapy (cART) inferred HIV-infected cells to decay exponentially with constant rates correlated to their strength of virus production. To further define in vivo decay kinetics of HIV-1 infected cells experimentally, we assessed infected cell-classes of distinct viral transcriptional activity in peripheral blood mononuclear cells (PBMC) of five patients during 1 year after initiation of cART+ PBMC can be stratified into four distinct viral transcriptional classes. Two overlapping cell-classes of high viral transcriptional activity, suggestive of a virion producing phenotype, rapidly declined to undetectable levels. Two cell classes expressing HIV-RNA at low and intermediate levels, presumably insufficient for virus production and occurring at frequencies exceeding those of productively infected cells matched definitions of HIV-latency. These cells persisted during cART. Nevertheless, during the first four weeks of therapy their kinetics resembled that of productively infected cells.In a novel analytical approach patient-matched PCR for unspliced and multiply spliced viral RNAs was combined with limiting dilution analysis at the single cell level. This revealed that HIV-RNAWe have observed biphasic decays of latently HIV-infected cells of low and intermediate viral transcriptional activity with marked decreases in cell numbers shortly after initiation of therapy and complete persistence in later phases. A similar decay pattern was shared by cells with greatly enhanced viral transcriptional activity which showed a certain grade of levelling off before their disappearance. Thus it is conceivable that turnover/decay rates of HIV-infected PBMC may be intrinsically variable. In particular they might be accelerated by HIV-induced activation and reactivation of the viral life cycle and slowed down by the disappearance of such feedback-loops after initiation of cART. Current combination antiretroviral therapy (cART) does not attack virus-infected cells themselves but targets viral replication at major steps in the viral life cycle . Thus, t+ cells [Later phases of decay were thought to reflect expanded life-spans of virus producing macrophages or memory T-lymphocytes . In addi+ cells -8.+-T-lymphoctyes [6 lymphocytes [Mathematical models based on plasma viremia only indirectly allow inferring kinetics of latently infected cells which lack virus production. Direct quantification of latently infected cells ex vivo has commonly been attained by viral outgrowth assays of resting CD4phoctyes . These bphocytes . Furtherphocytes  or antigphocytes .6 cells) observed in peripheral blood of patients on cART, even in the absence of detectable plasma viremia [This contrasts with comparatively high levels of cell-associated viral RNA  and multiply spliced (MsRNA) HIV-RNA species with limiting dilution end-point analysis of PBMC. Using this approach, we were able to dissect the population of HIV-RNA+ cells was performed to compute their frequencies which also allowed determining the average per-cell expression of HIV-RNA.Individually adjusted RT-PCR targeting HIV-1 nucleic acids was performed on serial dilutions of PBMC assessing HIV-DNA, UsRNA, total MsRNA and MsRNA-tatrev or MsRNA-nef . In para+, UsRNA+, MsRNA+, vRNA+ cells per patient and per time-point; p < 0.0001) showed that total HIV-infected PBMC exceeded cells expressing viral RNA, which revealed a preponderance of transcriptionally silent provirus in peripheral blood. Moreover, cells expressing UsRNA were invariably more frequent than cells expressing MsRNA and the latter were more frequent than cells positive for UsRNA-ex. These findings provide evidence for the existence of cells expressing solely UsRNA and cells expressing MsRNA and presumably also UsRNA and a third very rare population of cells positive for vRNA-ex.As shown in figure + cells 9-fold decrease; p = 0.0004; figure + cells  Several classes of HIV-infected cells differing in their viral transcriptional activity co-occur before therapy. After initiation of cART cells with lower RNA content appear to outlast cells expressing higher levels of viral RNA.ii) Three cell classes can be dissected directly using limiting cell-dilution, by virtue of their hierarchical distribution of frequencies: a class which expresses solely UsRNA, one class expressing MsRNA and presumably also UsRNA and a class of cells positive for vRNA-ex.iii) Cells expressing MsRNA may host one or more subcategories of infected cells with lower and higher viral transcriptional activity.+ cells namely, ILow , IIMedium , IIHigh  and IIIExtra(ongoing extracellular virion shedding).To account for the observed complexity of HIV-RNA expression in PBMC, we designed a simple model to resolve and identify cell categories of different transcriptional states. In particular, the data presented above suggest the coexistence of four main classes of HIV-RNAHIV-1 infected cells containing solely UsRNA. The existence of this cell class is deduced from our observations that UsRNA-positive cells were invariably more frequent than cells expressing MsRNA.HIV-1 infected cells expressing MsRNA at low levels. Evidence for this class of cells is based on significant differences in per-cell MsRNA content in PBMC from patients on cART as compared to untreated patients. It is highly likely that such cells express UsRNA because MsRNAs are obligatorily derived from primary unspliced HIV-transcripts [nscripts .HIV-1 infected cells with elevated viral transcription. Significantly higher relative expression of both UsRNA and MsRNA in untreated versus treated patients, suggests frequent presence of cells exhibiting Tat/Rev-mediated transcriptional activation [tivation ,21 at baCells carrying virion-enclosed HIV-1 RNA. Such cells to a major extent represent productively infected cells in a state of ongoing or recent burst of viral shedding as previously demonstrated by their association with activated viral transcription [cription ,15,16.+ specimens of total RNA extracts, relative per-cell expression of UsRNA and MsRNA in the three transcriptional categories ILow, IIMedium and IIHigh could be calculated as outlined in figure Applying distinct criteria as compiled in table Low, IIMedium and IIHigh. Median viral RNA expression ranged from 3.7 HIV-RNA copies/cell in class-ILow to 15 copies/cell in class-IIMedium and to 333 copies/cell in class-IIHigh. Notably, in class-IIMedium UsRNA expression was approximately four times higher than MsRNA expression , whereas class-IIHigh showed an inverse pattern with MsRNA expression equaling or slightly exceeding UsRNA expression . Thus class-IIMedium and class-IIHigh displayed different viral transcriptional signatures.Analysis of viral RNA per-cell contents figure  confirmeLow, IIMedium and IIHigh were compared in specimens obtained prior to and during therapy. In the stratum with basal viral transcription of exclusively UsRNA (ILow), per-cell viral RNA contents did not differ between baseline and cART  indicating that this cell-class did not comprise additional subcategories. Similarly, in class IIMedium cells, per cell content of UsRNA plus MsRNA did not reveal a statistically significant difference when baseline samples were compared to specimens obtained during cART . Since samples obtained during cART also comprised study week 2, when plasma viremia had not yet stabilized, we also performed comparison of baseline samples to cART without the specimens from week 2. Similarly this analysis did not reveal statistically significant differences between the two groups . Thus, viral RNA expression in class-IIMedium cells did not experience evident changes during the course of antiretroviral therapy.To further validate our stratification of HIV-infected PBMC, relative RNA contents of UsRNA and MsRNA in IHigh at baseline  as compared to on-therapy samples .Conversely, viral RNA content was 5× higher (Mann Whitney test p < 0.0001) in class-IIMedium and IIHigh was still not sufficient to delineate the full scale of viral transcriptional patterns. On a biological level, this finding provides evidence that class-IIHigh in untreated patients may harbor a subcategory of HIV-infected cells expressing hundreds of viral RNA copies per cell which likely represents productively infected lymphocytes. Due to limitations in sample size and resolution, transcriptional class-IIHigh could not be further dissected. However, we observed that cells expressing significant amounts of vRNA-ex  of 1.6 ± 0.2 d and a second phase with a half-life of 8.1 ± 2.3 d and suppression of viremia predominantly below levels of 50 RNA copies/ml after 12 weeks of treatment figure . The fac1/2 = 71 ± 60 d) decreases, in general more than 90% of HIV-1 RNA+ cells decayed rapidly after therapy initiation. HIV-infected cell categories of elevated transcriptional activity  became frequently undetectable after initiation of cART. However, their overall kinetics did not unequivocally match plain single phase exponential decay.Whereas the total number of HIV-1 infected cells, as assessed by HIV-1 DNA levels, experienced comparably modest (74 ± 7%) and slow  was similar to that of class-ILow (3.2 ± 0.9 d) and class-IIMedium: (4.1 ± 0.66 d). Hence, HIV-RNA+ cells displaying basal viral transcription decayed more slowly than productively infected class-IIIExtra cells but with similar rates as class-IIHigh.Similarly, decays of class-I+ cells of class-IIHigh and class-IIIExtra were largely outnumbered by cells of low viral activity (class-ILow plus class-IIMedium >95% of HIV-RNA+ cells)  figure . Furthern figure  decrease+ cells to four transcriptional categories, namely productively infected cells carrying virions at their surface (class-IIIExtra), cells with elevated viral transcriptional activity fuelled by Tat/Rev- [High), cells expressing low levels of MsRNA and intermediate amounts of UsRNA (class-IIMedium) and cells expressing solely UsRNA (class-I). The latter two classes match a strict definition of HIV-latency [In the present study highly sensitive, patient matched quantification of HIV-1 transcripts in total PBMC was combined with endpoint analysis at a single cell level. This enabled us to extend a previous analysis , in whicTat/Rev-  and NEF-Tat/Rev-  positive-latency ,22 since-latency ,22 and b-latency .Low viral RNA expression remained unchanged in a comparison of specimens from treated and untreated patients. Similarly, class-IIMedium HIV-RNA content appeared constant throughout the course of therapy, whereas viral RNA expression of class-IIHigh cells was significantly different in specimens before and during ART. This suggests that a highly active subgroup in this stratum was present in the absence of treatment which conceivably was identical or overlapping with the productively infected class-IIIExtra.Class-IThe stratification of viral RNA expressing PBMC as exercised in the present study provided only an approximate categorical model for the continuous patterns of HIV-1 transcription likely occurring in vivo. Furthermore, it is well possible that the cell classes characterized by different cell-frequencies and viral transcriptional activity may not reflect totally distinct and independent populations of infected cells. Rather they may represent cells 'caught' in different stages of the viral gene life cycle  was virtually identical to the 1st phase half-live of free plasma virus (1.6 ± 0.2 d). The half-lives of class-IIHigh cells were slightly extended (5.4 ± 2.3 d). Unexpectedly kinetics of class-IIHigh and, to some extent also of class-IIIExtra showed hyperbolic rather than plain negative exponential shapes. This finding may in part be explained by potential heterogeneities of cell classes as defined in our analysis. This applies in particular to class-IIHigh cells which likely comprise both class-IIIExtra cells and a cell category of lower transcriptional activity escaping immunological clearance for longer time periods presumably due to moderate expression of viral antigens as predicted by mathematical models based on kinetics of plasma viremia [All four HIV-RNA viremia . Indeed, 1.6 ± 0. d. The h 5.4 ± 2. d. UnexpLow and class-IIMedium kinetics proceeded similar to the course of class-IIHigh cells. Whereas it was anticipated that they would show slow but steady exponential decline [+ T-cells from acutely infected patients were examined by viral outgrowth assays. In consideration of the in vivo scarcity of provirus inducible by in vitro outgrowth assays [+ latently infected cells may shift from rapid to slower decay during the course of antiretroviral treatment appears to hold true also for cell subsets inducible by in vitro outgrowth assays. Such apparent variability in turnover of latently infected cells may be interpreted in two ways:Markedly contrary to initial expectations, class-I decline , we obse decline  and Stra decline  in whichh assays , the celHigh, also cell-class-ILow and class-IIMedium may be heterogeneous in terms of virological subcategories but also in reference to cellular lineage and/or in terms of their current state in the viral life cycle. Thus, class-ILow and class-IIMedium may comprise cell-types with rapid turnover and long lived cells. Whereas the latter may be allocated to resting HIV-infected CD4+ T-cells [As discussed for class-II T-cells , the mai T-cells ,37.Low and class-IIMedium cells , which were significantly correlated with each other . This suggests that HIV-RNA+ latently infected cells decayed with different kinetics than the majority of HIV-1 infected cells harboring nonintegrated viral DNA. Thus, the fast turnover of unintegrated HIV-DNA may not entirely account for the rapid initial decay of HIV-RNA+ cells.However, HIV-1 DNA levels, which have been reported to be dominated by nonintegrated molecules in PBMC of untreated patients , showed Therefore it is conceivable that turnover rates of HIV-1 infected cells may be intrinsically variable. In particular, they may be influenced by their cellular and humoral environment, which differs under conditions of cART and during viremia. Attenuation of antigenic stimulation due to cART results in declines of cellular ,40 and hThe present study was highly comprehensive in terms of PCR-targets and sample size despite limitations in patient number and in statistical resolution between different cell classes. Thus, it revealed important novel features of HIV-1 transcription and its relation with viral dynamics in vivo.+ cells could be identified and assigned to distinct viral transcription patterns. Among these cell classes, latently infected cells represent the majority of HIV-1 infected PBMC both in the absence of treatment and during cART. This emphasizes the crucial role that latent HIV-1 reservoirs play not only in the maintenance of viral persistence during cART but, as precursors of productively infected cells, also under conditions of ongoing viral replication.In summary, at least four categories of HIV-RNAForemost, we have gained evidence suggesting that at least two types of latently HIV-1 infected cells after initiation of cART decay with kinetics similar to those of productively infected cells. These rapid decays may be shaped by cellular and humoral immune responses against HIV and by HIV-induced activation which prevail before and early after initiation of cART but wither away when viral replication is stably suppressed. Verifying the hypothesis that decay of latently HIV-infected cells may not be constant but prone to influences from their cellular and humoral environment and identifying the factors governing the turnover of latently infected cells may help to devise novel therapeutic strategies to pursue the as yet elusive aim of eradicating HIV-1.5 cells/aliquot) to avert cell loss in dilutions. Cells were stored at -80°C as dry pellets.Five chronically HIV-1 infected therapy naïve patients initiating cART according to standard treatment guidelines  were enrolled after informed consent was obtained. The study was approved by the ethics committee of the University Hospital Zürich. Patients were treated with zidovudine plus lamivudine (300 mg BID), and ritonavir boosted lopinavir (100/400 mg BID), except for patient 103 who replaced ritonavir/lopinavir by efavirenz (daily 1 × 600 mg) at week 32. Median (range) baseline clinical parameters were 147 CD4 T-lymphocytes/μl blood and 158'000  viral RNA copies/ml plasma. PBMC were purified as described   using two protocols in parallel ,15. Totagag gene were used to quantify UsRNA, vRNA-ex and HIV-DNA [PCR primers and fluorescent probes mapping to the  HIV-DNA . PCR str HIV-DNA  utilizin HIV-DNA . Some sp HIV-DNA  .+ specimens near the detection limit of the test, quantification of HIV-RNA was censored to avoid gross overestimation: If only one duplicate PCR was positive and/or nominal copy numbers were below 1 copy per reaction, it was assumed that 1 RNA copy had been present per tube.RNA copy-numbers were calculated as the mean of duplicate PCR measurements, extrapolated to copy numbers per tube (5–10-fold multiplication) and normalized to the input of PBMC. In HIV-RNA+ cells were calculated by 50% endpoint analysis [+ cells. Rarely this resulted in nominally negative values which were censored to 1 copy per cell, to allow further analyses using logarithmic transformation.Stratification and assignment of specimens to different transcriptional classes were executed according to criteria compiled in table analysis  accordinanalysis . SpecifiStatistical comparisons between groups were performed using nonparametric tests; the Mann-Whitney test for unpaired data-sets, Wilcoxon signed rank tests for paired analyses and, when adjustment for multiple testing was applied, one way Anova and a Friedman post test analyzing all possible combinations was applied.Low, IIMedium, IIHigh: HIV-infected cells with low, intermediate and expression levels of HIV-RNA respectively; sem: standard error of the mean; d: days.cART: combination antiretroviral therapy; PBMC: peripheral blood mononuclear cells; UsRNA: unspliced HIV-RNA; MsRNA: multiply spliced HIV-RNA; MsRNA-total: MsRNA encoding tat, rev and nef; MsRA-tatrev: MsRNA encoding Tat and Rev; MsRNA-nef: MsRNA encoding Nef; vRNA-ex: virus encapsidated cell-associated unspliced HIV-RNA; IThe authors declare that they have no competing interests.MF designed and developed the algorithm for single cell transcriptional analysis, carried out the data analysis, conceived the study and wrote the manuscript. BJ participated in sequence analysis, design of PCR primers and drafting of the manuscript. BN participated in performing the laboratory work for PCR and sequence analysis. PK participated in data analysis, drafting the manuscript and development of single cell transcriptional analysis. RH participated in patient-recruitment and -care. VvW participated in statistical analysis and drafting the manuscript. MA participated in statistical analysis. RW participated in drafting the manuscript and designing the present study. HFG participated in conceiving the study, and participated in its design and coordination and helped to draft and finalize the manuscript. All authors read and approved the final manuscript.


A polymerase chain reaction (PCR)-based subtractive hybridization technique was used to identify transformation-related genes in malignant melanoma. Melanoma biopsies were compared with tissues of benign melanocytic naevi and 549 gene fragments were screened using arrayed filters. Thirty-eight clones were confirmed to be differentially expressed representing 30 different genes (18 melanoma-specific and 12 naevus-specific genes). To further confirm differential gene expression, Northern blot analyses with six of the 30 genes as probes were performed. All six were differentially expressed in benign and malignant melanocytic lesions, specifically dbpB/YB-1, 67-kDa laminin receptor, CAGH-3, 71-kDa heat shock protein and two unknown genes. The expression levels of these genes were then analysed in 50 different tissues to determine their overall expression profile. In conclusion, the technique of PCR-based subtractive hybridization in combination with arrayed filters allows detection of differences in gene expression even in tissues from which high-quality RNA is hard to isolate. The genes identified in this study are of interest because of their potential role in the pathogenesis of malignant melanoma. © 2000 Cancer Research Campaign


NTR neurotrophin receptor. Trk receptors promote cell survival and differentiation while p75NTR induces, in most cases, the activity of JNK-p53-Bax apoptosis pathway or suppresses intracellular survival signaling cascades. Robust Trk activation blocks p75NTR -induced apoptosis by suppressing the JNK-p53-Bax pathway. The aim of this exploratory study was to investigate the expression levels of neurotrophin receptors, Trks and p75NTR, and the activation of JNK pathway in human astrocytomas and in adjacent non-neoplastic brain tissue.Neurotrophins are growth factors that regulate cell growth, differentiation and apoptosis in the nervous system. Their diverse actions are mediated through two different transmembrane – receptor signaling systems: Trk receptor tyrosine kinases  and p75NTR and phosphorylated forms of JNK (pJNK) and c-Jun (pc-Jun) were used. The labeling index (LI), defined as the percentage of positive (labeled) cells out of the total number of tumor cells counted, was determined.Formalin-fixed paraffin-embedded serial sections from 33 supratentorial astrocytomas  were immunostained following microwave pretreatment. Polyclonal antibodies against TrkA, TrkB, TrkC and monoclonal antibodies against p75NTR receptor expression was found in a small percentage of tumor cells (~1%) in some tumors. The endothelium of tumor capillaries showed conspicuous immunoreactivity for TrkB receptor. Trk immunoreactivity seemed to be localized in some neurons and astrocytes in non-neoplastic tissue. Phosphorylated forms of JNK (pJNK) and c-Jun (pc-Jun) were significantly co-expressed in a tumor grade-dependent manner (p < 0.05). Interestingly, a statistically significant (p < 0.05) reverse relationship between Trk receptors LIs and pc-Jun/pJNK LIs was noted in some glioblastomas multiforme.Moderate to strong, granular cytoplasmic immunoreactivity for TrkA, TrkB and TrkC receptors was detected in greater than or equal to 10% of tumor cells in the majority of tumors independently of grade; on the contrary, p75In the context of astrocytomas, Trk receptors  expression may promote tumor growth independently of grade. Furthermore, activation of JNK pathway may contribute to progression towards malignancy. Considering the fact that regional tumor heterogeneity may be a limiting factor for immunohistochemical studies, the significance of the reverse relationship between Trk receptors and pc-Jun/pJNK LIs with respect to biological behavior of human astrocytomas requires further evaluation. NTR neurotrophin receptor, a member of tumor necrosis factor (TNF) receptor superfamily  play a crucial role in function of developing and adult vertebrate nervous system -3. TheseNTR functions as a ligand-stimulated apoptotic receptor inducing the activity of the JNK-p53-Bax pathway or suppressing Ras/PI-3k/Akt activation. Robust Trk activation blocks p75NTR -induced apoptosis by suppressing the JNK-p53-Bax pathway [NTR but not to Trk receptors suggesting that pro-neurotrophins can elicit apoptosis via p75NTR even in cells expressing survival-promoting Trk receptors [Trk receptors promote cell survival via several intracellular signaling cascades e.g. Ras/PI-3k/Akt pathway -4. In moeceptors .The major downstream target of JNK [c-Jun N-terminal kinases (JNKs)/Stress-activated protein kinases (SAPKs) MAP kinases] is the c-Jun transcription factor which is phosphorylated at serine residues 63 and 73 . The axiNTR [Gliomas of astrocytic origin are the most common primary brain tumors of adults . AstrocyNTR . The abeNTR -18 as weNTR ,19-21. RNTR ,23.NTR and phosphorylated forms of JNK (pJNK) and c-Jun (pc-Jun) in serial sections of human astrocytomas of different grade of malignancy.The aim of this study was to investigate immunohistochemicaly the expression levels of neurotrophin receptors TrkA, TrkB, TrkC, p75Astrocytic gliomas (n = 33) were obtained from patients who had undergone surgery at Department of Neurosurgery of the Patras University Hospital  from 1991 to 1996. The formalin-fixed, paraffin-embedded archival tissue blocks were retrieved, and the matching hematoxylin and eosin (H & E) – stained slides were reviewed and screened for representative tumor regions by a neuropathologist. Tumors were evaluated by routine methods for histopathology including immunohistochemical staining for glial fibrillary acidic protein (GFAP) and Ki-67 index as proliferation marker and graded according to the diagnostic criteria of the WHO classification system . The tum63 and anti-phospho-JNK (G-7) , a mouse monoclonal antibody directed against the carboxy terminus of JNK1 phosphorylated on Thr183 and Tyr185 , were also purchased from Santa Cruz Biotechnology . A mouse monoclonal anti-nerve growth factor receptor (p75NGFR) antibody  directed against amino acids 1–160 of extracellular domain of p75NTR was purchased from Lab Vision Corporation .Anti-pan-Trk (B-3) , a mouse monoclonal antibody that reacts with a highly conserved carboxy terminus of TrkA gp140 and is reactive with TrkA, TrkB, and TrkC receptors, anti-TrkA (763), a rabbit polyclonal antibody against a carboxy terminal epitope of human TrkA (amino acids 763–777) , anti-TrkB (794) , and anti-TrkC (798) , rabbit polyclonal antibodies against carboxy terminal peptides (794–808 and 798–812 residues respectively) of gp145TrkB or gp145TrkC proteins were purchased from Santa Cruz Biotechnology . Antibodies recognize the full-length isoforms of Trk receptors and the catalytic intracellular tyrosine kinase domain. According to the manufacturer, antibodies do not cross react with each other. Anti-phospho-c-Jun (KM-1) , a mouse monoclonal antibody which detects c-Jun only when it is phosphorylated on SerNTR, pc-Jun and pJNK and an anti-mouse/rabbit poly HRP IHC Detection System  for detection of TrkA, TrkB and TrkC were used according to manufacturers' specifications. In brief, antigen retrieval was performed by placing the unstained slides in 0.01 mM citrate buffer (PH 6.0) and treating them in a microwave oven for 10 min. Overnight incubation at 4°C was performed for anti -pan-Trk, -p75NTR, -pc-Jun and -pJNK primary antibodies and 1 hour at room temperature incubation was performed for anti-TrkA, -TrkB and -TrkC primary antibodies. Antigen-antibody reaction was visualized routinely using the peroxidase enhancer as a chromogen. Mayer's hematoxylin nuclear staining was used as a counter stain. Negative controls included unrelated primary antibodies. As positive controls, specimens of inflammatory intestine known to express Trks and p75NTR [Immunohistochemical studies were performed on 4 μm thick serial sections. A standard biotin-streptavidin peroxidase procedure  for detection of pan-Trk, p75d p75NTR  and gliod p75NTR  were useThe results of immunohistochemical staining were evaluated independently by two observers. Using a final magnification of 400× (objective × eyepiece), ten non-overlapping fields were chosen at random and a total of 100 tumor cells were counted manually in every field with the aid of an ocular grid. To assess the fraction of immunolabeled cells in each case and each antibody, the labeling index (LI), defined as the percentage of positive (labeled) cells out of the total number of tumor cells counted, was determined. Labeled cells in tumor blood vessels/microvascular proliferation were excluded from the cell counts.Nonparametric methods were used for the statistical analysis of the results. Spearman correlation was used to assess significance of relationships between different LIs and between age and LIs. Spearman correlation was also used to assess significance of relationships between LIs and grade. Wilcoxon rank test was used to compare LIs between male and female. P values <0.05 were considered significant. Statistical analyses were carried out using the SPSS package (version 12.0).Moderate to strong Trk immunoreactivity was found in the majority of astrocytomas included in this study. Trk immunoreactivity was cytoplasmic with granular appearance Figures , 2. The In diffuse fibrillary astrocytomas  reactive astrocytes showed strong immunoreactivity for all Trk receptors. Regarding grade IV tumors (glioblastomas multiforme), Trk-positively stained cells had a tendency to aggregate around tumor blood vessels or in the vicinity bordering tumor necrosis. Elongated, bipolar cells as well as giant cells Figure  were intThere was no statistically significant correlation between Trk expression and histological grade . Trk expression was not significantly correlated with age . Furthermore, Trk expression was not different by gender .NTR immunoreactivity was confined to cytoplasm of a small number (~1%) of scattered or small clusters of tumor cells in a few tumors. Normal tissue was totally p75NTR -negative.p75Trk receptors, pc-Jun and pJNK expression was analyzed by examining adjacent  sections of each specimen. Only the nuclear immunoreactivity was evaluated for both pc-Jun and pJNK proteins. There was a statistically significant association between pc-Jun and pJNK expression . There was also a statistically significant positive correlation between pc-Jun/pJNK expression and grade of malignancy . pc-Jun/pJNK expression was significantly correlated with age . pc-Jun/pJNK expression was not different by gender . Normal tissue was pc-Jun and pJNK imunonegative. Regarding co-expression of Trk receptors and pc-Jun/pJNK in the same tumor cells three patterns were noted: Trk-positive – pc-Jun/pJNK-positive cells, Trk-positive – pc-Jun/pJNK-negative cells and Trk-negative – pc-Jun/pJNK-positive cells  showed a reverse relationship between Trk and pc-Jun/pJNK LIs. In other words, a high LI for Trk expression was associated with a low LI for pc-Jun/pJNK expression in the same tumor and vice versa Table . In thisThere is growing evidence relating neurotrophin receptors expression in pathogenesis of astrocytomas. Wang et al. (1998) have detected TrkA, TrkB and TrkC expression in neoplastic astrocytes but not in neoplastic oligodendrocytes and suggested that Trk expression may be useful for lineage marking but unrelated to any signaling cascade involved in the maintenance of a transformed phenotype . On the Recent data support that, in glial cells, neurotrophins, through their Trk receptors, may act as mitogens in response to injury  and promIn contrast to gliomas, tumors of neuroepithelial origin such as medulloblastomas ,20 and nNTR and in such instances pro-apoptotic responses of p75NTR e.g. activation of JNK pathway, are, in most cases, suppressed [NTR neurotrophin receptor in astrocytomas included in this study erases the possibility of apoptosis mediated by binding of pro-neurotrophins (pro-NGF and/or pro-BDNF) to p75NTR, even in the presence of survival-promoting Trk receptors [Trk receptors are often co-expressed with p75ppressed . The abseceptors , in thesHowever, JNK activation has been reported in human gliomas  and a grTrkB receptor expression detected in tumor vessels suggests that these receptors may be an important component of the angiogenic cascade required for the development of astrocytic tumors in view of recent data showing the neurotrophin-dependent proliferation of brain capillary endothelial cells .Finally, tumor cells acquire the ability to activate several pathways in order to survive and neurotrophin/Trk signal transduction axis as well as JNK pathway may regulate such survival possibilities. The complexity and cross talk between different signaling cascades may limit the potential therapeutic efficacy of targeting a single molecule. Therefore, it appears necessary to develop an expanded molecular sub-classification of these tumors and to consider combinatorial treatment regimens targeted at different pathways. However, astrocytomas are strikingly heterogeneous tumors in terms of protein profile even within a single tumor which may be a limiting factor in the immunohistochemical evaluation of protein expression levels in these tumors.NTR mediated apoptosis has no functional role in human astrocytomas. Activation of JNK pathway may contribute to progression towards malignancy. Further studies would elucidate the significance of the reverse relationship between Trk LIs and pc-Jun/pJNK LIs. As tumor cells activate several pathways in order to survive, future therapeutic approach may consider combinatorial treatment regimens targeted at different pathways.Trk  receptor overexpression may promote tumor cell survival and enhance tumor growth while p75All authors declare that there are no financial or non-financial competing interests  in relation to this manuscript.MA conceived of the study, carried out the design, participated in analysis of immunostaining, performed the statistical analysis, and wrote the manuscript; MK carried out the immunostaining, participated in analysis of immunostaining, and drafted the manuscript. GG supplied clinical data and reviewed the manuscript; TM supplied clinical data and reviewed the manuscript; JV carried out the pathological staging and helped in its design and critical review of the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


EGFR ligand expression was determined by Northern blot analysis, reverse transcription polymerase chain reaction, metabolic labelling and confocal microscopy. We found that amphiregulin (AR) was the most abundant EGFR ligand expressed in HCA-7 and Caco-2 cells. AR was localized to the basolateral surface and detected in basolateral-conditioned medium. Basolateral administration of neutralizing AR antibodies significantly reduced basal DNA replication. A single class of high-affinity EGFRs was detected in the basolateral compartment, whereas the apical compartment of polarized cells, and cells cultured on plastic, displayed two classes of receptor affinity. Basolateral administration of transforming growth factor alpha (TGF-α) or an EGFR neutralizing antibody also resulted in a dose-dependent stimulation or attenuation, respectively, of DNA replication. However, no mitogenic response was observed when these agents were added to the apical compartment or to confluent cells cultured on plastic. We conclude that amphiregulin acts as an autocrine growth factor in HCA-7 and Caco-2 cells, and EGFR ligand-induced proliferation is influenced by cellular polarity. © 1999 Cancer Research CampaignColonic enterocytes, like many epithelial cells in vivo, are polarized with functionally distinct apical and basolateral membrane domains. The aims of this study were to characterize the endogenous epidermal growth factor (EGF)-like ligands expressed in two polarizing colon cancer cell lines, HCA-7 Colony 29 (HCA-7) and Caco-2, and to examine the effects of cell polarity on EGF receptor-mediated mitogenesis. HCA-7 and Caco-2 cells were grown on plastic, or as a polarized monolayer on Transwell filters. Cell proliferation was measured by


During recent years N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers have been developed as targetable drug carriers. These soluble synthetic polymers are internalized by cells by pinocytosis and they can be tailor-made to include peptidyl side-chains degradable intracellularly by specific lysosomal enzymes. Thus they provide the opportunity fo achieve controlled intracellular delivery of anticancer agents. The anthracycline antibiotic daunomycin, and protein synthesis inhibitor puromycin, were bound to HPMA copolymers via several different peptide side-chains, including Gly-Gly, Gly-Phe-Leu-Gly and Gly-Phe-Phe-Leu. Incubation of polymer-drug conjugates with isolated lysosomal enzymes  showed that significant release of drug occurred over 20 h, more than 20% of daunomycin and more than 80% of puromycin being liberated. To test their pharmacological activity conjugates were incubated with either the mouse leukaemia L1210, or the human lymphoblastoid leukaemia CCRF in vitro. The conjugates tested were all less effective than free daunomycin, but they showed differential toxicity against L1210 depending on the aminoacid sequence of their drug-polymer linkage. Inclusion of fucosylamine-terminating side-chains into the HPMA copolymer structure increased the affinity of conjugates for the L1210 cell membrane and resulted in increased toxicity. In contrast HPMA-daunomycin conjugates with or without fucosylamine affected CCRF cells equally, but this cell line was more sensitive than the mouse leukaemia to both free and polymer-bound daunomycin. Incubation of L1210 cells in polymer-bound daunomycin for 72 h, followed by plating cells out in low density in drug-free medium, showed that a concentration of polymer-bound drug (184 micrograms ml-1) could be selected to achieve a cytotoxic effect.


Serum IGF-Ilevels were lower in diabetic than in nondiabetic animals,and rose after stimulation only in the nondiabetic animals.Furthermore, hepatic GH resistance and IGF-I mRNA levelsand IGF-I peptide were increased in nondiabetic animalsin response to GH stimulation, whereas the low levels per seof all these parameters in diabetic mice were unaffected. The study shows that STZ diabetic mice demonstrate a substantialpart of the clinical features of type 1 diabetes in humans,including GH hypersecretion and GH resistance. Accordingly,it is proposed that STZ diabetic mice may be a bettermodel of the perturbations of the GH/IGF-I axis in diabetesthan STZ diabetic rats.The growth hormone (GH) and insulin-like growth factorI (IGF-I) axis were studied in streptozotocin (STZ) diabeticand nondiabetic female mice following intravenous(IV) injection of the GH secretagogue (GHS) ipamorelin orsaline. On day 14, blood samples were obtained before and10 minutes after the injection. Livers were removed andfrozen for determination of the mRNA expressions of theGH receptor, GH-binding protein, and IGF-I, and hepaticIGF-I peptide. Serum samples were analyzed for GH andIGF-I. Following ipamorelin injection, the GH levels werefound to be 150 ± 35


A disturbed energy balance has been demonstrated in lung cancer patients. Both an enhanced resting energy expenditure (REE) and a decreased energy intake contribute to weight loss. Enhanced systemic levels of inflammatory mediators were found to be related to the enhanced REE in lung cancer. The aim of the present study was to investigate energy metabolism and systemic levels of inflammatory mediators in small-cell lung carcinoma (SCLC) patients before and after treatment with chemotherapy. Hypermetabolism and an enhanced inflammatory response have already been demonstrated in SCLC by our group before. Twelve newly diagnosed SCLC patients were consecutively included in the study. REE was measured by indirect calorimetry and body composition was determined by bioelectrical impedance (BIA) before and 1 month after treatment. To assess the inflammatory state the acute-phase proteins, C-reactive protein (CRP) and lipopolysaccharide-binding protein (LBP), both soluble tumour necrosis factor (TNF) receptors, (sTNF-R)-55 and sTNF-R75, and soluble intercellular adhesion molecule (sICAM)-1 were measured in plasma before and 1 month after treatment. CRP was assessed by turbidemetry, whereas the other inflammatory parameters were measured by enzyme-linked immunosorbent assay (ELISA). A significant reduction in REE was found irrespective of therapeutic outcome, whereas body weight and body composition remained stable. The acute-phase proteins CRP and LBP were reduced significantly after treatment with chemotherapy, whereas both sTNF receptors and sICAM-1 remained enhanced. No correlation, however, existed between the decrease in REE and the decrease in the acute-phase proteins. In conclusion, chemotherapeutic treatment attenuates the tumour-related metabolic derangements and acute-phase response.


I.p. immunization with gamma-irradiated hepatoma cells induces resistance to s.c. tumour-cell challenge in syngeneic WAB/Not rats. Mild heat treatment of these cells (greater than 41 degrees C for 30 min) destroyed this immunoprotective effect, but did not abolish tumour-specific antibody production in treated rats. The binding of syngeneic and alloantibodies to surface antigens expressed on hepatoma cells was unaffected by heat treatment. Thus, heat-treated gamma-irradiated hepatoma cells retain a serologically defined tumour-specific antigen but are unable to elicit immunoprotection. By examining the incorporation of radioactive precursors into DNA, RNA and protein in heat-treated cells, it was determined that above 41 degrees C there was a significant decrease in metabolic activity. It is postulated that for the effective induction of transplantation immunity to tumours, tumour-specific antigens should be present on the surface membranes of a metabolically active cell. This hypothesis accounts for the absence or marked reduction of immunoprotection induced by inviable or glutaraldehyde-treated cells, isolated cell membranes and soluble tumour extracts which retain serologically defined tumour-specific antigens.