Cancer-associated fibroblasts of the prostate promote a compliant and more invasive phenotype in benign prostate epithelial cells.

Reciprocal interactions between prostate epithelial cells and their adjacent stromal microenvironment not only are essential for tissue homeostasis but also play a key role in tumor development and progression. Malignant transformation is associated with the formation of a reactive stroma where cancer-associated fibroblasts (CAFs) induce matrix remodeling and thereby provide atypical biochemical and biomechanical signals to epithelial cells. Previous work has been focused on the cellular and molecular phenotype as well as on matrix stiffness and remodeling, providing potential targets for cancer therapeutics. So far, biomechanical changes in CAFs and adjacent epithelial cells of the prostate have not been explored. Here, we compared the mechanical properties of primary prostatic CAFs and patient-matched non-malignant prostate tissue fibroblasts (NPFs) using atomic force microscopy (AFM) and real-time deformability cytometry (RT-FDC). It was found that CAFs exhibit an increased apparent Young's modulus, coinciding with an altered architecture of the cytoskeleton compared with NPFs. In contrast, co-cultures of benign prostate epithelial (BPH-1) cells with CAFs resulted in a decreased stiffness of the epithelial cells, as well as an elongated morphological phenotype, when compared with co-cultures with NPFs. Moreover, the presence of CAFs increased proliferation and invasion of epithelial cells, features typically associated with tumor progression. Altogether, this study provides novel insights into the mechanical interactions between epithelial cells with the malignant prostate microenvironment, which could potentially be explored for new diagnostic approaches.

ERBIN deficiency links STAT3 and TGF-ß pathway defects with atopy in humans.

Nonimmunological connective tissue phenotypes in humans are common among some congenital and acquired allergic diseases. Several of these congenital disorders have been associated with either increased TGF-ß activity or impaired STAT3 activation, suggesting that these pathways might intersect and that their disruption may contribute to atopy. In this study, we show that STAT3 negatively regulates TGF-ß signaling via ERBB2-interacting protein (ERBIN), a SMAD anchor for receptor activation and SMAD2/3 binding protein. Individuals with dominant-negative STAT3 mutations (STAT3mut ) or a loss-of-function mutation in ERBB2IP (ERBB2IPmut ) have evidence of deregulated TGF-ß signaling with increased regulatory T cells and total FOXP3 expression. These naturally occurring mutations, recapitulated in vitro, impair STAT3-ERBIN-SMAD2/3 complex formation and fail to constrain nuclear pSMAD2/3 in response to TGF-ß. In turn, cell-intrinsic deregulation of TGF-ß signaling is associated with increased functional IL-4Rα expression on naive lymphocytes and can induce expression and activation of the IL-4/IL-4Rα/GATA3 axis in vitro. These findings link increased TGF-ß pathway activation in ERBB2IPmut and STAT3mut patient lymphocytes with increased T helper type 2 cytokine expression and elevated IgE.

Loss of B Cells in Patients with Heterozygous Mutations in IKAROS.

BACKGROUND: Common variable immunodeficiency (CVID) is characterized by late-onset hypogammaglobulinemia in the absence of predisposing factors. The genetic cause is unknown in the majority of cases, and less than 10% of patients have a family history of the disease. Most patients have normal numbers of B cells but lack plasma cells. METHODS: We used whole-exome sequencing and array-based comparative genomic hybridization to evaluate a subset of patients with CVID and low B-cell numbers. Mutant proteins were analyzed for DNA binding with the use of an electrophoretic mobility-shift assay (EMSA) and confocal microscopy. Flow cytometry was used to analyze peripheral-blood lymphocytes and bone marrow aspirates. RESULTS: Six different heterozygous mutations in IKZF1, the gene encoding the transcription factor IKAROS, were identified in 29 persons from six families. In two families, the mutation was a de novo event in the proband. All the mutations, four amino acid substitutions, an intragenic deletion, and a 4.7-Mb multigene deletion involved the DNA-binding domain of IKAROS. The proteins bearing missense mutations failed to bind target DNA sequences on EMSA and confocal microscopy; however, they did not inhibit the binding of wild-type IKAROS. Studies in family members showed progressive loss of B cells and serum immunoglobulins. Bone marrow aspirates in two patients had markedly decreased early B-cell precursors, but plasma cells were present. Acute lymphoblastic leukemia developed in 2 of the 29 patients. CONCLUSIONS: Heterozygous mutations in the transcription factor IKAROS caused an autosomal dominant form of CVID that is associated with a striking decrease in B-cell numbers. (Funded by the National Institutes of Health and others.).

DNA hypermethylation in prostate cancer is a consequence of aberrant epithelial differentiation and hyperproliferation.

Prostate cancer (CaP) is mostly composed of luminal-like differentiated cells, but contains a small subpopulation of basal cells (including stem-like cells), which can proliferate and differentiate into luminal-like cells. In cancers, CpG island hypermethylation has been associated with gene downregulation, but the causal relationship between the two phenomena is still debated. Here we clarify the origin and function of CpG island hypermethylation in CaP, in the context of a cancer cell hierarchy and epithelial differentiation, by analysis of separated basal and luminal cells from cancers. For a set of genes (including GSTP1) that are hypermethylated in CaP, gene downregulation is the result of cell differentiation and is not cancer specific. Hypermethylation is however seen in more differentiated cancer cells and is promoted by hyperproliferation. These genes are maintained as actively expressed and methylation-free in undifferentiated CaP cells, and their hypermethylation is not essential for either tumour development or expansion. We present evidence for the causes and the dynamics of CpG island hypermethylation in CaP, showing that, for a specific set of genes, promoter methylation is downstream of gene downregulation and is not a driver of gene repression, while gene repression is a result of tissue-specific differentiation.

Atmospheric oxidation capacity sustained by a tropical forest.

Terrestrial vegetation, especially tropical rain forest, releases vast quantities of volatile organic compounds (VOCs) to the atmosphere, which are removed by oxidation reactions and deposition of reaction products. The oxidation is mainly initiated by hydroxyl radicals (OH), primarily formed through the photodissociation of ozone. Previously it was thought that, in unpolluted air, biogenic VOCs deplete OH and reduce the atmospheric oxidation capacity. Conversely, in polluted air VOC oxidation leads to noxious oxidant build-up by the catalytic action of nitrogen oxides (NO(x) = NO + NO2). Here we report aircraft measurements of atmospheric trace gases performed over the pristine Amazon forest. Our data reveal unexpectedly high OH concentrations. We propose that natural VOC oxidation, notably of isoprene, recycles OH efficiently in low-NO(x) air through reactions of organic peroxy radicals. Computations with an atmospheric chemistry model and the results of laboratory experiments suggest that an OH recycling efficiency of 40-80 per cent in isoprene oxidation may be able to explain the high OH levels we observed in the field. Although further laboratory studies are necessary to explore the chemical mechanism responsible for OH recycling in more detail, our results demonstrate that the biosphere maintains a remarkable balance with the atmospheric environment.

Kallikrein 4 (hK4) and prostate-specific antigen (PSA) are associated with the loss of E-cadherin and an epithelial-mesenchymal transition (EMT)-like effect in prostate cancer cells.

Prostate-specific antigen (PSA) and the related kallikrein family of serine proteases are current or emerging biomarkers for prostate cancer detection and progression. Kallikrein 4 (KLK4/hK4) is of particular interest, as KLK4 mRNA has been shown to be elevated in prostate cancer. In this study, we now show that the comparative expression of hK4 protein in prostate cancer tissues, compared with benign glands, is greater than that of PSA and kallikrein 2 (KLK2/hK2), suggesting that hK4 may play an important functional role in prostate cancer progression in addition to its biomarker potential. To examine the roles that hK4, as well as PSA and hK2, play in processes associated with progression, these kallikreins were separately transfected into the PC-3 prostate cancer cell line, and the consequence of their stable transfection was investigated. PC-3 cells expressing hK4 had a decreased growth rate, but no changes in cell proliferation were observed in the cells expressing PSA or hK2. hK4 and PSA, but not hK2, induced a 2.4-fold and 1.7-fold respective increase, in cellular migration, but not invasion, through Matrigel, a synthetic extracellular matrix. We hypothesised that this increase in motility displayed by the hK4 and PSA-expressing PC-3 cells may be related to the observed change in structure in these cells from a typical rounded epithelial-like cell to a spindle-shaped, more mesenchymal-like cell, with compromised adhesion to the culture surface. Thus, the expression of E-cadherin and vimentin, both associated with an epithelial-mesenchymal transition (EMT), was investigated. E-cadherin protein was lost and mRNA levels were significantly decreased in PC-3 cells expressing hK4 and PSA (10-fold and 7-fold respectively), suggesting transcriptional repression of E-cadherin, while the expression of vimentin was increased in these cells. The loss of E-cadherin and associated increase in vimentin are indicative of EMT and provides compelling evidence that hK4, in particular, and PSA have a functional role in the progression of prostate cancer through their promotion of tumour cell migration.

Global air pollution crossroads over the Mediterranean.

The Mediterranean Intensive Oxidant Study, performed in the summer of 2001, uncovered air pollution layers from the surface to an altitude of 15 kilometers. In the boundary layer, air pollution standards are exceeded throughout the region, caused by West and East European pollution from the north. Aerosol particles also reduce solar radiation penetration to the surface, which can suppress precipitation. In the middle troposphere, Asian and to a lesser extent North American pollution is transported from the west. Additional Asian pollution from the east, transported from the monsoon in the upper troposphere, crosses the Mediterranean tropopause, which pollutes the lower stratosphere at middle latitudes.

The Indian Ocean experiment: widespread air pollution from South and Southeast Asia.

The Indian Ocean Experiment (INDOEX) was an international, multiplatform field campaign to measure long-range transport of air pollution from South and Southeast Asia toward the Indian Ocean during the dry monsoon season in January to March 1999. Surprisingly high pollution levels were observed over the entire northern Indian Ocean toward the Intertropical Convergence Zone at about 6 degrees S. We show that agricultural burning and especially biofuel use enhance carbon monoxide concentrations. Fossil fuel combustion and biomass burning cause a high aerosol loading. The growing pollution in this region gives rise to extensive air quality degradation with local, regional, and global implications, including a reduction of the oxidizing power of the atmosphere.

Prediction of visual function after cataract surgery. A prospectively validated model.

OBJECTIVE: To develop a model to predict visual functional improvement after cataract extraction with intraocular lens implantation based on preoperative data. DESIGN: A prospective study with serial evaluations of visual function preoperatively and at 3 and 12 months after surgery. SETTING: The General Eye Service of the Massachusetts Eye and Ear Infirmary. Boston, Mass, and 33 ophthalmology practices in Boston. PATIENTS: Patients (N = 426; ages, > or = 65 years) who were undergoing cataract surgery. METHODS: Twelve-month improvement in visual function was measured by using the Activities of Daily Vision Scale (ADVS). Ordinal logistic regression was used to identify correlates of improved ADVS scores in 281 patients (derivative set). Potential factors included the preoperative visual acuity, preoperative ADVS score, four chronic ocular diseases, eight medical conditions, and demographic characteristics. Five predictors were identified and used to construct a prediction rule. The accuracy of the prediction rule was evaluated in an independent group of 145 patients (validation set). RESULTS: Postoperatively, 40% of the 281 patients in the derivative set had substantial improvement in their ADVS scores, and 53 (19%) had some improvement. Predictors of improvement included younger age (P < .001), a poorer preoperative ADVS score (P < .001), posterior subcapsular cataract (P = .09), and absence of age-related cataract (P = .09), and absence of age-related macular degeneration (P = .07) and/or diabetes (P = .006). When applied to the independent sample of 145 patients, these five characteristics classified the patients into three groups in which the probabilities of substantial improvement were 85%, 34%, and 3%, thus verifying the discriminatory power of the prediction rule. CONCLUSIONS: Preoperative data can identify patients who are likely to have improvements in visual function after cataract surgery. Such findings may be useful in the selection of patients for this high-volume procedure.

Improved visual function and attenuation of declines in health-related quality of life after cataract extraction.

OBJECTIVE: To measure the effect of cataract extraction and lens implantation on elderly persons' health-related quality of life and on their ability to perform visual activities. DESIGN: Evaluations of health status were conducted preoperatively and at 3 and 12 months after surgery on patients scheduled for cataract extraction. SETTING: Patients were enrolled from the General Eye Service of the Massachusetts Eye and Ear Infirmary and 33 Boston practices. PATIENTS: The cohort consisted of 464 patients aged 65 years or older who were identified from the surgical schedule of the Massachusetts Eye and Ear Infirmary. At 3 to 12 months after surgery, 458 (99%) of the participants were successfully contacted. Health-related quality of life data were available for 419 (90%) to assess changes after surgery. MAIN OUTCOME MEASURES: Ophthalmologic examinations were performed preoperatively and during the follow-up period. The Activities of Daily Vision Scale (ADVS) and the Medical Outcomes Study 36-item short from (SF-36) were administered before surgery and at 3 and 12 months postoperatively to assess changes in health status. RESULTS: At 12 months after surgery, 95% of patients had improved Snellen visual acuity, 80% had improved ADVS scores, but only 36% had improved SF-36 physical functioning. Average scores on seven of eight SF-36 subscales worsened at 12 months. Patients with improved ADVS scores had significantly smaller declines across all SF-36 dimensions except for role limitations due to emotional problems. CONCLUSION: Improved visual function after cataract surgery was associated with better health-related quality of life, suggesting that age-related declines in health may be attenuated by improvements in visual function.

Interferon-gamma induces the synthesis and activation of cytosolic phospholipase A2.

Both IFN-alpha/beta and IFN-gamma have recently been demonstrated to induce a rapid but transient activation of phospholipase A2 (PLA2) in BALB/c 3T3 fibroblasts and a human neuroblastoma cell line. We report that IFN-gamma induces the synthesis and prolonged activation of cytosolic phospholipase A2 (cPLA2) in a human bronchial epithelial cell line (BEAS 2B). Treatment of the cells with IFN-gamma (300 U/ml) increased the release of [3H]arachidonic acid (AA) from prelabeled cells with a maximal effect at 12 h after stimulation. The increased [3H]AA release was inhibited by the PLA2 inhibitor p-bromophenacyl bromide (10(-5) M). Calcium ionophore A23187 (10(-5) M) further increased the [3H]AA release from the IFN-gamma-treated cells. Subcellular enzyme activity assay revealed that IFN-gamma increased PLA2 activity in both the cytosol and membrane fractions with a translocation of the cPLA2 to cell membranes in a Ca(2+)-free cell lysing buffer. Treatment with IFN-gamma also induced the release of 15-HETE, an arachidonic acid metabolite. Immunoblot showed that IFN-gamma induced the synthesis of cPLA2 protein. Nuclear run-on assay demonstrated that IFN-gamma initiated cPLA2 gene transcription within 15 min, and this effect was sustained at 4 h and returned to near control level at 12 h. The cPLA2 mRNA level was assayed by reverse transcription and PCR. IFN-gamma was found to increase the cPLA2 mRNA after 2-24 h treatment. Furthermore, the IFN-gamma induced cPLA2 mRNA increase was blocked by inhibitors of protein kinase C and calcium/calmodulin-dependent protein kinases, suggesting the involvement of these protein kinases in IFN-gamma-induced gene expression of cPLA2. This study shows that IFN-gamma induces the synthesis and prolonged activation of cPLA2.

Leukotriene B4 and interleukin-8 in human immunodeficiency virus-related pulmonary disease.

STUDY OBJECTIVE: To investigate the pathogenesis of lung injury in Pneumocystic carinii pneumonia and nonspecific interstitial pneumonitis (NIP), common pulmonary complications of human immunodeficiency virus (HIV) infection. The efficacy of corticosteroid therapy in P carinii pneumonia and the observation that bronchoalveolar lavage (BAL) neutrophilia predicts a poor prognosis support the premise that the lung injury of P carinii pneumonia is due to the host's inflammatory response to the infection. DESIGN: In vitro measurements on previously collected BAL fluid samples. SETTING: The Clinical Center of the National Institutes of Health, a research hospital and tertiary care referral center. PATIENTS: Five normal volunteers, 5 asymptomatic HIV-positive patients, 10 HIV-positive patients with NIP (5 asymptomatic and 5 with respiratory symptoms), and 19 HIV-positive patients with P carinii pneumonia. MEASUREMENTS AND RESULTS: BAL leukotriene B4 (LTB4), interleukin 8 (IL-8), and phospholipase A2 (PLA2) were measured. IL-8 and PLA2 were elevated in patients with P carinii pneumonia, and IL-8 correlated with BAL fluid absolute neutrophil count. LTB4, IL-8, and PLA2 levels were elevated in patients with NIP; LTB4 and PLA2 levels correlated with absolute neutrophil count, and IL-8 correlated with alveolar-arterial oxygen pressure difference. IL-8 was elevated in the asymptomatic HIV-positive patients, and there was a trend toward elevation of PLA2 in this group. CONCLUSION: IL-8 appears to play a role in the pathogenesis of lung injury in P carinii pneumonia and may be the principal neutrophil chemotaxin in this disease; PLA2 may also be involved in the pathogenesis of P carinii pneumonia. Both LTB4 and IL-8 may be involved in the recruitment of neutrophils and subsequent lung injury of NIP. These data suggest that there are varying mechanisms by which inflammatory cells are recruited to the lung in different HIV-related lung diseases.

Production of eicosanoids in response to endothelin-1 and identification of specific endothelin-1 binding sites in airway epithelial cells.

The effect of endothelin-1 (ET-1) on arachidonate metabolism in the respiratory epithelium was investigated in primary cultures of feline tracheal epithelial cells. Subconfluent epithelial cell cultures were stimulated by ET-1, and eicosanoid generation was determined by high performance liquid chromatography (HLPC) of 3H-labeled arachidonic acid (AA) metabolites and by radioimmunoassay (RIA) of corresponding nonradiolabeled HPLC elution. The HPLC chromatograms of [3H]AA-prelabeled samples revealed that ET-1 (10(-5) M) augmented the release of prostaglandin (PG) E2, 12-hydroxyeicosatetraenoic acid (HETE), PGF2 alpha, and AA. RIA of corresponding nonradiolabeled HPLC elution demonstrated a significantly increased release of PGE2, PGF2 alpha, and 12-HETE as well as 5-HETE in response to ET-1 stimulation. 5-HETE release from ET-1-stimulated cells was further identified by gas chromatography/mass spectrometry (GC/MS). The stimulating effect of ET-1 on AA metabolism was dose dependent (10(-5) to 10(-7) M) and peaked within 1 h with a progressive decline over the subsequent hours. Using 125I-labeled ET-1 as radioligand, the presence of specific binding sites for ET-1 was demonstrated in cultured feline tracheal epithelial cells. ET-1 binding reached equilibrium within 1 h at 37 degrees C. Scatchard analysis suggested the existence of two saturable binding sites, with the estimated equilibrium dissociation constant (Kd) of 35.3 pM and maximal binding capacity (Bmax) of 15.0 fmol/10(7) cells for the higher affinity binding site and Kd of 205.9 pM and Bmax of 35.0 fmol/10(7) cells for the lower affinity binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of the 'Activities of Daily Vision Scale'. A measure of visual functional status.

To develop a method for the evaluation of visual function in subjects with cataracts, the authors identified 20 visual activities and categorized them into five subscales (distance vision, near vision, glare disability, night driving, and daytime driving) that comprised the Activities of Daily Vision Scale (ADVS). Each subscale in the ADVS was scored between 100 (no visual difficulty) and 0 (inability to perform the activity because of visual difficulty). In 334 subjects scheduled for cataract extraction (mean age 75 +/- 9 years, 67% women), ADVS scores (mean +/- standard deviation) for each subscale ranged from 44 +/- 31 for night driving to 72 +/- 24 for near vision activities. When administered by telephone, inter-rater reliability coefficients (r) were 0.82 to 0.97 (P < 0.001) for each of the subscales, and test-retest reliability was 0.87 for the scale overall. Cronbach's coefficient alpha was very high for both the in-person (alpha = 0.94) and telephone (alpha = 0.91) formats. Criterion validity, the correlation between visual loss and ADVS score, was -0.37 (P < 0.001) when the ADVS was administered in person and -0.39 (P < 0.001) when it was administered by telephone. Content validity as assessed with factor analysis showed that 88% of the variance of the principal components weighted on one factor. The authors conclude that substantial visual disability is not captured by routine visual testing and that the ADVS is a reliable and valid measure of patient's perception of visual functional impairment.

Reduction of pulmonary surfactant in patients with human immunodeficiency virus infection and Pneumocystis carinii pneumonia.

We assessed qualitative and quantitative differences in surfactant lipid composition of bronchoalveolar lavage (BAL) fluid in patients with acquired immune deficiency syndrome (AIDS) and Pneumocystis carinii (PC) pneumonia. Five normal volunteers and 27 patients with human immunodeficiency virus (HIV) infection underwent BAL for evaluation of possible pulmonary infection. Bronchoalveolar lavage studies in eight patients were negative for PC organisms, and 19 were positive. Pneumocystis carinii pneumonia was graded (mild vs moderate to severe) by initial alveolar-arterial oxygen gradient. Bronchoalveolar lavage fluid was centrifuged, the lipids were extracted from the supernatant, and total lipid profiles of dephosphorylated glycerolipids were analyzed as trimethylsilylether derivatives by high temperature gas-liquid chromatography. Phospholipase A2 levels were determined using a radiolabeled E coli membrane method. Compared to the normal volunteers (109 +/- 13 micrograms/5 ml) and the PC negative group (107 +/- 13 micrograms/5 ml), total BAL lipid was reduced for both the mild PC pneumonia group (73 +/- 10 micrograms/5 ml) and the moderate to severe PC pneumonia group (46 +/- 4 micrograms/5 ml). There was a parallel reduction of diacylglycerol lipids: normal volunteers, 52 +/- 7 micrograms/5 ml; PC negative, 52 +/- 9 micrograms/5 ml; mild PC pneumonia, 35 +/- 7 micrograms/5 ml; and moderate to severe PC pneumonia, 15 +/- 2 micrograms/5 ml. Phospholipase A2 activity in moderate to severe PC pneumonia was twice that of the PC negative patients, and 30 times that for normals. The data demonstrate a marked diminution in surfactant glycerophospholipid in patients with AIDS and PC pneumonia and suggest a potential role for surfactant abnormality in the pathophysiology of this disease.