Currently in the U.S. it is recommended that tuberculosis screening and treatment programs be targeted at high-risk populations. While a strategy of targeted testing and treatment of persons most likely to develop tuberculosis is attractive, it is uncertain how best to accomplish this goal. In this study we seek to identify geographical areas where on-going tuberculosis transmission is occurring by linking Geographic Information Systems (GIS) technology with molecular surveillance.This cross-sectional analysis was performed on data collected on persons newly diagnosed with culture positive tuberculosis at the Tarrant County Health Department (TCHD) between January 1, 1993 and December 31, 2000. Clinical isolates were molecularly characterized using IS6110-based RFLP analysis and spoligotyping methods to identify patients infected with the same strain. Residential addresses at the time of diagnosis of tuberculosis were geocoded and mapped according to strain characterization. Generalized estimating equations (GEE) analysis models were used to identify risk factors involved in clustering.Evaluation of the spatial distribution of cases within zip-code boundaries identified distinct areas of geographical distribution of same strain disease. We identified these geographical areas as having increased likelihood of on-going transmission. Based on this evidence we plan to perform geographically based screening and treatment programs.Using GIS analysis combined with molecular epidemiological surveillance may be an effective method for identifying instances of local transmission. These methods can be used to enhance targeted screening and control efforts, with the goal of interruption of disease transmission and ultimately incidence reduction. Mycobacterium tuberculosis strains (TB), in combination with traditional surveillance, has yielded insights into tuberculosis transmission . One hundred and seventy-one (32.4%) patients were African-American, 165 (31.3%) were Caucasian, 109 (20.7%) were Hispanic, and 82 (15.6%) were Asian. African-Americans with tuberculosis were significantly more likely to have a clustered strain . Alternatively, Asians , and Hispanics  were significantly more likely to have a unique strain. Three hundred and twenty-nine (67.4%) of the patients were males, and of these, 214 (65.0%) had clustered strains; 78 of 159 females (49.1%) had clustered strains. Males were more likely than females to have a strain that matched at least one other person in Tarrant County . Persons with previous experience of homelessness were strongly associated with clustering suggesting a high rate of on-going transmission among this population.  (Table Three hundred and twenty-one (65.7%) patients were born in the United States. Of those, 235 (73.2%) had clinical isolates that matched the isolate from at least one other person living in Tarrant County. One hundred and sixty-seven patients were born outside of the United States. Of those, 57 (34.1%) clinical isolates that matched the isolate from at least one other person living in Tarrant County. U.S. born individuals were significantly more likely to be genotypically clustered than foreign-born counterparts . The birth country of foreign-born patients varied. Of those born outside of the U.S, 77 (46.1%) were born in Latin America, 47 (28.1%) in Southeast Asia, 14 (8.4%) in Sub-Saharan Africa, 12 (7.2%) in Pacific Asia, 11 (6.6%) in South Asia, and 6 (3.6%) in Europe.Evaluation of the spatial distribution of number of cases within zip-code boundaries displayed a distinct geographical distribution of disease. The average incidence for the entire county during the study period was 5.9 cases per 100,000. Zip code 1 recorded the highest incidence of 94.3 cases per 100,000 populations, followed by zip code 2 with an average incidence of 55.2 cases per 100,000 population  occurred in the same zip code with the highest incidence. Similarly, zip code 2 on the southeast border of zip code 1, recorded the second highest proportion of persons with molecular clustered TB isolates with 76.6% of all reported cases clustered. Cases reported in zip code 1 were more than six times as likely  than any other zip code to have isolates that match at least one other person living in Tarrant County.In zip code 3, we observed a morbidity that was more than triple the county average . Unlike other high morbidity areas, zip code 3 had a strong preponderance of unique strain distribution. In this zip code, 17 out of 26 (65.4%) patients had isolates that did not match any other patient in Tarrant County. Cases reported in this zip code were 70% less likely  to have a clustered strain, suggesting that the high rates of tuberculosis did not result from local on-going transmission.The number of cases in the United States is at its lowest point in history, with 15,075 cases reported in 2002 . The rolThe use of molecular strain characterization methods in conjunction with traditional surveillance has led to the recognition of a number of risk factors associated with on-going transmission, and has identified numerous outbreaks of tuberculosis undetected by conventional approaches -27. ThesThese finding are similar to those reported in Los Angeles where locations, specifically homeless shelters were identified as important sites of tuberculosis transmission . SimilarWe identified that 55% of our patients were clustered and 47% attributable to ongoing community transmission. This differs from a study conducted in a high incidence area of South Africa, where 72% of cases were clustered and 58% attributable to ongoing community transmission . Our lowAlthough the majority of the tuberculosis morbidity within the developed world is strongly influenced by imported tuberculosis from high prevalence countries ,36, the There are some limitations to this research approach. This is based on secondary data, which includes variables collected from a cross-sectional period of time. Although each case is an incident case at the time of diagnosis, under this cross-sectional design, exposure and disease outcomes are assessed simultaneously. In addition patients with tuberculosis may have moved shortly before their diagnosis. However, this should not cause systematic error (bias) or result in an association of clustering with specific locations, because these events would be expected to produce a random misclassification. Also persons exposed within certain zip codes may go on to reside elsewhere and later develop the disease, and result in an underestimate of the morbidity and that may be reflected in calculating associations. Finally, genotyping results were not available for a proportion of TB cases in this study. Some unique isolates might have clustered if some of the missing isolates had been available or if other cases with the same strain moved or are located outside the study area . We therWhen using this approach TB control programs must select the appropriate geographical boundary to examine transmission in their area. For example, using zip codes may be too large a boundary in very populated metropolitan areas. Census block groups may provide greater resolution in determining localized transmission.6110 RFLP is recognized as the most discriminatory method for genotyping M. tuberculosis isolates, the discriminatory ability of the technique decreases when there are fewer than 6 IS6110 insertions in the genome. In this case, spoligotyping was used for further strain discrimination. However, it is still possible that some isolates classified as being the same strain based on identical genotypes may represent distantly related, but distinct, strains. Moreover, demonstration that particular patients have the same strain supports, but does not irrefutably prove, direct transmission between these patients as opposed to another source of infection. Conversely, strains continue to evolve, and the resulting genotypic differences over time can result in assigning isolates from cases of direct transmission to distinct strain lineages. Given that a small minority of the isolates had fewer than 6 IS6110 bands (18.2%) or differed by the presence or absence of one band in an otherwise conserved pattern (3.7%), we believe that estimates of the degree of clustering and the size of clusters are conservative.Nor are the molecular techniques used without limitation. Patients are clustered according to their isolates having the same genotype. While ISUsing GIS analysis combined with molecular epidemiological surveillance can be an effective method for identifying tuberculosis transmission not identified during standard contact tracing methods. The application of these methods can be utilized in countries where contact tracing is routinely performed. These methods can enhance targeted screening and control efforts, with the goal of interruption of disease transmission and ultimately incidence reduction. This study demonstrates that using existing health data, GIS can identify previously undetected TB transmission. These results were used to design new targeted screening efforts . StudiesCI Confidence IntervalGEE Generalized Estimating EquationsGIS Geographic Information SystemsHIV Human Immuno-deficiency VirusIDW Inverse Distance WeightingNCTCG North Central Texas Council of GovernmentsOR Odds RatioRFLP Restriction Fragment Length PolymorphismTB TuberculosisTCHD Tarrant County Health DepartmentTDH Texas Department of HealthStudy concept and design: PM, MB, SWAcquisition of data: PM, TQ, KJ, DD, GB, SWAnalysis and interpretation of data: PM, MB, TQ, JO, SWDrafting of the manuscript: PM, SWCritical revision of the manuscript for important intellectual content: SW, PM, JO, TNStatistical expertise: KS, MB, PMObtained funding: TQ, SWAdministrative, technical or material support: GBThis work was supported in part by the Centers for Disease Control and Prevention, National Tuberculosis Genotyping and Surveillance Network Cooperative Agreement U52/CCU600497-18, and Tuberculosis Epidemiologic Studies Consortium 200-2001-00084.


Escherichia coli and other bacteria capable of rapid growth is commonly attributed to selection for high translational power. Conversely, the absence of such a correlation in some slowly growing microbes has been interpreted as the absence of selection for translational power. Because codon bias caused by translational selection varies between rapidly growing and slowly growing microbes, we investigated whether observed differences in translational power among microbes could be explained entirely by differences in the degree of codon bias. Although the data are not available to estimate the effect of codon bias in other species, we developed an empirically-based mathematical model to compare the translation rate of E. coli to the translation rate of a hypothetical strain which differs from E. coli only by lacking codon bias.Translational power is the cellular rate of protein synthesis normalized to the biomass invested in translational machinery. Published data suggest a previously unrecognized pattern: translational power is higher among rapidly growing microbes, and lower among slowly growing microbes. One factor known to affect translational power is biased use of synonymous codons. The correlation within an organism between expression level and degree of codon bias among genes of E. coli and slowly growing microbial species. Using empirical codon-specific in vivo translation rates for 29 codons, and several scenarios for extrapolating from these data to estimates over all codons, we find that codon bias cannot account for more than a doubling of the translation rate in E. coli, even with unrealistic simplifying assumptions that exaggerate the effect of codon bias. With more realistic assumptions, our best estimate is that codon bias accelerates translation in E. coli by no more than 60% in comparison to microbes with very little codon bias.Our reanalysis of data from the scientific literature suggests that translational power can differ by a factor of 5 or more between While codon bias confers a substantial benefit of faster translation and hence greater translational power, the magnitude of this effect is insufficient to explain observed differences in translational power among bacterial and archaeal species, particularly the differences between slowly growing and rapidly growing species. Hence, large differences in translational power suggest that the translational apparatus itself differs among microbes in ways that influence translational performance. Translational power is the rate of protein synthesis of a cell or culture, normalized to the amount of biomass invested in the protein synthesis machinery. We are introducing the term 'translational power' to describe precisely the same concept  that was originally defined as 'ribosome efficiency' -3. In rerate at which a process occurs. Physicists and engineers use a distinct term, 'power,' to refer to the rate of energy consumption or the rate at which work is performed . F. FμP/R iC, implying a constant? The two quantities in the numerator, the mass of RNA in a ribosome and the average mass of an amino acid, are indeed constant or nearly constant, both within a strain at different growth rates, and across strains. However, despite the constant ribosomal fraction of RNA reported in reference [E. coli from 1.7 hr-1 to 0.28 hr-1 [E. coli and a slowly growing strain. The rRNA fraction is reported as 84% for H. cutirubrum at specific growth rates of both 0.10 hr-1 and 0.05 hr-1, after the authors made the deliberately generous assumption that messenger RNA comprises 5% of the total RNA [R. prowazekii at a specific growth rate of ~0.07 hr-1, after a correction is made for 2–3% messenger RNA [What of the term we have labeled eference , other d.28 hr-1 , a resul.28 hr-1 ,65. Thisotal RNA . The rRNnger RNA . These dE. coli and all other bacterial and archaeal species for which we could find both the protein content and the RNA content of cultures growing at known rates. Throughout this work, E. coli is represented by the Bremer and Dennis data [E. coli in many other studies. Similarly, comparisons between E. coli and 2 closely related species of enteric bacteria, S. enterica and E. aerogenes, are made using only a single representative study for the latter strains, chosen from among several published reports. For the remaining species, only a single published study was available for comparison, except for one species represented by two studies, both of which are included. For strains not grown at 37°C, we assume that the growth rate, but not the macromolecular content, would be altered by growth in the same medium at a different temperature [E. coli, the comparison is mathematically identical whether the temperature correction is applied to E. coli or to the comparison strain.)Table nis data , which aperature . The groperature .  One of three measures was used to gauge the level of investment in the translational apparatus, depending on the quantity measured in the original study. The possible measures were the number of ribosomes per cell volume, the ratio of protein to ribosomal RNA, or the ratio of protein to total RNA. Values of these quantities for E. coli were interpolated between adjacent data points to estimate the growth rate at which E. coli made the same investment in the translational apparatus as the comparison organism. The translational power of the comparison organism at the fastest available growth rate was then expressed as a percentage of the translational power of E. coli at the 'same investment' growth rate. A comparison at similar investment levels reflects the expectation that the selective pressure to maximize translational power increases with the biomass invested in the apparatus [E. coli growth rate , the disparities in translational power would be greater for most of the comparisons shown.The comparisons in Table pparatus ,58. If tCi codons of type i are translated during a single cell generation, so that the sum over all sense codons C = ΣCi is the total number of codons translated during a cell generation.  If we define ci = Ci/C as the proportion of all codons of type i in the proteome and ri as the average translation rate of codons of type i, the total time required for replication of the proteome  will beConsider a cell in which a total of R# is the average number of ribosomes active in translation over the cell cycle and the sum is over all sense codons. Codon bias in favor of rapidly translated codons will reduce gp in comparison to uniform codon use. If a mutation changes the fitness of an organism from w to w', the benefit of the mutation is typically described as s, where w'/w = 1 + s. By analogy, and considering gp to be inversely related to fitness, we can express the translation rate benefit of codon bias aswhere C) is the same in Uni as in E. coli by hypothesis. With the restrictive condition that the abundance of each individual component of the translational apparatus is unchanged in Uni, ribosome content (R#) will be the same also. Hence, the C/R# term of gp in Equation (8) cancels from both the numerator and denominator of Equation (9) for sbias, leading toThe protein content  for both Uni and E. coli, again invoking the restrictive stipulation that the abundance of each individual tRNA species is unchanged. If rate constants for the interaction of each codon with each of its cognate tRNA species were known, we could calculate the optimal tRNA abundance distribution for the codon frequencies of Uni, and infer the resulting codon-specific translation rates [in vivo codon-specific translation rate data are available only as codon averages, including translation from all tRNA species cognate to each codon. Hence, rate constants specific to each codon-cognate tRNA pair cannot be calculated from the available data for the codons translated by multiple tRNA species, and thus we cannot calculate an optimal tRNA abundance distribution for Uni. Instead, we have constrained Uni to maintain the same tRNA distribution and codon-specific translation rates as E. coli. Insofar as the E. coli rates reflect an allocation of tRNA abundance that would be sub-optimal for Uni (as we argue in the Discussion section), our approach will tend to overestimate of the benefit of codon bias in E. coli, a conservative error for our purposes.We will use the same codon-specific translation rates  either with Uni having strictly uniform use of synonymous codons, or with Uni assigned a set of low bias codon frequencies Table . The lowpallidum . By relatRNA/Rshift) of Curran and Yarus [in vivo translational kinetics. The original publication transposed values reported for two arginine codons, CGC and CGA [subsequent to CGA [To represent codon-specific translation rates, we use the relative rate data . Following Björk, we assume that 40% of the tRNAs for glutamate, glutamine and lysine with uridine in the anticodon wobble position are modified to mnm5Se2U and thus read codons ending in A or G; the balance of these tRNA species are assumed to have mnm5S2U in the wobble position and read A-ending codons only [et al. [To calculate the total abundance of cognate tRNA for each codon, we assign cognate specificity largely according to Björk , and useet al. . We diffons only . The abu [et al. , since t [et al. . We showE. coli, assuming optimality  according to theory developed by Solomovici et al. [We address the incompleteness of codon-specific translation rate data in several ways. In Scenario 1, we assume that the effects of biased use of YNN codons on translation rate can be used to represent the effects of bias over all codons, without assigning particular translation rates to the unmeasured codons. However, since the YNN codons are almost half of all sense codons but only account for about a third of all expression Table , they mui et al. .sbias. Hence, in Scenario 1 we apply Equation (10) over YNN codons, with the calculated translation time for non-proline YNN codons weighted by a factor of 3.2, which scales the expression level of these codons to the expression level of all non-proline codons. In other words, we assume the effects of codon bias on translation rate among the 25 non-proline YNN sense codons are representative of the effects of codon bias among all 57 non-proline sense codons, whereas the translation rates measured for proline codons are applied only to themselves.The 29 YNN codons encode 10 amino acids, 9 of which have multiple codons. For 7 of these 9 amino acids, the most common synonym is the codon with the fastest translation rate. One of the remaining amino acids is serine, for which the two fastest-translated codons are the two most abundant, although in reverse order, with relatively small differences between the two in both rate and abundance. Only proline appears to be anomalous; the 2 most abundant codons encode over 90% of all proline residues in the proteome , but supet al. [2 = 0.56) of a quadratic relationship passing through the origin between the codon frequency and translation rate data of Table ci = 0.205 ri - 0.522 ri2. We use this equation to predict translation rates from codon frequency for all RNN codons, as shown in Table Curran and Yarus noted that among highly expressed genes, there is a significant tendency for rapidly-translated codons to be used frequently, although the relationship appears to be nonlinear . We obseet al.  at the hci = 0.352 ri - 1.611 ri2 (R2 = 0.81). Among the 10 preferred codons, we include UGG, the sole tryptophan codon, and UUG, the preferred leucine codon within the UUR split box although not the preferred leucine codon overall. We then apply this equation to predict translation rates from codon frequencies for 12 preferred RNN codons, including AUG, the sole methionine codon, and AGG and AGC, the preferred arginine and serine codons within their respective split boxes, although not the preferred codons overall. For non-preferred RNN codons, translation rate is predicted by multiplying the predicted rate for the preferred synonym (within the full or split box) by the ratio of the square roots of the codon frequencies for the non-preferred and preferred codons:The preceding scenario applied to the YNN codons tends to predict translation rates among synonymous alternatives that are not as disparate as those actually observed. Furthermore, the fit of a functional relationship between codon frequency and translation rate among YNN codons is better when only preferred codons are considered, instead of all codons. Hence, we fit a quadratic relationship passing through the origin to data from 10 preferred non-proline YNN codons, obtaining 2 = 0.57). The predicted rates for RNN codons and the measured rates for YNN codons  are assigned the slowest relative translation rate observed among YNN codons (ri = 0.6 for the rare leucine codon CUA). Second, the translation rates for preferred RNN codons within full or split boxes (except AGG) are estimated according to the regression equation described for Scenario 3. Finally, the translation rates for non-preferred codons (except AGA and AUA) are predicted from the preferred synonym using the ratios of the most disparate translation rates observed empirically among synonymous alternatives, treating split boxes and full boxes of the translational code separately. The most extreme ratio observed among translation rates in a split box is 3.375, for glutamate codons in the study of Sorensen and Pedersen [x ≤ y ≤ 24 in ratios of the form 1:x:y:24 failed to find any that greatly increased the estimated benefit beyond that using the leucine ratios, data not shown.) Although this scenario is based on extreme observations, applying these 3 rules to the non-proline YNN codons leads to a correlation of predicted and measured translation rates (R2 = 0.67) somewhat better than that obtained under Scenario 2 or Scenario 3. The predicted rates for RNN codons and the measured rates for YNN codons . However, the empirical codon frequencies of Dong et al. [2 = 0.70) than with the empirical relative rates of Curran and Yarus [2 = 0.31). A good correlation between the predicted translation rates and the empirical codon frequencies is expected, since the codon frequencies were used to generate the predictions. However, the poor correlation between predicted and empirical translation rates could reflect the inadequacies in any of 3 areas: 1) the assumptions of Solomovici et al. [et al. [E. coli is not perfectly optimized for maximal translation rates  the i et al. , and/or eference ), eitherNonetheless, the disparity between the relative rates of synonymous preferred and non-preferred codons for most amino acids are greater with the predicted rates of Scenario 5 than with the observed rates. Hence, Scenario 5 will generate a higher estimate of the translation rate benefit of codon bias than would a strict application of the empirical codon-specific translation rates.  The predicted translation rates for all codons Table  are usedLD conceived of the project, collected and analyzed the data, developed the mathematical model, and drafted the manuscript. TMS helped plan the project, critiqued the work as it progressed, and edited the manuscript.


This study tested the hypothesis that human macrophages have the ability to modify oxidation products in LDL and oxidized LDL (oxLDL) via a cellular antioxidant defence system. While many studies have focused on macrophage LDL oxidation in atherosclerosis development, less attention has been given to the cellular antioxidant capacity of these cells.Compared to cell-free controls (6.2 ± 0.7 nmol/mg LDL), macrophages reduced TBARS to 4.42 ± 0.4 nmol/mg LDL after 24 h incubation with LDL (P = 0.022). After 2 h incubation with oxLDL, TBARS were 3.69 ± 0.5 nmol/mg LDL in cell-free media, and 2.48 ± 0.9 nmol/mg LDL in the presence of macrophages (P = 0.034). A reduction of lipid peroxides in LDL (33.7 ± 6.6 nmol/mg LDL) was found in the presence of cells after 24 h compared to cell-free incubation (105.0 ± 14.1 nmol/mg LDL) (P = 0.005). The levels of lipid peroxides in oxLDL were 137.9 ± 59.9 nmol/mg LDL and in cell-free media 242 ± 60.0 nmol/mg LDL (P = 0.012). Similar results were obtained for hydrogen peroxide. Reactive oxygen species were detected in LDL, acetylated LDL, and oxLDL by isoluminol-enhanced chemiluminescence (CL). Interestingly, oxLDL alone gives a high CL signal. Macrophages reduced the CL response in oxLDL by 45% (P = 0.0016). The increased levels of glutathione in oxLDL-treated macrophages were accompanied by enhanced catalase and glutathione peroxidase activities.Our results suggest that macrophages respond to oxidative stress by endogenous antioxidant activity, which is sufficient to decrease reactive oxygen species both in LDL and oxLDL. This may suggest that the antioxidant activity is insufficient during atherosclerosis development. Thus, macrophages may play a dual role in atherogenesis, i.e. both by promoting and limiting LDL-oxidation. Oxidative modification of low density lipoprotein (LDL) plays a major role in the pathogenesis of atherosclerosis. The first stage of atherogenesis is characterized by an influx and accumulation of LDL in the intima, followed by recruitment of blood-derived monocytes and lymphocytes to the developing lesion . SubsequHowever, cells may also provide a protective effect by reducing oxidation products present in LDL and oxLDL. Murine macrophages effectively block LDL oxidation by mechanisms which include metal ion sequestration . Recent Four selenium-dependent glutathione peroxidases (GPx) have been identified sofar: cytosolic GPx (cGPx), gastrointestinal GPx (GI-GPx), plasma GPx (pGPx), and phospholipid hydroperoxide GPx (PHGPx) ,13. The This study tested the hypothesis that human macrophages have the ability to modify oxidation products in LDL and oxLDL. We also analyzed the activity of cellular antioxidant defenses such as catalase, glutathione peroxidase, and superoxide dismutase in these cells. We used early macrophages as cell culture model to mimic newly recruited macrophages into the intima.6 cells per well. Non-adherent cells were removed after 1 h. RPMI 1640 containing 100 μg/mL LDL or oxLDL was incubated at 37°C in 5% CO2 in the presence or absence of macrophages. By definition, monocytes are denoted macrophages when they are attached, thus the cells used in this study are considered early human monocyte-derived macrophages (HMDM).Mononuclear cells were isolated by the Ficoll-Hypaque procedure   from buf6 cells/mL in Krebs-Ringer Bicarbonate buffer supplemented with glucose (KRG) . To obtain non-viable macrophages, cells were stored at +4°C for 16 h. Trypan blue exclusion test confirmed that 100% of the cells were non-viable.For chemiluminescence experiments, monocytes were allowed to adhere to cell culture flasks for 1 h. Adhered macrophages were then detached by incubation with PBS containing 5 mM EDTA and 2% fetal calf serum for 20 minutes at +4°C . Cells w4/mg LDL. Oxidation was terminated through the addition of 0.5 mmol/L EDTA. The oxLDL was purified on a PD-10 column with PBS as elution buffer and sterilized by filtration through a 0.22 μm filter. Native LDL was acetylated as described [Fresh human EDTA-plasma was obtained from healthy male donors after overnight fasting. LDL (density 1.019–1.063 g/L) was isolated by sequential ultracentrifugation . Before escribed . Oxidati5 cells. As a specific inhibitor of hydrogen peroxide, 30 μg catalase (Roche AB) was added in some experiments to oxLDL without cells.The chemiluminescence (CL) assay was performed at 37°C and the CL detected for at least 100 minutes with a luminescence counter  . The CL Thiobarbituric acid-reactive substances (TBARS) were determined by the method of Yagi . Fluores2O2eq) were analyzed in the LDL-containing media incubated with or without macrophages. The assay is based on the oxidation of ferrous ions to ferric ions by hydrogen peroxide at acidic pH . The ferric ion binds to the indicator dye xylenol-orange to form a stable complex which is measured at 560 nm.Levels of hydrogen peroxide equivalents (HApolipoprotein B (Apo B) concentration was determined in cell culture media by immunoprecipitation enhanced by polyethylene glycol at 340 nm . ApoB analyses were performed on a Konelab 20 autoanalyser .The intracellular levels of glutathione and the activity of GPx were measured in crude extracts from macrophages incubated with either LDL or oxLDL. The cells were washed twice with ice-cold PBS and harvested in 0.5 mL lysis buffer. For glutathione peroxidase measurements, this buffer contained 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1 mM dithiothreitol. For glutathione measurements, the cells were lysed in 0.5 mL ice-cold 5% metaphosphoric acid. The lysate was spun down at 3000 × g, and the supernatants stored at -80°C.The presence of glutathione peroxidase was determined with a colorimetric assay (Bioxytech GPX-340) and glutathione (GSH) was measured with Bioxytech GSH-400, both from OXIS International Inc. The assay GPX-340 measures the functional activity of the GPx. In functional terms, all four types of GPx  appear similar in catalytic activity ,25.GPx----> ROH + GSSG(oxidized glutathione)+ H2OROOH + 2GSH ----+ + H+ --------> 2GSH + NADP+GSSG + NADPH2O2 was measured in cell lysates at 240 nm. One unit of catalase activity was defined as the rate constant for the reaction using purified catalase (Roche AB) as standard.Catalase activity in macrophages was quantified by the method of Aebi . DecompoIn the cell lysates, the CuZn-superoxide dismutase (SOD) and Mn-SOD enzymatic activities were measured with a direct spectrophotometric method . ExtraceData were expressed as means ± standard error. Statistical analysis was performed using Student's paired t-test or ANOVA. P values < 0.05 were considered statistically significant.2O2 in LDL-containing culture media after 2 h and 24 h incubation with or without macrophages.To study the effect of macrophages on LDL oxidation, we measured TBARS, LPO, and H2O2eq increase with time from 116 ± 31 nmol/mg LDL after 2 h, to 270 ± 46 nmol/mg LDL after 24 h (P = 0.009) , suggesting both LDL and oxLDL are oxidized during culture conditions. H2O2eq is significantly decreased in both LDL (P = 0.038) and oxLDL (P = 0.035) after incubation with macrophages. In macrophage-treated LDL, the H2O2eqcontent is decreased to 14.8 ± 2.9 nmol/mg LDL after 24 h, which is similar to levels found in non-treated LDL (0 h). Taken together, these results suggest a cell-mediated loss of oxidation products in both LDL and oxLDL in the presence of macrophages.Compared to cell-free controls (6.2 ± 0.7 nmol/mg LDL), there was a significant reduction of TBARS by macrophages to 4.42 ± 0.4 nmol/mg LDL after 24 h incubation with LDL (P = 0.022) Fig. . After 209) Fig. . Levels Apo B was analysed to study if the cell mediated decrease in oxidation products was due to a decrease of LDL or oxLDL in the cell culture media. In the presence of macrophages, the content of apo B in native LDL is reduced by 7 % after 2 h (P = 0.013) and 11 % after 24 h (P = 0.025) Fig. . In the 5cells) or without cells at 37°C. OxLDL alone had a CL response of approximately 400 mV after 2 h   and GPx (P = 0.0002) Figs  and 6B. 48) Fig. .Neither LDL nor oxLDL significantly affect the CuZn-SOD or Mn-SOD activity in macrophages Table . However2O2 participate in the oxidation of LDL [in vitro. We have recently shown that hypoxia enhances both macrophage-mediated LDL oxidation and the expression of the putative LDL-oxidizing enzyme 15-lipoxygenase-2 [Oxidation of LDL is a crucial event in the pathophysiology of atherosclerosis. Reactive oxygen species such as Hn of LDL . Althouggenase-2 . While m2O2eqcontent decreases 64% in cell culture media after 24 h compared to cell-free controls. These results are in agreement with an earlier study where human macrophages reduce the content of cholesteryl ester hydroperoxides in LDL by 43% [2O2 both in LDL and oxLDL. Our data also suggest that oxidation products accumulate in LDL and in oxLDL during regular cell culture conditions in the absence of cells. This has to be taken into consideration when cell culture data using LDL are interpreted.This study shows that macrophages play an important role in limiting lipid oxidation products that accumulate in LDL and oxLDL. Since early macrophages are used in this study, this may resemble newly recruited macrophages entering into the arterial intima. Regarding oxLDL, macrophages decrease TBARS levels by about 30%, LPO decreases 43%, and the HL by 43% . When thThe chemiluminescence technique is generally used to detect cellular ROS production. The amplifying molecule isoluminol reacts with ROS to produce an excited state intermediate that emits light upon relaxation to the ground state . Our dat2O2 or soluble alkyl peroxides, by coupling its reduction of H2O with oxidation of glutathione [2O2-induced oxidation and activation of NFκB in transfected rabbit smooth muscle cells [in vivo relevance, since an increased activity of glutathione peroxidase is found in the artery wall of cholesterol-fed rabbits [There are a number of cellular defences against oxidative stress, such as superoxide dismutase, catalase, and glutathione-related enzymes. In this study we sought to define the antioxidative activity of macrophages. All glutathione peroxidases reduce Htathione . We fountathione . Interesle cells . Our res rabbits .2O2 and lipid peroxides induce catalase gene expression in cultured rabbit endothelial cells, rabbit macrophages, and human smooth muscle cells [Catalase is a representative antioxidant enzyme and previous studies show that oxidants such as Hle cells . This stle cells . AdditioEC-SOD occurs in high concentration in both non-diseased  and atheCells can tolerate mild oxidative stress, which triggers the antioxidant defence system in an attempt to restore the oxidant- antioxidant balance . We did in vivo. Thus, it is evident that macrophages play a dual role in atherogenesis, i.e. both by promoting and limiting LDL-oxidation. It remains to be determined during which stage of lesion development these individual characteristics pertain.This study suggests that oxidative stress induced by oxLDL could be balanced by a cellular antioxidant defence by newly recruited macrophages to sites of LDL oxidation. As oxidation of LDL is implicated in the development of atherosclerosis, and oxidation products of LDL are present in advanced atheromatous lesions, this may suggest that the antioxidant activity is insufficient A strategy to intervene with the development of atherosclerosis would be to increase the endogenous intracellular antioxidant capacity of cells, which would remove and detoxify oxidized LDL. Interestingly, recent results show that Lovastatin increases hepatic catalase activity in cholesterol-fed rabbits . In lighAll authors have contributed to the design of the study, the data analysis, and the writing of the manuscript. The final version of the manuscript has been read and approved by all authors prior to submission. Each author's specific contribution was as follows; LMH.: study design, macrophage cell culture, CL analysis, LDL treatment. C.U.: macrophage cell culture, TBARS, LPO, GpX, and catalase analyses. A.K.: data interpretation, writing, and editing. D.v.R: CL analysis. S.L.M: SOD analyses. C.D.: setting up the CL method. O.W.: study coordination and data interpretation.


Transforming growth factor beta (TGFβ), a multifunctional cytokine, plays a crucial role in the accumulation of extracellular matrix components in lung fibrosis, where lung fibroblasts are considered to play a major role. Even though the effects of TGFβ on the gene expression of several proteins have been investigated in several lung fibroblast cell lines, the global pattern of response to this cytokine in adult lung fibroblasts is still unknown.We used Affymetrix oligonucleotide microarrays U95v2, containing approximately 12,000 human genes, to study the transcriptional profile in response to a four hour treatment with TGFβ in control lung fibroblasts and in fibroblasts from patients with idiopathic and scleroderma-associated pulmonary fibrosis. A combination of the Affymetrix change algorithm (Microarray Suite 5) and of analysis of variance models was used to identify TGFβ-regulated genes. Additional criteria were an average up- or down- regulation of at least two fold.nuclear co-repressor 2, SMAD specific E3 ubiquitin protein ligase 2 (SMURF2), bone morphogenetic protein 4, and angiotensin II receptor type 1 (AGTR1), and confirmed the microarray results by real time-PCR. Western Blotting confirmed induction at the protein level of AGTR1, the most highly induced gene in both control and fibrotic lung fibroblasts among genes encoding for signal transduction molecules.Exposure of fibroblasts to TGFβ had a profound impact on gene expression, resulting in regulation of 129 transcripts. We focused on genes not previously found to be regulated by TGFβ in lung fibroblasts or other cell types, including Upregulation of AGTR1 occurred through the MKK1/MKK2 signalling pathway. Immunohistochemical staining showed AGTR1 expression by lung fibroblasts in fibroblastic foci within biopsies of idiopathic pulmonary fibrosis.This study identifies several novel TGFβ targets in lung fibroblasts, and confirms with independent methods the induction of angiotensin II receptor type 1, underlining a potential role for angiotensin II receptor 1 antagonism in the treatment of lung fibrosis. Transforming Growth Factor beta (TGFβ) is a multifunctional cytokine that regulates a variety of physiological processes, including cell growth and differentiation, extracellular matrix production, embryonic development and wound healing . AlteredIn the lung, TGFβ is consistently linked with progressive fibrosis -5. IncreLung fibroblasts are the main cell type responsible for excessive extracellular matrix synthesis and deposition in fibrosing lung disorders . TGFβ moangiotensin II receptor type 1, the most highly TGFβ-induced gene among those encoding for signal transduction molecules.We therefore quantified gene expression by oligonucleotide microarrays of adult lung fibroblasts  in response to TGFβ, and identified several novel TGFβ targets among the wide variety of genes regulated by this cytokine. Of these, we particularly focused on Primary adult lung fibroblasts were cultured from three control samples (unaffected lung from patients undergoing cancer-resection surgery) and from open-lung biopsy samples of lung fibrosis patients, three with idiopathic pulmonary fibrosis (IPF)  and threAt the end of the treatment period with or without TGFβ, total RNA was harvested , quantified, and integrity was verified by denaturing gel electrophoresis.Preparation of RNA samples for chip hybridization followed Affymetrix  protocols. Each RNA sample derived from an individual fibroblast line was hybridized on a separate microarray chip. Hybridization of cRNA to Affymetrix human U95Av2 chips, containing approximately 12,000 well characterized human genes, signal amplification and data collection were performed using an Affymetrix fluidics station and chip reader, following Affymetrix protocol. Scanned files were analyzed using Affymetrix Version 5.0 software (MAS5). Chip files were analyzed by scaling to an average intensity of 150 per gene, as recommended by Affymetrix. Reproducibility was assessed using two pairs of RNA samples from the same control line, TGFβ-treated/untreated; the concordance correlation coefficients were of 0.979 and 0.983, respectively.TGFβ response was analyzed by using a combination of the MAS5 Affymetrix change algorithm and of ANOVA models. According to Affymetrix criteria, in each TGFβ-treated/medium only pair, genes were defined as differentially regulated (either up or down) by TGFβ only when identified as significantly increased (I) or decreased (D) as determined by the Affymetrix change algorithm, with a change p value<0.001, and were detected as Present  at least in the samples with the highest count (i.e. medium only in the case of D and TGFβ in the case of I). Genes were defined as TGFβ-responsive in normal human lung fibroblasts when they fulfilled all of the following three conditions: a) they were detected as TGFβ-regulated by Affymetrix criteria (see above) in at least two of the three control pairs; b) they showed a mean fold change after TGFβ of at least 2 (or lower than 0.5) in control fibroblasts; c) either a two-way ANOVA including only control fibroblasts detected a significant (p < 0.05) increase or decrease in control fibroblasts after TGFβ or they were also found to be responsive in at least four of the six fibrotic fibroblast lines and a significant effect (p < 0.05) of treatment (with TGFβ) was detected by a repeated measure ANOVA model including all the samples and adjusting for individual samples, disease, and interaction between treatment and disease. All statistical analyses were performed on log transformed data to reduce inequalities of variance. Thus, the latter ANOVA model could detect genes which were equally up- or down-regulated in normal and fibrotic fibroblasts, taking advantage of the larger number of samples, while the first model  could detect changes possibly occurring in controls but not in fibrotic cell lines.Except for unknown genes, all gene symbols and names are given according to the nomenclature proposed by the Human Genome Organization (HUGO) Gene Nomenclature Committee.18) and random decamers (dN10) using M-MLV reverse transcriptase (Promega) for 1 hour at 37°C. The cDNA was diluted to 100 μl with DEPC-treated water and 1 μl was used per real-time PCR reaction. A set of eight standards containing a known concentration of target amplicon was made by PCR amplification, isolation by gel electrophoresis through a 2% agarose gel followed by gel purification using QIAquick PCR purification spin columns (Qiagen). The concentration of the amplicon was measured by spectrophotometry and diluted in DEPC-treated water containing transfer RNA (10 μg/ml) to make standards of 10 fold dilutions from 100 pg/ μl to 0.01 fg/ μl. The target was measured in each sample and standard by real-time PCR using FastStart DNA Master SYBR Green (Roche Applied Science) as described by the manufacturer, in half the reaction volume (10 μl). Samples and standards were amplified for 30 to 40 cycles with the appropriate primers  at least in duplicate. The amount of target in the sample in picograms was read from the standard curve and values were normalised to 28S ribosomal RNA . The oligonucleotide primer sequences are listed (5'-3'): angiotensin II receptor type1 (AGTR1) primers: forward TGC TTC AGC CAG CGT CAG TT and reverse GGG ACT CAT AAT GGA AAG CAC; SMAD specific E3 ubiquitin protein ligase 2 (SMURF2): forward AAC AAG AAC TAC GCA ATG GGG and reverse GTC CTC TGT TCA TAG CCT TCT G; nuclear receptor co-repressor 2 (NCOR2): forward CAG CAG CGC ATC AAG TTC AT and reverse GTA ATA GAG GAC GCA CTC AGC; bone morphogenetic protein 4 (BMP4) primers: forward CTA CTG GAC ACG AGA CTG GT and reverse GAG TCT GAT GGA GGT GAG TC.Real time PCR (RT-PCR) was performed to confirm selected novel TGFβ targets in lung fibroblasts. Adult lung fibroblast lines [three control and three fibrotic (IPF)] were treated with or without TGFβ (4 ng/ml) for four hours. Total RNA was isolated from treated and untreated samples using Trizol (Life Technologies) and the integrity of the RNA was verified by gel electrophoresis. Total RNA (1 microgram) was reverse transcribed in a 20 μl reaction volume containing oligonucleotide dTs  were serum-deprived overnight, and exposed to either 4 ng/ml of activated TGF-β1 (R&D Systems) or serum-free culture-medium with the addition of 0.1% BSA for 24 hours. To determine the signalling pathways through which TGFβ induces AGTR1, lung fibroblasts were treated with specific inhibitors 30 minutes before treatment with TGFβ. These included the dual MKK1/MKK2 inhibitor U0126 (10 μM) and predominant MKK1 inhibitor PD98059 (50 μM), known to inhibit MKK2 only weakly , as wellThe distribution of staining for AGTR1 was assessed by immunohistochemistry in surgical lung biopsies from four patients with idiopathic pulmonary fibrosis (IPF), meeting the diagnostic criteria of the American Thoracic Society/European Respiratory Society Consensus Classification , and in 2O2 as substrate . Sections were then washed in tap water, counterstained with Carrazzis hematoxylin, and mounted with Gelmount  for examination using an Olympus BH-2 photomicroscope. Controls included an exchange of primary antibodies with goat matched antibodies. To confirm staining specificity, sections were also incubated with either nonimmune rabbit IgG control or secondary antibody only.Slides were then briefly rinsed in PBS, blocked with 10% normal goat serum for 20', incubated with rabbit polyclonal anti-human AGTR1 antibody  for one hour at room temperature. After washing with PBS, sections were incubated with biotinylated goat anti-rabbit IgG diluted in PBS (1:200) for 30 minutes, rinsed, and finally incubated with Vectastain Elite STR-ABC reagent (Vector Laboratories) for 30 minutes. After washing, sections were visualized using 3-amino-9-ethylcarbazole chromogen and H.According to the criteria outlined in the methods, a four hour treatment with TGFβ was found to regulate 129 transcripts in human lung fibroblasts. TGFβ-responsive transcripts included genes with roles in gene expression, matrix formation, cytoskeletal remodelling, signalling, cell proliferation, protein expression and degradation, cell adhesion and metabolism. A complete list of TGFβ-regulated genes is provided see . The comWe did not observe a substantial degree of difference in the response to TGFβ between the two fibrotic groups (idiopathic pulmonary fibrosis and scleroderma-associated pulmonary fibrosis) and control lung fibroblasts. Once the criteria outlined in the methods section and the p-value for interaction with treatment had been taken into account, there were no significant differences in the response to TGFβ among the three groups except for two genes, KIAA0261 (probe N: 40086_at), an unknown gene more upregulated in IPF (median fold change 2.2) than in scleroderma-associated pulmonary fibrosis (1.5) and in controls (1.3), and BTG1 (probe N: 37294_at), which was only slightly more downregulated in scleroderma-associated pulmonary fibrosis (fold change:0.4) than in IPF (0.6) and in controls (0.7). As both the number of genes and the magnitude of the differences were minimal, they were not considered meaningful and were not investigated further. Among genes responding significantly to TGFβ in control lung fibroblasts, as assessed by ANOVA analysis, none changed in opposite directions in either of the fibrotic groups. All the genes that responded significantly in the control group alone, were also TGFβ-responsive when analysis was extended to include the fibrotic cell lines. Furthermore, none of these genes responded differently to TGFβ between the two fibrotic groups, which are thus presented together in Tables For the purpose of this study, we will concentrate on genes involved in transcriptional regulation, cytoskeletal/extracellular matrix organization, and signal transduction Tables  and 2.JUNB. Other TGFβ targets in lung fibroblasts identified by this study included Smad co-activators RUNX1 and CBFB, recently implicated in the targeted subnuclear localization of TGFβ-regulated Smads [FOXO1A, NPAS2, and TIEG (TGFβ-inducible early growth response), while ZFP36L2, a zinc finger transcription factor linked to cell proliferation induction, was repressed by TGFβ. Serum response factor (SRF) and MKL1 were also induced by TGFβ. Transcriptional repressors induced by TGFβ included Ski, which together with Sno interacts with Smad molecules to inhibit transcription and may contribute to terminating TGFβ response [TCF8, a previously reported TGFβ target in fetal lung fibroblasts [NCOR2 (or SMRT) and BHLHB2, which repress transcription by recruiting histone deacetylases [musculin (MSC).TGFβ regulated a wide array of transcription factors Table , includied Smads ,23. Tranresponse  and TCF8roblasts . Other tetylases , and musconnective tissue growth factor (CTGF) were upregulated, while we observed inhibition of bone morphogenetic protein 4 (BMP4), a member of the TGFβ superfamily whose activity has recently been shown to be inhibited by CTGF through direct binding [Most genes in this category were known TGFβ targets. As expected, transcripts involved in promoting extracellular matrix formation and cell adhesion such as  binding .elastin (ELN), collagens (COL4A1), plasminogen activator inhibitor (PAI1 or SERPINE1) and PLOD2, an enzyme which stabilizes collagen cross-links  was upregulated by TGFβ. Genes involved in cytoskeletal organization induced by TGFβ included known target tropomyosin (TPM1). Interestingly, smoothelin, a smooth muscle gene recently reported to be highly induced by TGFβ in fetal lung fibroblasts [TGFβ also induced matrix genes including roblasts , was alsSMAD7 and downregulation of SMAD3 [SMURF2, a recently identified E3 ubiquitin ligase, which negatively regulates TGFβ signalling by targeting both TGFβ receptor-Smad7 complexes and Smad2 for ubiquitin-dependent degradation [betaglycan, a membrane anchored proteoglycan which increases the affinity between TGFβ and type I and II receptors. Interestingly, TGFβ upregulated activin A type I receptor, a receptor for TGFβ family member activin, whose stimulation induces fibroblast-mediated collagen gel contraction [ARHB and RADD (Ras-related GTP-binding protein), involved in cytoskeleton remodelling, were also upregulated by TGFβ. TGFβ also induced Dickkopf1 (DKK1), a potent inhibitor of Wnt/beta-catenin signalling.Among signalling molecules Table , known tof SMAD3 ,16. Noveradation ,28. At ttraction . Membersangiotensin II receptor 1 (AGTR1) in lung fibroblasts; conversely, the gene encoding for vasodilatory peptide adrenomedullin (ADM) was inhibited by TGFβ.Of particular interest was the novel observation that TGFβ upregulated JUN-B, SMAD7, connective tissue growth factor, elastin, and SERPINE1 [angiotensin II receptor type 1 (AGTR1), SMURF2, a gene involved in terminating TGFβ signalling, NCOR2, a transcriptional co-repressor and BMP4, a member of the TGFβ family. Compared to untreated samples, we confirmed that TGFβ upregulated AGTR1 , SMURF2, , NCOR2 , and downregulated BMP4 , with no difference in the response between control and fibrotic fibroblasts  , as its involvement is likely to significantly amplify the pro-fibrotic actions of TGFβ. The ligand for this receptor is angiotensin II, a vasoactive peptide which has been linked to fibrogenesis in the kidney and in the heart [Among the novel TGFβ targets identified by microarray analysis in lung fibroblasts, we focused our attention on the induction of he heart ,40. Recehe heart ,42. Elevhe heart . Comparehe heart ,43. Bloche heart . Intereshe heart .In addition to Smad molecules, the classic signalling pathway used by TGFβ family members, TGFβ also signals through the mitogen-activated protein kinase (MAPK) signalling pathways . In thisThe functional effects of AGTR1 stimulation in lung fibroblasts are only partially known. Although two isoforms of angiotensin II receptor exist, AGTR1 and AGTR2, the effects described so far of angiotensin II on lung fibroblasts are ascribed to the type 1 receptor. AGTR1 has been found to mediate mitogenesis in human lung fibroblasts  and extrAGTR1 was found to localize to fibroblasts within fibroblastic foci in IPF/UIP biopsies. An increase in AGTR1 staining has been reported in the fibrotic regions surrounding the bronchioles in chronic obstructive pulmonary disease . The finOur findings confirm that in response to TGFβ, both control and fibrotic lung fibroblasts are potent effector cells expressing a very wide range of genes that are likely to contribute to the fibrotic process. In particular, we have shown that TGFβ has the capacity to influence the expression of angiotensin II receptor type 1 both at the mRNA and at the protein level. In view of the known induction of TGFβ by angiotensin II , our finEAR participated in the design and interpretation of the study, carried out the cell culture work and participated in the microarray work, performed immunohistochemistry staining, and drafted the manuscript. DJA participated in the design and coordination of the study and in the preparation of the manuscript, SH performed the RT-PCR assays, XSW carried out the Western Blot analysis, PS performed the statistical analysis and participated in the interpretation of results and preparation of the manuscript, GBG participated in the microarray work, AUW participated in the interpretation of results, SV participated in cell line selection and clinical characterization, AGN reviewed fibrotic lung biopsies and interpreted immunohistochemistry staining, CD and CMB contributed towards the overall organizational setup for the study of lung fibroblast lines and participated in the interpretation of results, AL and JDP participated in the preparation of the manuscript, KIW conceived of the study and participated in the design, RdB participated in study design, interpretation and coordination. All authors read and approved the final manuscript.Complete list of genes regulated by a four hour treatment with TGFβ in control and fibrotic fibroblasts This data set contains all the genes up- or down-regulated by a four hour treatment with TGFβ (according to the criteria described in the methods) in control and fibrotic lung fibroblasts. Fibrotic lung fibroblast fold ratios are the average of the fold ratios for lung fibroblasts from idiopathic pulmonary fibrosis and pulmonary fibrosis associated with systemic sclerosis. Genes are sub-grouped into functional classes. Affymetrix probe set numbers, approved gene symbols, gene names and GenBank accession numbers are provided in the table.Click here for file


Asthma is characterized by type 2 T-helper cell (Th2) inflammation, goblet cell hyperplasia, airway hyperreactivity, and airway fibrosis. Monocyte chemoattractant protein-1 (MCP-1 or CCL2) and its receptor, CCR2, have been shown to play important roles in the development of Th2 inflammation. CCR2-deficient mice have been found to have altered inflammatory and physiologic responses in some models of experimental allergic asthma, but the role of CCR2 in contributing to inflammation and airway hyperreactivity appears to vary considerably between models. Furthermore, MCP-1-deficient mice have not previously been studied in models of experimental allergic asthma.Aspergillus antigen-induced model of Th2 cytokine-driven allergic asthma associated with airway fibrosis to mice deficient in either MCP-1 or CCR2. Previous studies with live Aspergillus conidia instilled into the lung revealed that MCP-1 and CCR2 play a role in anti-fungal responses; in contrast, we used a non-viable Aspergillus antigen preparation known to induce a robust eosinophilic inflammatory response.To test whether MCP-1 and CCR2 are each required for the development of experimental allergic asthma, we applied an Aspergillus antigen. Surprisingly, mice deficient in either MCP-1 or CCR2 had responses to Aspergillus antigen similar to those seen in wild-type mice, including production of Th2 cytokines.We found that wild-type C57BL/6 mice developed eosinophilic airway inflammation, goblet cell hyperplasia, airway hyperreactivity, elevations in serum IgE, and airway fibrosis in response to airway challenge with We conclude that robust Th2-mediated lung pathology can occur even in the complete absence of MCP-1 or CCR2. Monocyte chemoattractant protein-1  and its receptor, CCR2, have been the focus of intense interest due to increasing awareness of their association with debilitating human diseases, including asthma  and pulmAspergillus fumigatus conidia to A. fumigatus sensitized mice. Airway fibrosis was significantly increased in mice treated with MCP-1 neutralizing antibody and in CCR2-deficient mice. However, these increases in fibrosis were seen in the setting of impaired clearance of conidia and a markedly increased neutrophilic inflammatory response, suggesting that the increased fibrosis might be attributable simply to an impaired antifungal response. Previous studies involving other models of allergic asthma applied to CCR2-deficient mice did not examine whether airway fibrosis occurred in these models or whether development of fibrosis was dependent on CCR2 expression [In addition to Th2 inflammation, airway fibrosis is another important feature of human asthma. Blease and colleagues ,29 examipression -27. ConsAspergillus antigen model of Th2-cytokine-driven allergic asthma associated with significant airway fibrosis and measured pulmonary inflammation, cytokine production, AHR and fibrosis.In this study, we hypothesized that the effects of MCP-1 are mediated through CCR2 and that MCP-1 and CCR2 are independently required for the development of experimental allergic asthma. To test this hypothesis, we subjected mice deficient in either MCP-1 or CCR2 to an Mcp-1+/+ and Mcp-1-/- mice [Ccr2+/+ and Ccr2-/- mice [Mcp-1 or Ccr2 genes was confirmed by PCR. Similar numbers of male and female six-week-old mice were used for the study. The UCSF Institutional Animal Care and Use Committee approved all experimental protocols.Breeding pairs of -/- mice  and Ccr2-/- mice  were genAspergillus fumigatus antigen preparation consisted of a mixture of culture filtrate (300 μg protein/mouse) and mycelial extract (80 μg protein/mouse) in PBS . Culture filtrates and mycelial extract were prepared as described previously [Aspergillus antigen intranasally five times at four-day intervals. Control mice were given 50 μl of PBS according to the same schedule as Aspergillus antigen-treated mice. All measurements and samples were obtained from mice four days after the final Aspergillus antigen administration, which was 20 days after the first challenge. Our group has previously found that airway reactivity measured four days after the final Aspergillus antigen challenge was similar to reactivity measured at earlier time points  [The 200, was reported.Mice were anesthetized and paralyzed by intraperitoneal injection of etomidate (28 mg/kg)  and pancuronium bromide (0.1 mg/kg) . A tracheal cannula was inserted via a midcervical incision and the mice were ventilated using a Harvard model 683 rodent ventilator  . Using a whole body plethysmograph, airflow resistance was calculated during baseline breathing and in response to serially increasing doses of intravenous acetylcholine chloride  . The log of the concentration of acetylcholine (μg/gm) required for a 200% increase in total lung resistance, designated log PCAfter completion of the airway physiology measurements, the lungs were lavaged five times with 0.8-ml aliquots of sterile PBS. The lavage fluid was pooled and centrifuged, and the cell pellet was treated with red-blood-cell lysing buffer . After being washed, the samples were resuspended in PBS. Total leukocytes were counted using a hemacytometer. Differential cell counts were determined by cytocentrifugation and Diff-Quik staining  followed by microscopic examination of at least 300 cells.Aspergillus antigen. Lungs were then removed en bloc and the left mainstem bronchus was firmly sutured closed. The left lung was removed by cutting the left mainstem distal to the suture. It was then frozen in liquid nitrogen and stored at -70°C until processed for hydroxyproline content. The right lung was inflated to 20 cm water pressure with 10% neutral buffered formalin  and fixed in 10% formalin for more than 48 h. Fixed lungs were embedded in paraffin, sectioned at 5 μm thickness, and stained with either hematoxylin and eosin (H&E), periodic acid Schiff (PAS), or trichrome by the Pathology Department of San Francisco General Hospital using standard protocols. The proportion of peribronchial inflammatory cells that were eosinophils was determined by counting inflammatory cells surrounding airways with lumens of 100–200 μm (measured on the short axis) on H&E stained sections. We analyzed 500 total cells (100 cells from each of five airways) for each animal studied.Thoracic lymph nodes were harvested from mice exposed to To prepare single-cell suspensions for cytokine analyses, isolated lymph nodes were gently minced using a syringe plunger and cells were passed through 70-μm cell strainers. Red blood cells were removed by hypotonic lysis at room temperature. Lymph node cells were counted, centrifuged, and resuspended in RPMI medium 1640  supplemented with FCS (10% vol/vol) , penicillin (100 U/ml) , streptomycin (100 μg/ml) , phorbol 12 myristate 13-acetate (PMA) (25 ng/m) , and ionomycin (1 μg/ml)  to a final concentration of 5 million cells per ml. Cells were then aliquoted into 96-well plates and incubated at 37°C. After 40 h, cell supernatants were harvested and stored at -70°C until they were analyzed. ELISA for IL-4, -5, -13 and IFN-γ was performed on stimulated lymph node cell supernatant per the manufacturer's protocols .Aspergillus antigen protocol. Four days after the final Aspergillus antigen administration, lungs from Aspergillus antigen- and PBS-treated mice were lavaged two times with 0.6-ml aliquots of sterile PBS. The samples were centrifuged and the supernatants were collected and stored at -70°C until analysis. ELISA for MCP-1 was performed on cell-free BAL fluid per the manufacturer's protocol .For quantitation of MCP-1 levels in BAL fluid, C57BL/6 wild-type mice were treated with the previously described Aspergillus antigen- or PBS-treated mice after airway responsiveness measurement. Serum total IgE concentration was determined by a sandwich ELISA using complementary antibody pairs for mouse IgE (clone R35-72 and R35-118) obtained from Pharmingen  according to the manufacturer's instructions. Color development was achieved using streptavidin-conjugated horseradish peroxidase  followed by addition of HRP substrate .Sera were obtained from blood collected by cardiac puncture from Lungs were analyzed for hydroxyproline content as previously described  with sliStatistical significance for treatment effect was determined by analysis of variance with post-ANOVA t tests corrected for multiple comparisons using Bonferroni adjustment. These statistical analyses were performed using statistical software STATA 5.0  and R   than in PBS-treated mice (5.8 ± 1.3 pg/ml), (P = 0.01).We used a model system that involved repeated intranasal challenges with Aspergillus antigen induction of MCP-1 was accompanied by a significant degree of lung inflammation as assessed by BAL fluid cell counts and lung histology. In wild-type mice, Aspergillus antigen induced a >20-fold increase in BAL fluid cell numbers . The numbers of macrophages, lymphocytes and neutrophils were not significantly different from those in Aspergillus antigen-treated wild-type mice  alone, we compared airway reactivity of PBS- and Aspergillus-antigen-treated mice 4 days after the final challenge as described in the methods section. Measurements from this time point were previously found to be comparable to those from earlier time points [Mcp-1-/-, and Ccr2-/- mice since preliminary experiments showed similar airway reactivity between PBS-treated wild-type, Mcp-1-/-, and Ccr2-/- mice (not shown). Aspergillus-antigen-treated wild-type, Mcp-1-/-, and Ccr2-/- mice each had significantly lower PC200 values than did PBS-treated controls (P < 0.001), indicating the development of AHR .To determine airway reactivity to acetylcholine in mice exposed to e points . In the AHR Fig. . AlthougAspergillus-antigen challenge results in increased mucus production, we analyzed lung histology by PAS-staining. As shown in Fig. Aspergillus-antigen-treated mice from all three groups showed accumulation of PAS-stained material in epithelial cells  MCP-1- and CCR2-deficient mice had concentrations of the cytokines IL-4, IL-5, IL-13 and IFN-γ generally similar to those in wild-type mice  but not later  [et al. [Our results stand in contrast to some previous reports showing important roles for MCP-1 or CCR2 in other models of allergic asthma ,27,33. Avalbumin ,26,33 orvalbumin ,34,35, sallenge) . Geneticallenge) . Previouallenge) -27, wherAspergillus antigen challenge. We found that Aspergillus antigen-induced monocyte recruitment (as measured by counting BAL fluid macrophages) was intact in both MCP-1- and CCR2-deficient mice. While intact alveolar macrophage recruitment in response to airway instillation of Saccharopolyspora rectivirgula has been reported in CCR2-deficient mice [in vivo models have demonstrated requirements for MCP-1 and CCR2 in monocyte/macrophage recruitment [Aspergillus antigen model. In support of this observation, a recent microarray-based analysis of gene expression changes in a similar asthma model found that 14 different chemokines (including MCP-1/JE) were induced by Aspergillus antigen challenge [Aspergillus antigen. While there was marked eosinophil recruitment to the lungs in MCP-1- and CCR2-deficient mice, there was a trend toward fewer eosinophils than in wild-type mice. Since MCP-1 is not a chemoattractant for eosinophils (which lack CCR2), this trend suggests that MCP-1 may have indirect effects on eosinophil recruitment in this model. A more dramatic decrease of eosinophil recruitment has been seen following neutralization of MCP-1 in another model, but that effect was associated with other signs of impaired Th2 immunity [Aspergillus antigen occurred even in the complete absence of either of these molecules.Of note, neither MCP-1 nor CCR2 was critical for inflammatory cell migration to the lungs after ent mice , other iruitment ,39-42. Ohallenge . Howeverimmunity . AlthougAspergillus antigen challenge, diminished Th2 cytokine production has been reported in studies of MCP-1 neutralization or deletion in different models [In contrast to our results indicating a robust Th2 response in MCP-1- and CCR2-deficient mice after t models ,33,44,45t models ,28, decrt models , or havet models . As mentt models  are operAspergillus conidia were accompanied by neutrophilic inflammation and the inability of CCR2-deficient mice to clear the organism normally [Aspergillus organisms in the airway may have altered the fibrotic response. Other studies involving different experimental systems have suggested that MCP-1 and CCR2 may directly or indirectly contribute to the development of fibrosis. Gharaee-Kermani et al. [Aspergillus antigen challenge, similar to wild-type mice. Our result, in contrast to the reported requirement for CCR2 in the development of bleomycin-induced pulmonary fibrosis, suggests that different cell types and mediators may be operational in allergen-induced airway fibrosis than those observed in bleomycin-induced lung fibrosis.The role of MCP-1 and CCR2 in the development of allergen-induced airway fibrosis has not been extensively explored. Previous findings of increased pulmonary fibrosis in CCR2-deficient mice compared to wild-type mice after treatment with normally ,29. Consi et al.  found thi et al. ,49; howei et al. , in contAspergillus antigen-driven model of allergic airway disease. Previous studies have demonstrated roles for MCP-1 and CCR2 in other models of inflammation and fibrosis, including different allergic airway disease models [In conclusion, this study demonstrates that pulmonary inflammation, Th2 immune responses, Th2-mediated airway pathology, and lung fibrosis are remarkably intact despite the complete absence of MCP-1 or CCR2 in an e models ,27,33. TAHR, airways hyperreactivity; BALF, bronchoalveolar lavage fluid.LLK conceived of the experiment, carried out all experiments and prepared the manuscript. MWR assisted in collection and analysis of mouse samples. XLB performed all mouse airway measurements. SC and XH performed antigen challenge and assisted in collection and analysis of mouse samples. IFC and BJR provided the targeted knock-out mice, provided expert advice and interpretation of the study's results. DJE participated in the study's design, coordination and final revisions of the manuscript. All authors read and approved the final manuscript.


Colorectal cancers displaying high-degree microsatellite instability (MSI-H) have an improved prognosis compared to microsatellite stable (MSS) cancers. The observation of pronounced lymphocytic infiltrates suggests that MSI-H cancers are inherently more immunogenic. We aimed to compare the gene expression profiles of MSI-H and MSS cancers to provide evidence for an activated immune response in the former.® HG-U133A chip. Data was analysed on GeneSpring software version 6.0. Confirmatory real-time RT-PCR was performed on 28 MSI-H and 26 MSS cancers. A comparison of 29 MSI-H and 104 MSS cancers identified 2070 genes that were differentially expressed between the two groups [P < 0.005]. Significantly, many key immunomodulatory genes were up-regulated in MSI-H cancers. These included antigen chaperone molecules , pro-inflammatory cytokines (Interleukin (IL)-18, IL-15, IL-8, IL-24, IL-7) and cytotoxic mediators . Quantitative RT-PCR confirmed up-regulation of HSP-70 [P = 0.016], HSP-110 [P = 0.002], IL-18 [P = 0.004], IL-8 [0.002] and Granulysin [P < 0.0001].We analysed tissue from 133 colorectal cancer patients with full consent and Local Ethics Committee approval. Genomic DNA was analysed for microsatellite instability in BAT-26. High-quality RNA was used for microarray analysis on the AffymetrixThe upregulation of a large number of genes implicated in immune response supports the theory that MSI-H cancers are immunogenic. The novel observation of Heat Shock Protein up-regulation in MSI-H cancer is highly significant in light of the recognised roles of these proteins in innate and antigen-specific immunogenicity. Increased mRNA levels of pro-inflammatory cytokines and cytotoxic mediators also indicate an activated anti-tumour immune response. Colorectal cancer remains a leading cause of cancer death in the Western world despite recent advances in surgery, radiotherapy and chemotherapy . ImmunotApproximately 15–20% of sporadic colorectal cancers and nearly all large bowel malignancies in the Hereditary Non-Polyposis Colorectal Cancer (HNPCC) syndrome are characterised by widespread microsatellite instability . Microsain vitro experiments . This suggests that our observations do not simply reflect the more pronounced lymphocyte infiltrates of MSI-H colorectal cancer although we accept that this remains a possibility.Certainly, our results provide new evidence to support the notion that MSI-H colorectal cancers are immunogenic and new insights into the pathways that may be involved. Further focussed study of these cancers may clarify the immunology of colorectal cancer and, specifically, may provide useful targets for directing immunotherapeutic strategies.The Local Ethics Committee approved this study and all patients gave informed consent prior to surgery. Tissue was available from a bank of 223 colorectal cancers resected at The Royal London Hospital between December 1997 and March 2003. Tissue samples of tumour and normal mucosa were taken within 20 minutes of resection and snap frozen in liquid nitrogen. Normal mucosa and tumour DNA was extracted and used in PCR reactions to amplify the mononucleotide BAT-26 marker, as described elsewhere [32]. Products were separated and visualised on micro-fabricated chips to identify tumours displaying bandshifts characteristic of a high degree of microsatellite instability [33].® kit  and quality was assessed on the Agilent Bioanalyser 2100. Only 129 samples, from our tumour bank, that yielded high quality mRNA with minimal degradation and clear 18S/28S ribosomal peaks, were included in the analysis. Preparation of in vitro transcription (IVT) products, oligonucleotide array hybridization and scanning were performed according to Affymetrix®  protocols. In brief, 5 μg of total RNA from each colon tumour and T7-linked oligo-dT primers were used for first-strand cDNA synthesis. IVT reactions were performed in batches to generate biotinylated cRNA targets, which were chemically fragmented at 95°C for 35 minutes. Fragmented biotinylated cRNA (10 μg) was hybridized at 45°C for 16 hours to Affymetrix® high density oligonucleotide array human HG-U133A chip, which contains 22,283 probe sets representing more than 14,500 well-substantiated human genes. The arrays were washed and stained with streptavidin-phycoerythrin . Signal amplification was performed using a biotinylated anti-streptavidin antibody. The array was scanned according to the manufacturer's instructions . Scanned images were inspected for the presence of obvious defects (artefacts or scratches) on the array. Defective chips were excluded and the sample was re-analysed. To minimize discrepancies due to variables such as sample preparation, hybridization conditions, staining, or array lot, the raw expression data was scaled using Affymetrix® Microarray Suite 5.0 software. The trimmed mean signal of all probe sets on the HG-U133A chip was adjusted to a user-specified target signal value (1500) for each array for global scaling. No specific exclusion criteria were applied.Total RNA was prepared from samples using an RNeasyth percentile of all values on that chip. For "per gene normalisation" the data for a given gene is normalised to the median expression level of that gene across all samples. The data sets are then assigned to the two groups MSI-H and MSS, and the expression profiles of the two groups were compared using unpaired t-tests  and multiple testing corrections to identify genes that were differentially expressed between them.Comparative analysis between expression profiles for MSI-H and MSS samples was carried out on GeneSpring™ software version 6.0 . The "Cross gene error model for deviation from 1.0" was active. Gene expression data was normalised in two ways: "per chip normalisation" and "per gene normalisation". For "per chip normalisation" all expression data on a chip is normalised to the 50Nine genes were further analysed using quantitative real-time RT-PCR. To compare similar groups each MSI-H tumour was matched to an MSS tumour from patients of similar age, the same side of the colon and, where possible, same Dukes' Stage. Sample RNA was extracted, quantified and quality-controlled as for microarray analysis. High-quality RNA was available on 28 tumours of the 29 tumours with MSI-H. These were matched to a group of 26 MSS cancers Table . Three aMSI = Microsatellite instabilityMSI-H = High degree microsatellite instabilityMSS = Microsatellite stable (including low degree instability)AB carried out RT-PCR experiments and analysis, microarray data analysis and drafted the manuscript.SA participated in microarray experiments and carried out microarray data analysis.RH, FH, XH and PS coordinated and performed microarray experiments.RF performed histopathological analysis of cancers.SB and SD conceived the study, supervised the project and drafted the manuscript.All authors read and approved the final manuscript.2070 genes differentially expressed between MSI-H and MSS colorectal cancers . 4788 genes differentially expressed between MSI-H and MSS colorectal cancers . 542 genes differentially expressed between MSI-H and MSS colorectal cancers .Click here for file2184 genes differentially expressed between MSI-H (HNPCC profiles excluded) and MSS colorectal cancers . 1436 genes upregulated in sporadic MSI-H cancers (with fold change). 748 genes upregulated in MSS cancers (with fold change). 4874 genes differentially expressed between MSI-H and MSS colorectal cancers .Click here for file


Biochemical testing for pheochromocytoma by measurement of fractionated plasma metanephrines is limited by false positive rates of up to 18% in people without known genetic predisposition to the disease. The plasma normetanephrine fraction is responsible for most false positives and plasma normetanephrine increases with age. The objective of this study was to determine if we could improve the specificity of fractionated plasma measurements, by statistically adjusting for age.An age-adjusted metanephrine score was derived using logistic regression from 343 subjects (including 33 people with pheochromocytoma) who underwent fractionated plasma metanephrine measurements as part of investigations for suspected pheochromocytoma at Mayo Clinic Rochester (derivation set). The performance of the age-adjusted score was validated in a dataset of 158 subjects (including patients 23 with pheochromocytoma) that underwent measurements of fractionated plasma metanephrines at Mayo Clinic the following year . None of the participants in the validation dataset had known genetic predisposition to pheochromocytoma.The sensitivity of the age-adjusted metanephrine score was the same as that of traditional interpretation of fractionated plasma metanephrine measurements, yielding a sensitivity of 100% . However, the false positive rate with traditional interpretation of fractionated plasma metanephrine measurements was 16.3%  and that of the age-adjusted score was significantly lower at 3.0%  (p < 0.001 using McNemar's test).An adjustment for age in the interpretation of results of fractionated plasma metanephrines may significantly decrease false positives when using this test to exclude sporadic pheochromocytoma. Such improvements in false positive rate may result in savings of expenditures related to confirmatory imaging. Pheochromocytoma is a rare tumor of the adrenal medulla or sympathetic ganglia, which can secrete excessive catecholamines . Signs aReasons for false positive fractionated metanephrine test results have been explored and alternatives for further evaluation of patients with positive test results have been proposed. It is known that acetaminophen may interfere with measurements of fractionated plasma metanephrines using the Lenders' method , so thisOur objective was to determine whether an adjustment of fractionated plasma metanephrines for age may improve the specificity of interpretation of these measurements when biochemically excluding sporadic pheochromocytoma, and thus result in costs savings in confirmatory imaging studies.The age-adjusted fractionated plasma metanephrine score was derived from a previously described dataset of 349 subjects (including 33 people with pheochromocytoma) who underwent measurement of fractionated plasma metanephrines as part of an evaluation of suspected pheochromocytoma  . The diaThe technique of Lenders  was used to measure fractionated plasma metanephrines . The tra2 was used for categorical variables and Student's t-test for independent samples was used for continuous variables). Variables which were different between both groups at a significance level of 0.1 were then entered into a multivariable logistic regression model predicting pheochromocytoma. Age was the only variable of statistical significance distinguishing true positive from false positive fractionated plasma metanephrine measurements in the derivation set. Thus, we forced age with measurement values of normetanephrine and metanephrine fractions in a multivariable logistic regression model predicting pheochromocytoma in the derivation set . The formula for this age-adjusted fractionated plasma metanephrine score is shown below:Clinical characteristics of subjects in the derivation set who did not have pheochromocytoma but had measurements of the normetanephrine or metanephrine fraction above the upper reference limits  were compared to those without pheochromocytoma who had true negative tests (χ-4.188 + -0.07(age) + 4.516(metanephrine) + 3.129(normetanephrine).2 = 4.73, df = 8, p = 0.79 and the Cox and Snell r2 = 0.38 showing significant model goodness-of-fit. A positivity cut-off age-adjusted metanephrine value of = -1.4752 was chosen as it carried an acceptable sensitivity of = 90.9%  and specificity of 96.8%  in the derivation set. The sensitivity level of over 90% was chosen because such a sensitivity level was believed to be clinically reasonable and at this level, specificity was still acceptable. We were aware that the lower the cut-off, the higher the sensitivity, but this would be at the expense of specificity.Age was in years, metanephrine fraction in nmol/L and normetanephrine fraction in nmol/L in this formula. In the derivation set, the Hosmer and Lemeshow test had χThe formula for adjustment of age was applied to fractionated plasma metanephrine measurements in the second dataset  for testing of sensitivity and specificity in a population in whom sporadic pheochromocytoma was sought. In the validation set, individuals with known genetic predisposition to pheochromocytoma were excluded. Sensitivities were calculated by division of subjects with true positive test results by all the subjects with pheochromocytoma, and specificities were calculated by division of subjects with true negative test results divided by all subjects without pheochromocytoma. For sensitivities, specificities, and likelihood ratios, 95 percent confidence intervals (CI) were calculated using Wilson's method  . The speWe investigated whether use of an age-adjusted fractionated plasma metanephrine measurement could result in cost savings in imaging expenditures, compared to use of fractionated plasma metanephrine measurements interpreted in a conventional fashion, for detection of sporadic pheochromocytoma in a hypothetical tertiary care hypertensive population. We thus performed a decision analysis, with resource implications defined by costs of confirmatory imaging (CT and MRI), interpreted from a third party payer perspective. In the decision analysis, we compared algorithm "A" in which biochemical testing consisted of measurement of fractionated plasma metanephrines with these measurements interpreted relative to the 95% reference range (defined by a normetanephrine fraction above 0.9 nmol/L or a metanephrine fraction above 0.5 nmol/L) to algorithm "B", in which fractionated plasma metanephrine measurements were interpreted by using the age-adjusted metanephrine score. The sensitivity and specificity of biochemical tests was based on data from the validation set. In each algorithm, all patients with positive biochemical testing would undergo confirmatory imaging. The imaging protocol for patients with positive biochemical tests in either strategies began with computerized tomography ([CT] with and without intravenous contrast) of the abdomen, then if negative, I-131 or I-123 metaiodobenzylguanidine (MIBG) scintigraphy (efficacy for I-131 and costs for I-123 shown). The horizon (endpoint) of the analyses was positive diagnosis or exclusion of pheochromocytoma, for hypothetical hypertensive patients subjected to each strategy. The outcome of the analyses was the number of patients with pheochromocytoma expected to be detected by each strategy. The costs of false positive biochemical tests were reflected only in the costs of subsequent imaging and not in potential costs of needless surgery or its possible complications.The diagnostic efficacy of imaging studies was based on respective estimates from the literature: The sensitivity of CT imaging of the abdomen was assumed to be 98% with a specificity of 70% . The senAll costs were reported in 2002 US dollars. Costs of imaging investigations were obtained from the Mayo Clinic Rochester Business Office. For the purpose of the decision analysis model, the prevalence of pheochromocytoma in the hypertensive population that would typically be screened was assumed to be 0.5% .The derivation set (from which the age-adjusted score was developed) consisted of 349 consecutive subjects (including 33 people with pheochromocytoma) who underwent fractionated plasma metanephrine measurements as well as 24-hour urinary total metanephrine measurements with or without 24-hour urinary catecholamine measurements in testing for pheochromocytoma at Mayo Clinic Rochester. In the derivation set, 8 of the 33 individuals had clinically-diagnosed genetic syndromes predisposing to pheochromocytoma . The 316 individuals in the derivation set who did not have pheochromocytoma underwent such testing the following reasons: refractory hypertension , spells , adrenal mass , previous pheochromocytoma or known genetic predisposition to pheochromocytoma . The mean age of subjects with pheochromocytoma was 48 years , whereas the mean age of subjects without pheochromocytoma was 52 years . Six of the 316 subjects without pheochromocytoma in the derivation set did not have a plasma metanephrine fraction recorded secondary to "interfering substances" and therefore were excluded from the analyses. In the derivation set, the sensitivity of traditionally interpreted fractionated plasma metanephrine measurements (using 95% reference ranges) was 93.9%  (31/33 subjects), with a specificity of 85.2%  (264/310 subjects). Baseline characteristics of individuals in the derivation set without pheochromocytoma were compared for individuals who had true negative fractionated metanephrine measurements (n = 264) to those who had false positive results (n = 46) . Of the patients with pheochromocytoma, none were known to be genetically predisposed to pheochromocytoma and 14/23 were women (61%). The mean age of subjects with pheochromocytoma was 50 years , whereas the mean age of subjects without pheochromocytoma was 55 years . Of the 135 subjects without pheochromocytoma, 83 (62%) were women. Reasons for measurement of fractionated plasma metanephrines in the subjects without pheochromocytoma were as follows: hypertension , spells with or without hypertension , an incidentally discovered adrenal mass , and previously surgically cured pheochromocytoma .In the validation set, the sensitivity of the age-adjusted metanephrine score was the same as the traditional interpretation of fractionated plasma metanephrine measurements at 100% . The specificity of the traditional interpretation of fractionated plasma metanephrine measurements was 83.7%  and the specificity of the age-adjusted plasma metanephrine score was 97.0% . Thus, the false positive rate with traditional interpretation of fractionated plasma metanephrine measurements was 16.3%  and with the age-adjusted score it was significantly lower at 3.0%   was followed by CT imaging for all positive biochemical tests and if CT imaging was negative, then MIBG (I-123 or I-131) would be performed , 499/500 patients with pheochromocytoma  would be expected to be detected ; furthermore 94,510/99,500 of subjects without pheochromocytoma would be reassured with a negative diagnosis . In algorithm "A", 16,718 individuals would undergo CT scanning and 11,363 individuals would undergo I-123 or I-131 MIBG imaging. The total cost of imaging for algorithm "A" would be estimated to be 45.7 million dollars.If algorithm "B"  would be used in 100,000 subjects with hypertension, 499/500 patients with pheochromocytoma (99.8%) would be expected to be detected and 98592/99,500 individuals without pheochromocytoma would be reassured with a negative test result . In algorithm "B", 3,485 individuals would undergo CT scanning and 2,100 individuals would undergo I-123 or I-131 MIBG imaging. The cost of imaging for algorithm "B" would be approximately 9.0 million dollars. Thus, use of the age-adjusted plasma metanephrine score for biochemical testing for sporadic pheochromocytoma in a hypothetical population of 100,000 tertiary care hypertensive patients could result in a cost savings of 36.7 million dollars with equal detection of pheochromocytoma cases, relative to using the same biochemical testing but interpreting fractionated plasma metanephrine measurements in a traditional fashion.We agree with observation by Eisenhofer and colleagues that when it comes to measurement of fractionated plasma metanephrines for exclusion of pheochromocytoma, "false-positive results remain a problem" , particuOf note, the sensitivity of the age-adjusted metanephrine score was superior in the validation set (100%) to that observed in the original dataset from which it was derived (91%). An explanation for this finding may be that the validation set included only people who were at risk for sporadic pheochromocytoma , whereas genetically predisposed individuals were included in the original derivation dataset. We have previously observed that fractionated plasma metanephrine measurements may be normal in genetically-predisposed individuals with small pheochromocytomas . MoreoveOur study is subject to several limitations. Firstly, limited clinical data on each studied individual were collected so variables that could be of interest such as: body mass index, creatinine-clearance, and rates of diabetes mellitus were not recorded. Furthermore, without autopsy confirmation, one cannot be absolutely certain that individuals labelled as not having a pheochromocytoma did not have an occult paraganglioma or pheochromocytoma. However, we believe that reasonable clinical criteria were used in excluding pheochromocytoma in our study. Another limitation is that we used an assay for measurement of fractionated plasma metanephrines that may be have been subject to interference with acetaminophen , whereasIs calculation of an age-adjusted metanephrine score practical for use in daily clinical practice? In this day of palm pilots and desktop computers, we believe that it may be feasible for clinicians to enter the formula for age-adjustment into standard desktop spreadsheet software  and perform such adjustments in the clinic. Alternatively, laboratories can provide age adjusted values to physicians when reporting test results. Thus, we do believe that calculation of an age-adjusted score is feasible to assist physicians in interpretation of fractionated plasma metanephrine measurements. Indeed, such calculations may be less cumbersome and may generate fewer healthcare expenditures than alternative procedures such as supplemental clonidine-suppression testing or collection of 24-hour urinary measures.Our observations should, however, be validated in another population outside of Mayo Clinic. Of particular note, our findings are applicable only to the screening of pheochromocytoma in low risk subjects who do not have known genetic predisposition to disease. In high risk, genetically predisposed individuals, mild elevations of normetanephrine or metanephrine measurements may be clinically significant and should prompt imaging.An adjustment for age in interpretation of results of fractionated plasma metanephrine measurements may significantly improve the high false positive rate seen with this test when aiming to exclude sporadic pheochromocytoma. This improvement in specificity may result in savings in expenditures related to confirmatory imaging. Additional research is needed to investigate the generalizability of these findings in other clinical centres.Dr. Sawka is a Skeletal Health Scholar funded, in part, by the Canadian Institutes of Health Research. Dr. Sawka was also a Fellow in Health Economics at McMaster University, partly funded by an unrestricted educational grant from Hoffmann-La Roche.The other co-authors have no competing interests to declare.All co-authors reviewed the manuscript and made suggestions for revisions. The project idea was conceived by A.M. Sawka. Analyses were performed by A.M. Sawka, with input from Dr. Thabane. The manuscript was written and revised by Dr. Sawka.The pre-publication history for this paper can be accessed here: