Functional decline threatens independent living and is common among individuals diagnosed with cancer, especially those who are elderly. The purpose of this study was to explore whether dietary and exercise practices are associated with physical function status among older cancer survivors.Mailed surveys were used to ascertain data on physical function, dietary fat, fruit and vegetable (F&V) consumption, and exercise among elderly diagnosed with early stage (I-II) breast (N = 286) or prostate cancer (N = 402) within the past 18 months.Sixty-one percent of respondents reported diets with <30% of energy from fat, 20.4% reported F&V intakes of 5+ daily servings, and 44.6% reported regular vigorous exercise. Significant, independent associations were found between physical functioning and reported dietary fat intake, F&V consumption, and exercise. A simultaneous multiple regression model controlled for age, race, gender, time since diagnosis and concurrent health behaviors yielded the following estimates: (1) 0.2 increase in the SF-36 physical function subscale (PFS) score with each reported 1% decrease in percent energy from fat (p < .0001); (2) 0.9 increase in the SF-36 PFS score for each reported serving of F&V/day (p = .0049); and (3) 15.4 increase in the SF-36 PFS score with a positive response for regular vigorous exercise (p < .0001).Results of this cross-sectional survey suggest that regular vigorous exercise and consumption of diets low in fat and rich in F&Vs are associated with higher levels of physical functioning among older cancer survivors. Interventions that promote healthful lifestyle change may deliver considerable benefit within this ever increasing and vulnerable population. There are 9.6 M cancer survivors in the US today, and 61% are age 65 or older . UnfortuWe explored associations between lifestyle factors and physical functioning among elderly cancer survivors who were screened for Project LEAD (Leading the Way in Exercise And Diet), a home-based, diet and exercise intervention trial . BrieflyT-tests and chi-square analyses were performed to determine if respondents differed from non-respondents on age, time since diagnosis, race and gender, and to test for differences between male and female respondents. Pearson correlation coefficients indicated associations between F&V intake and percent dietary fat, and physical function scores. T-tests were used to explore associations between exercise and function. Linear regression analyses permitted an examination of associations between physical function and health behaviors controlling for several likely confounders including gender, age, race, time since diagnosis and concurrent health behaviors. Given the homogeneity in stage at diagnosis, stage information was omitted from this analysis.Between August 2000 and May 2003, 2,431 cases were identified and physician approval for contact was granted for 2,034 cases. Incomplete address information existed for 24 of these cases, yielding 2,010 viable posted surveys, of which 688 complete surveys were returned (34% response rate). Characteristics of respondents and non-respondents are provided in Table Data regarding physical function and lifestyle behaviors are provided in Table Modest correlations were obtained between the indicators of exercise and diet, with positive agreement noted between F&V intake and exercise (Pearson ρ = .12/p = .003) as well as F&V intake and dietary fat (ρ = .42/p < .0001). An inverse association was noted between dietary fat and exercise (ρ = -.08/p = .05). In bivariate associations, all three behaviors were significantly associated with SF-36 physical functioning scores (p < .05): F&V intake (ρ = .09), dietary fat (ρ = -.10), and vigorous exercise (ρ = .37). In multivariable linear regression analyses, with the SF-36 PFS score serving as the dependent variable and controlling for age, race, gender, time since diagnosis, and concurrent health behaviors, the simultaneous associations of the three indicators remained statistically significant (independent) and yielded the following estimates: (1) a 0.2 increase in the SF-36 PFS score with each reported 1% decrease in percent energy from fat (p < .0001); (2) a 0.9 increase in the SF-36 PFS score for each reported serving of F&V/day (p = .0049); and (3) a 15.4 increase in the SF-36 PFS score with a positive response for regular vigorous exercise (p < .0001).In comparing these cross-sectional data on lifestyle behaviors of older cancer survivors to normative data reported on healthy elders, as well as to previous data reported on general populations of breast and prostate cancer survivors (where a dearth of data have been reported on older cancer survivors per se), we find both differences and similarities. Like data that exist on healthy elders (age 65+) responding to the 2000 Behavioral Risk Factor Surveillance System (BRFSS) survey , or findA potential explanation for the higher physical function scores exhibited within our sample may relate to the higher prevalence of healthful lifestyle practices which in turn may increase physical function. This links back to the primary thrust of this study, which was to determine evidence for a link between functional status and health behavior.Our results suggest that regular vigorous exercise and a diet rich in F&Vs and low in fat is associated with higher levels of physical functioning among elderly recently diagnosed with cancer. Although the association between exercise and improved function has been reported repeatedly in other studies among elders ,23, thisFurthermore, only one study to date has reported the link between diet composition and physical function. Ortega et al. found that diets low in fat and high in F&Vs were associated with higher levels of physical functioning among a sample of upper socio-economic, elderly male Spaniards at risk for cardiovascular disease . This prThe primary limitations of this study relate to respondent bias and cross-sectional design. Our response rate of 34% is indeed less than the 59% we have achieved in previous mailed surveys among similar populations . Given tFindings of this study support the recent American Cancer Society diet and exercise guidelines for survivors , which cF&V: Fruits and VegetablesSF-36: Short Form-36 Health SurveyPFS: Physical Function SubscaleThe author(s) declare that they have no competing interests.WDW, ECC, MCM, CFP and HJC conceived of the study, and participated in its design. DCS participation in the coordination of the study, as well as data cleaning and interpretation of findings. RS performed the statistical analysis. All authors read and approved the final manuscript.


Several previous epidemiological studies have shown a relation between drinking water quality and death in cardiovascular disease whereas others have not found such a relationship. An intervention study was undertaken to evaluate the effect of water with added magnesium and natural mineral water on blood pressure.A group of 70 subjects with borderline hypertension was recruited and consumed 1) a water low in minerals, 2) magnesium enriched water or 3) natural mineral water, in a random, double blind fashion during four weeks.Among persons with an initial low excretion of magnesium or calcium in the urine, the urinary excretion of magnesium was increased in the groups consuming the two waters containing magnesium after 4 weeks. A significant decrease in blood pressure was found in the group consuming mineral water at 2 and 4 weeks.The results suggest that minerals taken in water are significant for the body burden and that an intake of mineral water among persons with a low urinary excretion of magnesium or calcium may decrease the blood pressure. Further studies should investigate the extent of mineral deficiency in different populations and the efficiency of different vehicles for supplying minerals, particularly magnesium and calcium. A relation between mortality from ischaemic heart disease (IHD) and drinking water characteristics was first shown in Japan in 1957  and reviIn a case-control study, an inverse relation was found between the amount of magnesium in drinking water and death from acute myocardial infarction and for females also between the amount of calcium and death . Diets rRegarding individual minerals, several studies have been reported where hypertensive subjects were treated orally with nutritional doses of magnesium . The resEpidemiological studies on cardiovascular disease suggest that drinking water is an important vehicle for the supply of minerals . This isThe present intervention study was undertaken to determine the effect of minerals in water on one of the major risk factors for cardiovascular disease – blood pressure. Subjects with slightly elevated blood pressure consumed water with different levels of minerals. Serum and urinary levels of minerals were measured as a marker of intervention and blood pressure was measured before and after the intervention.Female and male subjects, aged 45 – 64 years (n = 70) were recruited by advertising in local newspapers. Inclusion criteria were living in an area with low magnesium content in the drinking water, systolic pressure 15 mm above normal values for their age, diastolic pressure above 90 mm Hg, and within 20% of ideal body weight. Exclusion criteria were hypertension target organ damage, chronic diseases , pregnancy, and taking oral contraceptives or regular intake of mineral supplements. Subjects with a diastolic pressure above 100 mm Hg were advised to consult a physician for treatment. A few persons decided not to seek a physician's advice and choose to participate anyway. The Ethical committee at the Medical faculty, University of Gothenburg, approved the study.Blood pressure was measured using standardized techniques before the intervention, at 2 weeks and at the end at 4 weeks. Two separate recordings were made (diastolic pressure as Korothoff phase 5) after 5 minutes of supine rest. The blood pressure is reported as the average of these recordings.idem). Magnesium and calcium levels in urine were expressed as the creatinine ratio.Blood samples were taken before and after the intervention to determine the serum concentration of magnesium, calcium, sodium, creatinine and potassium . Before and after the intervention period, 24 hours urine samples were collected and the amounts of magnesium, calcium, and creatinine were determined . Table Table In spite of the random allocation to the different waters, it was found that the group consuming water C comprised a larger number of persons with a high initial systolic pressure. In the groups receiving waters A and B, none of the subjects had systolic blood pressures above 170 before the intervention. The subjects drinking water C were divided into those with an initial systolic pressure above and below 170 mm. For the group with the higher pressure (n = 6), there was a decrease in the systolic pressure before and at 4 weeks p = 0.023) but no difference at 2 weeks or for diastolic pressure. The results from the remainder of the group are also shown in Table  but no dThe study is of exploratory character, based on a relatively small number of subjects and should be interpreted with care. There is also a lack of some data that retrospectively would have been of interest such as sodium in the urine and the effect of water with only calcium added. We do not think, however, that this has any influence on the major conclusions from the study.The intervention with the two waters with added magnesium influenced the body burden in terms of an increased excretion of magnesium in urine. This is consistent with findings from previous intervention studies ,17 althoThe intervention with water containing high amounts of several minerals decreased the blood pressure significantly in contrast to water with magnesium only where no significant effect was detected. This does not exclude that an effect could have been found with the latter water, had the intervention time been longer. On the other hand, the finding supports the concept that interventions should be performed under conditions similar to the ones present in normal environments, rather than with one specific agent. This could also explain the lack of an effect in previous studies where single minerals have been given as reviewed in the introduction.In summary, the results suggest that waterborne minerals constitute a supply for the body burden, that the urinary excretion can be used as a physiologically relevant indicator of the body burden of magnesium and calcium, and that the supplementation of magnesium together with other minerals may reduce blood pressure among persons with a low body burden of magnesium and calcium, either due to an insufficient intake through food or water, or through some metabolic/clinical disturbance. Additional studies are needed to explore this further.The study was supported by an unconditional grant to Gothenburg University from the Nestlé Water Institute, Vittel, France. The authors had full freedom for data analysis, manuscript preparation and submittance to a journal. RR has not received any fees or salaries for this work, including article processing charges, nor does he own any shares or has any other financial interest in the company. MA was an employee at the Water Institute at the time of the study.RR and MA jointly developed the research plan. RR conducted the field study. RR and MA jointly analyzed the data and wrote the manuscript.The pre-publication history for this paper can be accessed here:


Chronic obstructive pulmonary disease and emphysema are a frequent result of long-term smoking, but the exact mechanisms, specifically which types of cells are associated with the lung destruction, are unclear.+ and CD8+ T lymphocytes that expressed chemokine receptors CCR5 and CXCR3 (both markers of T helper 1 cells), but not CCR3 or CCR4 (markers of T helper 2 cells). Lung lymphocytes in patients with chronic obstructive pulmonary disease and emphysema secrete more interferon gamma—often associated with T helper 1 cells—and interferon-inducible protein 10 and monokine induced by interferon, both of which bind to CXCR3 and are involved in attracting T helper 1 cells. In response to interferon-inducible protein 10 and monokine induced by interferon, but not interferon gamma, lung macrophages secreted macrophage metalloelastase , a potent elastin-degrading enzyme that causes tissue destruction and which has been linked to emphysema.We studied different subsets of lymphocytes taken from portions of human lungs removed surgically to find out which lymphocytes were the most frequent, which cell-surface markers these lymphocytes expressed, and whether the lymphocytes secreted any specific factors that could be associated with disease. We found that loss of lung function in patients with chronic obstructive pulmonary disease and emphysema was associated with a high percentage of CD4These data suggest that Th1 lymphoctytes in the lungs of people with smoking-related damage drive progression of emphysema through CXCR3 ligands, interferon-inducible protein 10, and monokine induced by interferon. The underlying changes that cause smoking-related damage are unclear. This paper suggests that there is a skew towards increased T helper 1 lymphocytes, which may drive progression of this damage A rho smoke ,9,10,11.ho smoke . Furtherho smoke . It has ho smoke .Chemokines, their receptors, and cell adhesion molecules regulate migration of immune cells into inflamed tissue ,16,17,18In this study we determined the dominant T helper phenotype in lung samples from ex-smoker individuals with moderate to severe COPD and emphysema and control individuals with no evidence of smoking-related lung disease. Analysis of chemokine receptor expression on isolated peripheral lung lymphocytes from ex-smokers with COPD/emphysema indicated that both CD4 and CD8 T helper cells are strongly polarized to the Th1 phenotype compared to T cells isolated from lung tissue of normal individuals or individuals with non-smoking-related obstructive lung disease. The same cells spontaneously secreted more IFN-γ and CXCR3 receptor ligands MIG and IP-10 in the COPD and emphysema group than in the group without emphysema. Further, IP-10 and MIG, but not IFN-γ, upregulated macrophage metalloelastase  from isolated lung macrophages. Together, our findings reveal the strong association between COPD/emphysema- and Th1-driven adaptive immunity, suggesting a link to lung destruction mediated by IFN-γ, MIG, and IP-10.Twenty-eight non-atopic ex-smoker individuals see  undergoiAll participants were recruited from the surgical clinic at the Michael E. DeBakey Veterans Affairs Medical Center and the Methodist Hospital, and were undergoing lung resection for diagnostic or therapeutic purposes . Study pHigh-resolution CT (two in emphysema group and two in control group) or conventional CT analysis was used to detect emphysema, characterized by the presence of areas of low attenuation contrasted with surrounding normal lung parenchyma ,33. CT s7 cells for future analysis.Lung lymphocytes were isolated by modifying established protocols, using a combination of mechanical fragmentation, enzyme digestion, and centrifugation procedures described previously ,36,37. VThe following monoclonal antibodies were purchased from BD Biosciences Pharmingen : FITC-, Cy5-, and PE-conjugated anti-CD4, -CD8, -CD3, -CD14, -CD69, -CXCR3, -CCR3, -CCR4, and -CCR5. For enzyme-linked immunosorbent assay studies, anti-human antibodies to IFN-γ, IL-4, IP-10, MIG, I-TAC, and the appropriate secondary reagents were purchased from R&D Systems .7 cells/ml, and 50 μl of cells was incubated with antibodies to CD3 and CD4 or CD8.Phenotypic characterization of T cells was done by two-color flow cytometry  using combinations of the following monclonal antibodies: FITC-conjugated anti-CD4, -CD8, and -CD14; PE- and Cy5-conjugated anti-CCR4, -CCR3, -CCR5, and -CXCR3. Freshly isolated lung lymphocytes were resuspended to 1 × 10Lung lymphocytes were cultured in the presence or absence of phorbol myristate acetate (PMA)/ionomycin and brefeldin A for 12 h. Cells were harvested, fixed with formaldehyde, permeabilized with saponin, and intracellularly labeled for IFN-γ and IL-4, in addition to staining for surface CD69, CD4, and CD8 according to the manufacturer's recommendations .Lung lymphocytes were isolated from surgical tissue and cultured in vitro in triplicate for 4 d. Supernatants were collected and stored at –80 °C for future analysis. Standard antibody-based enzyme-linked immunosorbent assay was used to measure supernatant concentrations of IP-10, MIG, IL-4, and IFN-γ according to the manufacturer's instructions (R&D Systems and BD Biosciences Pharmingen).Peripheral blood mononuclear cells and lung macrophages were isolated by positive selection using immunomagnetic beads conjugated with anti-CD14, and cultured in serum-free medium  prior to overnight stimulation with 0, 50, 250, or 500 ng/ml of IFN-γ, IL-4, MIG, I-TAC, and IP-10. Supernatants were collected, and MMP12 was detected using anti-human MMP12 (R&D Systems) by Western blotting according to the manufacturer's instructions.+ lung macrophages stimulated overnight with rIP-10 (500 ng/ml) in the presence or absence of blocking anti-CXCR3 antibodies . Two-step real-time reverse transcription PCR was used to determine the relative expression of mRNA using the ABI Perkin Elmer Prism 5700 Sequence Detection System  as described previously [Total cellular RNA was extracted from CD14eviously .Paraffin-embedded, and fresh-frozen lung sections (5 μm) were immunostained using monoclonal antibodies against human MMP12 (R&D Systems) or non-immune antisera by an immunoperoxidase protocol  and counterstained with hematoxylin as recommended by the manufacturer.p < 0.05 was considered statistically significant.The Mann-Whitney test  and Student's T test (two-tailed) were used to compare differences between the two groups of subjects. n = 10) and emphysema (n = 18) groups, and did not discriminate between these populations . Together, these data indicate that a strong type 1 bias is characteristic of the T cells isolated from the peripheral lung of participants with COPD and emphysema and that this immune phenotype correlates with the lung destruction that is characteristic of this disease. Further, we have shown for the first time, to our knowledge, that CXCR3 expression, a marker of Th1 inflammation, extends to lung monocytes and macrophages.Although human lung macrophages are not known to express CXCR3, we suspected based on the immunohistochemical localization of this chemokine receptor that CD14or CXCR3 . In addi+/CD8+ lung lymphocytes. Surface staining for CD4 was not feasible with this protocol; however, the percentage of CD8−/IFN-γ+ cells was approximately equal to that of CD8+/IFN-γ+ cells . This information confirmed our finding that the predominant Th1 bias in COPD/emphysema reflects the microenvironment unique to the lungs of ex-smoker individuals. The prevalence of asthma among people who smoke is currently not known, but in order to study COPD/emphysema in a population without other confounding variables, people who might have had asthma were excluded, and thus our findings are restricted to non-asthmatic individuals with emphysema.+ and CCR8+ T cells express IL-4, and CCR4 expression was prominent in people with severe atopic dermatitis, which decreased upon abatement of disease activity [+/CXCR3+ T cells in the airway epithelium and submucosa [+ T cells in emphysema patients and the functional interplay between Th1-related chemokines and elastolytic MMPs.Our use of T cell chemokine receptor expression analysis to determine recruitment of lung T cells is not without precedent. Analysis by immunohistochemistry of airway mucosa of people with atopic asthma after antigen challenge revealed that large numbers of CCR4activity ,43. Immuubmucosa . We exteIn addition to detailing surface chemokine receptor expression, we have functionally confirmed the marked Th1 bias of peripheral lung T cells, demonstrating that either at rest or following stimulation, these cells secrete IFN-γ and not IL-4. Our findings therefore confirm the utility of chemokine receptor expression patterns in the initial assessment of T cell effector phenotype.Destruction of lung parenchyma in emphysema is thought to occur through excessive proteolysis mediated by the elastin-degrading enzymes MMP2, MMP9, and MMP12 from the MMP family, and by neutrophil elastase from the serine proteinase family ,46. CytoAlthough this was an entirely human study, our findings show remarkable parallels with studies performed in mice. MMP12 deficiency has been shown to protect mice against emphysema after chronic exposure to cigarette smoke, implying that MMP12 may be the key proteinase in the development of emphysema in this species ,50. StudMany people develop long-term lung problems after smoking, including a condition called emphysema. At the very end of the airways are tiny air sacs. In healthy people, the air sacs stretch and relax easily on breathing in and out. But in people with emphysema, the air sacs fill up with air but can't empty out properly, so air gets trapped, making breathing difficult. While the symptoms of emphysema can be treated, there are no treatments that can reverse the damage to the lung.The researchers studied two groups of patients, all ex-smokers who had been admitted to a hospital to have part of their lung removed—some because of cancer, some for other reasons. The researchers studied the lung samples and looked to see exactly what type of immune cells the patients with emphysema had in their lungs and found that most of the immune cells were of one particular type. The researchers also showed that the immune cells could tell other lung cells to produce chemicals that can damage the lung.Lung damage in emphysema may not be caused directly by toxins in cigarette smoke. Instead, if you have emphysema, your body may react to the toxins and produce a special kind of immune cell that is key in causing the lung damage. So perhaps if doctors can find a way to change how this cell behaves, it might be possible to reduce or limit the lung damage. Obviously, not smoking, or stopping smoking, is the best way to prevent COPD or emphysema.The study is quite small, which means that the results may not be completely accurate; in particular, the study did not include detailed information from patients who had never smoked. So it is too soon to say for sure whether these special immune cells really are the link between smoking and lung damage in emphysema. Researchers will need to study many more patients with emphysema as well as people who have never smoked.Two places to start are the patient Web pages of the following professional organizations.http://www.yourlunghealth.org/diseases_conditions/copd/American Association for Respiratory Care: http://www.brit-thoracic.org.uk/public_content.asp?pageid=9&catid=21&subcatid=177The British Thoracic Society:


Osteosarcoma is a highly malignant bone neoplasm of children and young adults. It is characterized by extremely complex karyotypes and high frequency of chromosomal amplifications. Currently, only the histological response (degree of necrosis) to therapy represent gold standard for predicting the outcome in a patient with non-metastatic osteosarcoma at the time of definitive surgery. Patients with lower degree of necrosis have a higher risk of relapse and poor outcome even after chemotherapy and complete resection of the primary tumor. Therefore, a better understanding of the underlying molecular genetic events leading to tumor initiation and progression could result in the identification of potential diagnostic and therapeutic targets.We used a genome-wide screening method – array based comparative genomic hybridization (array-CGH) to identify DNA copy number changes in 48 patients with osteosarcoma. We applied fluorescence in situ hybridization (FISH) to validate some of amplified clones in this study.PRDM16), 6p21.1 , 8q24, 12q14.3 (IFNG), 16p13 (MGRN1), and 17p11.2 . We validated some of the amplified clones by FISH from 6p12-p21, 8q23-q24, and 17p11.2 amplicons. Homozygous deletions were noted for 32 clones and only 7 clones showed in more than one case. These 7 clones were mapped to 1q25.1 (4 cases), 3p14.1 (4 cases), 13q12.2 (2 cases), 4p15.1 (2 cases), 6q12 (2 cases), 6q12 (2 cases) and 6q16.3 (2 cases).Clones showing gains (79%) were more frequent than losses (66%). High-level amplifications and homozygous deletions constitute 28.6% and 3.8% of tumor genome respectively. High-level amplifications were present in 238 clones, of which about 37% of them showed recurrent amplification. Most frequently amplified clones were mapped to 1p36.32 (This study clearly demonstrates the utility of array CGH in defining high-resolution DNA copy number changes and refining amplifications. The resolution of array CGH technology combined with human genome database suggested the possible target genes present in the gained or lost clones. Osteosarcoma (OS) is a primary malignant tumor of bone arising from primitive bone-forming mesenchymal cells and it accounts for approximately 60% of malignant bone tumors in the first two decades of life . These tChromosomal aberrations in osteosarcoma are highly complex and characterized by high frequency of amplifications. These amplifications may result in the overexpression of genes and contribute to the genomic instability in osteosarcoma. The identification of genes within the amplified sites is crucial for understanding the biology and clinical behavior of osteosarcoma. Until, recently gene amplification has been detected by PCR, southern blot analysis or FISH-based approach using gene specific probes. These techniques are inherently restricted to the previously known amplified genes in the genome. In contrast, genome-wide screening of amplified chromosomal regions with CGH has become an important tool for the detection of amplified regions in the tumor genome. So far published chromosomal CGH studies in osteosarcoma have identified several high-level chromosomal amplifications at 1p22, 1p31, 1q21, 1q23, 2q24, 3p25, 3q26, 6q24.3, 4q12, 5p14-p15, 5q33, 6p12-p21, 6q24.3, 7p21-p22, 8q12-q23, 10p21, 10q11.1, 10q22, 11q13, 11q23, 12p13, 12q12-q15, 17p11.2, 17q21, 18q22, 19p13.1 and 20p11.2 [A total of 48 tumors from 42 patients  were collected from the Texas Children's Cancer Center, Houston, TX  and Memorial Sloan Kettering Cancer Center, New York . All tissues in this study were obtained after IRB approved informed consents were signed. The age at diagnosis ranged from 5 years to 71 years at diagnosis. The histological information of 42 patients is presented in Table The array used in this study consists of 967 human BACs, which were spaced approximately 3 megabase across the whole genome. These arrays were obtained from Spectral Genomics, Houston, TX. The experiments were performed according to the manufacturer's protocol. Arrays were pre-hybridized with human Cot-I DNA  and salmon testes DNA to block the repetitive sequences on BACs. One microgram of normal DNA (reference) and tumor DNA (test) was labeled with cy5-dUTP and cy3-dUTP respectively, by random priming. To avoid dye bias, we performed dye swap experiments for each sample. The probe mixture is dissolved in hybridization mixture, denatured, cooled, and mounted with 22 × 60 mm coverslip. Hybridizations were performed in sealed chambers for 16–20 hours at 60°C. After post hybridization washes, arrays were rinsed, dried with compressed air, and scanned into two 16-bit TIFF image files using Gene Pix 4000A two-color fluorescent scanner  and quantitated using GenePix software .After scanning of the slide, the fluorescent intensities of the green and red channels were background subtracted. The resulting values were normalized by intensity based local weighted regression method (Lowess) to correct for systematic bias in dye labeling and fluorescent intensity . Then thp-values of the each clone by comparing to the clone with median variance using chi-square distribution . The clones that have p-value greater than preset cutoff 0.9 were considered as invariant clone set, i.e. clones that do not vary significantly in all experiments. Then the mean and standard deviation of the log ratios of these invariant clones in each experiment were calculated. The clones with log ratios that exceed mean +/- 2 × SD of the invariant set were considered gains and losses, respectively. For amplification and homozygous deletions, clones were defined to have at least 2 fold of the upper bound threshold and 4-fold of lower bound threshold, respectively. The gene(s) present in the clones were identified using UCSC browser  by downloading gene table (refFlat) from human gene assembly, July 2003. We search the candidate genes based on linear mapping position, which include 100 kb up and downstream from the clone center position. The supplemental data for this article is available at: We developed a new analytical method called invariant analysis to define the significant copy number changes. This method is designed to: i) increase the power of the analysis by combining all the cases in our dataset to define an invariant population (unchanged population); and, ii) to address the signal to noise differences among individual cases due to sample and hybridization variability. Our goal is to define a set of unchanged clones that can be used to calculate the upper and lower bound thresholds of the log ratios for the unchanged population in each experiment. First, we calculated the variance of each clone from all the experiments. We computed the . The experiments in each of the two groups, amplification and normal, used for comparison were defined based on the invariant analysis (see above). The clones that have p <0.001 were considered as significant. We chose a stringent cutoff to minimize the multiple testing problem.Significant clones in 6p, 8q, 12q and 17p amplicons were calculated using 2-sample t-test with randomized variance model FISH was performed to validate and quantify chromosomal amplicons using clones from 6p12-p21 , 8q24.3 (RP11-89K10), and 17p11.2-p12  clones, and centromeric clone from 6 (pEDZ6) were labeled with Spectrum Red or Spectrum Green  by nick translation. Hybridization and FISH analysis was performed as described previously .To define the gains and losses in our experiments, we used invariant analysis for the first time to describe genomic changes by array CGH. In this method, we defined an invariant clone set that has low variance of log ratios among all the array experiments. After the mean and standard deviation of the log ratios in the invariant set of each experiment were calculated, clones that have higher or lower log ratios than the mean +/- 2SD of the invariant set (upper bound and lower bound) were used to define gains and losses. We chose to use this method because it addresses some of the shortcomings of the modeling method, such as using all information provided in a set of experiment to determine the unchanged population instead of using one experiment at a time. However, the variation of each experiment is accounted for because the thresholds are calculated using the invariant set from each experiment. It also does not require a separate reference set for comparison. Finally, it provides an adjustable cutoff to optimize the thresholds to the training data, if provided.The amplified and homozygously deleted clones were defined to have at least 2 fold of the upper bound and 4-fold of lower bound, respectively. Figure Copy number changes excluding clones from sex chromosomes were involved in a significant fraction of most tumor genome. The estimated average genomic distance between clones was ~3–4 Mb. The frequency of clones showing gains (79%) was greater than losses (66%). High-level amplifications and homozygous deletions constitute 28.6% and 3.8% of tumor genome respectively. The most frequently deleted clones were identified from the chromosomal regions 2q31.1, 3p14.1, 4p16.2, 6q12, 6q21, 6q27, 7q35, 10p15.1, 10q22-q23, 10q25-q26, 11q25, 13q12.2, 13q14.3, 13q22.1, 17p13.3 and 17q12 (Table Homozygous deletions were noted for 32 clones (3.8%). Recurrent homozygous deletions were noted for 7 clones that are were mapped to 1q25.1 (4 cases), 3p14.1 (4 cases), 13q12.2 (2 cases), 4p15.1 (2 cases), 6q12 (2 cases), 6q12 (2 cases) and 6q16.3 (2 cases). Figure ROR1), 1p36.1 (PRDM16), 1q21, 1q23 (TNFF6), 2q24, 3p25, 3q26.1, 4p16.3, 5p14, 5q33, 6p11.2-p21, 7p21, 8q12.1, 8q24.13, 10p21, 10q11.1, 10q22 (KCNMA1), 11q13, 11q23 (GRIK4), 12q12, 12q13-q15, 12q21-q21.33, 17p11.2-p12, 17q21 (NGFR), 18q22, and 19p13.1 (NFAT). Of these amplified sites, 6p11.2-p21, 8q12.1, 8q24.13, 12q12, 12q13-q15, 12q21-q21.33, 16p13 and 17p11.2-p12 were frequent.Previous studies using CGH have identified several chromosomal amplification sites in osteosarcoma. Because of the limitation of the method, it fails to pinpoint the precise site of amplicon. However, the present study by array CGH has identified 238 clones (28.6%) with high-level amplifications. Recurrent amplifications were noted in ~37% of the total amplified clones  cases analyzed. High-level amplification of the clones from same region was noted in 25% of the cases by array CGH. We found that most of the cases with amplification of 6p12-p21 displayed either increased or slightly varying degree of copy number increase across the 6p12-p21 region. The combined log ratios from all the cases defined the boundaries of amplification between RP3-329A5 and RP11-79F13. The amplicon spans approximately 9.4 Mb with amplification peak for clone RP11-81F7. Further, we used FISH to validate 6p amplicon on tumor metaphase and interphase cells from cases 274, 364, 426 and 527. Increased copy numbers for clones RP11-91E11, AL391415, RP11-81F7, RP11-79I2, RP11-90H17 and RP11-79F13 were noted in interphase cells with maximum copy number increase for clone RP11-81F7 Figure . This waMost cases with 8q gain, displayed varying degree of copy number increase predominantly from 8q12.1 (16.9%), 8q21.13 (29%), and 8q24.3 (35%). High-level amplifications were also noted from 8q12.1  tumors analyzed by array CGH. Three distinct amplicons – AMP1 (12q12), AMP2 (12q14.1) and AMP3 (12q21.33) were noted across the entire long arm of chromosome 12 Figure . Of thesAmplification of 17p11.2 was noted in 27% of the cases analyzed by array CGH. The amplicon was composed of three clones RP11-64B12 (p = 0.0000014), RP11-89K6 (p = 0.00000005) and RP11-189D22 (p = 0.0000001) and covering 3.7 Mb region on the short arm of chromosome 17 Figure . We usedThis study represents the first application of genome-wide copy number changes by array CGH in osteosarcoma. Recent studies in breast, renal and bladder cancer showed the potential assessment of this technology in detecting high-resolution copy number changes ,11,14. Tp53 significantly correlates with genome-wide DNA instability and seems to represent a major genetic factor contributing to the extremely high levels of genomic instability found in high-grade osteosarcomas [Gene amplification is an important genetic mechanism in human cancers, as it clearly associated with tumor progression and has a prognostic significance and has even provided a target for therapeutics ,18. Thessarcomas .CDC5L, HSPCB, and NFKBIE, and HGNC and MRPL14 are the target genes from 6p12-p21 amplicon. Of these genes, CDC5L may be an important gene in cancer because of its role as a positive cell cycle regulator for G2/M transition[HSPCB was shown recently by cDNA microarray studies on osteosarcoma [Our analysis have identified frequently amplified clones from 6p11.2-p21, 8q12.1, 8q24.13, 12q12, 12q13-q15, 12q21-q21.33, 16p13 and 17p11.2-p12. Amplification of clones from 6p12-p21 region was noted in 25% of the cases analyzed. This was consistent with the previously published results by CGH. By array CGH, we refined the 6p amplicon to 9.4 Mb with amplification peak for clone RP11-81F7. We recently demonstrated the origin of 6p amplicon as consequence of tandem duplication of clones RP11-81F7 and RP11-79F13 [ransition. ConsistNSE2 (breast cancer membrane protein 101 kDa) gene.High-level amplifications were also noted from 8q12.1 (RP11-550I15 – 6.3%), 8q21.13 (RP11-89H1 – 6.3%), 8q24.3 (RP11-89K10 – 6.3%) and RP11-637F16 (12.5%). There were no candidate genes present in clones RP11-550I15, RP11-89H1 and RP11-637F16, but clone RP11-89K10 contained GLI, CHOP, SAS, HMGI-C, CDK4, HDM2, and PRIM1 from 12q13-q15 region in osteosarcoma [IFNG from AMP2 , which is physically mapped close to the HDM2 oncogene locus[IFNG strongly suppresses osteoclastogenesis by interfering with the RANKL-RANK signaling pathway. IFNG induces rapid degradation of the RANK adaptor protein, TRAF6, resulting in strong inhibition of the RANKL-induced activation of the transcription factor NFKB and JNK [ELK3 and PCTAIRE protein kinase 2 (PCTK2). ELK3 is a member of the ETS-domain transcription factor family and the protein is activated by signal-induced phosphorylation [PCTK2 belongs to the cdc2/cdkx subfamily of the ser/thr family of protein kinases and play an important role in the regulation of the mammalian cell cycle [CCND2, ETV6, and KRAS2 from 12p region [High-level amplification of clones on 12q revealed three distinct sites of amplifications – AMP1 (12q12), AMP2 (12q14.1) and AMP3 (12q21.33). Pervious studies have shown the amplification osarcoma ,23. The ene locus. Previou and JNK . The AMPrylation . The proll cycle . High-lep region .PP3A, SMCR5, DRG2, FL11, MYCD, SOX 17, ELAC2, and PMP22). Recent studies have shown the amplification of some of the genes identified in the present study  from 17p11.2-p12 in high-grade OS by semi-quantitative PCR and cDNA microarrays [Amplification of 17p11.2 was noted in 27% of the cases analyzed by array CGH. Our array CGH analysis has identified three clones with high-level amplifications that spans ~3.7 Mb region on 17p11.2. Several candidate genes were identified within these clones  in an atypical myoproliferative disorder [NUFIP1P) and BAI3 gene (brain-specific angiogenesis inhibitor gene), which is to homologous to BAI1 and shown to suppress glioblastoma [The present array CGH analysis has identified seven recurrent clones exhibiting homozygous deletions from 1q25.1, 3p14.1, 13q12.2, 4p15.1, 6q12, 6q12 and 6q16.3. These chromosomal regions were consistent with previously reported studies by loss of heterozygosity (LOH) and CGH ,31. The disorder . Homozygblastoma .In summary, high resolution array-based CGH revealed large number of chromosomal aberrations previously identified in osteosarcoma by chromosomal CGH and conventional cytogenetic methods. The present study allowed precise identification of smaller DNA copy number alterations, which suggest the presence of specific target genes in osteosarcoma. Although this study suggested several possible target genes from amplified regions from 6p, 8q, 12q and 17p, but these genes should be validated by other molecular and immunohistochemical approaches on well-defined large patient samples. Further, interaction or association studies between small genomic losses and gains will facilitate the identification of new genetic pathways in the pathogenesis of osteosarcoma.None declared.TKM and KJ have contributed towards the data analysis. LP, ML, RG, and CL were assisted in sample collection and clinical information of the patients. X-YL has involved in array CGH experiments and data collection. CPH has involved in extracting the gene information from BAC clones. SS has provided the arrays used in this study. PHR was involved in the planning, and organization of the project.The pre-publication history for this paper can be accessed here:


Small-cell neuroendocrine carcinoma in the duodenum is an extremely rare neoplasm with poor prognosis.A 57-year-old man presented with sudden onset gastrointestinal bleeding and fainting attacks. Duodenoscopy and hypotonic duodenography revealed a 3 × 3 cm protruding tumor with ulcerations situated opposite the ampulla of Vater in the second part of the duodenum. Local excision of the tumor was performed, followed by adjuvant chemotherapy with 5-fluoro uracil and leucovorin. Examination of the tumor by immunohistochemistry and electron microscopy indicated it to be neuroendocrine in nature, expressing synaptophysin and AE1/AE3, and containing dense core granules. The patient showed no sign of recurrence and has been disease-free for more than 48 months after surgery.Most cases of small-cell neuroendocrine carcinoma in the duodenum show rapid progression of the disease, and even radical surgery with or without chemotherapy do not prevent death. We report a rare subtype of small-cell neuroendocrine carcinoma. This subtype appears to have a much better prognosis, and may be amenable to local excision, if the lesion is away from the ampulla of Vater. Duodenal Neuroendocrine tumors constitute 5% of all gastrointestinal neuroendocrine tumors ,2. Most A 57-year-old man presented with sudden gastrointestinal tract bleeding and episode of fainting. Duodenoscopy Figure  and hypoA laparotomy was performed. As there was no serosal invasion or regional lymphadenopathy wide local excision of the tumor was performed. On gross examination, the tumor showed two ulcerations and two different morphological components Figure  and 2b. 2) and leucovorin (20 mg/m2) were administered. The patient showed no sign of recurrence and is disease-free 48 months after surgery.The patient was discharged three weeks after operation with uneventful postoperative period. Four cycles of monthly adjuvant chemotherapy with 5-fluoro uracil (5-FU) (325 mg/mNeuroendocrine carcinomas (NEC) in the duodenum are extremely rare, and are classified as either 'small-cell' or 'non small-cell' types. The small-cell NEC occurring in the duodenum and elsewhere in gastrointestinal tract are similar to the small-cell carcinoma of the lung ,8. Only The natural course of small-cell NECs in the duodenum is still not clear. Most cases reported in the literature show rapid progress of the disease, with radical surgery and/or chemotherapy not altering the clinical course, and thus a poor prognosis. In six of the previous eight cases, patients underwent pancreaticoduodenectomy for removal of the tumor, while the remaining two did not undergo surgery because of multiple liver metastasis or poor general condition. In spite of radical resection with or without adjuvant chemotherapy, most cases showed rapid recurrence and metastasis. Of the eight reported cases, one was unusual as it occurred in a middle-aged female with rapid progress of the disease but effective response to adjuvant chemotherapy using 5FU, tumor necrosis factor, and interferon this patient survived for more than eighteen months . The preThis report identifies a new subtype of small-cell NEC in the duodenum. This subtype appears to have a much better prognosis, and may be amenable to local excision, if the lesion is away from the ampulla of Vater.None declared.NS, MT, MK, KY, KK, HN took part in the operation, performed the literature search and drafted the manuscript for submission. HN supervised the preparation of the manuscript and edited the final version for publication. TS, KS performed pathological investigations and contributed to the pathological content of the manuscript.All authors read and approved the manuscript.


Contrast-induced nephropathy is an important cause of acute renal failure. We assess the efficacy of acetylcysteine for prevention of contrast-induced nephropathy among patients undergoing intravascular angiography.We conducted a systematic review and meta-analysis of randomized controlled trials comparing prophylactic acetylcysteine plus hydration versus hydration alone in patients undergoing intravascular angiography. Studies were identified by searching MEDLINE, EMBASE, and CENTRAL databases. Our main outcome measures were the risk of contrast-induced nephropathy and the difference in serum creatinine between acetylcysteine and control groups at 48 h.higher incidence in six of the latter studies). The pooled odds ratio for contrast-induced nephropathy with acetylcysteine relative to control was 0.54  and the pooled estimate of difference in 48-h serum creatinine for acetylcysteine relative to control was -7.2 μmol/L . These pooled values need to be interpreted cautiously because of the heterogeneity across studies, and due to evidence of publication bias. Meta-regression suggested that the heterogeneity might be partially explained by whether the angiography was performed electively or as emergency.Fourteen studies involving 1261 patients were identified and included for analysis, and findings were heterogeneous across studies. Acetylcysteine was associated with a significantly reduced incidence of contrast-induced nephropathy in five studies, and no difference in the other nine (with a trend toward a These findings indicate that published studies of acetylcysteine for prevention of contrast-induced nephropathy yield inconsistent results. The efficacy of acetylcysteine will remain uncertain unless a large well-designed multi-center trial is performed. Contrast-induced nephropathy is a leading cause for acquired acute reductions in kidney function ,2. DespiThe pathophysiology of contrast-induced nephropathy remains incompletely understood. However, current evidence suggests that contrast media induce prolonged vasoconstriction and medullary ischemia coupled with generation of free radicals and oxidative injury to tubular cells -11.Acetylcysteine, a thiol-containing anti-oxidant, has been hypothesized to prevent contrast-induced nephropathy. The potential benefit of acetylcysteine is believed to be mediated by its properties as a scavenger of free-radical species and by increasing the synthesis of nitric oxide, a potent vasodilator, in response to ischemic or other toxic injury in the kidney . Given tWe identified published randomized controlled trials of acetylcysteine for prevention of contrast-induced nephropathy during intravascular angiography using both electronic and manual search strategies. We supplemented this by scanning the reference lists of all identified articles, reviewing selected conference proceedings, and by contacting experts in the field. All languages and types of publications were considered eligible. The comprehensive literature search was initially performed in April 2003 and updated in June 2004 to identify any potential new studies that may have appeared.MEDLINE , EMBASE  and CENTRAL  databases were searched via OVID using an approach recommended for systematic reviews of randomized trials . PubMed Two individuals (SMB and WAG) independently evaluated identified articles for eligibility on the basis of four inclusion criteria: 1) study design , 2) target population (patients undergoing intravascular angiography), 3) intervention  and 4) outcome .Two reviewers (SMB and WAG) independently extracted data from all primary studies fulfilling eligibility criteria. Any discrepancies in extracted data were resolved by consensus. Data extracted included identifying information, focus of the study, details of study protocol and demographic data. The primary outcome measures were the incidence of contrast-induced nephropathy and change in serum creatinine. The secondary outcome measure was requirement for renal replacement therapy. Authors of the studies were contacted for additional information when applicable.et al [Two reviewers (SMB and WAG) independently assessed methodological quality of individual studies. Any disagreements were resolved by consensus. Items used to assess study quality were methods of randomization, any blinding, use of a placebo, reporting of losses to follow-up or missing outcome assessments, and evidence of important baseline differences between the groups -18. An oet al .The presence of heterogeneity can compromise the interpretation and validity of meta-analyses and can result from significant differences in methodology, study populations, interventions, outcomes, or chance . A priorData from all of the selected randomized controlled trials were combined to estimate the pooled odds ratio (OR) with 95% confidence intervals (CIs) using a random-effects model as described by Der Simonian and Laird ,21. The A total of 66 unique citations were identified by our initial search strategy Figure . After tAll the randomized controlled trials were published in the years 2002 through 2004. Tables The definition of contrast-induced nephropathy was variable across studies. Four studies defined contrast-induced nephropathy as a > 44.2 μmol/L increase in serum creatinine from baseline ,29,32,37The reported incidence of contrast-induced nephropathy was variable across studies. Table Table Meta-regression was performed to assess a number of clinical and study quality factors that may have led to heterogeneity across studies. Interestingly, these analyses suggest that the heterogeneity may be partially explained by whether the angiography procedures were performed electively or as emergency, because studies where all enrolled patients were undergoing elective procedures had significantly lower odds ratios than did studies where emergency cases were included .Other meta-regression analyses demonstrated that the heterogeneity could not be accounted for by differences in patient age , baseline serum creatinine , volume of contrast media  or diabetes mellitus . Likewise, heterogeneity was not accounted for by differences in study quality including use of blinding , concealment of randomization , use of placebo  or overall Jadad score .There was some evidence to suggest possible publication bias according to Begg's test  and a trend with Egger's test . Figure Our meta-analysis of 14 peer-reviewed studies of patients undergoing intravascular angiography may lead some to conclude that the administration of acetylcysteine causes a reduced incidence of contrast-induced nephropathy. However, such a conclusion may be premature based on data published to date because our systematic review reveals considerable heterogeneity of findings across trials. Furthermore, our meta-analysis of post-treatment creatinine values does not reveal any truly meaningful difference in serum creatinine levels at 48 h between the acetylcysteine and control groups. Finally, insufficient data are available to allow inferences to be drawn about the efficacy of acetylcysteine on clinically meaningful endpoints such as dialysis, length of hospitalization or mortality.et al that recently received considerable attention, and that rather firmly concluded that acetylcysteine is beneficial [et al, and more directly address the relevance of this heterogeneity in the overall interpretation of study results. Two other meta-analyses have also recently been published, and similarly concluded that acetylcysteine is beneficial; however, these studies also failed to adequately address the issue of the considerable heterogeneity across studies [This meta-analysis has several features that distinguish it from a similar meta-analysis by Birck neficial . First, neficial . Fourth, studies ,42. Coll studies . Many wi studies ,45 make The presence of heterogeneity and/or publication bias can compromise the interpretation of meta-analyses and result in erroneous and potentially misleading conclusions ,43. A stdefinitely efficacious based on data published to date. Our firm conclusion based on this meta-analysis of published trails is that although the data seem quite promising, the efficacy of acetylcysteine has not been definitively proven.There are many parallels between the intravenous magnesium story and our meta-analysis findings for acetylcysteine. The marked heterogeneity of findings across studies, and the finding of funnel plot asymmetry (indicating possible publication bias), ought to be viewed as strong cautionary points against making firm conclusions about the efficacy of acetylcysteine. And while it is true that acetylcysteine is inexpensive, easy to use and has a favorable side-effect profile, it is probably premature to conclude scientifically that it is To isolate potential sources of heterogeneity we performed a meta-regression analysis exploring several clinical and study quality factors. There was no evidence of association between effect size and baseline serum creatinine, volume of contrast media, or diabetes mellitus, all independently identified risk factors for development of contrast-induced nephropathy ,53. HoweFunnel plot asymmetry is often interpreted to indicate publication bias. However, it is important to consider that this asymmetry may also be due to other sources of bias that deserve further examination. In particular, fundamental disparities in study design, inconsistencies in methodological quality and differences in the definition of primary outcomes may have contributed to funnel plot asymmetry. Our meta-regression analysis explored the potential role of several study quality factors, and none were identified as statistically significant predictors of apparent acetylcysteine efficacy across trials. Nonetheless, it is quite possible that other unmeasured study quality factors may have contributed to biased results and accompanying funnel plot asymmetry.Contrast-induced nephropathy continues to be an active subject matter for clinical investigation ,56. A deAll of the above leads us to conclude that while acetylcysteine appears to be safe and inexpensive, its efficacy for the prevention of contrast-induced nephropathy remains unproven. The results of the trials that we reviewed to date should be viewed as early promising evidence of benefit, and suggest that it is now perhaps reasonable to use acetylcysteine in routine care because of its relative ease of use and safety. However, its true efficacy will remain uncertain unless a definitive well-designed multi-center trial is performed. Such a clinical trial will be most relevant if it addresses a priori clinically meaningful endpoints of renal insufficiency, rather than surrogate endpoints based on changes in creatinine levels alone, and further considers stratification on hypothesized important subgroups that may benefit such as those with a low ejection fraction .The authors declare that they have no competing interests.SMB developed the study protocol, conducted literature search, screened articles for eligibility, extracted data, analyzed data, interpreted results, wrote and revised the manuscript. WAG contributed to protocol development, screened articles for eligibility, extracted data, analyzed data, interpreted results, and provided critique of successive drafts of the manuscript. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Microarrays are a powerful tool that have multiple applications both in clinical and cell biology arenas of common lung diseases. To exemplify how this tool can be useful, in this review, we will provide an overview of the application of microarray technology in research relevant to common lung diseases and present some of the future perspectives. Microarray technology is rapidly becoming a standard technology used in research laboratories all across the world. Since its first application in the mid 1990s -7 with oDNA microarrays, microscopic arrays of large sets of DNA sequences immobilized on solid substrates, are valuable tools in areas of research that require the identification or quantitation of many specific DNA sequences in complex nucleic acid samples . They ar1. Determination of transcriptional programs of cells for a given cellular function  or when they are exposed to certain conditions leading to activation, inhibition or apoptosis.2. Compare and contrast transcriptional programs to aid diagnosis of diseases, predict therapeutic response and provide class discovery and sub-classification of diseases.3. Identification of genome-wide binding sites for transcriptional factors that regulate the transcription of genes.4. Prediction of gene function.5. Identification of new therapeutic targets .6. Development of public databases that will help us understand the functioning of complex biological systems.7. Genetics of gene expression: Although this is a relatively new study field, it is advancing rapidly with major implications in complex clinical traits by the identification of promising candidate genes. Thus, we briefly review the current implementations of this novel approach highlighting its necessity in the research field. Treating mRNA transcript abundances as quantitative traits and mapping gene expression quantitative trait loci for these traits has been pursued in gene-specific ways. Unlike classical quantitative traits, the genetic linkages associated with transcript abundance permits a more precise look at cellular biochemical processes. Schadt et al.  describeDNA microarrays are used to estimate the levels of mRNA in the cell. The process can be described in three steps:Array construction: Currently, there are two widely used microarray technologies:1) • In situ synthetized oligonucleotide (20–25 mers) microarrays-mainly oligonucleotides synthetized by photolithography or ink-jet technology on a glass surface.• Spotted, in glass or nylon membrane matrices, microarrays-mostly created by robotic printing of pre-prepared cDNAs or oligonucleotides (polymerase chain reaction-PCR-generated products from cDNA libraries or clone collections) ,15.Sample preparation and array hybridization: The next step in the microarray experiment is to prepare the material that will be hybridized with the microarray. Gene expression is measured by the amount of mRNA therefore it must be extracted from the sample cells or tissues. For high-density microarrays one has to convert mRNA into cRNA, whereas for spotted arrays one can use mRNA, cDNA, or cRNA. The RNA needs to be labeled so that the detecting machinery can measure the quantity of RNA present. In oligonucleotide microarrays mRNA is extracted from experimental samples and is labeled with a fluorescent oligonucleotide (biotin). The biotin labeled cRNA and each sample is hybridized to a separate array, the array is scanned and absolute expression levels are obtained for each probe by using a dedicated laser scanner. In contrast, in spotted microarrays, mRNA is extracted from a sample and a control and one is labeled with cy-5 (red fluorescent dye) and the other with cy-3 (green fluorescent dye). Expression values are based on the competitive hybridization of the two samples being directly compared on a single array. Conventionally, in radioactive nylon membrane arrays RNA probes are labeled with P33 or P32 dCTP during a reverse transcription reaction. The great advantage of nylon microarrays is that they require relatively small amounts of PCR products because radioactive targets have a higher intrinsic detectability whereas in glass arrays the quality of RNA is not as critical as it is in nylon arrays. Therefore nylon arrays are mainly used when sample material is scarce or a small number of genes need to be assayed [2)  assayed ,17.3) Image analysis and data acquisition: The laser causes excitation of fluorescently or radioactively labeled cDNA probes. Only the spots representing mRNAs in the sample give emission signals. The emission is measured using a scanning confocal laser microscope for fluorescently labeled arrays or a flat-bed scanner for radioactive nylon arrays and finally data is analyzed by appropriate software. In spotted microarrays using fluorescent probes if particular mRNA from the sample is in abundance, the spot with a complementary probe will be red; if the concentration of the particular mRNA is higher in the control, the spot will be green. If both samples contain the same amount of a given mRNA, the spot will be yellow  is a refractory and lethal interstitial lung disease characterized by fibroblast proliferation, extracellular matrix (ECM) deposition and progressive lung scarring. The incidence of IPF is estimated at 15–40 cases per 100.000 per year, and the mean survival from the time of diagnosis is 3–5 yr regardless of treatment ,21. The -/-), thus protected from pulmonary fibrosis, and wild type mice. Interestingly a simple hierarchical cluster analysis identified most of the known TGF-β1 inducible genes preferentially induced in wild type mice. The latter finding provides support for the hypothesis that β6 knockout mice are protected from pulmonary fibrosis as a consequence of failure to activate TGF-β. The great importance of this study results from the identification and the global availability of several genes that are likely to be directly relevant to the fibrotic process. However the inability of microarray technology to detect genes that are not included in the array, identify critical proteins that participate in biological responses and ascribe changes in gene expression in specific cellular types limit the scientific rigidity of the data derived and highlights the necessity for combined application of novel approaches.In another study Kaminski et al.  used oliFurthermore driven by the same perspective idea to investigate the gene expression pattern in bleomycin-induced pulmonary fibrosis, Katsuma et al.  construcin vivo was established in a separate set of confirmational experiments in rats where it was revealed an overexpression of the ID by myofibroblasts within the fibrotic regions. These novel suggestions have major impact on our understanding of the crucial role of TGF-β1 as a fibroblast differentiation factor in response to fibrogenic stimuli. Nonetheless the present study does not determine the precise role of ID in regulating fibroblast responses to TGF-β1. To do so this study should be coupled with independent methods using ID blocking strategies. Moreover the use of incomplete arrays that detect only the included genes, the variability of cellular composition of tissues studied even from the same organ and the inability of this technology to distinguish changes in cellular composition from transcriptional changes pose major limitations to the global application of these results. Furthermore these observations offer plausible explanations for the lack of similar effects of TGF-β1 in previous studies.One of the most informative studies scrutinizing the global gene expression profile of fibroblasts in response to one of their most potent activators, TGF-β1, has been published by Chambers et al.  Gene expMoreover, bleomycin-induced pulmonary fibrosis rat model was extensively studied by Liu et al.  with theThese preliminary results suggest that this technology could identify unexpected molecular participants in IPF and might help in the development of novel targets for improved treatment. The method may also allow molecular fingerprinting that could improve the ability to identify subclassifications of pulmonary fibrosis that might be more informative than the current classification based primarily on histologic and radiographic patterns . NonetheAsthma is one of the most serious allergic diseases associated with both genetic and environmental factors such as allergens, respiratory tract infections, and atmospheric pollutants. Most asthma is associated with atopy, a predisposition to generate immunoglobulin (Ig)-E against environmental allergens . HoweverMicroarray technology offers a new opportunity to gain insight into global gene expression profiles in asthma, leading to the identification of asthma associated genes. Several experimental models have been used for this purpose although no animal disease model is identical to human disease. Zou et al. were theBrutsche et al.  designedSeveral morphologic changes in the airways of patients with asthma have been attributed to the Th-2 produced cytokines such as IL-13 and IL-5. However the molecular mechanisms underlying the contributions of these cytokines to asthma remain largely unknown.in vivo and in vitro.Towards this direction Lee et al.  applied One of the greatest disadvantages of microarrays and at the same time challenges for most of the investigators is the objective difficulty dealing with the results of the experiments resulting from the large quantities of information. Currently, the hurdle faced is the routine interpretation of this information to identify among thousands of dysregulated genes, those who are informative, causal and specific to the phenotypic change of interest. Thus, Temple et al.  comparedMicroarrays apart from their remarkable effectiveness in identifying novel gene expression patterns can also be used to clarify physiological mechanisms underlying the actions of numerous drugs, such as those applied in the management of patients with asthma. Several studies have utilized microarray technology to assess the gene expression profile of cells and tissues before and after treatment with commonly applied drugs such as corticosteroids. Two of them are reviewed here.Recently, Hakonarson et al.  in additMicroarray analysis performed by Laprise et al.  indicateMast cells represent key cells in the initiation and progression of asthma, releasing several mediators of inflammation, such as certain cytokines and chemokines. The past few years several studies have been focused on the identification of new mast cell products through the gene expression analysis. In one of them published by Sayama et al.  applicatFurthermore, Nakajima et al.  in theirMicroarrays using nylon membrane radioactive cDNAs have already been applied in the research field of asthma and much good work has been done with this technology.Brutsche et al.  applied One of the first gene-profiling studies highlighting the potential role of chemokines and their receptors in the pathogenesis of asthma was conducted by Syed et al.  They useIn another study Banerjee et al.  in theirAccumulated evidence from these analyses revealed that microarray analysis can be a powerful tool for identifying mediators of allergic asthmatic disease through a genomic-based strategy using non-human primates and provide us a novel large scale of differentially expressed genes. Additionally, authors compared different cellular models sharing similar experimental paradigm to focus on the most likely informative genes and filter out the bystanders. The application of this approach further streamlined the pivotal role of microarrays in determining transcriptional responses of genes to several inflammatory cytokines and in identifying gene expression patterns and important mediators associated with the initiation and the progression of asthma. Moreover, microarrays coupled with separate set of experiments have provided the investigators with useful knowledge concerning the efficacy of the already applied drugs in the treatment of asthma and helped them to understand their anti-inflammatory role in terms of physiology and molecular biology. Nevertheless, most of the studies exhibited essential weaknesses generated by the heterogeneity of samples studied and compared and by the disability of microarrays to quantify gene expression and yield information about transcriptional responses and post-translational protein modifications. Therefore more in depth analysis of the microarray results is needed in combination with novel approaches that will help us focus on the specific genes and elucidate their role in the cellular function and the pathogenesis of asthma Tables ,3.Chronic obstructive pulmonary disease (COPD) is a chronic disease characterized by progressive airflow obstruction, chronic cough and dyspnea in advanced stages, caused by smoking, environmental, and hereditary factors. It is associated with two clinical entities, chronic bronchitis and emphysema. In nowadays, the invention and application of microarray technology offers scientists the opportunity to gain a better understanding on the pathophysiology of COPD through the identification of novel gene expression patterns, leading to illumination of genes candidates for modern therapeutical approaches .It is already known that chronic bronchitis can be induced by several types of environmental pollutants such as diesel exhaust particles (DEP). Though recently a microarray study has been published by Koike et al.  addressiMicroarray approach is already being applied in respiratory clinical pharmacology with the identification of genes {Yamanaka et al. } that caOne of the major limitations in our attempt to elucidate the exact role of specific cell types in the pathogenesis of COPD is the compact anatomy of the lung which makes unraveling specific cell type gene expression changes difficult, requiring immunoelectron microscopy or laser capture microdissection. The first study to perform quantitative cell type-specific gene expression analysis using the pioneering technology of laser capture microdissection in human tissue samples coupled with RT-PCR and cDNA approach was recently published by Fuke et al.  Authors Several inflammatory cytokines have been implicated in the pathogenesis of emphysema, including TNFa, a molecule with versatile pathogenetic mechanisms. As a means to investigate some of them that culminate to lung-pathology, Vuillemenot et al.  applied Despite the clear link of smoking to the risk for chronic bronchitis, only 15–20% of cigarette smokers develop COPD,  suggestiOne of the most exciting aspects of microarrays is their use as tools for actively introducing serendipity to one's research . In expePresently, very few studies dealing with the role of HOX genes in the adult respiratory system are available in the literature. Golpon et al.  investigCollectively these results suggest that microarray analysis with its ability to highlight gene expression profiles on a large scale and coupled with progressive technologies and independently validated data has led researchers to shed further light into transcriptional programs regulating emphysema and to the identification of common mediators and molecular pathways involved in the pathogenesis of both COPD and pulmonary fibrosis, indicating novel targets for therapeutic interventions and useful genetic markers assessing susceptibility to COPD. Although limitations such as inconsistency between findings derived by microarray approach and independent studies, lack of functional changes assessment and significant data variability can be detectable in these studies, however evidence derived from these analyses is valuable and heavily informative Table .4. Novel interactions between factors (antioxidant genes) previously associated with ALI and factors (surfactant proteins) previously not associated with ALI emerged from the application of functional genomics during nickel-induced ALI. Data derived from this experiment and partially confirmed by Northern blot analysis and nuclease protection assays is valuable and consistent with the ongoing attempts to treat ALI with exogenous surfactant-associated proteins [Acute lung injury (ALI), a severe respiratory syndrome, develops in response to numerous insults. This syndrome that responds poorly in therapeutic interventions and has a poor prognosis has been associated with a myriad of mediators including cytokines, reactive oxygen and nitrogen species, growth factors and proteolytic enzymes ,55. Despproteins  in combiAlthough the fibroproliferative response to lung injury occurs in high frequency in patients with ALI, the mechanisms of this response are largely unknown. One of the most meaningful and informative studies addressing this important issue was recently published by Olman et al . AuthorsALI is frequently associated with endotoxemia and is characterized by the accumulation in the lungs of large populations of neutrophils activated to produce proinflammatory mediators. Many studies had demonstrated a critical role of the endotoxemia-induced activation of NF-κB in neutrophils in the development of ALI ,60. One To gain a more comprehensive understanding on the complex interplay between several inflammatory cytokines involved in the pathogenesis of pulmonary edema, several group of investigators {Cher et al. , SabbadiThe study of Perkowski et al.  has beenOne of the first gene-profiling studies to address an important disease process, such as sepsis at a multisystem level, was that of Ward et al.  Using DNIn summary, these studies exhibit the crucial role of a novel molecular technology in discovering, through global analysis of gene expression, genes previously identified only by their DNA sequence. Although the array analysis provides in some studies a comprehensive overview of gene expression in the lung during ALI ,58,61,64Sarcoidosis is a chronic systemic disorder characterized by the presence of non-caseating granulomas and accumulation of T-lymphocytes and macrophages in multiple organs . The mecCollectively these findings not only reveal the importance of the microarray platforms in identifying gene expression patterns that give the scientists the opportunity to elucidate the pathophysiological processes of complex diseases, such as sarcoidosis but also illuminate some of their origin disadvantages.The last five years has seen the emergence of a novel technology applied in almost every aspect of respiratory research, a technology that has also great future perspectives and may provide scientists with numerous avenues of investigation that have clinical implications. Since nowadays, microarray technology has been successfully used for the identification of potential target genes for therapeutic intervention in IPF, mechanisManaging and mining the huge amount of data generated by microarray experiments still remains a major challenge and limitation for most investigators. Whether gene expression changes are considered primary vs. reactive for a given disease is a complicated issue, and one can only begin to judge that if the methods and approaches used to generate the data are of sufficient scientific rigidity. The diversity and scope of the data require the creation of multidisciplinary teams consisting of physicians, biologists and bioinformaticians  . Thus, wBecause of the statistical issues raised by microarray technology, it is necessary for any meaningful interpretation that the data is replicated using independent methodological criteria, preferably with separate samples rather than the tissue or RNA used to derive the original targets. A rapid high through-put method for confirmation of microarray data is quantitative RT-PCR. Alternatively, Northern blots or ribonuclease protection assays provide the benefit of direct quantification. So far some studies have started to adapt these approaches Figure  but therCurrently, the use of microarray technology in respiratory research is limited by the tissue sample, incomplete available arrays and the analysis of data generated from this technology. Clinical use of microarrays technology is still in its infancy and remains exploratory. For these array-based methods to become truly revolutionary, they must become an integral part of the daily activities of the typical molecular biology laboratory and biomedical institute. There is plenty of room for technical improvements, further development, and more widespread acceptance and accessibility. Optimistically thinking we expect that over the next few years the pattern of development and use of microarrays will be on a similar trajectory to that seen for computers and other high-tech electronic devices, which started out as exotic and expensive tools in the hands of the few developers and then moved quickly to become easier to use, more available and less expensive. Alternatively authors believe that so far microarrays have not added much to our understanding and the possibility to live up to the great 'hype' that was generated belongs to the distant future.Our view is that the application of these approaches will improve dramatically the effectiveness and reliability of microarray technology in studies of diseases of complex organs like the lung, and will have a major impact on our understanding of molecular pathogenesis for the foreseeable future. Whether our hopes will be fulfilled or disproved remains to be seen.ADA: Adenosine DeaminaseALI: Acute Lung InjuryASM: Airway Smooth Musclea-SMA: a-Smooth Muscle ActinBAL: Bronchoalveolar lavageCAGE: Composite Atopy Gene ExpressionCOPD: Chronic Obstructive Pulmonary DiseaseCCR7: Chemokine Receptor 7DEP: Diesel Exhaust ParticlesECM: Extracellular MatrixeQTL: expression quantitative trait locusG.I: Gene IntensityIg: ImmunoglobulinID: Inhibitor of DifferentiationI/D: Intensity/Background ratioIPF: Idiopathic Pulmonary FibrosisMBP: Major Basic ProteinMCs: Mast CellsMCP: Monocyte Chemoattractant ProteinMMP: Matrix MetalloproteinaseNF-κB: Nuclear Factor-Kb2: snake venom phospholipase A2nsPLART-PCR: real time-polymerase chain reactionTGF-b: Transforming Growth Factor-bTNF: Tumor Necrosis FactorUIP: Usual Interstitial PneumoniaVEGF: Vascular Endothelial Growth FactorThe authors declare that they have no competing interests.AT, GP and DB were involved with the study conception. AT performed the data acquisition and interpretation. DB and GP were involved in revising the article for important intellectual content. All authors read and approved the final manuscript.


Endosymbiotic bacteria live within a host species. There are many and diverse examples of such relationships, the study of which provides important lessons for ecology and evolution Symbiosis, an interdependent relationship between two species, is an important driver of evolutionary novelty and ecological diversity. Microbial symbionts in particular have been major evolutionary catalysts throughout the 4 billion years of life on earth and have largely shaped the evolution of complex organisms. Endosymbiosis is a specific type of symbiosis in which one—typically microbial—partner lives within its host and represents the most intimate contact between interacting organisms. Mitochondria and chloroplasts, for example, result from endosymbiotic events of lasting significance that extended the range of acceptable habitats for life. The wide distribution of intracellular bacteria across diverse hosts and marine and terrestrial habitats testifies to the continued importance of endosymbiosis in evolution.PLoS Biology, the publication of the full genome sequence of the reproductive parasite Wolbachia allows the first genomic comparisons across this range , and their impressive genetic diversity that spans several major bacterial groups. More recently, studies in genomics of obligate mutualists—and now The distinct lifestyle of endosymbionts has clear effects on rates and patterns of molecular evolution. Compared to free-living relatives, endosymbionts are thought to have reduced effective population sizes due to population bottlenecks upon transmission to host offspring and, in the case of obligate mutualists that coevolve with their hosts, limited opportunities for gene exchange. The nearly neutral theory of evolution  predictsIt is increasingly clear the distinct lifestyle of endosymbionts also shapes the architecture and content of their genomes, which include the smallest, most AT-rich bacterial genomes yet characterized . A commoBuchnera of three aphid host species, Wigglesworthia of tsetse flies, and Blochmannia of ants , and a relatively young mutualist of weevils (Sitophilus oryzae primary endosymbiont [SOPE]) show that genome streamlining in the endosymbionts may preclude extracellular existence, and highlight modifications in metabolic pathways to complement specific host physiology and ecology .Facultative mutualist: A beneficial symbiont that associates with the host, but can also live apart from it. Examples include Rhizobium spp. that associate with legumes, but also have a free-living stage to their life cycle.Obligate mutualist: A beneficial symbiont that lives exclusively with its host and depends on the host for survival. Examples include many nutritional endosymbionts of insects, which cannot survive outside of the insect host cell. These associations are reciprocally obligate when the host cannot survive without the endosymbiont.Parasite: A symbiont that has a negative effect on host fitness, in contrast to a mutualist, which increases host fitness.Reproductive parasite: A symbiont that manipulates host reproduction to its own benefit, but at the expense of host fitness. Reproductive parasites typically bias offspring toward infected females.Symbiosis: An association between two more species.


In a previous study we demonstrated the existence of numerical and functional alterations of γδ T cells in healthy elderly. Recently, we analysed the involvement of γδ T lymphocytes in malignant melanoma, describing a lower frequency of circulating γδ T cells, an altered pattern of cytokine production, and an impaired in vitro expansion of these cells in primary cutaneous melanoma patients.In this study we investigated the existence of numerical and functional alterations of circulating γδ T cells in young/adult and old melanoma patients, comparing the data obtained with age-matched healthy subjects.+ T cells was significantly and similarly reduced in young/adult and old melanoma patients and in old healthy subjects in comparison with young healthy donors. The decrease was due to a reduction of Vδ2 T cells whereas the number of Vδ1 T cells was not affected. A higher percentage of γδ+ T cells producing TNF-α was found in old healthy donors, whereas a reduced number of TNF-α-producing γδ+ T cells was present in melanoma patients independently by age. No significant difference was observed in IFN-γ production. After a 10-day in vitro culture, both the percentage and the expansion index of γδ T cells, and in particular of Vδ2 subset, were significantly and similarly reduced both in young/adult and old melanoma patients, and in healthy aged people, in comparison with young/adult healthy subjects.We demonstrated that the number of circulating γδOur study demonstrates that the numerical and functional impairment of γδ T cells found in melanoma patients is not correlated with age and that it has characteristics very similar to the alterations of γδ T cells found in old healthy subjects. We suggest that a similar impairment of γδ T cell population may be related to the increased susceptibility to tumors present in the elderly as well as in the pathogenesis of malignant melanoma. In huAlthough little is known about the physiologic significance of γδ T cells, their marked reactivity toward mycobacterial and parasitic antigens as well as tumor cells suggests that γδ T cells play a role in the anti-infectious and anti-tumoral immune surveillance ,4. Few dOn the basis of the pivotal role that γδ T cells may have in the elderly and in the immune response against melanoma we tried to find out a possible correlation in the alteration of γδ T cells between aged people and melanoma patients. In this study we evaluated the peripheral representation, the in vitro expansion, and cytokine production γδ T cells from young/adult and old melanoma patients, comparing the results with those obtained in age-matched healthy controls.Human peripheral blood was obtained from 9 young , 12 old  melanoma patients, and 10 young , and 13 old  healthy donors. Healthy subjects were volunteers in good and stable clinical conditions, and had laboratory parameters in the physiological range. We excluded subjects in poor health with degenerative diseases or in therapy with drugs interfering with the immune system. Melanoma patients have been admitted to the Dermatology Unit of the I.N.R.C.A. Hospital of Ancona. Melanoma patients were in good health other than for the existence of melanoma as checked on the basis of clinical and laboratory parameters. The investigations were performed after approval by a local institutional review board. A written informed consent was obtained from each subject. Diagnosis of melanoma was histologically confirmed. All patients brought cutaneous primary non-metastatic melanoma and were staged according to the new American Joint Committee on Cancer staging system for cutaneous melanoma . A blood2+ and Mg2+- free phosphate buffered saline  and resuspended in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 μg/ml)  at a concentration of 1.5 × 106/ml. Mononuclear cells were cultured in the complete medium supplemented with 100 U/ml of IL-2 . Phosphoantigen-specific stimulation of γδ T cells was performed using the nonpeptidic antigen isopentenylpyrophosphate . The cells were incubated at 37°C in an atmosphere of 95% air, 5% carbon dioxide, at 90% relative humidity in 24 well plates.Fresh peripheral blood mononuclear cells (PBMC) were fractionated on Ficoll-Paque  and separated by density gradient centrifugation . Cells from the interface of the gradients were washed twice with CaPBMCs were analysed for cell phenotype through double staining with the following monoclonal antibodies (mAbs): anti-CD3 (PE) and anti-pan γδ T cells (FITC), or anti-TCR Vδ1 or anti-TCR Vδ2. The phycoerythrin (PE) -conjugated monoclonal antibody anti-CD3 was purchased from EuroClone . The fluorescein isothiocyanate (FITC) -conjugated anti-pan TCR γδ, anti-TCR Vδ1, and anti-TCR Vδ2 were purchased from Endogen . IgG1 (Becton Dickinson) was used as isotype control.6 PBMCs were washed in PBS containing 0,1% NaN3 plus 5% FBS and labelled with 5 μl of anti-CD3 or anti-TCR Vδ1 MoAbs or 2.5 μl of anti-pan TCR γδ or anti-TCR Vδ2 for 30 min in ice. At the end of the incubation, cells were washed in PBS containing 0,1% NaN3, resuspended in PBS (Gibco) and immediately analysed with a Coulter XL flow cytometer.0.5 × 106 stimulated cells were stained with the anti-pan TCR γδ mAb for 30 min at 4°C. Fixation-permeabilization of cells was performed in PBS/2% paraformaldehyde for 15 min at 4°C, followed by incubation for 30 min at room temperature in the dark with PE-conjugated anti-human IFNγ mAb or anti-human TNF-α mAb diluted in PBS, 1% BSA, and 0.05% saponin. Cells were finally washed twice in PBS, 1% BSA, and 0.01 % saponin and analysed on a XL flow cytometer (Coulter).Mononuclear cells were stimulated with IPP and IL-2 for 18 h, and GolgiPlug  was added during the last 12 h of culture to block intracellular transport processes and allow cytokine accumulation. 0.5 × 10PBMC were cultured for up to 10 days in the complete medium supplemented with 100 U/ml of IL-2 and 30 μg/ml of IPP to determine a phosphoantigen-specific stimulation of γδ T cells. After 1 wk of culture, the volume corresponding to half the culture medium was replaced by fresh medium. On day 10 of culture viable cells were determined by trypan blue exclusion and used for FACS analysis. The viability was always greater than 98% as determined by trypan blue exclusion. The expansion of γδ T cells was followed by cytometric analysis through double staining of stimulated cells with anti-CD3 (PE) and anti-pan γδ, or anti Vδ1, or anti Vδ2 T (FITC) mAbs. The absolute number of γδ T cells in each culture was calculated as follow:  × /100. The γδ T cell expansion index was then calculated by dividing the absolute number of γδ T cells in stimulated cultures by the absolute number of γδ T cells before culture .P < 0.05). The statistical analysis was performed with SigmaStat software version 1.03 .Data were analysed for statistical significance by using parametric or nonparametric tests according to the distribution of the data. Comparisons of variables among groups were made by one-way analysis of variance (ANOVA) or Kruskal-Wallis one-way ANOVA on ranks. When significant differences were found, the differences among groups were made by the Student-Newman-Keuls method or Dunn's method. Difference between means was considered significant at the 5% level . As shown in Table Peripheral blood lymphocytes from 9 young/adult and 12 old melanoma patients and 10 young and 13 old healthy subjects were analysed for the percentage and the absolute number of γδ T cells through double staining with anti-CD3 and anti-γδ mAbs. As shown in Table Since it has been demonstrated that activated γδ T cells produce TNF-α and IFN-γ, we studied the intracellular production of these cytokines in one-day stimulated γδ T cells from healthy subjects and melanoma patients. As shown in Fig. The expansion of γδ T cells was evaluated after 10 days of culture in the presence of IPP and low dose interleukin-2 (IL-2). Both the proportion of γδ T cells, evidenced by double staining FACS analysis, and their relative increase in comparison with the γδ T cell number found on day 0 (expansion index), were evaluated. As shown in Fig. We and others have demonstrated an impaired potential of γδ T cells in aged people, as evidenced by the reduction of the absolute number of circulating γδ T cells, in particular of the Vδ2 T subset, an altered pattern of cytokine production, an impaired in vitro expansion of these cells, and an increased expression of the early activation marker CD69, in aged people in comparison with young subjects ,17,18. RWe demonstrated that both the number of circulating γδ T cells and their in vitro expansion were decreased in melanoma patients and that the impairment did not correlate with the age of patients. Young/adult and old melanoma patients had a similar derangement of γδ T cells, and this impairment had numerical and functional characteristics like to those found in old healthy subjects. This evidence stresses the relevant role that this lymphocyte population may exert, either directly or through the regulation of T cell-mediated specific responses , both inThe reduction of γδ T cell number well correlated with the decrease of the Vδ2 T cell subset, i.e., the most frequent subset of circulating γδ T cells . The Vδ2Under normal conditions, γδ T cells respond to antigen challenge by secreting large quantities of TNF-α and IFN-γ ,21 whichIn conclusion, we demonstrate that the numerical and functional derangement of γδ T cells which we have found in melanoma patients, is not correlated with age of donors, and that old patients with cutaneous primary melanoma have an impairment of γδ T cells similar to that found in old healthy subjects. This evidence suggests a link between γδ T cell deterioration and the low protection against infections and tumor diseases present in the elderly, as well as the inefficacious immune defense against melanoma, both in young/adult and old ages.