The prevalence of mental disorders is so high that members of the public will commonly have contact with someone affected. How they respond to that person  may affect outcomes. However, there is no information on what members of the public might do in such circumstances.In a national survey of 3998 Australian adults, respondents were presented with one of four case vignettes and asked what they would do if that person was someone they had known for a long time and cared about. There were four types of vignette: depression, depression with suicidal thoughts, early schizophrenia, and chronic schizophrenia. Verbatim responses to the open-ended question were coded into categories.The most common responses to all vignettes were to encourage professional help-seeking and to listen to and support the person. However, a significant minority did not give these responses. Much less common responses were to assess the problem or risk of harm, to give or seek information, to encourage self-help, or to support the family. Few respondents mentioned contacting a professional on the person's behalf or accompanying them to a professional. First aid responses were generally more appropriate in women, those with less stigmatizing attitudes, and those who correctly identified the disorder in the vignette.There is room for improving the range of mental health first aid responses in the community. Lack of knowledge of mental disorders and stigmatizing attitudes are important barriers to effective first aid. Surveys in many countries have found that mental disorders have a high prevalence and are a major cause of disability in the population -3. For eHow people initially respond to others with a mental disorder may influence their recovery. For example, it is known that many people with mental disorders get no professional help  and thatThere has been no previous research on mental health first aid knowledge in the population. Previous mental health literacy surveys have assessed knowledge and beliefs about mental disorders and their treatment , but theIn 2003–2004 a household survey was carried out of Australian adults aged 18 or over by the company AC Nielsen. Households were sampled from 250 census districts covering all states and territories and metropolitan and rural areas. Up to 5 call backs were made to metropolitan selections and 3 to non-metropolitan selections. To achieve a target sample of 4,000 interviews with adults aged 18 years or over, visits were made to 28,947 households. The outcome of these visits was: no contact after repeated visits 14,630; vacant house or lot 306; refused 7,815; person sampled within household temporarily unavailable 1,132; no suitable respondent in household 287; did not speak English 383; incapable of responding 213; and unavailable for the duration of the survey 181. The achieved sample was 3998 persons, with 1001 receiving the depression vignette, 999 the depression with suicidal thoughts vignette, 997 the early schizophrenia vignette, and 1001 the chronic schizophrenia vignette.The interview was based on a vignette of a person with a mental disorder. On a random basis, respondents were shown one of four vignettes: a person with major depression, one with major depression together with suicidal thoughts, a person with early schizophrenia, and one with chronic schizophrenia. All vignettes were written to satisfy the diagnostic criteria for either major depression or schizophrenia according to DSM-IV and ICD-10. The vignette with depression and the one with early schizophrenia were written to satisfy these diagnostic criteria at a minimal level, so that we could ascertain the public's reaction to cases of a developing disorder which had reached the point where intervention was needed. The vignette of the person with depression together with suicidal thoughts was identical to the depression vignette in all respects except the suicidal thoughts and was designed to assess how this symptom affected the public's response. The chronic schizophrenia vignette was designed to assess the response to someone with a severe long-standing disorder, where acceptance seemed less likely. Respondents were also randomly assigned to receive either male ("John") or female ("Mary") versions of the vignette. These vignettes (John version) are shown in Table After being presented with the vignette, respondents were asked a series of questions to assess their recognition of the disorder in the vignette, their beliefs about treatment and long-term outcomes, beliefs about causes and risk factors, stigmatizing attitudes, awareness of mental disorders in the media, contact with people like those in the vignette, and the health and sociodemographic characteristics of the respondent. The questions relevant to the present paper are described below.other people believe. Please indicate how strongly you agree or disagree with the following statements. Most people believe that people with a problem like John's could snap out of it if they wanted. Strongly agree, Agree, Neither agree nor disagree, Disagree, Strongly disagree". Sociodemographic characteristics recorded included age group (coded as 18–39, 40–59 and 60+ years), gender, and education .To assess recognition of the problem in the vignette, respondents were asked: "What would you say, if anything, is wrong with John?". Responses of "depression" were counted as correct for the first two vignettes above, and responses of "schizophrenia" or "psychosis" for the second two. To assess mental health first aid responses, respondents were asked the open-ended question: "Imagine John is someone you have known for a long time and care about. You want to help him. What would you do?". Answers were recorded verbatim by the interviewer. To assess contact with people like those in the vignette, respondents were asked: "Has anyone in your family or close circle of friends ever had problems similar to John's?"; "Have they received any professional help or treatment for these problems?'; "Have you ever had problems similar to John's?"; "Have you received any professional help or treatment for these problems?"; and "Have you ever had a job that involved providing treatment or services to a person with a problem like John's?". Those that said "yes" to these questions were respectively labelled in the analyses reported below as "carers", "consumers" or "professionals". To assess stigma, respondents were asked a series of nine questions designed to elicit their attitudes towards the person in the vignette  and nine items concerning what they thought others in the community would believe about the person in the vignette (perceived stigma) . PersonaResponses were coded according to the categories identified from an earlier study where the same question was administered as part of a randomized controlled trial of Mental Health First Aid . ResponsA. Encourage professional help-seekingB. Listen to / talk to / support personC. Listen to / talk to / support familyD. Assess problem / assess risk of harmE. Give or seek informationF. Encourage self-helpA. Encourage professional help-seeking, were subcoded into multiple categories to identify the type of professional help recommended. These categories were:Responses coded into category A1. GP / doctor unspecifiedA2. CounsellorA3. PsychiatristA4. PsychologistA5. Mental health team / servicesA6. Other mental health professionalsA7. Unspecified professionals and other professionalsA8. Accompany person (eg. Offer to go with him/her)A9. Contact help on their behalfExamples of responses coded into category B included "support, understanding and caring, someone to talk to him", "talk to her about it", "listen", "be there for him".Responses coded into category C were the same for category B, but referred to giving support, listening to, or talking to the sufferer's family. For example, "talk to her family", "contact relatives", "ask advice of parents", "support his parents".Responses coded into category D included "keep an eye on her, make sure she is safe", "make a contract with her so if she wants to harm herself she rings me first", "find out what is the real problem behind the behaviour, focus on the problem".Responses coded into category E included "ring the local health authority to get advice", "talk to other people who have been in the situation", "speak to health professionals and get best advice", "get some brochures from community health and give them to him". Responses coded into category F included "I suggest that he have a holiday/exercise/change jobs", "try and help him get into something he is interested in", "support groups", "do something for herself to get out of the situation".Inter-rater reliability of the coding was assessed by a second rater who independently coded 100 responses which were randomly selected using the SPSS Select Cases procedure .Inter-rater reliability of the content coding was assessed using kappa. Kappa values were interpreted according to Altman  as folloThe frequency of codings was analysed by pooling across male and female versions of each vignette and percent frequencies calculated. Percentages were calculated applying survey weights to give better population estimates. Standard errors of these percentages were estimated using the Complex Samples procedure in SPSS 12.0 . This prMultiple logistic regressions were then conducted to examine the levels of association between participants suggesting particular treatment options and their sociodemographic and mental health experience attributes. The following predictor variables were included: age group; consumer, carer and professional status, including whether or not professional help was obtained; and levels of perceived stigma and personal stigma. Two vignette measures – type of vignette provided and whether respondents correctly identified the problem portrayed in that vignette – were also included in the analyses. Each logistic regression was also adjusted to take into account of sampling weights and clustering method applied in this survey. These analyses were undertaken using STATA 8 .Inter-rater reliability was assessed for a randomly chosen 100 responses. Kappa was very good or good for encourage professional help-seeking (0.89), listen/talk/support person (0.70), listen/talk/support family (1.00), encourage seeing doctor (0.98), encourage seeing counsellor (0.93), encourage seeing psychiatrist (0.94), encourage seeing psychologist (0.88), and accompanying the person to a professional (0.95). It was moderate for give or seek information (0.48), encourage seeing unspecified and other professionals (0.56), and contact professional on their behalf (0.56). Kappa was fair for encourage self-help (0.34) and poor for assess problem/risk of harm (0.15). Kappa could not be calculated because of zero frequencies from the first rater for the categories of mental health team/services and other mental health professional.To better understand the reasons for the fair and poor agreement with two of the codes, positive and negative agreement were examined separately . In bothTable Table Table Table Table Table The most common first aid responses were found to be encouraging professional help-seeking and listening/talking/supporting the person. Nevertheless, these responses were far from universal, with 32–44% not mentioning professional help and 27–34% not mentioning listening/talking/supporting. Given the likely helpfulness of these first aid responses, they need greater promotion in the community.Other first aid responses were mentioned only by a minority. Of particular concern is the low percentage assessing risk of harm for the person in the depression/suicidal vignette. Asking about suicidal intentions is often recommended as a response ,14, althEncouraging self-help was another minority response, but was associated with stigma and lack of recognition of the mental disorder in the vignette. Respondents appear to have suggested self-help as an alternative to professional help, rather than as a complement to it. We have previously reviewed the evidence on self-help interventions for depression and anxiety disorders and found that some have support ,17. SuchWhen correlates of first aid responses were examined, most variables had at least one significant association. However, the variables that most often predicted first aid responses were female gender, low personal stigma and correct recognition of the disorder in the vignette. The latter two predictors indicate potential barriers to providing first aid. Respondents who saw the person in the vignette as having negative attributes were less likely to respond by encouraging professional help-seeking or providing personal support. Efforts to reduce stigma in the community may therefore facilitate greater first aid. People who did not recognize the disorder showed a similar pattern of responses. These people lack knowledge of mental disorders, at least to the extent of being able to apply a psychiatric label. Therefore community education about how to recognize these disorders may also facilitate helpful first aid responses.One approach to improving public responses to people with a mental disorder is an individual training course in mental health first aid . Such a The study has two limitations which must be acknowledged. The major one is that the study has assessed intended first aid to a hypothetical person in a case vignette. Whether these intentions would be implemented in practice is unknown. Intentions might be seen as placing an upper limit on responses, such that if a respondent fails to state an intention, this is unlikely to be seen in practice. An alternative approach would have been to ask the respondent how they had treated actual people they knew with mental health problems. However, the disadvantage of this alternative would have been the lack of standard situations. A second limitation is that the inter-rater reliability of coding some of the first aid responses was low. Conclusions about these responses must be viewed with caution. On the other hand, the strengths of the study are the large representative sample, the open-ended responses which did not constrain the respondents to particular alternatives, and the ability to compare responses to a series of standard scenarios.There is room for improving the range of mental health first aid responses in the community. Lack of knowledge of mental health and stigmatizing attitudes are important barriers to effective first aid.The author(s) declare that they have no competing interests.AFJ was involved in securing funding for the survey, had a major role in the design of the survey and the interview questionnaire, carried out the descriptive statistical analysis and had a major role in writing the manuscript.KAB provided research assistance with the survey, coded all the responses, and wrote the method relevant to this coding.KMG was involved in the design of the study, developed the stigma scales and wrote some of the manuscript.BAK developed the coding scheme, was the second rater for inter-rater reliability, and wrote some of the manuscript.RAP did the regression analyses and wrote the section of the Method describing this.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The clinical course of breast cancer is difficult to predict on the basis of established clinical and pathological prognostic criteria. Given the genetic complexity of breast carcinomas, it is not surprising that correlations with individual genetic abnormalities have also been disappointing. The use of gene expression profiles could result in more accurate and objective prognostication.BRCA2, DNMT3B and CCNE1) as an independent prognostic marker . This "poor prognosis" signature was then tested on an independent panel of ERα-positive breast tumors from a well-defined cohort of 104 postmenopausal breast cancer patients treated with primary surgery followed by adjuvant tamoxifen alone: although this "poor prognosis" signature was associated with shorter relapse-free survival in univariate analysis (P = 0.029), it did not persist as an independent prognostic factor in multivariate analysis (P = 0.27).To this end, we used real-time quantitative RT-PCR assays to quantify the mRNA expression of a large panel (n = 47) of genes previously identified as candidate prognostic molecular markers in a series of 100 ERα-positive breast tumor samples from patients with known long-term follow-up. We identified a three-gene expression signature (Our results confirm the value of gene expression signatures in predicting the outcome of breast cancer. Breast carcinoma is the most common female cancer and is showing an alarming year-on-year increase. Most patients do not die as a result of the primary tumor but from metastatic invasion. The mean 5-year relapse-free survival rate is about 60% overall, but differs significantly between patients with forms that rapidly metastasize and those with less aggressive forms.i.e. age, menopausal status, lymph-node status, macroscopic tumor size, histological grade and estrogen receptor status, fail to accurately predict clinical behavior.Current clinical, pathological and biological parameters, ERBB2, CCDN1, MYC, UPA and PAI1 [Breast cancer initiation and progression is a process involving multiple molecular alterations, many of which are reflected by changes in gene expression in malignant cells. Many clinical studies have attempted to identify correlations between altered expression of individual genes and breast cancer outcome, but often with contradictory results. Examples of such genes include and PAI1 . It is tThe recent development of effective tools for monitoring gene expression on a large scale is providing new insights into the involvement of gene networks and regulatory pathways in various tumor processes . It has BRCA2, DNMT3B and CCNE1) associated with poor clinical outcome. We then tested this "poor prognosis" signature on an independent panel of ERα-positive breast tumor samples from a well-defined cohort of 104 postmenopausal breast cancer patients treated with primary surgery followed by adjuvant tamoxifen alone with known long-term follow-up.In this study, we used real-time quantitative RT-PCR assays to quantify the mRNA expression of 47 candidate prognostic molecular markers in a series of 100 ERα-positive breast tumor samples. We identified a three-gene expression signature  were pre- or post-menopausal (37 and 63 patients, respectively). Sixty patients received adjuvant therapy, consisting of chemotherapy alone in 14 cases, hormone therapy alone in 15 cases, and both treatments in 31 cases. The standard prognostic factors are presented in Table The second series consisted of 104 post-menopausal women whose breast tumors were excised at Centre René Huguenin from 1980 to 1994. The patients (mean age 70.9 years, range 54–86) all received post-operative adjuvant hormone therapy consisting of tamoxifen (20 mg daily for 3–5 years) and no other treatment. The standard prognostic factors are reported in Table Complete clinical, histological and biological information was available for the two series of breast cancer patients; no radiotherapy or chemotherapy was given before surgery, and full follow-up took place at Centre René Huguenin. The histological type of the tumor and the number of positive axillary nodes were established at the time of surgery. The malignancy of infiltrating carcinomas was scored according to Scarff Bloom and Richardson's (SBR) histoprognostic system.Both series of tumor samples were placed in liquid nitrogen until total RNA extraction immediately following surgery.Quantitative values are obtained from the cycle number  at which the increase in fluorescent signal associated with an exponential growth of PCR products starts to be detected by the laser detector of the ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems, Foster City, CA) using the PE Biosystems analysis software according to the manufacturer's manuals.TBP (Genbank accession NM_003194) encoding for the TATA box-binding protein (a component of the DNA-binding protein complex TFIID) as an endogeneous RNA control, and normalized each sample on the basis of its TBP content.The precise amount of total RNA added to each reaction  and its quality (i.e. lack of extensive degradation) are both difficult to assess. We therefore also quantified transcripts of the gene TBP gene, termed "Ntarget", were determined by the formula: Ntarget = 2sampleΔCt , where ΔCt value of the sample was determined by subtracting the average Ct value of the target gene from the average Ct value of the TBP gene.Results, expressed as N-fold differences in target gene expression relative to the target values of the samples were subsequently normalized such that the Ntarget value of the tumor sample which contained the smallest amount of target gene mRNA in each tumor series would equal a value of 1.The NTBP and the 47 target genes were chosen with the assistance of the computer programs Oligo 5.0 . We conducted searches in dbEST, htgs and nr databases to confirm the total gene specificity of the nucleotide sequences chosen for the primers and probes, and the absence of single nucleotide polymorphisms. In particular, the primer pairs were selected to be unique when compared with the sequences of the closely related family member genes or of corresponding retropseudogenes. To avoid amplification of contaminating genomic DNA, one of the two primers or the probe was placed at the junction between two exons. Agarose gel electrophoresis allowed us to verify the specificity of PCR amplicons. The list of the 47 target genes tested in this study is indicated in Table Primers and probes for Total RNA was extracted from frozen tumor samples by using the acid-phenol guanidinium method. The quality of the RNA samples was determined by electrophoresis through agarose gels and staining with ethidium bromide, and the 18S and 28S RNA bands were visualized under ultraviolet light.Reverse transcription of total RNA was done in a final volume of 20 μL containing 1X RT buffer (500 μM each dNTP, 3 mM MgCl2, 75 mM KCl, 50 mM Tris-HCl pH 8.3), 20 units of RNasin RNase inhibitor (Promega, Madison, WI), 10 mM DDT, 100 units of Superscript II RNase H- reverse transcriptase (Invitrogen, Cergy Pontoise, France), 3 μM random hexamers  and 1 μg of total RNA. The samples were incubated at 20°C for 10 min and 42°C for 30 min, and reverse transcriptase was inactivated by heating at 99°C for 5 min and cooling at 5°C for 5 min.® PCR Core Reagents kit or the SYBR® Green PCR Core Reagents kit (Perkin-Elmer Applied Biosystems). The thermal cycling conditions comprised an initial denaturation step at 95°C for 10 min and 50 cycles at 95°C for 15 s and 65°C for 1 min.All PCR reactions were performed using a ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems). PCR was performed using either the TaqManU test  and the Spearman rank correlation test (link between 2 quantitative parameters). Differences between the two populations were judged significant at confidence levels greater than 95% (p < 0.05).The distributions of the gene mRNA levels were characterized by their median values and ranges. Relationships between mRNA levels of the different target genes and comparison between the target gene mRNA levels and the clinical parameters were estimated using nonparametric tests: the Mann-Whitney Y axis against 1 – the specificity  on the X axis, considering each value as a possible cutoff value. The AUC (area under curves) was calculated as a single measure for the discriminate efficacy of a molecular marker. When a molecular marker has no discriminative value, the ROC curve will lie close to the diagonal and the AUC is close to 0.5. When a test has strong discriminative value, the ROC curve will move up to the upper left-hand corner (or to the lower right-hand corner) and the AUC will be close to 1.0 (or 0).To visualize the efficacy of a molecular marker to discriminate two populations , we summarized the data in a ROC (receiver operating characteristic) curve . This cuHierarchical clustering was performed using the GenANOVA software .Relapse-free survival (RFS) was determined as the interval between diagnosis and detection of the first relapse .Survival distributions were estimated by the Kaplan-Meier method , and theThe results for the 47 genes are summarized in table BRCA2, DNMT3B, CCNE1, HMMR/RHAMM, MKI67, TERT and CCND1. The prognostic performance of these 7 genes was also assessed using ROC-AUC analysis. BRCA2 emerged as the most discriminatory marker of relapse status (ROC-AUC, 0.696). The mRNA expression of this gene, as well as DNMT3B, CCNE1, HMMR/RHAMM, MKI67 and TERT, was higher in patients who relapsed than in patients who did not relapse, while only CCND1 mRNA expression was lower in patients who relapsed.Seven genes showed significantly different expression according to relapse status (P < 0.05), namely i.e. the genes with the highest ROC-AUC values (BRCA2, DNMT3B and CCNE1, ROC-AUC: 0.696, 0.690 and 0.674, respectively), the patient population fell into two subgroups (65 and 35 subjects, respectively) with significantly different relapse-free survival curves  in univariate analysis, us Table  and the us Table . Only thBRCA2, DNMT3B and CCNE1 mRNA levels were significantly higher in patients who relapsed than in those who did not relapse.The results for each of the three genes are summarized in table On hierarchical clustering of the samples, the three-gene expression signature dichotomized the 104 patients into two subgroups (n = 30 and n = 74, respectively) of similar sizes to those of the initial patient population (n = 35 and n = 65, respectively).The "poor prognosis" signature was again associated with shorter relapse-free survival in this independent tumor series  in univariate analysis,  in univaze Table , only SBBRCA2, DNMT3B and CCNE1) with independent prognostic significance in breast cancer . This "poor prognosis" signature was then tested on an independent set of 104 ERα-positive breast tumors from a well-defined cohort of postmenopausal breast cancer patients treated with primary surgery followed by adjuvant tamoxifen alone. It was found to be significant in univariate analysis (P = 0.029), but not in multivariate analysis (P = 0.27). We have previously published individual data for 18 of these 47 genes, namely ERBB1-4 [MYC [TERT [CCND1 [CGB, CGA, ERα, ERβ, PR, PS2 [AR [DNMT3B [PAI1, PAI2 and UPA [We used real-time quantitative RT-PCR assays to quantify the mRNA expression of 47 genes previously identified as candidate prognostic molecular markers in 100 ERα-positive breast tumor samples. We identified a three-gene expression signature , and for distinguishing among closely related family member genes or alternatively spliced specific transcripts (e.g. the gene cluster p14/ARF, p16/CDKN2A and p15/CDKN2B as in the present study). Finally, real-time quantitative RT-PCR assay is a reference in terms of its performance, accuracy, sensitivity and throughput for nucleic acid quantification, and is more appropriate for routine use in clinical laboratories, being simple, rapid and yielding good inter-laboratory agreement and statistical confidence values.These discrepancies may be due to the clinical, histological and ethnic heterogeneity of breast cancer, but also to the fact that breast tumors consist of many different cell types – not just tumoral epithelial cells, but also additional epithelial cell types, stromal cells, endothelial cells, adipose cells, and infiltrating lymphocytes. Real-time RT-PCR requires smaller starting amounts of total RNA (about 1–2 ng per target gene) than do cDNA microarrays, making it more suitable for analyzing small tumor samples, cytopuncture specimens and microdissected samples. Real-time RT-PCR also has a linear dynamic range of at least four orders of magnitude, meaning that samples do not need to contain equal starting amounts of RNA. Real-time RT-PCR is also more suitable than cDNA microarrays for analyzing weak variations in gene expression and weakly expressed genes , confirming that the ERBB2 mRNA expression level is not a major prognostic factor in breast cancer; (b) ESR1/ERα mRNA levels were not different between the two subgroups (ROC-AUC, 0.530), suggesting that the ESR1/ERα mRNA expression level in ERα-positive tumors is not predictive of outcome.The comparison of median target gene mRNA levels between patients who did and did not relapse provided two interesting results: (a) CCNE1), DNA methylation (DNMT3B) and DNA damage repair (BRCA2). This gene expression signature is an interesting candidate for routine clinical use, especially as the three genes encode well-characterized proteins for which specific antibodies are already commercially available. Furthermore, the three protein products are amenable to pharmacological control.The three-gene expression signature predictive of subsequent relapse status comprised genes involved in cell cycle control . We also observed a strong positive link between BRCA2 and MKI67, which encodes the proliferation-related Ki-67 antigen (Spearman rank correlation test: r=+0.670, P < 10-7). The observed strong associations between BRCA2, HMMR/RHAMM and MKI67 mRNA expression explain why four- and five-gene expression signatures, comprising HMMR/RHAMM alone or together with MKI67, showed no additional prognostic value relative to the three-gene signature. control . We founBRCA2 expression ex vivo are in keeping with reports from several authors [BRCA2 mRNA expression is upregulated in rapidly proliferating cells in vitro. Our results are also in agreement with those of Egawa et al. [BRCA2 expression carries a poor prognosis in breast cancer. This link between BRCA2 overexpression and poor outcome should be taken into account when evaluating future BRCA2-based therapeutic approaches to breast cancer.Our results for  authors ,34 showia et al.  showing DNMT3B, the third gene in our expression signature, codes for one of the three functional DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) that catalyze the transfer of methyl groups to the 5-position of cytosine (DNA methylation). We previously showed that, among the three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B), only DNMT3B overexpression is associated with poor outcome in breast cancer [DNMT3B (like DNMT3A) is known to be a de novo methylator of CpG sites. Abnormal DNA methylation is thought to be a major early event in the development of tumors characterized by widespread genome hypomethylation leading to chromosome instability and localized DNA hypermethylation; the latter may be important in tumorigenesis by silencing tumor suppressor genes [Finally, t cancer . DNMT3B or genes .BRCA2, DNMT3B and CCNE1) with prognostic significance. The practical value of this signature remains to be validated in large prospective randomized studies.In conclusion, by studying the expression of 47 genes previously identified as candidate prognostic markers in breast cancer, we identified a three-gene expression signature (ERα, estrogen receptor alpha; RT-PCR, reverse transcriptase-polymerase chain reaction.Real-time RT-PCR have been carried out by ST and IG. IB and RL interpreted the result, performed bioinformatics and statistical analyses.


Non-neuronal cells, including those derived from lung, are reported to express nicotinic acetylcholine receptors (nAChR). We examined nAChR subunit expression in short-term cultures of human airway cells derived from a series of never smokers, ex-smokers, and active smokers.At the mRNA level, human bronchial epithelial (HBE) cells and airway fibroblasts expressed a range of nAChR subunits. In multiple cultures of both cell types, mRNA was detected for subunits that constitute functional muscle-type and neuronal-type pentomeric receptors. Two immortalized cell lines derived from HBE cells also expressed muscle-type and neuronal-type nAChR subunits. Airway fibroblasts expressed mRNA for three muscle-type subunits (α1, δ, and ε) significantly more often than HBE cells. Immunoblotting of HBE cell and airway fibroblast extracts confirmed that mRNA for many nAChR subunits is translated into detectable levels of protein, and evidence of glycosylation of nAChRs was observed. Some minor differences in nAChR expression were found based on smoking status in fibroblasts or HBE cells. Nicotine triggered calcium influx in the immortalized HBE cell line BEAS2B, which was blocked by α-bungarotoxin and to a lesser extent by hexamethonium. Activation of PKC and MAPK p38, but not MAPK p42/44, was observed in BEAS2B cells exposed to nicotine. In contrast, nicotine could activate p42/44 in airway fibroblasts within five minutes of exposure.These results suggest that muscle-type and neuronal-type nAChRs are functional in airway fibroblasts and HBE cells, that prior tobacco exposure does not appear to be an important variable in nAChR expression, and that distinct signaling pathways are observed in response to nicotine. Nicotine, the addictive component of tobacco smoke, signals through its family of receptors, the nicotinic acetylcholine receptors (nAChR). Acetylcholine is the endogenous ligand for these receptors, and has been found in many tissues outside of the nervous system. Non-neuronal nAChR have also been identified in tissues such as the skin, vasculature, and nasal mucosa . nAChR aThe ionic permeability of nAChR is dependent upon the subunit composition of the receptor, with some receptors showing preference to either calcium or sodium . HoweverThe primary route of exposure to nicotine is through inhalation, either by active smokers or non-smokers exposed to environmental tobacco smoke. Through inhalation, the lung, in particular, would be exposed to pharmacological doses of nicotine. In addition, receptor inactivation is likely to occur in sensitive receptors, due to the extended length of time that smokers use tobacco ,5. This In vitro data has indicated that airway epithelial cells release GM-CSF upon exposure to nicotine, and activate Akt, a signaling molecular important in cell survival [Using radiolabeled agonist, we have shown that saturable nicotine binding sites exist in the lung . Other psurvival ,9. Howevsurvival  airway tIn this study, a series of 37 short-term human bronchial epithelial cultures, 25 airway fibroblast cultures, and 2 immortalized bronchial epithelial cell lines were examined by RT-PCR for nAChR expression. We also examined protein expression by immunoblot to determine which subunits are most highly expressed and to determine if appropriate combinations are present at the protein level to form functional receptors. We determined that the nAChR present are functional by examining calcium influx after agonist exposure, and blockade by antagonists. We also show that exposure of airway cells to nicotine leads to activation of downstream signaling pathways. Finally, we examined the nAChR present in HBE and airway fibroblasts derived from smokers, ex-smokers, and never smokers to determine if alterations in nAChRs based on tobacco exposure can be detected.HBE cells are cultured from airway biopsies using standard methodology in serum-free medium . BrieflyBEAS2B were all purchased from ATCC and cultured according to ATCC instructions. IB3-1 cells were derived from a cystic fibrosis patient and cultured in Hams medium with 10% serum .All chemicals used were from Sigma (St. Louis, MO) and all supplies were from either Fisher Scientific (Pittsburgh, PA) or PGC Scientific (Frederick, MD), unless otherwise indicated.RNA is isolated from cultures using standard guanidinium thiocyanate method . RNA is 12–18 (Invitrogen) was annealed to 1 μg total RNA and reverse transcribed with Superscript II (Invitrogen). The reaction contained 1 μg RNA, 500 ng Oligo dT12–18, 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 1 mM each dNTP, 200 U superscript. Briefly, total RNA was incubated with oligo dT12–18 at 70°C for 10 min. The cDNA produced was then used as a template for PCR using specific primers. Table Primers were developed to unique regions of each subunit and were tested on human muscle and brain RNA purchased from Clontech . Optimized protocols were then used on RNA from airway cells. All primers span introns and do not amplify DNA. GAPDH or actin is always used as a positive control for RNA integrity. Oligo dT50 μg protein with loading buffer was denatured using 50 mM DTT and heated at 80°C for 15 min. Protein was loaded onto 10% Bis-Tris gels (Invitrogen). Brain lysate  and lysates from myotubes cultures (gift, Z.Z. Wang, U. Pittsburgh) were used for positive control. Protein was transferred to PVDF (Biorad, Hercules, CA) or Multiblots (ISC Bioexpress, Kaysville, UT). PVDF membranes were blocked for 1 hr with 5% blocker (Biorad) in TBS-T (2.7 mM KCl, 138 mM NaCl, 20 mM Trizma, pH 7.4, 0.1% Tween-20 (Biorad)). Primary antibody was diluted in carrier protein, 5% blocker for PVDF or 0.5% casein (Pierce) for Multiblots and incubated at 4°C overnight, see Table Airway cells were grown in 24-well dishes, with 10,000 cells per well. After 24 hours, medium was replaced with serum-free, prewarmed medium spiked with calcium-45, with a final specific activity of approximately 60 μCi/mM calcium. Drug was added to the wells as indicated, in triplicate. If nAChR antagonists or channel blockers were used, they were added 20 min before the addition of agonist. The calcium ionophore A23187 (Molecular Probes, Eugene OR) was used as a positive control for calcium influx. After incubation at 37°C, plates were put on ice and washed 3 times with ice-cold PBS. Lysis buffer (1% SDS, 0.3 N NaOH) was added. Lysates were transferred to scintillation vials, scintillation fluid added, and counted. Results are presented as percent control, with untreated control normalized to 100%.For phospho-PKC, phospho-p42/44, and phospho-p38, 10 minute exposure to 20 ng/ml EGF was used as the control. These conditions have been published as optimal for signaling by EGF in several published articles ,17. ImmuDifferences in expression frequency of nAChR subunits at either the RNA or protein level were analyzed by Fisher's Exact Test. In all other experiments, differences from control were determined using Student's T-test. All p-values reflect two-tailed tests.Using RT-PCR, we repeatedly detected mRNA for nAChR subunits in short-term cultures of human airway cells from bronchial biopsies. Figure The subunits that are expressed by HBE cells could potentially combine to form muscle-type (α1/ β1/ δ /ε) heteropentamers, neuronal α7 or α9 homopentamers, and neuronal heteropentamer receptors α3/ α5/ β2 or β4 and α6/β2 or β4. By examining the pattern of mRNA expression in each individual culture, combinations were observed that would produce a functional muscle-type receptor in 7 of 33 (21%) of cultures, a functional α3-containing neuronal type receptor in 10 of 28 (35%) of cultures, α6-containing neuronal type receptor in 13 of 28 (46%), α7 homopentamer receptors in 25 of 37 (68%), and homopentamer α9 receptors in 27 of 29 cultures (93%). Only 2 of 35 (6%) HBE cultures did not express at least one functional nAChR subunit combination.Two immortalized airway epithelial cell lines also expressed mRNA for many of these nAChR subunits, including muscle-type subunits Figure , Table 3Airway fibroblasts also expressed nAChR Table . We founThe mRNA expression of four nAChR subunits is significantly different when comparing airway fibroblasts and HBE cells. The muscle-type receptor subunits α1, δ, and ε are all expressed more frequently in airway fibroblasts (present in 96%, 96%, and 92%, respectively, of airway fibroblast cultures) than in HBE cells (α1 present in 32% of cultures, δ in 70% of cultures, and ε present in 58% of cultures, p < 0.02 for each subunit). In addition, the β3 subunit is also expressed more frequently in fibroblast cultures than in HBE cells (8/25 compared to 1/28, p < 0.01). The subunit combinations that could form functional receptors were also examined and compared between cell types. Consistent with the individual subunit data, the combination of all subunits for the muscle-type receptor is expressed significantly more frequently in human airway fibroblasts (74%) than in HBE cultures (21%)(p = 0.0001).The nAChR subunit α9 was frequently expressed by both HBE cells and airway fibroblasts. Although we find this nAChR frequently and it may have physiological significance, it is unlikely that signaling through this receptor would be responsible for the immediate downstream effects seen in our studies, which focus on the effects of nicotine, since nicotine does not act as an agonist for this receptor type.We next examined nAChR subunit protein expression using immunoblotting. All the cultures examined expressed protein for nAChR subunits, in general agreement with mRNA expression data . Figure We examined functionality of the AChR on HBE cells by measuring calcium influx. After treatment with nicotine, extracellular radioactive calcium Ca-45) is internalized by BEAS2B cells and short-term HBE cultures .To test the specificity of this response, we used the nicotinic antagonists α-bungarotoxin and hexamethonium with BEAS2B cells and nicotine. α-Bungarotoxin will block muscle-type nAChR as well as α7 homopentamers and hexamethonium will block neuronal heteropentamer nAChR such as α3- and α6- containing receptors. We used ionophore alone and with the nicotinic antagonists as a control in these experiments. Antagonists had no effect on ionophore-induced calcium influx (data not shown). In this experiment we found that α-bungarotoxin could completely prevent the calcium-45 influx seen after nicotine treatment, while hexamethonium could only slightly inhibit the effect of nicotine in BEAS2B cells  responds to nicotine because it is commonly activated by calcium influx. Calcium influx is an immediate effect of nicotine exposure to BEAS2B cells Figures  and 5. TUsing phosphorylation as a marker of activation, we find that PKC and p38, but not p42/44 are activated after treatment with nicotine in BEAS2B Figure . As showIn contrast, signaling experiments done with short-term airway fibroblast cultures show that nicotine caused phosphorylation of p42/44 within 10 minutes of exposure Figure . After dTogether, these data indicate that the muscle-type (α1/β1/δ/ε) nAChR is consistently present on airway epithelial cells, while airway fibroblasts consistently demonstrate both a muscle-type and an α7 homomeric nAChR. Normal airway epithelial cells may also sometimes express the neuronal α3/α5/β2 or α6/β2 nAChR. The nicotinic receptors are functional, regulate calcium influx upon ligand binding, and lead to downstream activation of the signaling pathways MAPK or PKC when bound by nicotine. Downstream effects can be blocked by use of nicotinic antagonists.We examined the relationship of prior smoking to nAChR expression on airway cells. To do so, we compared subunits expressed at the mRNA and protein level in HBE cultures from never smokers, active smokers, and ex-smokers to determine if long-term exposure to nicotine was a factor in the type of nAChR expressed. As shown in Table Interestingly, in airway fibroblasts mRNA patterns for combinations of neuronal heteropentamers containing the α3 subunit were downregulated with smoking Table . Cultureet al. [et al. [et al. [Recent evidence suggest that endogenous acetylcholine is a local signaling molecule in non-neuronal tissue, and that nicotinic acetylcholine receptors are found outside the nervous system. Our data show that HBE cells can express mRNA for the neuronal α3/α5/β2 or β4, α6/β2 or β4, and α7 and α9 pentamers, as well as the muscle type α1/β1/δ/ε nAChR. Previous data in the human airway examined small numbers of HBE cultures for a limited number of nAChR subunits. Maus et al.  used RT- [et al. . Our datInstead, our results suggest that the muscle-type nAChR present in HBE cells may have a functional role in HBE cells that has not previously been considered. The muscle-type receptor was more recently characterized than the neuronal type and previous literature never examined HBE cultures for the muscle-type receptor ,10,12. TSimilarly to HBE, mRNA and protein for nAChR subunits are commonly expressed by airway fibroblast cultures. Airway fibroblasts have never been examined for the presence of nicotinic receptors. Dermal fibroblasts have been shown to express mRNA for some receptors, although they were not examined for muscle-type receptors , and ginCalcium influx is a hallmark of the opening of the nAChR ion channel. An increase in intracellular calcium from the extracellular milieu can occur either by a direct influx of calcium through the nAChR channel, as occurs in α7 receptors, or by an influx of sodium that leads to depolarization of the cell and the opening of L-channels, as occurs after agonist binding to heteropentamer nAChR ,25. We dNicotine has previously been shown to affect signaling in human airway cells, and acetylcholine, the endogenous ligand, causes proliferation of HBE cells ,9. In ouOver the same time period, the MAPK family member p38 but not p42/44 is phosphorylated, a required step for activation. The activation of p38 is associated with regulation of apoptosis in response to cellular stress. The MAPK family kinases, such as p38, are not known to be directly activated by calcium; however there are several indirect pathways that lead to rapid phosphorylation of these pathways. These include signaling through the calcium/calmodulin-dependent protein kinases CaMKI, CaMKII, and CaMKIV as well as the calcium-activated signaling molecule PYK2.et al [This finding is in contrast to signaling activated by nicotine in airway fibroblasts. These cells phosphorylate p42/44 immediately upon treatment with nicotine. Like p38, p42/44 is not directly activated by calcium, but could be phosphorylated by calcium-activated signaling molecules. However, the pathways that lead to phosphorylation of different MAPK family members in the two cell types in response to nicotine have not been elucidated. It is possible that the nAChR type differences are responsible for the differential MAPK effects. For example, the α7 receptor is highly expressed on airway fibroblasts but not on HBE cells. The additional nAChR type on these fibroblasts may change the signaling pathways activated by cells in response to nicotine. This is consistent with a previous study by Jull et al . This stet al . A similFinally, we found that smoking had only modest effects on nAChR expression in the airway. Previous studies in the brain indicate that certain nAChR may be increased in frequency in smokers as compared to non-smokers , and a pIn contrast, there was a significant decrease in the frequency of the expression of the functional combination of subunits for the α3-containing receptors in airway fibroblasts of smokers and ex-smokers compared to never-smokers. This nAChR type is a sodium channel that undergoes inactivation upon long-term exposure to agonist, and, in airway fibroblasts, appears to be downregulated with long-term exposure to nicotine. This change in receptor expression remains even after exposure to nicotine ceases, as evidenced by the reduced frequency of expression in ex-smokers.We have shown that short-term cultures of normal airway fibroblasts as well as normal human bronchial epithelial cells from a number of different human donors consistently express functional nAChR and that these cell types differ in the type of nAChR they express. It is likely that the muscle-type nAChR plays a major role in the response of HBE cells to nicotine, and that the neuronal heteropentamers play a more minor role. Calcium influx as well as initiation of downstream signaling pathways indicate that receptors are functional and that both human bronchial epithelial cells and airway fibroblasts respond to nicotine, and those signaling responses may differ due to the difference nAChR present on the cell type. Together, these data suggest that exposure of the human airway to nicotine through tobacco smoke may have physiological consequences on airway homeostasis involving both the airway mucosa and the underlying submucosal mesenchymal cells. As such, nicotine may act to promote lung disease by acting to change cell growth and apoptosis. In airway fibroblasts this may leading to thickening of the airway wall seen in the pathogenesis of COPD. In the bronchial epithelium this may lead to preneoplasia or development of frank cancer.nAChR: nicotinic acetylcholine receptorHBE: human bronchial epithelial cellPKC: protein kinase CMAPK: mitogen activated protein kinaseBEGM: bronchial epithelial growth mediumEGF: epidermal growth factorP42/44: Extra-cellular signal regulated kinase isoforms 1 and 2DLC designed and performed the majority of the experiments and data analysis, and wrote the manuscript. TMH designed and performed RT-PCR with the assistance of MJS. JDL and NAC contributed the tissues that were grown into primary cultures and provided information on smoking history. AGD cultured the primary cells. JMS conceived of the study and participated in its design and coordination. All authors read and approved the manuscript.


DNA-directed synthesis represents a powerful new tool for molecular discovery. Its ultimate utility, however, hinges upon the diversity of chemical reactions that can be executed in the presence of unprotected DNA. We present a solid-phase reaction format that makes possible the use of standard organic reaction conditions and common reagents to facilitate chemical transformations on unprotected DNA supports. We demonstrate the feasibility of this strategy by comprehensively adapting solid-phase 9-fluorenylmethyoxycarbonyl–based peptide synthesis to be DNA-compatible, and we describe a set of tools for the adaptation of other chemistries. Efficient peptide coupling to DNA was observed for all 33 amino acids tested, and polypeptides as long as 12 amino acids were synthesized on DNA supports. Beyond the direct implications for synthesis of peptide–DNA conjugates, the methods described offer a general strategy for organic synthesis on unprotected DNA. Their employment can facilitate the generation of chemically diverse DNA-encoded molecular populations amenable to in vitro evolution and genetic manipulation. A method is presented that makes possible the use of standard organic reaction conditions and common reagents in the presence of unprotected DNA, an important step in enabling DNA-directed chemical synthesis and drug discovery A number of strategies have been proposed recently to enable the in vitro selection and evolution of chemical libraries . These nOne approach takes advantage of hybridization to induce proximity between reactants covalently attached to oligonucleotides. “Reading” is accomplished by the hybridization of the reactant conjugates to a DNA template, whereas synthetic execution results from the reactants being positioned closely together. The strategy has been demonstrated for several types of chemistries . HoweverRather than tailoring reactions to the narrow window of hybridization conditions, DNA reading and chemical transformation can be carried out in chronologically distinct steps . The DNAWe first had to choose a solid-phase material that exhibited several critical properties: reversible, efficient binding and release of unprotected DNA; robust solvent integrity; and resistance to chemical modification. The first requirement narrowed our focus to resins that noncovalently bind DNA. A number of resins were tested and excluded due to poor bind–release properties (diethylaminoethyl [DEAE] silica, Macro-Prep ceramic hydroxyapatite, and quaternary amine anion exchange resins). Others exhibited extensive compression in organic solvent (Sephacryl S-1000, macroporous methacrylate) or poor reswelling during organic to aqueous solvent transitions (Poros 50 HQ). Reverse-phase resins were excluded because they would presumably not retain DNA in many organic solvents.2O, methanol (MeOH), dimethyl sulfoxide, N,N-dimethylformamide (DMF), ethyl acetate, and dichloromethane. Lastly, Sepharose has been used previously as a material for solid-phase synthesis (DEAE Sepharose, a tertiary amine anion exchange resin, exhibited excellent bind and release properties. By a high performance liquid chromatography (HPLC) assay, oligonucleotides were immobilized and eluted quantitatively in small volumes . Single-ynthesis . All subWe studied 9-fluorenylmethyoxycarbonyl (Fmoc)–based peptide synthesis because it has a well-established solid-phase precedent and offers a challenge in diverse chemical functionality A. As a DN-hydroxysuccinimide, 1-hydroxy-7-azabenzotriazole (HOAt), or N-hydroxybenzotriazole -3-ethylcarbodiimide hydrochloride (EDC) out-performed other activating reagents examined; other reagents gave poor coupling yields (benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate [PyBOP]), resulted in the formation of undesired side products (2-[1H-benzotriazole-1-yl]-1,1,3,3-tetramethyluronium tetrafluoroborate [TBTU]), or led to poor recovery of DNA (dicyclohexylcarbodiimide [DCC] and diisopropylcarbodiimide [DIC]). Thirty-minute EDC coupling reactions were typically less than 50% efficient without the addition of acylation catalysts such as triazole . Of thesHOAt see . ImportaHOAt see , which mHOAt see .To examine whether the coupling conditions generalized to longer DNA fragments, we used an aminated 340-base ssDNA as the support. After coupling, the eluted amino acid–DNA conjugates were digested with nuclease P1, a 3′-to-5′ exonuclease that cleaves all but the 5′ phosphodiester bond of our ssDNA constructs. The 5′-terminal nucleotide, which maintains the linker and synthetic peptide product, was separated from the other nucleoside monophosphates by HPLC and verified by electrospray ionization mass spectrometry. Amino acid coupling to the 340-base ssDNA proceeded with efficiencies comparable to those observed with oligonucleotides (data not shown). In some cases, the sensitivity of this HPLC assay was increased using fluorescence detection. For these experiments, we synthesized a fluorescent lysine derivative, Fmoc-Lys(coumarin)-OH  esters, which are normally cleaved with trifluoroacetic acid, could be removed at pH 6.5 in an aqueous solution at 70 °C. This gentle condition offers a convenient approach for acid deprotection.The carboxylic acid side chains of aspartic and glutamic acid are usually protected as esters during Fmoc peptide synthesis. Sterically bulky esters are required to suppress piperidine-induced imide formation, which leads to undesired side chain peptide bonds. We discovered that the conventional tert-butyl ester deprotection did not proceed through intramolecular imide formation, we coupled either Fmoc-Asp(tBu)-OH or Fmoc-Asp-OtBu to Leu-NC20, followed by Fmoc-Phe-OH. The main and side chain isomers of these tripeptide–DNA conjugates were resolved by HPLC after removal of the t-butyl group. Interconversion of the isomers during deprotection was undetectable (less than 5%). When the experiment was repeated using a 10 mM NaOH solution for tert-butyl ester deprotection (where imide formation would be expected), interconversion of the side and main chain isomers was observed. These results indicate that the thermolytic deprotection maintains the regiochemistry of the initial peptide bonds. At this time, we have little other data that speak to the mechanism of deprotection.To verify that the thermolytic Protection of the primary amine side chain of lysine prevents the formation of branched peptides. 2-Nitrobenzenesulfonamide (nosylamide) protection was particularly attractive as a means for lysine protection because the protecting group is base-stable and removed under conditions known to be DNA-compatible . The lysArginine does not absolutely require side chain protection . Howevertert-butyl esters. However, the trityl group is not ideal in aqueous conditions because of its hydrophobicity and extreme lability at high temperatures. To offer a more robust solution, we sought an H2O-compatible histidine protecting group. The 2,4-dinitrophenyl group, widely used in Boc peptide synthesis, is more hydrophilic than trityl and is removed under the nosyl-deprotection conditions. Unfortunately, 2,4-dinitrophenyl is not stable to the piperidine used for Fmoc removal  group of His(Trt) is rapidly removed under the thermolytic conditions used to deprotect  removal . After tHistidine racemization is a well-recognized concern in peptide synthesis. To assay the extent of racemization occurring during histidine coupling, we synthesized the oligonucleotide–dipeptide conjugate His(CNP)-Ala-NC20 using either L- or D-Fmoc-His(CNP)-OH. The diasteriomeric dipeptide–oligonucleotide products were resolvable by reverse-phase HPLC. Neither L-His nor D-His coupling resulted in detectable racemization. The experiment was repeated using L- and D-Fmoc-His(Trt)-OH with the same result.tBu)-OH was employed as the protected form of cysteine. The tert-butyl thioether coupled efficiently (Fmoc-Cys(Siciently  and was n amino acids exceeded (0.9)n (To characterize multistep syntheses , we prepd (0.9)n . The HPLd (0.9)n , ruling Beyond the physical and chemical characterization of the peptide–oligonucleotide conjugates, it was important to examine their behavior in a biochemical setting. Thus, the [Leu]enkephalin pentapeptide was synthesized on an aminated 340-base ssDNA support, which was subsequently converted to duplex form. The conjugate exhibited a peptide-dependent electrophoretic mobility gel shift when incubated with the 3-E7 antibody , demonstDNA-directed synthesis requires “chemical translation” . Rather The proximity approach to chemical translation uses hybridization to induce proximity-driven chemical transformations. Because the DNA “reading” and chemical execution steps are simultaneous, reactions are necessarily performed in aqueous solutions with solute, pH, and temperature conditions that promote DNA–oligonucleotide hybridization. These conditions limit the generality, efficiency, and speed of possible organic transformations.2O, MeOH, ethanol, isopropanol, DMF, dimethyl sulfoxide, dichloromethane, and ethyl acetate (data not shown). We also carry out reactions at high temperatures. For example, difficult peptide couplings are facilitated with elevated temperature, and the BME-mediated deprotection of lysine, arginine, histidine, and cysteine is carried out at 60 °C. We have recently used microwave-assisted methods  submonomer synthesis on unprotected DNA (data not shown). The chemistry used for peptoid synthesis is entirely different from peptide chemistry, illustrating the generality of the strategy. The potential for adapting other chemistries is essentially limitless. Wittig reactions, azide reductions, 1,3 dipolar cycloadditions, reductive aminations, Heck couplings, and a wide variety of other useful chemical transformations have been carried out in the presence of unprotected DNA without modification of DNA . Nuclease P1 (#27–0852-01), DEAE Sepharose Fast Flow (#17–0709-01), and Medium Grade G-25 Sephadex (#17–0033-01) were purchased from Pharmacia-LKB Technology . Xba1 was purchased from New England Biolabs (Beverly, Massachusetts, United States). DEAE Sepharose columns were poured in Empty TWIST synthesis columns (#20–0030, Glen Research, Sterling, Virginia, United States). Kendall Monoject syringes  and a Promega (Madison, Wisconsin, United States) manifold with chemically resistant PFTE stopcocks  were used. All other chemical reagents or solvents were purchased from either Sigma-Aldrich (St. Louis, Missouri, United States) or Fisher Scientific International (Hampton, New Hampshire, United States).Fmoc amino acids were purchased from Novabiochem , Chem-Impex International , or Fluka (Basel, Switzerland). EDC was purchased from Omega Chemical (Levis, Quebec, Canada). 2N-X-AGCAGGCGAATTCGTAAGCC, where X represents a C12 linker (NC20) or a longer PEG linker (NP20). NC20 was synthesized using the Glen Research 5′-Amino-Modifier C12 (#10–1922). NP20 was synthesized using the Glen Research Spacer Phosphoramidite 18 (#10–1918) followed by the 5′-Amino-Modifier 5 (# 10–1905).The internal control 10-base oligonucleotide had the sequence CGGACTAGAG. The reactive 20-base oligonucleotides had the sequence H260nm ≈ 224.5 mM−1cm−1) and were not considered in efficiency and yield determination. Reaction products were collected, concentrated to approximately 50 μM using centrifugal evaporation, and desalted over G-25 Sephadex. A mixture of 1 μl of desalted oligonucleotide and 1 μl of a freshly prepared saturated matrix solution was spotted on a matrix-assisted laser desorption/ionization target and allowed to air dry before mass spectrometry analysis. The matrix solution was made from 250 μl of H2O, 250 μl of acetonitrile, 25 mg of THAP, and 10 mg of ammonium tartrate. Peptide sequences (five or more amino acids) were verified by Edman degradation peptide sequencing.Coupling reactions were monitored by HPLC mobility shift using a C18 analytical column  and UV detection at 260 nm and 280 nm . Linear gradients from 0%–90% acetonitrile in 100 mM triethylammonium acetate (pH 5.5) were employed. Coupling efficiencies  and yields  were determined by integration of elution peaks from the 260-nm channel. Chromophores added or removed during reactions cause changes in extinction coefficients less than the sensitivity (5%) of our HPLC assay . Yields were determined by integration of elution peaks from the 260-nm channel, using the P1 digestion product of unreacted starting material as a reference. Approximately 1 nmol of material was required for accurate UV detection. Products were collected, concentrated by centrifugal evaporation, and applied to a C18 SepPak cartridge (#WAT023590, Waters, Milford, Massachusetts, United States) in 25 mM triethylammonium acetate (pH 5.5). The cartridge was washed with 3 ml of 25 mM triethylammonium acetate (pH 5.5) and 1 ml of H2O. The products were eluted with 1 ml of 50/50 MeCN/H2O, concentrated to 100 μl by centrifugal evaporation, and analyzed by electrospray ionization mass spectrometry. For coumarin-labeled products, fluorescence was monitored (320 nm excitation/380 nm emission) with a scanning fluorescence detector (Thermo Separation Products, FL2000), and less than 50 pmol of material was necessary for accurate fluorescence detection.DEAE elute buffer containing the peptide–DNA conjugates was neutralized and brought to 100 mM sodium acetate (pH 5.2) and 400 μM ZnSO5/pixel). The intensity of full-length control and [Leu]enkephalin bands were similar to within 1% (S/N approximately 600). Upon peak integration along the entire lane, the full-length band represented a similar percentage of total intensity in the control (83%) and [Leu]enkephalin (81%) samples. The data suggest that, in the worst case, 3% of the DNA could have been modified during the course of peptide synthesis.A 5′-aminated 340-base ssDNA support was generated as described . After l2O followed by 12 ml of DEAE bind buffer (10 mM acetic acid and 0.005% Triton X-100) using a syringe or a syringe barrel, a male-male luer adapter, and a vacuum manifold  using a syringe. Long DNA molecules (340mers) were eluted with 4 ml of 1.5 M NaCl, 10 mM NaOH, and 0.005% Triton X-100 heated to 80 °C.2O, and 7.5 μl of diisopropylethylamine. Fmoc-Asn-OSu required four couplings rather than two to achieve quantitative yields.The following process was carried out twice. The column, with DNA bound, was washed with 3 ml of DMF. Using two syringes see , the colAfter the second amino acid incubation, the column was washed with 3 ml of DMF. Fmoc deprotection was carried out as follows: 3 ml of 20% piperidine in DMF was applied to a 3-ml syringe barrel attached to the top of the column. 1.5 ml was pushed through the column, followed by a 3-min incubation. An additional 1 ml was pushed through the column, followed by a 17-min incubation. The procedure was completed with a final 3-ml DMF wash.2O and 3 ml of MeOH and then incubated for 30 min at room temperature with a freshly prepared 500-μl solution of 50 mM Fmoc–amino acid-OH, 50 mM EDC, and 5 mM HOAt in MeOH. These conditions were derived directly from n amino acids exceeded (0.9)n. Fmoc deprotection was carried out as described for succinimidyl ester coupling. See Supporting Information for a more detailed description of peptide coupling.The column was washed with 500 μl of HtBu), and His(CNP), the column was washed with 3 ml of DMF and subsequently incubated for 30 min with 700 μl of DMF containing 500 mM BME and 250 mM DBU while submerged in a 60 °C H2O bath. The column was then washed with 3 ml of DMF and 12 ml of DEAE bind buffer. Lys(Ns) can also be deprotected quantitatively with a DMF solution containing 5% thiophenol and saturated K2CO3 at 37 °C for 90 min. These conditions deprotect Arg(Ns) inefficiently, and have not been tested for Cys(StBu) or His(CNP).For Lys(Ns), Arg(Ns), Cys(StBu), Glu(tBu), and His(Trt), after HPLC purification, the tert-butyl ester and/or trityl containing oligonucleotide–peptide hybrid was incubated in a 20-mM MgCl2 solution at 70 °C, yielding quantitative deprotection in 3 h (Asp, His) or 12 h (Glu). Deprotection can alternatively be carried out before HPLC purification: after eluting from the DEAE Sepharose column, NaOAc (pH 5.2) and MgCl2 were added to final concentrations of 30 mM and 200 mM, respectively, and the solution was then incubated at 70 °C for the appropriate time. In contrast, acid deprotection on solid support was inefficient.For Asp and after  peptide coupling. Calculated masses are noted to the left of the mass peaks.(149 KB PDF).Click here for additional data file.Figure S2(A) Reaction scheme for synthesis of GLFYG-NC20. Coupling efficiencies for individual steps are noted in black, and absolute yields from NC20 are noted in red. MALDI-MS results for all species are denoted under each species as “Observed .” See Protocol S1 for precise coupling procedures.(B) HPLC analysis of sequential couplings during peptide synthesis monitored at 260 nm. Load and elutes from columns 1–5 are indicated. Sequential coupling efficiencies (black) were calculated by integration of recovered aminated DNA peaks. Absolute yields (red) were calculated by integration of intended product peak relative to load. A nonaminated 10-base oligonucleotide (10mer) was included as a control for nonspecific DNA loss and modification. Percent recovery of 10mer is noted in red. The HPLC analysis employed a 60-min gradient of 0%–45% MeCN in100 mM TEAA (pH 5.5).(368 KB PDF).Click here for additional data file.Protocol S1(1.0 MB PDF).Click here for additional data file.


Through major efforts to reduce costs and expand access to antiretroviral therapy worldwide, widespread delivery of effective treatment to people living with HIV/AIDS is now conceivable even in severely resource-constrained settings. However, the potential epidemiologic impact of treatment in the context of a broader strategy for HIV/AIDS control has not yet been examined. In this paper, we quantify the opportunities and potential risks of large-scale treatment roll-out.We used an epidemiologic model of HIV/AIDS, calibrated to sub-Saharan Africa, to investigate a range of possible positive and negative health outcomes under alternative scenarios that reflect varying implementation of prevention and treatment. In baseline projections, reflecting “business as usual,” the numbers of new infections and AIDS deaths are expected to continue rising. In two scenarios representing treatment-centered strategies, with different assumptions about the impact of treatment on transmissibility and behavior, the change in the total number of new infections through 2020 ranges from a 10% increase to a 6% reduction, while the number of AIDS deaths through 2020 declines by 9% to 13%. A prevention-centered strategy provides greater reductions in incidence (36%) and mortality reductions similar to those of the treatment-centered scenarios by 2020, but more modest mortality benefits over the next 5 to 10 years. If treatment enhances prevention in a combined response, the expected benefits are substantial—29 million averted infections (55%) and 10 million averted deaths (27%) through the year 2020. However, if a narrow focus on treatment scale-up leads to reduced effectiveness of prevention efforts, the benefits of a combined response are considerably smaller—9 million averted infections (17%) and 6 million averted deaths (16%). Combining treatment with effective prevention efforts could reduce the resource needs for treatment dramatically in the long term. In the various scenarios the numbers of people being treated in 2020 ranges from 9.2 million in a treatment-only scenario with mixed effects, to 4.2 million in a combined response scenario with positive treatment–prevention synergies.These analyses demonstrate the importance of integrating expanded care activities with prevention activities if there are to be long-term reductions in the number of new HIV infections and significant declines in AIDS mortality. Treatment can enable more effective prevention, and prevention makes treatment affordable. Sustained progress in the global fight against HIV/AIDS will be attained only through a comprehensive response. Combining the two approaches of prevention and treatment of HIV/AIDS could avert more than 29 million new HIV infections by 2020 In June 2001, heads of state and government convened a United Nations Special Session on HIV/AIDS and adopted unanimously the “Declaration of Commitment on HIV/AIDS” . Today, The theme of the 15th International AIDS Conference in Bangkok last summer was timely and relevant. “Access for All” calls for extending to all of those in need both sufficient resources and a set of proven interventions to prevent new infections and save lives through effective treatment. Recent developments in HIV treatment, with simple combination therapies priced at less than US$150 per year—unthinkable just a short time ago—were a major driver of discussions during the conference. Widespread access to effective antiretroviral therapy (ART) for people living with HIV/AIDS is now conceivable even in countries with severely limited resources.The World Health Organization and its partners in the Joint United Nations Programme on HIV/AIDS have defined an ambitious “3 by 5” target of 3 million people on ART—half of those in most urgent need—by the end of 2005. The potential epidemiologic impact of large-scale roll-out of treatment programs, however, remains uncertain. Experience to date is limited, and comes mostly from Western countries and Brazil. While declines in AIDS mortality in the industrialized world have been impressive ,7,8, man.In our previous analysis of the potential benefits of a comprehensive package of preventive interventions , we noteBaseline projections of HIV epidemics in sub-Saharan Africa have been developed by the Joint United Nations Programme on HIV/AIDS and the World Health Organization based on the most current data available, and in collaboration with epidemiologic experts and analysts within the countries assessed . These “To simulate the effects of prevention and treatment on HIV/AIDS incidence, prevalence, and mortality, we first adapted the analytic approach used in the previously described Goals model  to allowThe model includes underlying regional demography, acquisition of HIV and other sexually transmitted infections (STIs), progression from HIV to AIDS, and progression from AIDS to death. Annual risks of HIV infection in each risk group depend on the number of partnerships, the number of sex acts per partnership, HIV prevalence among partners and condom use. These risks are magnified by the presence of other STIs  and alsoThe regional models were calibrated as follows: first, plausible ranges were specified for model parameters governing sexual behavior and biological factors (e.g., transmission risks and cofactor effects of other STIs) based on review of published studies and survey results; second, multiple simulations were undertaken by sampling values from each of the ranges and recalculating the model for each set of sampled parameter values; third, model fit was assessed by comparing modeled prevalence for adult males and females separately to baseline projections through 2020; and fourth, the best-fitting parameter set in each regional model was selected for the purpose of scenario analysis see .Potential impacts of prevention efforts at a given coverage level were based on previously published estimates  for a coWe examined a range of alternative scenarios based on various levels and effectiveness of prevention interventions, with and without successful attainment of the 3 by 5 treatment target for sub-Saharan Africa:Risk behaviors are maintained at current levels, and no treatment scale-up occurs. This is simply the baseline scenario that produces a relatively stable prevalence rate over the duration of the projection, with the number of people living with HIV and the number of new infections rising slowly over time because of population growth.In two alternative scenarios, the 3 by 5 target of 50% coverage of those in need of treatment by the end of 2005 is attained, and scale-up continues to reach 80% ART coverage of those in need by 2010, maintained at 80% thereafter. In an “optimal ART effects” scenario, we assumed that treatment reduces transmissibility by 99%, and that those under treatment have 50% lower annual partnership numbers and two times higher condom use than other adults. With a response that focuses primarily on treatment, it is assumed that behavior in the general community of infected and uninfected adults is unchanged from the baseline. In an alternative “mixed ART effects” scenario, less optimistic assumptions were made: that treatment reduces transmissibility only to the same levels as in asymptomatic infected individuals (two-thirds reduction from no treatment), and that behavior in treated patients is the same as in other adults. To capture the possibility of behavioral disinhibition in response to treatment availability, we assumed that condom use declines by 10% in both treated patients and the general community, with other behaviors unchanged. The potential for disinhibition is suggested primarily by experience in some developed countries, where condom use increased dramatically in the populations at highest risk prior to the introduction of ART but then declined; the likelihood and magnitude of reductions in condom use in sub-Saharan Africa, where such prevention-induced changes generally are much less prominent today, might be questioned. We therefore considered in sensitivity analyses a variant of this scenario that excludes disinhibition but preserves all other assumptions.In the absence of wide availability of treatment, reflecting weaker political and social support for HIV control efforts, we modeled a scenario in which the comprehensive prevention package described previously  has onlyWe examined two scenarios combining treatment and prevention efforts, reflecting either optimistic or pessimistic possibilities. In the optimistic scenario, treatment strengthens prevention efforts. ART coverage is the same as in the two treatment-centered scenarios, with optimal assumptions about treatment impact on transmissibility and patient behavior. It is assumed that widespread availability of treatment enables the full impact of prevention efforts to be attained as described by Stover et al. . In a moAdditional scenarios could include pessimistic assumptions about limited ART scale-up levels and timing, emergence of large-scale drug resistance resulting from low adherence, or other possible unintended outcomes of wider treatment. Certainly, large-scale treatment efforts will demand close monitoring of adverse effects. However, experience with treatment programs in developing countries has been encouraging thus far, with reported adherence levels that are at least as high as those in developed countries ,19.In the baseline projections for sub-Saharan Africa, the annual number of new adult HIV infections rises from 2.4 to 3.7 million between 2004 and 2020, and adult AIDS mortality rises from 1.8 to 2.6 million . If scalWith less optimistic assumptions (treatment-centered response/mixed ART effects), the number of new infections rises, to 4.3 million per year by 2020 (a 14% increase); mortality trends are similar to the optimistic scenario in the short term, but worse in the long term, even compared to the baseline. Excluding the assumption of reduced condom use through disinhibition from the treatment-centered/mixed effects scenario has minimal effect on the results, lowering the number of new infections in 2020 by only 2% compared to the scenario that includes disinhibition.A prevention-centered response would have greater impact on the number of new infections, lowering annual incidence by more than half by 2020. The long-term mortality trend is more favorable in the prevention-centered scenario than in the treatment-centered scenario because of reduced incidence, but prevention would produce negligible mortality benefits in the near- and mid-term future in comparison to strategies that include ART. Alternative assumptions regarding overall effectiveness in a prevention-centered response produce results that scale as expected, with reductions in annual incidence of 34% to 64% and reductions in annual mortality of 20% to 42% by 2020.If treatment and effective prevention are scaled up jointly in a combined response, the benefits in terms of both infections and deaths averted could be substantially higher. In an optimistic scenario in which treatment programs support expanded prevention, the annual number of new infections would be 74% lower and annual mortality would be 47% lower by 2020, compared to baseline. It is worth noting that the long-term decline in AIDS deaths is driven more by prevention of new infections than by direct survivorship benefits from ART. In a pessimistic scenario in which a more narrow treatment focus limits effective prevention, the overall benefits are much more modest, with 26% and 16% reductions, respectively, in new infections and mortality by 2020 compared to the baseline.Prevalence rises by 7% in the optimal and by 27% in the mixed treatment-centered scenarios by 2020, as longer survival for treated patients offsets reductions in new infections through reduced transmissibility (and risk reductions among treated patients in the more optimistic scenario) . In scenThe total number of infections averted through a combined response would be 29 million over the period 2004 to 2020 if treatment enhances prevention, a benefit that is ten times greater than that of a strategy which focuses on treatment only, even with optimal assumptions, and 51% greater than that of a strategy which focuses on (less effective) prevention alone . If a trCombining treatment with prevention efforts will reduce the resource needs for treatment substantially in the long term . In the In this paper, we have examined the potential epidemiologic impact of global HIV/AIDS control efforts under a range of alternative scenarios reflecting varying implementation of strategies for prevention and treatment. Although we focus in particular on population health outcomes and epidemiologic trends, we recognize that there are numerous other social, economic, and individual health effects of interventions including ART that are beyond the scope of this analysis. We also restrict our focus in this paper to sub-Saharan Africa, where the overwhelming majority of people living with and dying from HIV/AIDS reside; however, our findings have broader applicability and more general implications in the worldwide fight against HIV/AIDS, which we highlight here.Effective prevention requires more than having sufficient funds to offer information and services. It also requires an environment that encourages people to internalize messages about risky behavior and to adopt actual behavior change, and allows people to utilize services such as testing and counseling without fear of stigma or discrimination. Stoneburner and Low-Beer have argued that the supportive social and political environment in Uganda allowed people to discuss AIDS with family members and close friends, which led to greater behavior change than in Kenya or Zambia where most people received information from mass media only . People Involving communities and family members in the delivery of treatment—for example, as treatment monitors—offers unique entry points for effective prevention activities and a lever for population-wide behavior change. Experience with community roll-out of treatment programs has shown, for example, that uptake of voluntary counseling and testing increased by 300% in one year of roll-out in Haiti, and by a factor of 12 in Khayelitsha, South Africa, after treatment introduction ,22. A stDuring most of the past 15 years, efforts to address the AIDS epidemic in sub-Saharan Africa have focused on prevention. There have been successes in some countries, but overall these efforts have not achieved their goals. The advent of vastly expanding treatment programs in the coming years, if opportunities to capitalize on broadened political support and community mobilization can be seized, offers the potential to enhance prevention effectiveness and avert many new infections and deaths.While prevention programs are unlikely to achieve full impact in the absence of treatment, so too is the impact of treatment programs reduced if vigorous prevention efforts are absent. Without effective prevention, the number of people requiring care and treatment will grow each year. As more and more people are kept alive with ART the treatment burden will become enormous unless effective prevention reduces the number of people becoming newly infected.Without effective prevention programs, we project that the number of people receiving treatment will grow to 6.3 million by 2010 and up to 9.2 million by 2020 in Africa alone to achieve 80% coverage of those in urgent need. Meeting this need would require a tremendous increase in financing, human capacity and infrastructure that might not be attainable.If effective prevention programs are combined with treatment programs, the same level of 80% ART coverage would be achieved by treating 5.8 million in 2010 and 4.2 million in 2020. In other words, the same goal could be attained at a far lower treatment cost and with a much greater chance of sustainability.Over the long term, it is effective prevention that will reduce the burden of illness due to AIDS and the number of people in need of ART. The lessons learned in the industrialized world have to be taken on board. The availability of treatment and the shift in focus away from very effective prevention programs has led to increases in unsafe sexual behavior, STIs, and HIV transmission in some settings ,10,11. TCountries in sub-Saharan Africa are faced with the most devastating epidemic of our times. We now have the unique opportunity to derive the maximum impact from available resources. The results from our analyses show how potential synergies between prevention and treatment could be translated into considerable health benefits at the population level. But synergies do not mean simply that prevention and treatment are pursued in parallel. When whole communities become involved in the scale-up of treatment access—as will be necessary to achieve the ambitious treatment targets defined by the 3 by 5 campaign—crucial opportunities can be created for increasing their involvement in prevention activities. Only if interactions with patients, family, and community members occasioned by the provision of treatment are also used to reinforce prevention, and only if prevention workers have an opportunity to refer those in need to care and treatment, will we move at last from slogans to impact.Figure S1(80 KB EPS).Click here for additional data file.Protocol S1(172 KB PDF).Click here for additional data file.Infections from HIV continue to increase, especially in sub-Saharan Africa. The World Health Organization has a plan to get more than 3 million people on treatment by 2005 (the “3 by 5” initiative); however, the overall effect of this plan on the population's health is uncertain, and will depend on the balance between treatment and prevention efforts.They tried to predict the number of new infections and deaths each year in sub-Saharan Africa from now until 2020 depending on whether control efforts focused on prevention, treatment, or both. What they found was that by far the most effective way of decreasing new infections and deaths was to combine the two approaches, and that by doing so more than 29 million new infections and 10 million deaths might be prevented compared with continuing at current levels of prevention and care.Despite the huge amounts of money directed at HIV/AIDS, because the problem is so vast, the resources are not enough. Hence it is important to target these resources effectively. Policy makers around the world could use information like this to decide where best to direct attention and funding to combat HIV/AIDS.http://www.unaids.org/wad2004/report.htmlJoint United Nations Programme on HIV/AIDS, AIDS epidemic update, December 2004: http://www.who.int/3by5/World Heath Organization, 3 by 5 Initiative: http://www.kff.org/hivaids/hivghpwgpackage.cfmGlobal HIV Prevention Working Group:


Vascular endothelial growth factor (VEGF)-C is implicated in lymphangiogenesis, however the exact role of VEGF-C in promoting lymphatic spread of cancer cells remains largely unknown.The expression of VEGF-C was immunohistochemically determined in 97 endoscopic biopsy specimens from 46 patients with submucosal gastric carcinoma (SGC). Nodal metastases including micrometastasis and isolated tumor cells (ITC) were evaluated by immunohistochemical staining for cytokeratin in 1650 lymph nodes, and tumor cells in these metastatic nodes were also examined for VEGF-C expression.In biopsy samples, VEGF-C was positively detected in 21 (47%) patients. Metastases were identified in 46 (2.8%) nodes from 15 (33%) patients. Metastases were detected in 39 nodes by hematoxylin-eosin (H&E) staining and in additional 7 nodes as ITC by immunohistochemical staining. The rate of lymph node metastases was significantly correlated with VEGF-C expression in biopsy samples (p < 0.05). The positive and negative predictive values of VEGF-C in biopsy specimens for nodal metastasis were 44 %(10/21) and 80% (20/25), respectively. Among the 46 metastatic nodes, tumor cells in 29 (63%) nodes positive patients expressed VEGF-C, whereas those in 17 (37%) nodes did not. VEGF-C expression was high in macronodular foci in medullary areas, whereas more than half of ITC or micrometastasis located in peripheral sinus lacked the expression of VEGF-C.Despite the significant correlation, immunodetcetion of VEGF-C in endoscopic biopsy specimens could not accurately predict the nodal status, and thus cannot be applied for the decision of the treatment for SGC. VEGF-C may not be essential for lymphatic transport, but rather important to develop the macronodular lesion in metastatic nodes. The incidence of early gastric carcinoma defined as being confined to the mucosa or submucosal layer has increased. In Japan, endoscopic mucosal resection (EMR) is now generally accepted for intramucosal cancers that are associated with a minimal risk of regional lymph node (LN) metastasis -4. For sVECF-C is known to bind VEGFR-3, which is specifically expressed on lymphatic vessels and stimulates lymphangiogenesis ,10. ManyRecently, small metastatic lesions have been detected genetically or immunohistochemically in various cancers, even though they were diagnosed as negative by conventional examination with H&E staining. Such lesions are designated as micrometastasis or isolated tumor cells (ITC). The biological and clinical significance of such minute nodal invasion of carcinoma cells is still controversial -38. In tForty-six patients with SGC diagnosed and treated by curative gastrectomy with standard lymph node dissection at the First Department of Surgery, Tokyo University Hospital, Tokyo, between 1994 and 2002 were included in this study. These patients were examined endoscopically prior to surgery; several pieces of tissue specimens were then sampled with routine biopsy forceps from various portions of the tumor. Formalin-fixed and paraffin-embedded sections of 97 biopsy specimens and 1650 dissected lymph nodes derived from these 46 patients were evaluated in this study. Additionally, all the resected primary tumors were histologically examined with H&E staining according to the Japanese Classification of Gastric Carcinoma . Tumors 2O2 and 0.05% diaminobenzidine tetrahydrochloride for 3 minute. Light counterstaining with Mayer's hematoxylin was performed. The 46 lymph nodes that showed the presence of carcinoma were also evaluated for the expression of VEGF-C with the same immunostaining method.The expression of VEGF-C was investigated with immunohistochemical staining using affinity purified goat polyclonal antibodies against VEGF-C (IBL, Fujioka, Japan). Sections (3-μm thick) of biopsy samples were deparaffinized in xylene, hydrated through a graded series of ethanol, and then immersed in 3% hydrogen peroxide in 100% methanol for 30 min to inhibit endogenous peroxidase activity. To activate the antigens, the sections were boiled in 10 mM citrate buffer, pH 6.0 for 30 minutes. After being rinsed in phosphate-buffered saline (PBS), the sections were incubated with normal rabbit serum for 10 min, and then incubated overnight at 4°C in humid chambers with the primary antibody to VEGF-C at 1/30 dilution. After three washes with PBS, the sections were incubated with biotinylated rabbit anti-goat immunoglobulin for 20 minute. After washing again with PBS, the slides were treated with peroxidase-conjugated streptavidin for 20 minutes, and developed by immersion in 0.01% HThe dissected lymph nodes were fixed in 10% formalin and embedded in paraffin. From each node, one 3-μm-thick section was prepared for H&E staining, and another three serial 5-μm sections were prepared for immunohistochemical staining with CAM 5.2 (Becton Dickinson, San Jose, CA), a mouse monoclonal antibody that reacts with human cytokeratin numbers 8 and 18 . The strMetastasis was defined as the presence of tumor cells, whether single or in small clusters detected by H&E or immunohistochemical staining. Metastatic lesions that were more than 2.0 mm in diameter were defined as macrometastasis, while micrometastasis was defined as a tumor deposit larger than 0.2 mm but less than 2.0 mm, and ITC was defined as a tumor deposit less than 0.2 mm in maximum diameter.All statistical calculations were carried out using StatView-J 5.0 statistical software (SAS Institute, USA). The relationship between clinical and pathological characteristics of patients and the expression of VEGF-C was examined by Fisher's exact test. Differences with a p value of less than 0.05 were considered to be statistically significant.Metastases were observed in 12 patients (26.1%) and 39 lymph nodes (2.4%) by H&E examination Fig. . Among tTable The biopsy specimens were divided into two categories by the staining pattern of VEGF-C, diffuse or focal staining of carcinoma cells as described previously . When diAmong the 97 biopsy samples from 46 patients, carcinoma cells were contained in only one biopsy sample in 14 patients, and in 2, 3 and 4 biopsy samples in 16, 13 and 3 patients, respectively. In all of the latter cases, carcinoma cells in biopsy samples derived from different places showed exactly the same staining pattern of VEGF-C, and thus VEGF-C-positive and -negative tumors could be clearly distinguished.In biopsy specimens, VEGF-C was positively detected in 21 (46%) cases. As shown in Table Nodal metastasis was detected in 10 (48%) of 21 VEGF-C-positive tumors, and the rate was significantly higher than that in VEGF-C-negative tumors as evaluated by biopsy specimens (5/25, 20%) (p = 0.047). However, the positive and negative predictive values of VEGF-C in biopsy for nodal status were 44% (10/21) and 75% (20/25), respectively, and 5 (20%) of 25 VEGF-C-negative tumors were accompanied with lymph node metastasis.Table Among 36 nodes from 10 patients who were determined as VEGF-C-positive in biopsy samples, tumor cells located in 10 (29%) nodes from 4 (40%) patients totally lacked the expression of VEGF-C. This finding clearly indicates that carcinoma cells that highly express VEGF-C are not always preferentially transported to the regional lymph nodes, even though the expression of this lymphangiogenic factor had a positive correlation with lymph node metastasis.More interestingly, the expression of VEGF-C was strongly correlated with the size of metastatic foci and the location of carcinoma cells in metastatic nodes Table . When caMany cancers metastasize to regional lymph nodes, and a positive nodal status often correlates with a poor prognosis of patients. However, the mechanisms of lymphatic metastasis have not been investigated in detail. Recent studies have demonstrated that the expression of VEGF-C is enhanced in various solid tumors, suggesting the possible contribution of VEGF-C to nodal metastasis, possibly through lymphangiogenesis ,42. NumbIn this study, therefore, we evaluated the expression of VEGF-C in biopsy samples in SGC. Our initial hypothesis was that VEGF-C expression can predict the accurate nodal status including micrometastasis/ITC, and thus may be useful to avoid the unnecessary gastrectomy in some SGC. Our results suggest that VEGF-C expression in biopsy specimens correlate with lymph node metastasis in SGC. However, the positive and negative predictive values were 44% and 80% respectively, and 20% of VEGF-C-negative tumors were node positive. This suggests that the immunodetection of VEGF-C in biopsy samples can not be used as clinical indicator to decide the treatment of SGC.Present study provides some interesting findings on the possible role of VEGF-C in nodal metastasis. Biopsy samples were obtained at preoperative endoscopy, fixed with formalin immediately after biopsy, and thus appear to reflect the in situ expression level of VEGF-C more precisely than surgically resected specimens. In our results, VEGF-C expression in biopsy samples showed a significant correlation with that in surgical specimens with 57% sensitivity and 91% specificity. However, more than half (60%) of the tumors categorized as VEGF-C negative in biopsy specimens were positive in surgical specimens, although VEGF-C-positive tumors in biopsy samples showed a good consistency with those in surgical specimens. This raises the possibility that VEGF-C expression may be somewhat upregulated by surgical manipulation.VEGF gene expression is regulated by a variety of stimuli, and hypoxia is known to be one of the most potent inducers of VEGF-A ,44. VEGFin vivo experiments using VEGF-C-transfected tumors have shown the same histological findings [Nonetheless, our data showed a significant correlation of VEGF-C expression in biopsy specimens with nodal metastasis, supporting a possible role of VEGF-C in lymphatic metastasis. As with hematogeneous metastasis, lymphatic metastasis of cancer cells is considered to be divided into several steps: invasion to lymphatic capillaries, movement into the lymphatic lumen with the lymphatic stream, attachment to the subcapsular sinus of lymph nodes, and invasion into the cortex. Lymphangiogenesis means the development and proliferation of new lymphatics from host vessels, but the ability of tumor cells to induce lymphangiogenesis and the presence of intratumoral lymphatic vessels are controversial. However, most malignant tumors are known to be associated with an increased number of lymphatic vessels in the peripheral area . In factfindings ,50. SincIn regional lymph nodes, tumor cells are thought to reach the peripheral sinus from afferent lymphatics. In fact, many metastatic cells were detected around the sinus area unless they developed into a macronodular lesion. Recently, small lesions have been divided into two categories; micrometastasis and isolated tumor cells (ITC), which are distinguished based on their size . MicromeIn our study, the metastatic lesions did not always express VEGF-C, and such small metastatic foci often lacked the expression of VEGF-C. Especially, ITC identified only with immunohistochemical staining are totally negative for VEGF-C. In addition, in 4 tumors with lymphatic invasion, none of the tumor cells located in the lymphatic vessels in the primary tumor expressed VEGF-C (data not presented). These unexpected results suggest that expression of VEGF-C in tumor cells is not relevant to the transportation to regional nodes once they enter lymphatic vessels.In contrast, most of the macrometastases or cancer cells invading the medullary area of metastatic nodes highly expressed VEGF-C. This phenomenon was quite interesting, though not fully explained by today's knowledge. This may suggest that proliferation and invasion in the internal area of metastatic nodes may partially require VEGF-C expression in tumor cells. Thus far, there is no definite report on the effects of VEGF-C on tumor cells. The VEGF-receptor 3 (VEGFR-3), a specific ligand of VEGF-C, was expressed only on certain tumor cells -54 and nIn summary, our retrospective study demonstrated that VEGF-C expression in tumor cells in biopsy specimens was significantly correlated with lymphatic metastasis in SGC, although the accuracy was not high enough to be used for clinical indicator. Metastatic tumor cells in micrometastasis or ITC located in marginal sinus often lacked the expression of VEGF-C, whereas macrometastasis located in the medullary area in metastatic nodes highly express VEGF-C. This suggests a possibility that expression of VEGF-C is not essential for lymphatic transport from primary tumor, but rather important to develop the macronodular lesion in metastatic lymph nodes.The author(s) declare that they have no competing interests.MI. Conceived of the study and wrote the original version of the manuscript.JK. Carried out the literature search and helped in drafting the manuscript.SK. Collected clinical and pathologic data and participated in manuscript preparationHN Helped to shape the idea for the study coordinated the study and edited the manuscript.All authors have read and approved the final manuscript.