The aim of this study was to examine the invasiveness of first trimester trophoblasts according to the secretion profile of MMP-2 and -9 at different gestational stages, and to test the similarity between primary trophoblast cell-culture and the JAR choriocarcinoma cell-line.First trimester trophoblasts were divided into two groups: 6–8 weeks (early) and 9–12 w (late) of gestation. The two trophoblast groups and JAR cells were cultured in medium, with various concentrations of forskolin and Epidermal Growth Factor (EGF). Proteolytic activity was detected by zymography and invasiveness was assessed by Matrigel invasion assay. Student's T-test was used for statistical analysis.In 6–8 w trophoblast, proMMP-2 was only slightly dominant over proMMP-9 (53.2% vs. 46.8% respectively), whereas in 9–12 w, proMMP-9 was clearly dominant over proMMP-2 (61.7% vs.38.3% respectively). In JAR cells proMMP-2 was strongly dominant (90.2% vs.9.8% respectively). In JAR cells forskolin significantly increased proMMP-2 and -9 secretion (128.5% +/- 12 and 183.2% +/- 27.9 of control, respectively). EGF had a dual effect on JAR cells: at 8 ng/ml both proMMP-2 and -9 were increased (133.5% +/-15 and 223.9% +/- 32.4 of control, respectively) while at 80 ng/ml both proMMP-2 and -9 were decreased (65.1% +/- 18.3 and 66.6% +/- 37 of control, respectively). Forskolin significantly increased both proMMP-2 and -9 secretion in 6–8 w and 9–12 w trophoblasts . EGF also significantly increased both proMMP-2 and -9 secretion in 6–8 w and 9–12 w trophoblasts . Both forskolin and EGF increased trophoblast cells invasiveness in all groups. The invasive ability of trophoblast cells, induced by forskolin, was reduced by MMP-2 antibody in: JAR cells, 6–8 w and 9–12 w trophoblasts. Likewise trophoblast invasion induced by EGF was reduced by MMP-2 antibody in all groups. However the invasive ability induced by forskolin or EGF was inhibited by MMP-9 antibody only in trophoblasts from 9–12 w.First trimester trophoblasts express differential gelatinase secretion profile according to the gestational week. In JAR and early trophoblasts (6–8 w) MMP-2 is the main gelatinase and the key enzyme in trophoblast invasion. Thereafter in late first trimester trophoblasts (9–12 w), both MMP-2 and -9 participate in trophoblast invasion. This study shows a differential, dynamic importance of each gelatinase in trophoblast invasion during the 1st trimester.Successful implantation depends on the ability of the embryo to degrade the basement membrane of the uterine epithelium and to invade the uterine stroma. Cytotrophoblastic cells (CTB) are derived from trophoectodermal cells of the blastocyst. CTB ensue to become either the villous cytotrophoblastic cells which will proliferate and differentiate by fusion to form the syncytiotrophoblast, or they will stream out of the syncytiotrophoblast to form mononuclear multilayered invasive extravillous cytotrophoblastic cells. The temporal and spatial regulation of trophoblast invasion is mediated in an auto-and paracrinic way by trophoblastic and uterine factors . Severalhoblasts -14. The 4 cells/well) were cultured in M-199 medium (Beit-Ha'Emek, Israel) containing 10% Fetal Calf Serum (FCS, Beit-Ha'Emek, Israel) and penicillin/streptomycin (Beit-Ha'Emek, Israel). Cell culture was maintained in a humidified atmosphere containing 5% CO2 at 37°C. After 24 hours of culture to facilitate cell attachment, the medium was removed, and M-199 medium with 1.5% serum supplemented with antibiotics was added. The cells were cultured with various concentrations of: a) Forskolin 1–100 μM, b) Epidermal Growth Factor (EGF), 0.8–80 ng/ml (Sigma). Control consisted of M-199 with 1.5% serum alone. Cells were cultured for an additional 48 hours, and media were removed for analysis of MMP secretion and stored at -20°C until use. Cell count was performed with XTT in order to normalize MMP secretion to cell number.The JAR (Jar, HTB 144) human choriocarcinoma line was established from a trophoblast tumor of the placenta . The JAR cells were a generous gift from Prof. Hochberg A Department of Biological Chemistry, Hebrew University, Jerusalem, Israel. JAR cells (1 × 105 cells/well in 96-well plates or in Transwell plates (Corning) with M-199 medium supplemented with 1.5% FCS and 1% penicillin/streptomycin and kept in 5% CO2 at 37°C. Inducers (10 μM forskolin or 8 ng/ml EGF (chosen as working concentrations after a dose-response study in JAR cell-line) were added to medium, and after 48–72 h media were collected for analysis of MMP secretion and cell count was performed. This method supplies a 95–98% purity of trophoblastic cells, including all trophoblastic sub-groups. We verified the purity of trophoblast cells by using immunohistochemistry with specific antibodies to cytokeratin 7 (positive) and vimentin (negative), commonly used for indication of trophoblast purity [Human trophoblast cells were obtained from legal abortions , with the approval of the local ethical committee (in compliance with the Helsinki Declaration) and the consent of the participating patients. Trophoblast cells were isolated as described previously in detail elsewhere ,9,15,16 Evaluation of cell proliferation was performed with XTT Reagent kit (XTT, cell proliferation kit, Beit-Ha'Emek, Israel) according to manufactures protocol. This is based on the activity of mitochondria enzymes in live cells, reducing tetrazolium salts, XTT, into colored formazan compounds, which can be detected colorimetric with a spectrophotometer at 450 nm (ELISA reader). Dye absorbance is proportional to the number of cells in each well.2, 0.2% Brij 35 and 50 mmol/l Tris, pH 7.5. The buffer was decanted and the gels stained with Coomassie Blue G in 30% methanol and 10% acetic acid for 10 min at room temperature on a rotary shaker. Stain was washed out with water until clear bands were seen. Areas where proteolytic activity degraded the gelatin were seen as absence of staining. Identification of each gelatinase band was done in accordance to their molecular weight and commercial standards . These bands (proMMP) were quantified using the BioImaging gel documentation system  endowed with TINA software (Raytest, Staubenhardt, Germany). MMP secretion was expressed as percent of control.In order to detect proteolytic activity in conditioned media (CM) collected after 48–72 h culture, substrate-gel-electrophoresis (Zymography) on gels containing gelatin as the substrate were used as was described by our previous manuscript [5 cells/well in 200 μl medium, while 500 μl medium was added to lower well. 1st trimester trophoblast were cultured in upper wells at a density of 2 × 105 cells/well in 100 μl medium. The same density of cells, in the absence or presence of activators, was seeded in a well without transwell and counted at time of the invasion assay, as reference of total cells. Preliminary studies found no significant matrigel-mediated changes in multiplication rates between 6–8 w and 9–12 w trophoblasts, whether seeded on matrigel or at plastic bottom of well (data not shown). Activators (10 μM Forskolin or 8 ng/ml EGF) and inhibiting MMP-2 or MMP-9 antibodies  were added to medium in upper and lower wells. Plates were incubated at 37°C for 36–48 hours, and then non-invaded cells on top of the transwell were scraped off with a cotton swab. The amount of invaded cells in the lower well as a percent of total seeded cells was evaluated with XTT Reagent kit. The percent of invasion was calculated as:Matrigel invasion assay was prepared in our laboratory with modifications as described in detail elsewhere ,18. BrieInvasion was expressed as Invasion Index (Percent of control).Results are expressed as mean ± SEM of 5–10 independent experiments, each treatment performed in duplicates. Statistical analysis was performed using the SPSS statistical software. Student's t-test and "one way analysis of variance" (ANOVA) were used when appropriate. P < 0.05 was considered significant.Immunohistochemistry was performed as previously described  using th4 cells/well), 1st trimester trophoblast cells 6–8 w and 9–12 w (1–2 × 105 cells/well) were incubated for 48 hours, then media collected and gelatinase secretion analyzed by zymography. Figure JAR cells (1 × 104 cells/well) were incubated 48 hours in the absence or presence of forskolin (1 μM, 10 μM or 100 μM) or EGF (0.8 ng/ml, 8 ng/ml or 80 ng/ml) and media was analyzed by zymography for gelatinase secretion. Fig JAR cells (1 × 104 cells/well), 1st trimester trophoblast cells 6–8 w and 9–12 w (1–2 × 105 cells/well) were incubated for 48 hours in the absence or presence of forskolin 10 μM. Figure JAR cells (1 × 104 cells/well), 1st trimester trophoblast cells 6–8 w and 9–12 w (1–2 × 105 cells/well) were incubated for 48 hours in the absence or presence of EGF 8 ng/ml. Figure JAR cells (1 × 105 cells/well), 1st trimester trophoblast cells 6–8 w and 9–12 w (2 × 105 cells/well) were incubated for 36–48 hours in the absence or presence of forskolin 10 μM on top of Transwell wells containing a transwell membrane coated with matrigel. Forskolin (10 μM) significantly enhanced trophoblast invasion in all cell groups. Forskolin increased cell invasion in JAR cells (110.6% ± 3.4, P < 0.05)   Fig. , in 6–8 05) Fig.  and in 905) Fig. . The add05) Fig.  and 6CThe addition of inhibitory MMP-9 antibody surprisingly increased invasion of control cells of 6–8 w and 9–12 w trophoblast (132.0 ± 8.3 and 134.9 ± 15.2, respectively, P < 0.05) as compared to control, and only in 9–12 w forskolin-induced cells caused a significant decreased invasion compared with induced cells alone (201.5% ± 6.3 versus 302.4% ± 56.0, respectively, P < 0.05). ANOVA post test confirmed the t test results Fig.  and 6C.5 cells/well), 1st trimester trophoblast cells 6–8 w and 9–12 w (2 × 105 cells/well) were incubated for 36–48 hours in the absence or presence of EGF 8 ng/ml on top of Transwell wells containing a transwell membrane coated with matrigel. The results of EGF resembled those of forskolin. EGF enhanced trophoblast invasion in all cell groups. EGF increased cell invasion in JAR cells (112.6% ± 2.9, P < 0.05)   Fig. , in 6–8 05) Fig.  and in 905) Fig. . In EGF-05) Fig.  and 7C.The addition of inhibitory MMP-9 antibody affected 9–12 w EGF–induced cells and caused a significant decreased invasion compared with induced cells alone (169.7% ± 18.1 versus 192.4 ± 10.5, respectively, P < 0.05), ANOVA post test confirmed the t test results Figure.  and 7C.The inhibitory affect of MMP-2/-9 antibody on control cells was described in the previous section.Trophoblastic invasion of the endometrium is highly regulated by interrelated reactions between invasion-promoting factors, such as cytokines, growth factors, MMP-2 and MMP-9, and invasion-inhibiting factors such as TIMPs.In the current study, the secretion and activity of MMP-2 and MMP-9 in human cytotrophoblastic cells from different weeks of gestation was measured and compared with a choriocarcinoma cell-line.st trimester villous tissue, and a dramatically decrease in MMP-2 levels in the second trimester. These results support our findings of a dynamic gelatinase secretion profile during the 1st trimester.A differential secretion profile of proMMP-2 and -9 was found between 6–8 w and 9–12 w as expressed by a shift in the relative proportion of each gelatinase. Elevated proMMP-9 secretion in 9–12 w was observed, compared with both 6–8 w and JAR cells. These results are consistent with those of Xu et al , who fouIn our study, MMP secretion was induced by two separate signal pathways: PKA and PTK. Several factors with importance in embryo implantation act via the cAMP-protein kinase A (PKA) signal transduction pathway, including human chorion gonadotropin (hCG), the primary signal of an implanting pregnancy . Forskolst trimester trophoblast (from both groups). This indicates that forskolin might influence trophoblast cells invasiveness by enhancing the secretion of gelatinases. Zymography measures all forms of MMP (active and inactive) and therefore does not reliably represent the true physiological activity, which is influenced by the presence of MMP inhibitors and activators. In order to examine the role of the gelatinases in the implantation process, we examined cell invasion after PKA stimulation in JAR cells, 6–8 and 9–12 w trophoblasts. Our results showed a significant increase in invasive ability in JAR cells and in early and late 1st trimester trophoblasts, after stimulation with forskolin. In order to detect the contribution of each gelatinase to this invasive process, inhibitory antibodies to MMP-2 and MMP-9 were added to cell culture and cell invasive ability examined. MMP-2 inhibitory antibody caused a significant decrease in cell invasion in JAR cells, 6–8 w and 9–12 w trophoblasts treated with forskolin, whereas MMP-9 inhibitory antibody only caused a decrease in 9–12 w trophoblasts. This indicates, that most probably MMP-2 and not MMP-9 is the key-enzyme in the invasion process of JAR cells and early 1st trimester trophoblasts stimulated by forskolin, whereas MMP-9 together with MMP-2 plays a role in late 1st trimester trophoblasts. Only a few published data describe the relationship between forskolin and trophoblast invasion. Human chorionic gonadotropin acts via cAMP, and is considered a sign of differentiation of trophoblasts to syncytiotrophoblast. The hCG receptor was shown to be expressed on invasive trophoblast and in choriocarcinoma cells, and hCG was found to increase in vitro invasion and migration of a trophoblastic cell line, an effect that was also mimicked by forskolin [In this study, forskolin was found to significantly enhance proMMP-2 secretion in JAR choriocarcinoma cell-line, 6–8 w and 9–12 w trophoblasts. Forskolin also enhanced proMMP-9 secretion, in choriocarcinoma cells and in 1orskolin . Severalorskolin  or inapporskolin  or gestaorskolin . To the st throughout third trimester [Epidermal Growth factor (EGF) plays a major role in placental implantation, growth and differentiation and is regarded a paracrinic factor modifying the implantation process. EGF acts on trophoblasts via a specific receptor (EGFR) from the tyrosine kinase receptor family . EGF is rimester In this study EGF stimulated secretion of proMMP-2 in JAR cells, 6–8 w and 9–12 w trophoblasts and also enhanced the secretion of proMMP-9 in JAR, in 6–8 w trophoblasts and in 9–12 w trophoblasts. Our results therefore indicate, that MMP-2 and MMP-9 secretion by trophoblastic cells may be stimulated through the PTK pathways during the first trimester. We noted that EGF at a high concentration (80 ng/ml), in contrast, decreased proMMP-2 and -9 secretion in JAR cells. This result corresponds with previous published data regarding a dual, concentration dependent effect of EGF on cell functions ,32.st trimester trophoblasts, after stimulation with EGF. In EGF-stimulated JAR cells and in 6–8 w and 9–12 w trophoblasts inhibitory MMP-2 antibody decreased cell invasion, whereas inhibitory MMP-9 antibody caused a significant decrease in invasion only in 9–12 w trophoblasts. We thereby showed, that in EGF stimulated cells as well, MMP-2 is the key-enzyme in the invasion process in vitro in JAR cell and in early 1st trimester trophoblasts, whereas in late 1st trimester trophoblasts both MMP-2 and MMP-9 have a role. EGF was found to induce changes in morphology and to increase invasive capacity of first trimester trophoblasts, whereas later gestational cytotrophoblasts (2nd trimester), whose invasive capacity is diminished, are much less affected [In order to examine the role of the gelatinases in the implantation process, we examined cell invasion after PTK stimulation in JAR cells, 6–8 and 9–12 w trophoblasts. Our results showed a significant increase in invasion ability in JAR cells and in early and late 1affected . EGF wasaffected .st trimester trophoblasts (6–8 w), MMP-2 is the major gelatinase participant in cell invasion, whereas in later 1st trimester trophoblasts (>9 w) both MMP-9 and MMP-2 most probably participate in cell invasion, however we cannot exclude the possibility of other MMP family members participating in this process. Isaka et al [st trimester placenta [in vitro [st trimester, as earlier discussed. The choice of pathway for stimulation of cell invasion may also contribute to a difference in results. We used stimulation through the PKA and PTK pathways; whereas other groups used the PKC pathway [In JAR control cells inhibitory, MMP-2 antibody also decreased invasion, whereas MMP-9 antibody had no affect, indicating that the basic invasive ability of these choriocarcinoma cells is mainly due to MMP-2 and not to MMP-9. Inhibition of MMP-2 in control cells also decreased invasion in 6–8 w trophoblast, but not in 9–12 w, indicating again the importance of MMP-2 in early (6–8 w) trophoblast invasiveness. Surprisingly inhibition of MMP-9 in 6–8 w and 9–12 w trophoblasts without treatment caused an increase in invasion. We speculate that this may be due to a release of other proteinases, including other MMPs, since MMP-9 is known to dimerize ,35 All iplacenta ,36, wherin vitro . We spec pathway ,8. Variost trimester trophoblasts in cell invasive ability and in MMP secretion profile and differ from late 1st trimester trophoblast in these parameters.We found, that MMP-2 is also the key-enzyme in JAR cell invasion; therefore JAR cells resemble early 1It has been documented, that JAR-trophoblast cells have the ability to invade in vivo ,38. We cst trimester. We suggest that MMP-2 is the key-enzyme in JAR and early 1st trimester (6–8 w) trophoblast invasion, whereas both MMP-2 and -9 are important for late (9–12 w) trophoblast invasion.We showed that forskolin and EGF stimulate proMMP-2 and -9 secretion from trophoblasts, and that there is a differential, dynamic importance of each gelatinase in trophoblast invasion during 1


NKX3.1 maps to chromosome band 8p21, which is a region frequently lost in prostate cancer, but not in TGCT. Mutations have not been reported in the NKX3.1 sequence, and the gene is hypothesized to be epigenetically inactivated. In the present study we examined the methylation status of the NKX3.1 promoter in relevant primary tumors and cell lines: primary TGCTs (n = 55), intratubular germ cell neoplasias (n = 7), germ cell tumor cell lines (n = 3), primary prostate adenocarcinomas (n = 20), and prostate cancer cell lines (n = 3) by methylation-specific PCR and bisulphite sequencing.Recent studies have demonstrated that the NKX3.1 protein is commonly down-regulated in testicular germ cell tumors (TGCTs) and prostate carcinomas. The homeobox gene Down-regulation of NKX3.1 expression was generally not caused by promoter hypermethylation, which was only found in one TGCT. However, other epigenetic mechanisms, such as modulation of chromatin structure or modifications of histones, may explain the lack of NKX3.1 expression, which is seen in most TGCTs and prostate cancer specimens. NK3 transcription factor related locus 1 (NKX3.1) is highly specific for the prostate and the testis [NKX3.1 is located in chromosome band 8p21 [Nkx3.1 leads to prostatic epithelial hyperplasia and dysplasia [NKX3.1 suppresses growth and tumorigenicity in human prostate carcinoma cell lines [NKX3.1 have been found [NXK3.1 is therefore believed to be epigenetically inactivated in the cases with loss of protein expression [The protein expression of the homeobox gene e testis ,5. NKX3.and 8p21 ,7, a regand 8p21 . In miceysplasia ,11, and ll lines . Howeverll lines -15. No gen found , and NXKpression . Only onNKX3.1 promoter in TGCTs and prostate adenocarcinomas to examine whether this mechanism may explain the commonly observed loss of NKX3.1 protein.During the last decade, epigenetic changes in cancer have been frequently reported and are now recognized to be at least as common as genetic changes . The besNKX3.1 sequence, and this was the same sample that was positive for methylation from the MSP analysis. Interestingly, all the samples that were sequenced, including the normal blood, unmethylated cell lines, and primary tumors, displayed some extent of methylation (the majority below 25%) at the cytosine in CpG number 21 (base 1914762, +1 bp from transcription start). We detected a possible polymorphism in base 1914730 (+33 bp from transcription start and 15 bp upstream of the coding sequence). In previous sequences this site has been described as a guanine (Gene bank accession number NT_023666, and AF24770). In the cell lines, 5/6 contained adenosine in this position, but all except the germ cell tumor cell lines NCCIT and TERA2 were heterozygotes. In contrast, all 5 primary tumors sequenced were homozygous for the adenosine allele.Only one out of 54 TGCTs and none of the prostate adenocarcinomas (n = 20), intratubular germ cell neoplasias (n = 7), normal testis tissues (n = 4), or the cell lines (n = 6) displayed methylation when analyzed with MSP Figure . Bisulphet al., submitted). This simultaneous down regulation of both protein and mRNA levels of NKX3.1 is consistent with epigenetic regulation, which is further supported by the fact that mutations have not been detected in the NKX3.1 gene [NKX3.1 promoter in TGCT and prostate cancer samples. However, with the exception of a single TGCT, the NKX3.1 promoter was unmethylated in the samples analyzed. The methylated TGCT was classified as a yolk sac tumor, and has also been demonstrated to have promoter hypermethylation of several other genes that are generally unmethylated in TGCTs . We therefore consider this sample not to be representative for the general TGCT epigenotype, nor for the general epigenetic profile of yolk sac tumors. Thus, we do not regard promoter hypermethylation as the general mechanism of NKX3.1 down-regulation neither in TGCT nor in prostate carcinomas.We have previously reported that the protein expression of NKX3.1 is virtually ubiquitously lost in TGCTs . This wa3.1 gene . DNA proNKX3.1, in contrast to PC-3 and DU-145, which do not express NKX3.1 since they lack a functional androgen receptor [NKX3.1 expression in PC-3 and DU-145 cells is not due to methylation. This was also the case with the germ cell tumor cell lines. From Western analysis, the cell line NCCIT had strong expression of NKX3.1 whereas both TERA1 and TERA2 had no expression (data not shown).We also studied cell lines since it can be argued that presence of normal cells as well as tumor heterogeneity may mask cancer specific methylation in primary tumors. LNCaP cells have previously been demonstrated to express receptor . The lacNKX3.1 that we detected 15 bases upstream of the coding sequence has to our knowledge not been described previously. It was identified by bisulphite sequencing of the cell lines and a subgroup of primary tumors, thus caution should be taken when concluding from these results, since regular sequencing analysis is the recommended approach for describing sequence changes. As the polymorphism is located in the promoter region of NKX3.1, it has no influence on the protein structure. However, it can still have a potential role in the transcriptional regulation of NKX3.1. A polymorphism in the coding sequence is also reported for NKX3.1 [The polymorphism in r NKX3.1 .All samples analyzed with bisulphite sequencing, including cell lines expressing NKX3.1, as well as non-expressing cell lines, demonstrated some degree of methylation in the cytosine in CpG number 21. As this site-specific methylation included only one CpG site, it is unlikely that it will have any regulating effect on gene expression. However, considering its intriguing location immediate upstream of the transcription start point, this possibility should not be excluded. There is also the possibility that the apparent methylation could be due to a less efficient bisulphite conversion for this site. In general, the bisulphite sequencing results showed that all cytosines at non-CpG sites were converted to thymine Figure , but seqNKX3.1 promoter is unmethylated, the simultaneous down-regulation of mRNA and protein levels in TGCTs and the absence of mutations still make other epigenetic mechanisms, such as modulation of chromatin structure or modifications of histones, possible explanations for loss of NKX3.1 expression in testicular- and prostate cancers.In summary, these data show that the previously reported down-regulation of NKX3.1 in TGCTs and prostate carcinomas is not caused by promoter hypermethylation. Even though the in situ; n = 7), normal testis tissue (n = 4), germ cell tumor cell lines (TERA1, TERA2, and NCCIT), prostate adenocarcinomas (n = 20), and prostate cancer cell lines (LNCaP, PC-3, and DU-145). The primary TGCTs include all histological subtypes: seminomas, embryonal carcinomas, teratomas, yolk sac tumors, and one choriocarcinoma, classified according to the WHO's recommendations [et al., submitted).Included in the present study are primary TGCTs (n = 55), intratubular germ cell neoplasias . The two fragments were amplified by the Fast Star DNA polymerase (Roche Ltd, Basel, Switzerland) in a reaction containing 1.5 mM Mg2+. We used a 58°C annealing temperature for both primer sets. Bisulphite treated DNA from normal blood (NB) and Sss1 methyltransferase (New England Biolabs Inc., Beverly, MA, USA) in vitro treated placenta DNA  represented the unmethylated positive control and the methylated positive control, respectively. Water, replacing bisulphite treated template, was the negative control in both reactions.The DNA samples were initially bisulphite modified ,25, whicNKX3.1 bisulphite sequence fragment (Gene bank accession number NT_023666 (minus strand), bases 1914526 to 1914961) was 436 bp long and covered 52 CpG sites in the promoter and first exon of the gene. We designed bisulphite sequencing primers (MedProbe) with the following sequences; sense: 5'ATTGGGGAAGGAGAGGGAATTG3', antisense: 5'CCTCTAACTCTAACTCTAACTCC3'. The Mg2+ content of the reaction was 1.3 mM, the enzyme used was HotStarTaq™ DNA polymerase , and the annealing temperature 52°C. The PCR fragments were eluted from a 2% agarose gel (BioRad Laboratories Inc, CA, USA) containing ethidium bromide, by the MinElute™Gel Extraction kit (QIAGEN), and sequenced with the dGTP BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA) in an ABI Prism 377 Sequencer (Applied Biosystems). The bisulphite sequencing results were scored according to Melki et al. where the amount of methylcytosine of each CpG dinucleotide is quantified by comparing the peak height of the cytosine signal with the sum of the cytosine and thymine peak height signals [Bisulphite sequencing allows a positive display of 5-methyl cytosines in the gene promoter after bisulphite modification as unmethylated cytosines appear as thymines, while 5-methylcytosines appear as cytosines in the final sequence . A subse signals .GEL performed the experimental analyses and statistics, interpreted the results, and drafted the manuscript. RIS did an independent scoring of the results, and contributed to manuscript preparation. MFF designed the primers used for the MSP and bisulphite treatment and contributed to manuscript preparation. VMA and RH were reference pathologists for the testicular cancer tissues and prostate tissues, respectively. FS was responsible for the Western Blot studies of the cell lines and participated in the writing of the manuscript. Parts of this work were done in the lab of ME who also contributed with scientific discussions. MRT provided the relevant selected series of primary prostate carcinomas with known genetic profiles, and contributed to manuscript preparation. RAL conceived the study, was responsible for its design and coordination, and contributed in the evaluation of the results and in preparation of the manuscript.


Nothofagus (southern beech), with an 80-million-year-old fossil record, has become iconic as a plant genus whose ancient Gondwanan relationships reach back into the Cretaceous era. Closely associated with Wegener's theory of “Kontinentaldrift”, Nothofagus has been regarded as the “key genus in plant biogeography”. This paradigm has the New Zealand species as passengers on a Moa's Ark that rafted away from other landmasses following the breakup of Gondwana. An alternative explanation for the current transoceanic distribution of species seems almost inconceivable given that Nothofagus seeds are generally thought to be poorly suited for dispersal across large distances or oceans. Here we test the Moa's Ark hypothesis using relaxed molecular clock methods in the analysis of a 7.2-kb fragment of the chloroplast genome. Our analyses provide the first unequivocal molecular clock evidence that, whilst some Nothofagus transoceanic distributions are consistent with vicariance, trans-Tasman Sea distributions can only be explained by long-distance dispersal. Thus, our analyses support the interpretation of an absence of Lophozonia and Fuscospora pollen types in the New Zealand Cretaceous fossil record as evidence for Tertiary dispersals of Nothofagus to New Zealand. Our findings contradict those from recent cladistic analyses of biogeographic data that have concluded transoceanic Nothofagus distributions can only be explained by vicariance events and subsequent extinction. They indicate that the biogeographic history of Nothofagus is more complex than envisaged under opposing polarised views expressed in the ongoing controversy over the relevance of dispersal and vicariance for explaining plant biodiversity. They provide motivation and justification for developing more complex hypotheses that seek to explain the origins of Southern Hemisphere biota. A phylogenetic analysis of Nothofagus species provides evidence for their transoceanic dispersal during the Tertiary, and helps resolve the debate about the origins of plant biodiversity in the Southern Hemisphere Nothofagus species are not suited for dispersal across large distances has played a major role in motivating the hypothesis that transoceanic distributions of Nothofagus  ago  ago . In cont Zealand ,22,23.Nothofagus from Australia to New Zealand. Whilst the extinct “ancestral” Nothofagus pollen type occurred in New Zealand prior to the breakup of Gondwana, Fuscospora pollen first appeared in New Zealand during the Palaeocene (65 Myr ago) and Lophozonia pollen first appeared during the late Eocene . Sixty-Myr ago; ,20. Hencon. Hill  has alsoispersal .trnL–trnF and atpB–psaI; see Nothofagus subgenera (Lophozonia, Fuscospora, and Nothofagus). Our aim has been to date divergence of extant species in the subgenera Lophozonia and Fuscospora. We have carried out relaxed molecular clock analyses using the methods of Sanderson [Nothofagus species, phylogenetic relationships between trans-Tasman species in both Lophozonia and Fuscospora can only be explained by mid- to late-Tertiary transoceanic dispersal.A contribution to the debate over the relative importance of vicariance and dispersal can be made by estimating the divergence times of extant species. However, DNA sequences of insufficient length have prevented robust molecular clock analyses from being undertaken. For this reason, we report the sequencing of a 7.2-kb chloroplast genome fragment covering the gene regions  for Nothofagus (subgenera or pollen groups (a) Lophozonia, (b) Fuscospora, and (c) Nothofagus) and outgroup Castanea sativa (not shown). In a sensitivity analysis of 60 substitution models, the tree shown in 8 model were used for further analyses. The latter was included because the Bayesian relaxed molecular clock (BRMC) approach as implemented in the program MULTIDIVTIME . Constraining these two nodes in this way produced unrealistic age estimates for all basal nodes. For example, using the BRMC method, which additionally required a prior expectation to be specified for the age of the root node , we estimated a more likely age for the root node at 191 Myr. For the PL approach, which does not require specification of a prior, we estimated the age of the root node at 634 Myr. Other basal nodes in both the Fuscospora and Lophozonia lineages were also much older than reasonably expected . Transoceanic dispersal appears the most likely explanation for the trans-Tasman sister relationships indicated in Fuscospora lineages. The age for node L4, which separates Australasian and South American Lophozonia, may also be consistent with vicariance. The BRMC method dates it at 34 Myr before present. However, the PL method estimates this node to be only 25 Myr old, an age too recent to be consistent with vicariance. Thus we regard our results for node L4 as equivocal. Nevertheless, southern beeches are likely to have been present in Antarctica 25 myr ago [Our findings from molecular clock analyses using five independent calibrations (for four nodes), suggest that the sister relationships of the Australasian  species within both  myr ago , and thuLophozonia, Fuscospora, and Nothofagus lineages.The robustness of our phylogenetic inferences has been investigated by varying the substitution model (60 symmetric models were used), estimating the variance of age estimates, and evaluating the influence of calibrations on divergence times. With the exception of the root node, the PL method consistently gave more recent age estimates than did the BRMC method. Both methods showed sensitivity to the number of calibration points used, a finding consistent with recent observations on the performance of relaxed molecular clock methods . In geneNothofagus can be explained by continental drift following the breakup of Gondwana and subsequent extinction of some species [Nothofagus species in New Zealand are not the direct descendants of the Fuscospora and Lophozonia southern beeches that reached New Zealand in the Palaeocene and Eocene eras, respectively [Ascarina and Laurelia in New Zealand [Thus the hypothesis that present-day distribution patterns of  species  can be rectively . This fi Zealand ,4.Nothofagus seeds. For the reasons that we outline in our introduction, it seems likely that only once the mechanisms of long-distance dispersal are understood will hypotheses based on DNA divergence time estimates be truly convincing. DNA sequence analyses have also suggested that long-distance dispersal and continental drift are both important for explaining distributions of the conifer Agathis (Araucariaceae) in the South Pacific [Agathis is not as strong as it is for Nothofagus, the findings from the molecular studies on these genera highlight the importance of considering more complex hypotheses of relationship in debates concerning the relative importance of dispersal and vicariance.The strength of our molecular analyses highlights the importance of future research into potential mechanisms of long-distance dispersal, and in particular reinvestigation of the transoceanic dispersal properties of  Pacific . AlthougatpB–psaI region and the trnL–trnF region) were determined for each of 11 accessions of Nothofagus (subgenera or pollen groups Lophozonia, Fuscospora, and Nothofagus) sampled in South America, Australia, and New Zealand (see C. sativa (an outgroup taxon from Fagaceae) and aligned using progressive multiple-sequence alignment: ClustalX version 1.81 [atpB gene and atpB–rbcL intergene region of Nothofagus.Chloroplast DNA sequences (7.2 kb comprising the land see . These gion 1.81 . This rehttp://darwin.uvigo.es/software/modeltest.html) plus F84, F84+I, F84+Γ8, and F84+I+Γ8. ML parameters of these models were estimated by PAUP* following the approach used in MODELTEST. These parameters were then used to conduct 60 individual ML heuristic searches in PAUP* with tree bisection-reconnection branch swapping and a neighbour-joining starting tree. ML bootstrap proportions were obtained after 100 replications, using the same search strategy and ML parameters as for the analysis of the original dataset.Phylogenetic analyses were conducted with PAUP* version 4.0b10  under thhttp://ginger.ucdavis.edu/r8s/) with the TN algorithm. The outgroup was excluded using the “prune” command. The degree of autocorrelation within lineages was estimated using cross-validation as suggested by Sanderson [8 model for each of the bootstrap resampled datasets using PAUP*. Divergence estimates were then calculated for each of the 100 bootstrap replicates using r8s to obtain standard deviations on each node by the “profile” command and the settings described above.Divergence dates were obtained using the PL method of Sanderson  as impleanderson , and theanderson . The proanderson  was usedhttp://abacus.gene.ucl.ac.uk/software/paml.html) was used to estimate the ML parameters of the F84+ Γ8 substitution model, using the ML topology previously identified. Second, the program ESTBNEW (ftp://abacus.gene.ucl.ac.uk/pub/T3/) was used to estimate branch lengths of the ML topology and the corresponding variance–covariance matrix. Finally, the program MULTIDIVTIME was used to run a Markov chain Monte Carlo for estimating mean posterior divergence times on nodes with associated standard deviations from the variance–covariance matrix produced by ESTBNEW. The Markov chain was sampled 10,000 times every 100 cycles after a burn-in stage of 100,000 cycles. We used a 75-Myr (SD = 37.5 Myr) prior [The BRMC approach was applied using the program MULTIDIVTIME as implemented in the Thornian Time Traveller (T3) package . First, r) prior  for the http://www.ncbi.nlm.nih.gov/) accession numbers for the sequences discussed in this paper are given in The GenBank (


The aims of this study were to determine whether observed geographic variations in breast cancer incidence are random or statistically significant, whether statistically significant excesses are temporary or time-persistent, and whether they can be explained by covariates such as socioeconomic status (SES) or urban/rural status?A purely spatial analysis found fourteen geographic areas that deviated significantly from randomness: ten with higher incidence rates than expected, four lower than expected. After covariate adjustment, three of the ten high areas remained statistically significant and one new high area emerged. The space-time analysis identified eleven geographic areas as statistically significant, seven high and four low. After covariate adjustment, four of the seven high areas remained statistically significant and a fifth high area also identified in the purely spatial analysis emerged.These analyses identify geographic areas with invasive breast cancer incidence higher or lower than expected, the times of their excess, and whether or not their status is affected when the model is adjusted for risk factors. These surveillance findings can be a sound starting point for the epidemiologist and has the potential of monitoring time trends for cancer control activities. This study is an observational epidemiological investigation of breast cancer incidence in Massachusetts. It examines geographic variations over a ten year period using both purely spatial and space-time models to determine whether observed fluctuations in incidence rates are random or whether fluctuations represent statistically significant deviations from randomness. This study examines whether apparent excesses are stable over time, or are temporary, and also determines whether excesses, high or low, can be accounted for by risk factors such as socioeconomic status (SES) or urban/rural status. This study demonstrates how surveillance data can be analyzed to identify those geographic areas that warrant closer attention, the basis for determining the need for public health action or to aid in assessing the effectiveness of intervention programs .Massachusetts has been included in studies of inter-region and intra-region variability of breast cancer incidence and mortality in the United States. Several of these studies aggregated breast cancer mortality data to the regional or county level -8. LadenA recent study on the geographic distribution of the proportion of late-stage breast cancer cases diagnosed in Massachusetts females between 1982 and 1986 aggregated cases to town, ZIP Code, and census tract levels . The towThe principal components analysis performed on the seven SES variables revealed two components. The two components accounted for about 80% of the variance among the seven economic measures with the loadings of each variable on the two components shown in Table Component scores for wealth and poverty were calculated for each tract. The scores were divided into quintiles and used in a Poisson regression to determine their capacity to predict breast cancer incidence. SES variables are scaled so that high wealth scores represent the most wealth and high poverty scores represent the most poverty.The initial Poisson regression of the wealth component showed that category 5, the quintile of the most wealth, had a higher associated risk for breast cancer. There was no increasing or decreasing trend when the fifth category was compared to the other four categories, but they were all lower than the fifth, so categories 1 to 4 were collapsed to create a dichotomous wealth variable. A Poisson regression showed that the highest wealth category had an 8.9% increase in incidence over the combined other categories.The analysis also revealed that breast cancer incidence was inversely related to poverty level, as shown in Table The purely spatial analysis ignores the time of diagnosis. Figure Four areas had significantly fewer cases than expected for the ten year period. Low A had 1068 observed cases and a RR of 0.74, indicating about 26% fewer cases than expected. Low B is west of Cape Cod with 521 observed cases and a RR of 0.77. The other two low areas, "C" and "D," in western Massachusetts had 11% and 19% fewer cases than expected, respectively.The remainder of Table When the purely spatial model is adjusted for the two SES variables representing wealth and poverty, seven of the high areas are no longer significant. High areas "3," "6" and "7" remain significant. High 6 has expanded to include more tracts and more cases but the RR is about the same. High areas "2" and "10" have merged into a larger area, High 11. Low A covers fewer tracts compared to the age-adjusted analysis, while Low B includes more tracts. A low area, labeled "F" in Table As compared to the analysis adjusting for SES alone, when the two SES variables and urban/rural status are all included in the purely spatial model, the results show little change, except that High 11 is slightly larger in geographic area and low areas "C" and "F" are no longer significant. Figure The results of the space-time age-adjusted analysis are somewhat similar to the purely spatial analysis in that seven of the ten areas of excess incidence from the purely spatial analysis were also statistically significantly high in the space-time analysis, along with all four of the low areas. However, as shown in the left side of Table Figure The two high areas, "4" and "5", around Boston that were statistically significant in the purely spatial analysis are also statistically significantly high for the entire study period. High 1 was elevated 16% from 1991 to 1997. In northeast Massachusetts, High 6 is statistically significant from 1992 to 1995 with a RR of 1.35. Another high area, High 3, from the purely spatial analysis is also statistically significant in the space-time analysis and represents a single tract in western MA. It is significant from 1989 to 1996 with 51 cases diagnosed and a RR of 3.05. High 9 from the purely spatial analysis was not significant in the age-adjusted space-time analysis. High 11, a large area in the southeast, includes most of High 2 and High 10 from the purely spatial analysis plus some additional tracts that had not been statistically significant before.Low A in Boston is statistically significant for the entire 10-year period. Low B, in southeastern MA, and Low C, in western MA, remain statistically significantly low from 1988 to 1994, and 1988 to 1996, respectively. Low D, east of Low C, is also statistically significantly low from 1988 to 1995.Adjusting the space-time analysis for urban/rural status has little effect, except to change the RR and p-values slightly. When the model includes both the wealth and poverty components as covariates, five areas are found to be excessively high, and one of these, High 7, is new to the space-time analyses. This small area had been identified in the purely spatial analysis and remained statistically significant after adjustment for SES. Low D is no longer statistically significant.When adjusting for SES and urban/rural status together, all the high and low areas from the SES and age-analysis, except for Low C, remain significant. These results are displayed in Figure We examined the possibility of a geocoding bias. Of 46,333 total cases, 4440 were randomly assigned to a census tract located within the reported town. These cases could not be assigned directly to a census tract because their records did not have a residential address. Cancer registries like the MCR collect a patient's usual residence at the time of diagnosis. For patients having only postal box addresses, if the MCR staff were unable to obtain a residential address at the time of diagnosis, we assigned these cases to tracts within the town of the mailing addresses, as described in Methods below. This should not be problematic in larger areas of excess or deficit since error would be confined to a town. In the smaller areas identified by the analyses, all had higher than expected case numbers. We examined the smaller areas of excess to determine how many of the observed cases had been randomly placed into a tract. To estimate how the RRs would be affected, the RRs were re-computed from the purely spatial age-adjusted analysis, while excluding these cases. High areas "2" and "3" did not contain any cases assigned to the tract randomly. In High 4, eight of the 126 cases observed were assigned at random; without these cases, the RR would be 1.63 rather than 1.75. High 5 had 31 of the 276 observed cases assigned randomly; without these cases, the RR would be 1.28 rather than 1.44. Thus, even if none of the randomly assigned cases were assigned to these smaller areas, the relative risks would not be altered substantially. However, MCR has studied High 7 and determined that several large apartment complexes were geocoded as being part of the census tract which makes up High 7 instead of placing it in the correct neighboring tract. Since High 7 does not have many females as part of the population, these geocoding errors may have made a large difference and therefore, this tract is not likely to have high breast cancer incidence.This study adjusted for age, SES, and urban/rural status. Other known risk factors could possibly explain the high areas uncovered. The following attributable risk percentages for such factors have been reported in the literature: 10.7% due to high alcohol intake, 15.0% due to low beta-carotene intake, 8.6% due to low vitamin E intake, 11.6% due to low levels of physical activity , 29.5% dThis study used aggregated data to compensate for the lack of individual level data and is therefore exposed to the ecological fallacy. Krieger has shown that this is not a serious problem . Also, iThe current study is part of the surveillance process, an observational epidemiologic investigation to provide reliable statistical modeling of the raw surveillance data so that program evaluation or planning can be focused on those variations in incidence rates with a low likelihood of being random. In the current study it is especially interesting to observe whether geographical areas, high or low, are affected by SES adjustment or not. It is also interesting to observe which geographical areas seem to be high or low for the entire study period, and which are high or low on a temporary basis.The first step when studying these high and low areas is to look closely at the geocoded data in these areas to ensure that there were not any geocoding-related errors. An example of a geocoding-related error was High 7. However, this problem should only be an issue in areas that only contain a small number of tracts. Roche et al. have made other suggestions as to what can be done when these areas are found to be truly elevated .Several areas (Highs "1," "4," "5," and "9" and Low C) are no longer significant with the adjustment for SES and urban/rural status. What is it about the high SES in these areas that affects the RR of these areas? It could be that late age at first birth has a substantial contribution since women of high SES delay childbearing to establish their careers. Note that the p-values and RRs are not correlated and both need to be studied. For ease of comparing statistics, refer to Tables High areas "3" and "6," as well as Low areas "A" and "B" do not change a great deal with SES adjustment, so these covariates are not having the same effects as they had above. Perhaps a case-control study could be designed on these areas to determine what other risk factors are driving this elevated incidence rate, such as genetic or behavioral factors.High 6 remained unaffected by adjustment for SES and urban/rural status. However, since it was only temporarily elevated for 4 years, perhaps this area should also be monitored to see if its incidence rate significantly elevates. However, High 11 should be investigated for other factors that may be elevating the risk since it remained elevated for 5 years near the end of the study period.The low areas should be assessed since cases might be going undetected if sufficient screening programs are not in place. If screening programs are in place, they may need to be altered and improved.Finally, it is possible for the populations in two different areas to experience the same amounts of cancer; yet if some cancers remain undetected in one area and are diagnosed in the second area, the second area will appear to have a higher incidence. This is why when breast cancer screening is aggressively promoted in an area with worrisome incidence, the cancer rate goes up for a period of time. Furthermore, not all areas have equal access to the latest in diagnostic equipment capable of detecting the most minute cancers or even pre-cancerous cells. A patient's cancer might be diagnosed if she went to a facility with the latest diagnostic equipment, but might not be diagnosed if she had gone to a different place. Incidence may also reflect not just differences in diagnosis rates, but also differences in treatment patterns. If women or physicians in one area tend to opt for more lumpectomies without radiation, the patients still have a chance to develop later cancers in that breast; if women or physicians in another area tend to opt for lumpectomies with radiation or mastectomies for the same stage of disease, they will not be having any subsequent cancers in that breast .Cases are from the Massachusetts Cancer Registry (MCR), and include all invasive breast cancer cases diagnosed in female residents between 1988 and 1997 (n = 46,333), using the standard definition of invasive breast cancer. Annual Census tracts were used to aggregate cases because they are more uniform than towns or Zip Codes in population size, provide a more sensitive analysis of densely populated areas, and are more homogeneous in their resident characteristics. However, 12.6% (n = 5832) of the cases diagnosed in 1988–1997 could not be assigned a reliable residential census tract because of inaccuracies or omissions in the address information provided to the MCR. In most of these cases, a mailing address had been provided and, even after extensive research, MCR staff could not assign a reliable residential address for the patient at the time of diagnosis. To assign the unassigned cases to census tracts, we compared town and census tract boundaries for 1196 four-digit census tracts. About 90 of the census tract codes included 2-digit suffixes to designate 2, 3, 4, or 5 distinct tracts. In this study, only the first four digits of such tract codes were used, thus combining some separate tracts into one. For example, census tracts 6002.01 and 6002.02 were combined into the 4-digit tract 6002. 1990 Census block numbering areas in Massachusetts are treated here as if they were census tracts. Nearly all of the 1196 four-digit 1990 tracts could be related to town boundaries, with one or more tracts located entirely within a town, or one or more towns entirely within a census tract. Two census tracts overlapped parts of multiple towns and for these it was possible to estimate the proportion of the total tract population living in each town. Unassigned cases were then assigned as follows: for towns completely contained in one tract, cases were assigned to that tract, accounting for 1392 cases. For a town containing two or more census tracts, cases were randomly assigned to tracts proportionate to the town's female population, accounting for 4440 cases. The allocation of cases should therefore be free from systematic error and any error should be localized to a particular town, while the broader state patterns remain correct.The spatial scan statistic  was usedProperties that make the spatial scan statistic suitable for geographic surveillance are: 1) it takes into account the uneven geographic distribution of cases and population densities, 2) it does not make assumptions about cluster size or location, 3) it adjusts for multiple testing – a common problem in testing multiple combinations of cluster locations and sizes, 4) it identifies the spatial or space-time locations where the null hypothesis is rejected, and 5) it can detect multiple clusters .Purely spatial analyses were performed first, ignoring time. The maximum spatial cluster size was first set to include up to 50% of the population for both excesses and deficits and then set at 10%, to test for excesses and deficits separately because testing at the 10% level can identify smaller, more defined areas. However, each area identified in an analysis had a likelihood associated with it that was compared to the 9,999 likelihoods from the initial 50% maximum spatial size test, thus accounting the inference for a multiplicity of statistical tests. Space-time analyses were then performed to determine whether the clusters from the purely spatial analysis were long term or temporary. The maximum temporal cluster size was set at 90% and also included purely spatial clusters with a temporal size of 100% for all space-time analyses. This allows for the entire time period all the way down to the smallest amount of time to be considered as the time window, ten years down to one year.All SaTScan analyses were performed on age-adjusted population counts. To calculate the age-adjusted expected counts, the 1990 and 2000 female population counts were combined into a weighted average of the two based on the years being analyzed, 1988 through 1997. This was done for each age group within each tract. The natural log of this weighted average was entered as the offset variable in a Poisson regression in SAS . There wAfter identifying statistically significant geographic areas and determining whether they were long term or temporary, the next step was to determine whether these areas would change when the model was adjusted for known risk factors. Socioeconomic factors have long been identified as risk factors for breast cancer ,24,25. UAn SES index was created following the method of Yost et al . Yost anA variable indicating the urban or rural status of each tract was derived from data available on the Massachusetts Institute for Social and Economic Research (MISER) . An urbaSaTScan  created TJS: PI, responsible for design, funding, of project with overall responsibility for implementing the project, including the final paper. LMD: Principal data analysis, responsible for final checks on accuracy of data and all analyses, including their written interpretation. MK: Co-PI, assisted in the overall design of the project and close collaborator on all analyses. MK also assisted a great deal in editorial decisions of drafts and of the final paper. DG: Co-PI, assisted in the design of the project, including funding and implementation. DG also consulted on editorial and technical issues. SG: Director of MCR, she was responsible for the overall integrity of the data, and ensuring that use of the data corresponded to the policies of the Massachusetts Dept of Public Health. She also assisted with editorial help on all drafts. MM is responsible for the geocoding of the data in the MCR. She was a constant help to this project as we tried multiple approaches to our analyses. MM was also very helpful in clarifying limitations of the work and also provided many editorial suggestions. All authors read and approved the final manuscript.


Chronic pain is a common and frequently disabling problem in older adults. Clinical guidelines emphasize the need to use multimodal therapies to manage persistent pain in this population. Pain self-management training is a multimodal therapy that has been found to be effective in young to middle-aged adult samples. This training includes education about pain as well as instruction and practice in several management techniques, including relaxation, physical exercise, modification of negative thoughts, and goal setting. Few studies have examined the effectiveness of this therapy in older adult samples.This is a randomized, controlled trial to assess the effectiveness of a pain self-management training group intervention, as compared with an education-only control condition. Participants are recruited from retirement communities in the Pacific Northwest of the United States and must be 65 years or older and experience persistent, noncancer pain that limits their activities. The primary outcome is physical disability, as measured by the Roland-Morris Disability Questionnaire. Secondary outcomes are depression , pain intensity (Brief Pain Inventory), and pain-related interference with activities (Brief Pain Inventory). Randomization occurs by facility to minimize cross-contamination between groups. The target sample size is 273 enrolled, which assuming a 20% attrition rate at 12 months, will provide us with 84% power to detect a moderate effect size of .50 for the primary outcome.Few studies have investigated the effects of multimodal pain self-management training among older adults. This randomized controlled trial is designed to assess the efficacy of a pain self-management program that incorporates physical and psychosocial pain coping skills among adults in the mid-old to old-old range. Chronic pain is a common problem in the elderly, and is often associated with significant physical disability and psychosocial problems . EstimatAs the United States population grows older, the public health problem of chronic pain and its sequelae will worsen. Projections show dramatic increases in this age group; approximately 25% of the population will be age 65 years or older in 2050. Moreover, by 2030 there will be an estimated 8 million people who are 85 years or older [There is substantial empirical evidence that attention to cognitive and behavioral factors, in addition to physiological factors, is necessary for the successful treatment of chronic nonmalignant pain ,5. EmpirDespite their documented efficacy in young to middle-aged samples -12, cognKeefe and colleagues  evaluateOne study that examined a cognitive-behavioral therapy in old-old adults (mean age 77.2 years) evaluated the efficacy of a 10-week CBT intervention (n = 11) versus an attention/support (AS) condition (n = 10) for nursing home residents . The CBTAs the U.S. population continues to age, retirement communities have gained popularity. A retirement community allows older adults with varying lifestyles and physical abilities to live in an environment that encourages independence while providing needed access to health and social resources . AlthougThe primary goal of this study is to evaluate the efficacy of a pain self-management group intervention (SMG), as compared with a control condition (BOOK), in decreasing physical disability, pain, pain-related interference with activities, and depression in older retirement community residents with chronic pain. In addition, we wish to determine the extent to which SMG participation is associated with changes in specific pain-related beliefs and coping strategies, and the extent to which changes in these process variables are associated with changes in outcomes . We plan to test the following hypotheses:1. At post-treatment and each follow-up, participants assigned to SMG, as compared with participants assigned to BOOK, will report less physical disability (primary outcome), and lower pain intensity, pain-related interference with activities, and depressive symptom severity (secondary outcomes).2. Participants assigned to SMG, as compared with participants assigned to BOOK, will show greater pre- to post-treatment increases in self-efficacy and use of adaptive pain coping strategies and greater decreases in catastrophizing. Significant differences between SMG and BOOK groups in pain-related beliefs and coping strategies will be maintained at 6-month and 1-year follow-ups.3. Pre- to post-treatment changes in specific pain-related beliefs (catastrophizing, self-efficacy) and coping strategies  will be associated significantly with changes in physical and social functioning, pain intensity, and depression over the same period among SMG participants. These changes in beliefs and coping strategies will be maintained at 6-month and 1-year follow-ups.Figure This is a currently ongoing randomized controlled trial. The study procedures and measures have been approved by the Swedish Medical Center institutional review board. Figure Participants (targeted enrollment n = 273) are recruited from residents living in one of the 34 participating retirement communities in Seattle, Washington and the surrounding area. Study inclusion criteria are: (1) 65 years of age or older, (2) pain > 3 months duration that interferes with regular activities; and (3) ability to read and complete study questionnaires in English. Exclusion criteria are: (1) current, active cancer and (2) surgery within the past 6 months or surgery planned in the next 6 months.Participants are recruited using newsletter announcements, flyers, brochures, and informational talks given at each facility. Retirement community residents who are interested in the study are screened to assess eligibility. Eligible residents who provide written informed consent are then asked to complete the baseline measures and to provide the name of their primary care provider (PCP), as well as permission to contact the PCP. PCPs of SMG group participants are sent a letter about the study and asked to contact the research nurse if there is any medical reason to restrict the resident's participation in the exercise portion of the study.After all participants from a facility have completed the baseline questionnaires, the facility is randomized to receive either the BOOK or the SMG. Randomization is done by facility, rather than by individual participant, for several reasons. First, it expands the number of participating facilities by making feasible recruitment from smaller facilities. If approximately 5% of residents were recruited from any one facility, then it would not be scientifically or financially sound to involve facilities with fewer than 200 residents in independent or assisted living. A pilot study indicated that the ideal self-management group size is 5–12 participants. If we randomized within facilities, at least 10 participants would need to be recruited from each facility to allow 5 SMG participants. However, if all participants within a facility are randomized to the same condition, then smaller facilities can participate. If more than 12 residents in a facility randomized to the SMG condition enroll in the study, more than one group is scheduled. A second advantage of randomization by facility is that there is little risk of participants in one condition talking with participants randomized to another condition about their experiences in the study. Thus, there is less treatment contamination and likelihood that participants from different conditions will compare treatments, a situation that can provoke dissatisfaction among participants who do not receive the treatment of their choice.® tubing for the performance of selected exercises, and two hot/cold gel packs.The SMG intervention, which consists of seven weekly 90-minute group sessions, includes the major components of empirically-supported self-management interventions ,24, refiA key component of this self-management group is the development of personalized pain management plans. Participants begin developing a plan during the first class and revise it each week as they learn and practice additional pain management skills. With the assistance of the facilitator and, at times, other group members, participants review pain control strategies that they have learned and practiced and choose one or several strategies that best meet their individual needs and interests. Participants identify specifically what they will do (in measurable terms) and define the parameters . Although each person develops his or her own plan, the plans incorporate the same repertoire of activities that are taught in the class. These plans are monitored weekly during the classes and during follow-up phone calls (described below).The Chronic Pain Workbook, 2ndEdition [Participants who are assigned to the BOOK condition receive a copy of dEdition . FacilitThe SMG group facilitator telephones each participant at 12, 16, 22, and 30 weeks after the final group session. During the booster phone calls, facilitators inquire about pain and functioning, current pain management plans, and successes and obstacles in meeting pain management goals, as well as provide encouragement and assistance in problem-solving obstacles encountered in pain management. BOOK participants receive follow-up phone calls at the same intervals to control for attention.Group facilitators are nurses and psychologists with expertise in geriatrics and/or pain management and experience in facilitating therapeutic groups. All are specifically trained according to the treatment protocol. We monitor group facilitator adherence to the self-management group protocol, as recommended by Waltz et al. . All facStudy measures were chosen based on psychometric properties, including sensitivity to change; brevity; and appropriateness for use with community-dwelling, older adults with chronic pain. They are described below and summarized in Table The following measures are administered at baseline to describe the sample and to explore whether these variables are associated with treatment response. We will compare the two study groups on these measures to determine whether they are comparable at baseline.During the screening process and baseline assessment, participants are asked a series of questions to elicit demographic and pain history variables, including age, race, ethnicity, gender, marital status, education level, sites and duration of pain, and prior and current pain treatments.The MMSE is a measure that is widely used to assess cognitive function, particularly in older adults. It consists of 30 items, and requires 5–10 minutes to administer. Items assess orientation, memory, attention, and calculation. The MMSE has been demonstrated to be valid and to have good test-retest reliability .The CI is an extensively used, valid, and reliable measure of comorbid medical conditions . The CI Participants complete the 8-item version of Lorig et al.'s Self- Efficacy Scale , which aThe CSQ is one of the most widely used measures of pain coping and catastrophizing ,39. MeasThe CPCI measures cognitive and behavioral coping strategies used by people to manage chronic pain. It contains 9 subscales: guarding, resting, asking for assistance, relaxation, task persistence, exercise/stretch, seeking support, coping self-statements, and medication use . The CPCPrior to learning the study condition to which they are randomized, participants are asked the degree to which they believe that each study condition will be helpful to them. They respond using a 0 to 10 scale, with 0 indicating "not helpful at all" and 10 indicating "extremely helpful."1. Class attendance. Group leaders document weekly class attendance. Total attendance is assessed as a percentage of classes the participant attended (out of 7).2. Reading log/usefulness. Both BOOK and SMG participants complete a form in which they report the amount read on each topic using a 0–5 scale . They also rate the usefulness of each section using a 0–5 scale .3. Goal attainment. Attainment of SMG participants' pain management goals is assessed using the Personal Pain Management Plan (PPMP). Each week, participants in the SMG group are asked to document the type and frequency of each activity they have chosen to utilize in the management of their chronic pain. They monitor and document the pain management activities that they actually performed over the week. Participants also document obstacles that they have encountered in trying to meet their goals and the solutions they have identified to overcome those obstacles. This form is printed on 2-page paper. The top copy is turned in each week and participants keep the bottom copy for their own records. The PPMP serves several purposes: 1) to assist participants to identify and follow through on their personalized goals; 2) to assess treatment adherence; and 3) to cross-validate data that are collected using the Chronic Pain Coping Inventory.Roland-Morris disability questionnaire (RMDQ): The RMDQ [The RMDQ  is widelThe RMDQ -55. AlthThe RMDQ . The RMDBrief pain inventory (BPI): The BPI is a widely-used, reliable, valid instrument that assesses pain history, location, intensity, and activity interference [rference ,57. For rference .Pain-related interference is a composite measure of the degree to which pain limits a person's general function . This vaGeriatric depression scale (GDS): The GDS [ The GDS  is a 30- The GDS ,61. The A mixed effects model will be used to analyze data using the participant as the unit of analysis and controlling for baseline value of the outcome as a covariate. A reasonably accurate approximation to this analysis could be obtained by the following procedure: first compute change scores (pre to post) for each person, then collapse to get the mean change score within each site, then do t-tests on these means. This simpler model was used for power calculations, since it allows standard software to be used.intra-class correlation. Effect size is defined as the mean change score of all individuals in the intervention group minus the mean change score of all individuals in the control group, divided by the standard deviation of change score within groups. Power calculations for the proposed study are based on estimates of 34 sites (17 intervention and 17 control), 6.4 participants per site (N = 218) providing data at 12 months (20% attrition rate).The plan to randomize by site, rather than by individual participant, required additional considerations in calculating statistical power. With this group-randomized design, power depends on the correlation of people within sites, or the Table site and person nested within site. Group (i.e., treatment or control) and Time will be fixed effects. The repeated measurements of physical disability at post-intervention, 6 months, and 1 year will be the outcome measure. The baseline value of physical disability will be included in the model as a covariate. Any baseline variables that are correlated with the outcome variable and/or differ between the two treatment groups (e.g., gender, age, comorbidity) will also be included as covariates in the analysis. If the main effect for group is significant, contrasts within this model will be used to test for treatment effect separately at each of the three outcome times. Secondary analyses will be similar, fitting a mixed effects model that uses one of the secondary outcomes (e.g., pain intensity, pain-related interference, and depression) in place of physical disability. The analysis of Hypothesis 1 will be by intent-to-treat.The test of hypothesis 1 compares the SMG and BOOK participants on the primary outcome  and secondary outcomes (pain intensity, pain-related interference with activities, and depressive symptom severity) at each of the 3 follow-up assessments. The analytic method that we will use to evaluate this hypothesis is the mixed effects analysis of covariance (ANCOVA), as proposed by Laird and Ware  and implHypothesis 2 involves comparing the SMG and BOOK groups on changes in process variables (pain-related beliefs and coping strategies). A mixed effects model, as described under hypothesis 1, will be used for these analyses. As for hypothesis 1, baseline variables that are predictive of outcome and/or differ between groups will be included as covariates in the analyses for hypothesis 2.Hypothesis 3 involves the correlation of changes in beliefs and coping to changes in the outcome variables. For each assessment time, change from baseline will be computed and scatter plots will be used to describe relationships, with Pearson and/or Spearman correlation coefficients used to summarize the strength of the association. Although we hypothesize that significant associations in changes will occur only in the SMG group, we also will perform exploratory analyses in the BOOK group to assess for these associations.In addition to performing major analyses to test study hypotheses, we will also perform exploratory analyses to examine whether there are subgroups of participants in whom the intervention had a particularly strong or a particularly weak effect. For example, we will explore whether: (1) there is a difference in response to therapy based on age group (young-old, mid-old, old-old); (2) men respond differently to therapy than women; and (3) pain severity at baseline is related to strength of treatment effect. These analyses will be performed using the ANCOVA described above, augmented by adding, for example, an indicator for female gender and the interaction term between gender and treatment group.Persistent pain is a common problem in older adults that can be debilitating. Self-management strategies that incorporate physical and psychosocial pain coping skills are effective in decreasing pain and improving function and mood in younger adults. Little is known, however, about the efficacy of this therapy for older adults, especially those in the mid-old to old-old range. Our randomized controlled trial assesses the efficacy of such a treatment program, as compared with a control condition, in decreasing pain and improving physical and psychosocial functioning in elderly retirement community residents with chronic pain.None declared.ME and JAT developed the intervention and conducted the pilot test of the self-management groups. KCC developed the analysis plan, conducted the power calculations, and wrote the related sections of the paper. CAK assisted in refining the intervention. ME and JAT wrote the initial description of the intervention and this article. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The assessment of tumor invasion of underlying benign stroma in neoplastic squamous proliferation of the larynx may pose a diagnostic challenge, particularly in small biopsy specimens that are frequently tangentially sectioned. We studied whether thresholds of an eosinophilic response to laryngeal squamous neoplasms provides an adjunctive histologic criterion for determining the presence of invasion.Eighty-seven(n = 87) cases of invasive squamous cell carcinoma and preinvasive squamous neoplasia were evaluated. In each case, the number of eosinophils per high power field(eosinophils/hpf), and per 10 hpf in the tissue adjacent to the neoplastic epithelium, were counted and tabulated. For statistical purposes, the elevated eosinophils were defined and categorized as: focally and moderately elevated (5–9 eos/hpf), focally and markedly increased(>10/hpf), diffusely and moderately elevated(5–19 eos/10hpf), and diffusely and markedly increased (>20/10hpf).In the invasive carcinoma, eosinophil counts were elevated focally and /or diffusely, more frequently seen than in non-invasive neoplastic lesions. The increased eosinophil counts, specifically >10hpf, and >20/10hpf, were all statistically significantly associated with stromal invasion. Greater than 10 eosinophils/hpf and/or >20 eosinophils/10hpf had highest predictive power, with a sensitivity, specificity and positive predictive value of 82%, 93%, 96% and 80%, 100% and 100%, respectively. Virtually, greater than 20 eosinophils/10 hpf was diagnostic for tumor invasion in our series.Our study suggests for the first time that the elevated eosinophil count in squamous neoplasia of the larynx is a morphologic feature associated with tumor invasion. When the number of infiltrating eosinophils exceeds 10/hpf and or >20/10 hpf in a laryngeal biopsy with squamous neoplasia, it represents an indicator for the possibility of tumor invasion. Similarly, the presence of eosinophils meeting these thresholds in an excisional specimen should prompt a thorough evaluation for invasiveness, when evidence of invasion is absent, or when invasion is suspected by conventional criteria in the initial sections. Invasive squamous cell carcinoma(SC) is the most common malignancy of the larynx. Distinget al recently reported that the presence of eosinophils is associated with invasion in the neoplastic squamous lesions in the female genital tract, and proposed that eosinophilia provided as adjunctive morphologic feature in identifying SC in the cervix and vulva[A moderate to marked stromal eosinophils that may infiltrate into the neoplastic epithelium has occasionally been reported in invasive carcinoma -4. Spiegand vulva,6. One oand vulva.The biopsy and resection specimens with available H&E stained slides of laryngeal SC and SCIS diagnosed at Roswell Park Cancer Institute from 1993 through 2000 were reviewed by two of the authors simultaneously (MZ and DT). Cases with prior radiation and/ or chemotherapy were excluded. All histology specimens at Roswell Park Cancer Institute were fixed in 10% formalin. Paraffin blocks of 5 μm thickness were cut and the sections were stained with conventional hematoxylin and eosin. For each biopsy and resection specimen, the original diagnosis was recorded and compared with the review diagnosis. Cases with any degree of invasion including "minimal invasion" or "microscopic invasion" in either a biopsy or resection specimen or both were classified as SC. Cases with SCIS only in a biopsy specimen that subsequently had invasion in the resection specimen were classified as SC. On the other hand, a resection specimen lacking invasion was required for a case to be classified as SCIS. For each of specimen, the high-power field(hpf) (Olympus BH2 ×10 ocular and ×40 objective lens) with a maximum number of eosinophils was identified and recorded as eos/hpf. Then, the eosinophils in the adjacent nine contiguous hpf were counted, added to those in the first, and recorded as eos/10hpf. Only nucleated cells with intensely red cytoplasmic granules were accepted as eosinophils, and care was taken to exclude red blood cells with superimposed mononuclear and polymorphonuclear inflammatory cells. And those that were confined to lymphovascular spaces were excluded. During the course of the study, it was noted that frozen section preparation results in the degranulation of eosinophils and leads to difficulty in their being recognized; however, under these conditions, collections extracytoplasmic typical red granules approximately the expected size of an eosinophil allows for their identification. As an internal control, non-neoplastic portions of the specimens, whenever available, were also evaluated.Frequency was computed for each eosinophil category in invasive and non-invasive squamous neoplasia specimens obtained from biopsy and excision. Chi-square test was utilized to examine the difference of frequency distribution between each elevated eosinophil category and the referent eosinophil category (0–4 eos/10hpf). This analysis was conducted independently for specimens obtained from biopsy and excision. Sensitivity, specificity, positive predictive value and negative predictive value were computed for eosinophil counts that were exceeding 10 eos/hpf or 20 eos/10hpf to evaluate if these two classifications of eosinophil count would suggest any significance of clinical implication from statistical point of view. P value less than 0.05 was used to determine statistical significance. Elevated eosinophils were defined and categorized as: focally and moderately elevated (5–9 eos/hpf), focally and markedly increased(>10/hpf), diffusely and moderately elevated(5–19 eos/10hpf), and diffusely and markedly increased (>20/10hpf), while 0–4 eosinophils/10hpf was used as a baseline. The recorded eosinophil counts were analyzed to determine whether there were thresholds of stromal versus neoplastic squamous eosinophils per 1 hpf and 10 hpf that were significantly associated with invasive tumor in both biopsy and follow-up excisional/resectional specimens.A total of 87 cases were evaluated and sixty-eight percent of the cases (n = 59) displayed a chronic inflammation in the stroma. Fifty-seven were biopsy specimens and 30 were ablative resection specimens. The diagnoses of 57 biopsy specimens were 35 invasive carcinoma, 4 minimal invasive carcinoma, 2 suspicious for invasion, and 16 were preinvasive squamous cell neoplasia. 27 of the biopsy specimens  were followed by ablative resections . The follow-up specimens confirmed 19 invasive carcinoma and six preinvasive carcinoma, and revealed an invasive carcinoma in the suspicious case. In this case, there were stromal nests of neoplastic squamous cells that were associated with 15 and 34 eosinophils per 1 and 10 hpf, respectively, and clinical image studied revealed an advanced disease (stage IIIa). Additionally, Other 3 ablative resection specimens  were initial operations. There were 2 invasive carcinoma and 1 preinvasive lesion.The distribution of eosinophil counts is summarized in Table There is no association of elevated tissue eosinophils with overall inflammatory response of the stroma in the specimens studied (p < 0.05). Specifically, among the 37 cases with > = 10 eosinophils/hpf, 24 cases displayed a non-specific inflammation, while 50 cases with <10 eosinophils/hpf, 35 displayed a non-specific inflammation. Among the 36 cases with > = 20 eosinophils/10hpf, 24 cases revealed a non-specific inflammation, while 51 cases with <20 eosinophils/10hpf, 34 revealed a non-specific inflammation. In fact, some invasive carcinomas (n = 6) virtually contained no chronic inflammatory background, but showed a marked elevated tissue eosinophila Fig. .The predictive values of tissue eosinophils in assessing stromal invasion in squamous neoplastic lesions of larynx are presented in Table Sections with adequate non neoplastic epithelium were available in twelve cases. Eight of them were absent for eosinophils. The highest counts for non-neoplastic epithelium were 4 eosinophils/hpf and 8 eosinophils/10hpf. Although it seems that non-neoplastic epithelium contains less eosinophils than squamous neoplasia, no statistical analyses were performed since the number of available non-neoplastic regions in this series was too small.For decades, pathologists have used a variety of histologic features, including desmoplastic stromal reaction, intrastromal foreign body reaction to keratin, and the presence of separate minute clusters of intrastromal neoplastic cells, to assess and identify invasion,9. HowevIn this series, a systematic study of eosinophils in tissues of squamous neoplasia of larynx suggests that elevated eosinophils are a morphologic marker for assessing tumor invasiveness. We observed that in the invasive squamous carcinomas eosinophils were significantly elevated focally and /or diffusely, statistically more frequent than in non-invasive neoplasia. The increased eosinophil counts (>10 hpf, and >20/10 hpf) in laryngeal biopsy and excisional specimens were all statistically significantly associated with stromal invasion. In contrast, values below both of these thresholds had a significant predictive value for the absence of invasion. The slight decrease in the correlation of >10 eosinophils/hpf with invasion in excisional specimens, relative to that in biopsy counterparts, may be attributed to the increased chance of observing microscopic clusters of eosinophils unrelated to invasion in the larger specimens.It is not surprising to observe inflammation in the specimens examined, likely due to several factors including the specific anatomic location and an overall inflammatory response of the stroma to the tumor, among others,9. HowevAlthough elevated eosinophil counts are statistically significantlly associated with stromal invasion in squamous cell carcinoma of larynx, occasionally the presence of high number of eosinophils were observed in the non-invasive counterpart tissues Table . In otheSince the first observation of malignancy with marked blood eosinophilia described by Rheinbach in 1893, eosinophilia has been described in human cancers from a variety of organs -12. In hThe mechanism of eosinophilic accumulation in cases of invasive carcinoma remains largely unknown. It has been suggested that such eosinophilic infiltration may be induced by a tumor-derived eosinophil chemotactic factor,19. A reIn conclusion, although the etiology of tissue eosinophils in invasive carcinoma is unknown, our study is the first to suggest that an elevated eosinophil count in the squamous neoplasia of larynx may serve as a morphologic feature associated with tumor invasion. The presence of more than individual eosinophils, specifically when the number of infiltrating eosinophils exceeds 10/hpf and or >20/10 hpf in a biopsy of larynx with squamous neoplasia, represents a histologic marker for the presence of tumor invasion. Similarly, the presence of eosinophils reaching these thresholds in an excisional specimen should prompt a thorough search for invasiveness when evidence of invasion is absent, or when invasion is suspected by conventional criteria in the initial sections. Although the present study assesses a quantitative parameter of tumor invasion, in our daily practice we find it useful that a readily appreciable elevation of tissue eosinophilia alerts us to search for possible invasiveness in tissue biopsy of laryngeal lesions.The author(s) declare that they have no competing interests.Drs. Said, Speiegel, Tan for study design; Drs. Said, Tan, for pathology evaluation; Drs. Alrwi, Douglas, Hicks, Loree, Riguel, Wiseman for surgical evaluation and clinical follow-up as well as card review. Dr. Yang for statistical analyses. Dr. Cheney for administrative and financial support.The pre-publication history for this paper can be accessed here:


Development and implementation of non-communicable disease (NCD) prevention polices in the developing countries is a multidimensional challenge. This article highlights the evolution of a strategic approach in Pakistan. The model is evidence-based and encompasses a concerted and integrated approach to NCDs. It has been modelled to impact a set of indicators through the combination of a range of actions capitalizing on the strengths of a public-private partnership. The paper highlights the merits and limitations of this approach. The experience outlines a number of clear imperatives for fostering an enabling environment for integrated NCD prevention public health models, which involve roles played by a range of stakeholders. It also highlights the value that such partnership arrangements bring in facilitating the mission and mandates of ministries of health, international agencies with global health mandates, and the non-profit private sector. The experience is of relevance to developing countries that have NCD programs running and those that need to develop them. It provides an empirical basis for enhancing the performance of the health system by fostering partnerships within integrated evidence-based models and permits an analysis of health systems models built on shared responsibility for the purpose of providing sustainable health outcomes. Non-communicable diseases exhort a considerable toll on individuals, societies and health systems ,2. LocatEstablished evidence highlights the potential to limit NCD mortality and morbidity through appropriate public health strategies aimed at disease prevention, risk factor control and health promotion . AddressNational Action Plan for Non-Communicable Disease Prevention, Control and Health Promotion in Pakistan (NAP-NCD) to achieve national goals for the prevention and control of NCDs [In Pakistan, the public-private tripartite partnership led by Heartfile (a non-profit NGO registered under the Societies Registration act of 1860 in Pakistan) and constituted additionally by the Ministry of Health and the WHO Pakistan office has recently released the  of NCDs . This paThe present exercise is the first opportunity to mount a truly 'national plan of action' aimed at preventing and controlling NCDs with the Governments' commitment to NCD prevention as a priority and to enlist a broad range in inputs from within Pakistan for addressing a challenging issue. The NAP-NCD outlines a concerted and comprehensive approach; one that incorporates both policies and actions. It is set within a long-term and life-course perspective and calls for an institutional, community and public policy level change. It has been designed to overcome the tendency to rely on a disjointed set of small scale projects, factoring integration at six levels: grouping NCDs so that they can be targeted through a set of actions, harmonizing actions, integrating actions with existing public health systems, incorporating contemporary evidence-based concepts, combining prevention and health promotion and harnessing the potential within partnerships.the term NCDs is technically reserved for a group of preventable diseases that are linked by common risk factors: cardiovascular diseases, some chronic lung conditions, cancer and diabetes fall within this category. However NAP-NCD also includes injuries and mental health within this framework as country requirements necessitate that these be addressed within a combined strategic framework through synchronized public health measures. There are many common grounds for combining public health actions to address these diseases.the NAP-NCD delivers an Integrated Framework for Action (IFA) ; this isthe approach adopted horizontally integrates NCD prevention with existing public health and social welfare infrastructure. It thus contributes to strengthening of the pubic health configuration and reorients health services to a more preventive orientation.NAP-NCD packages several contemporary and novel approaches. The population approach includes a behavioural research and social marketing-guided communication strategy and active role of local opinion leaders and educational institutions. Reorientation of health services includes scaling up of professional capacity and basic infrastructure in health care facilities and ensuring availability of, and access to, essential drugs at all levels of health care. The IFA packages a common population surveillance mechanism for all NCD's (with the exception of cancer). The model includes population surveillance of common risk factors and combines a module on population surveillance of injuries and mental health. The model has also been adapted for program evaluation.prevention is concerned with avoiding diseases whereas health promotion is about improving health and wellbeing. Both approaches are overlapping and complimentary and can be present in the same programme with similar activities and hold different meanings for two groups of targeted populations with different results. The public health approach to NCDs offers one of the best opportunities to combine prevention and health promotion to improve multiple positive outcomes; an approach NAP-NCD has capitalized on.this initiative created a mechanism for visible involvement and participation of relevant ministries, educational institutions, NGOs and leadership foci at a national consultation level and created avenues for their participation in the process that led to its development. In addition, all the key elements and advantages that stand to be gained from comprehensive grouping and maximizing on partnerships have been built upon: integration with the existing health system, inter-sectoral and intra-health-sector collaborations, linkage with the national health policy and partnerships with the private sector. NAP-NCD recognizes the scope of partnerships in public health activities and outlines a scope of interventions that are built on shared responsibility, allowing for agencies to participate according to their own missions, mandates, interests and resources. NAP-NCD fosters partnerships and interface arrangements between the public and private sectors so that the federal government is not solely responsible for getting these programmes out to the communities, but can rely on groups and national organizations that have complementary mandates. These partnerships are in harmony with national health priorities, complement state initiatives and are optimally integrated with national health systems.Reproductive health and communicable disease prevention and control have traditionally been priority areas for the Ministry of Health. Prevention and control of NCDs did not previously feature as part of the National Health Policy of 2001. There were therefore no specific programs in the national and provincial health departments and no budgetary line for NCD prevention up until the signing of the agreement, which lay the terms of reference for developing NAP-NCD . By leveThe next stage of implementation of NAP-NCD includes reorientation of health services and a comprehensive school health program. A proposal is already in the funding pipeline to seek additional resources from the governments' development budget and from donor sources.As part of its global mandate, WHO provides technical support to 'priority national programs' through its Joint Government/WHO Program Review Mission (JPRM) program; this is a regular biannual budgetary line. In addition, 'extra-budgetary' resources are provided for 'WHO priority programs' such as the polio campaign. In the year 2003, US $ 843 million were allocated for 192 countries under the former and 1.4 billion as part of the latter [For the year 2001–2003, Pakistan was allocated a budget of US $ 20 million . HoweverHeartfile has been planning and implementing national media campaigns and community-based projects for cardiovascular disease prevention incorporating social marking approaches ,24. AlthCurrently, the NGO draws support from donor funds through 'project aid'. In future, the NGOs funding is likely to be compromised with shifting donor focus on 'programme aid', as part of which, donors provide funds through national budgets. Partnering in this program therefore contributes to sustainability of the NGO as this provides a mechanism for sustained funding.The ingredients in this public health strategy are sound; however there are several limitations of this approach. Firstly, it needs to be supported by a clear, strong and sustained political commitment; secondly, the successful implementation of this plan requires the setting up of appropriate infrastructure and public health workforce with adequate capacity for core public health functions. This has implications for the need to build capacity and related infrastructure as a parallel process.The public-private partnership dimension of this plan emanates from within the overall 'development policy framework', which encourages private-sector participation in state activities. However it does have its own challenges. This experience therefore presents a clear imperative for addressing ethical, methodological, accountability, sustainability and governance issues in public-private and other multi-stakeholder arrangements -27.Useful lessons can be learnt from this experience both by developing countries and low resource settings that have NCD programs running and others that do not.Most developing countries have limited capacity for NCD prevention and control . There iThis example is also relevant to NGOs in other developing countries, who receive financial support from donors through project aid as it serves as an empirical basis for the integration of NGO activities with national plans and goals.The desired impact of this intervention is positive change in population health behaviours; therefore its ultimate success can be judged by changes in population outcomes, which can only be assessed over a period of time. However the process evaluation framework of the Action Plan outlined in the IFA is modelled to access how the program achieves its effects and includes the evaluation resource inputs, description of activities and intermediate outcomes. A review of the Logical Framework Analysis of the Action Plan and the IFA has shown progress against many of the process indicators .Notwithstanding that there are several limitations of this strategy, it does provide the empirical basis for an integrated response to NCD prevention and health promotion in a developing country setting. The IFA is an innovative tool, which helps to set country targets and defines integrated actions to meet those targets. However future efforts must also seek to integrate strategies with communicable disease control, particularly in areas where a life course approach is pursued; this will enable the development of sustainable public health systems for all disease. It is hoped that the findings from this program can be a basis for policy change.