Esca, a Grapevine Trunk Disease (GTD), is of major concern for viticulture worldwide. Our study compares the fungal communities that inhabit the wood tissues of vines that expressed or not foliar esca-symptoms. The trunk and rootstock tissues were apparently healthy, whether the 10 year-old plants were symptomatic or not. The only difference was in the cordon, which contained white rot, a typical form of esca, in 79% of symptomatic plants. Observations over a period of one year using a fingerprint method, Single Strand Conformation Polymorphism (SSCP), and the ITS-DNA sequencing of cultivable fungi, showed that shifts occurred in the fungal communities colonizing the healthy wood tissues. However, whatever the sampling time, spring, summer, autumn or winter, the fungi colonizing the healthy tissues of asymptomatic or symptomatic plants were not significantly different. Forty-eight genera were isolated, with species of Hypocreaceae and Botryosphaeriaceae being the most abundant species. Diverse fungal assemblages, made up of potentially plant-pathogenic and -protective fungi, colonized these non-necrotic tissues. Some fungi, possibly involved in GTD, inhabited the non-necrotic wood of young plants, but no increase in necrosis areas was observed over the one-year period. Grapevine trunk diseases (GTDs) that affect vineyards are of major concern for the wine industry. The three main diseases, eutypa dieback, esca disease and Botryosphaeria dieback, constitute a threat for the main vine-growing regions of the southern and northern hemispheres (i) From one year to another the same vines do not necessary express foliar symptoms, so when we consider a period of several years the number of “esca-foliar symptomatic vines at least once” is far more relevant. (ii) About 10% of vines of surveyed varieties expressed trunk diseases foliar symptoms, e.g. Sauvignon variety in the Aquitaine region. (iii) Approximately 13% of French vineyards are unproductive, mainly because of these three diseases. (iv) Esca moderately affects the phenolic composition of grapes and decreases the sensory quality of wines, suggesting a particularly dramatic increase in the economic importance of esca if no control methods are found (v) Regarding etiology, the symptoms that occur in the trunk, leaves and berries have been extensively described, indicating that if eutypa dieback symptoms markedly differ from those of esca and of BDA, differentiating between these two latter diseases often proves rather elusive, with Lecomte et al.In the last decade, the National Grapevine Trunk Diseases Survey was established in France in order to monitor and analyze the importance and severity of the trunk diseases (period 2003 to 2008). It was reported that 50 to 83% of the plots showed symptoms of esca et al.et al.et al. (2012) suggested that a second simplification could be made by not having to separate esca symptoms into mild or apoplectic forms. For the first time, the authors introduced a classification based on a graduated scale of severity, going from leaves showing some discoloration to total vine wilting. As regards the link between foliar and wood necroses, Maher et al.Initially, due to the complexity of its symptoms, five different diseases of esca complex were used in the literature: brown wood streaking, Petri disease, young esca, esca and esca proper i.e. central necrosis, black punctuated necrosis, white rot and sectorial necrosis, were associated with the multiplication of a few fungi. For instance, Fomitiporia mediterranea is predominant in white rot in Europe Phaeomoniella chlamydospora and Phaeoacremonium aleophilumP. chlamydospora is the most frequent in central necrosis P. aleophilum and P. chlamydospora, followed by F. mediterranea. The second process led to the development of light-colored, soft, sectorial necrosis, primarily caused by Eutypa lata , followed by F. mediterranea. However, the dynamics of necrotic development in woody tissues remains largely speculative et al.Pathogenic fungi are assumed to be responsible for all these necroses; they grow within the wood, decay it and slowly kill the vines. Development of specific necroses, (i) in these plants, the wood tissues of the trunks and rootstocks are mainly non-necrotic and apparently healthy, (ii) the mycoflora colonizing the various types of necroses has been extensively described (iii), as suggested by Maher et al.In this context, the objective of our experiment was to produce complementary data that would help in improving our understanding of esca development. So, we compared the fungal microflora inhabiting the apparently healthy wood tissues of young (10 year-old) esca-diseased and healthy vines for three reasons: Vitis vinifera) grafted on rootstock 101 14 MG and planted in a sandy-clay soil. Being interested by the differences between symptomatic and asymptomatic plants, we selected only plants that had previously expressed esca foliar symptoms at least twice over a period of 4 years (2005 to 2009). The control plants never expressed the foliar symptoms.Bordeaux Sciences Agro is responsible of Luchey-Halde vineyard. No specific permissions were required for the experimentations we have done. The sampling site was located at the Luchey-Halde vineyard in Pessac-Léognan, . This vineyard has been surveyed for the presence of esca foliar symptoms since its plantation in 2000. Experimentations were carried out on 10-year-old vines of the cultivar Cabernet Sauvignon , in the summer (June 2010), in the autumn (September 2010) and in the winter (January 2011).i.e. cordons, trunks and rootstocks, in order to verify the status of the wood, i.e. necrotic or healthy. Except for white rot, which is associated with esca, only non-necrotic tissues were sampled, in order to isolate cultivable fungi and to extract the fungal DNA.Our sampling method was vine-destructive, as the plants were first uprooted before their various parts were cut longitudinally, All the esca foliar-symptomatic and asymptomatic vines collected in 2010 and 2011, the samples consisted of 15 chips (around 5 mm in length) of non-necrotic woody tissues from the inner and outer parts of trunks and rootstocks.A surface sterilization method was used in order to eliminate the epiphytic fungi. The wood fragments were immersed in a 5% calcium hypochlorite solution for 30 seconds, washed in sterile distilled water, and dried on a sterile filter paper. For each vine part, 15 sterilized chips were placed on Malt Agar (3 pieces per plate) and incubated at 25°C in the dark. The fungal development was monitored over a 4-week period. When fungal colonies emerged from the wood tissues, mycelial fragments were transferred to new Malt Agar plates. The fungi that did not grow to more than 1 cm around the piece of wood over a period of one month were not isolated.Whenever possible, the taxonomic identification of the fungi was based on morphological and cultural features, and on examination of fruiting structures and conidia under the microscope. To identify the species of the most abundant genera, and other isolates whose sporulation was not observed, sequencing of the Internal Transcribed Spacer region of the rDNA was performed. It should be noted that the isolates to be sequenced were selected randomly.For each vine collected, 10 g of non-necrotic tissues were sampled in the inner and outer parts of trunks, in the inner part of rootstocks and cordons, and from the trunk and stock barks. When white rot tissue was identified (only in esca-foliar symptomatic vines), 10 g were also collected. All these samples were ground in liquid nitrogen with a one-ball mill of Dangoumau type, and kept at −80°C prior to DNA extraction.DNA was extracted from 60-mg aliquots of woody tissues using the Indvisorb Spin Plant mini Kit  according to the manufacturer’s instructions. The DNA extracts were then quantified with a nanodrop , and homogenized at a concentration of 10 ng/µl.−1 of BSA (New Englang BioLabs), 0.2 µM of each primer, Pfu Turbo buffer 1x and 0.05 unit of Pfu Turbo DNA polymerase (Stratagene/Agilent Technologies). The cycling parameters were 95°C for 2 min, followed by 35 cycles at 95°C for 1 min, 56°C for 1 min, 72°C for 1 min and final extension at 72°C for 10 min. The PCR products (∼250 bp) were visualized by 2% TBE agarose gel electrophoresis prior to SSCP analysis.The pair of primers recognizing the mitochondrial large subunit rDNA gene, ML1-ML2 from White et al. SSCP analyses were performed on an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems) equipped with four 36-cm long capillaries. One microliter of a PCR product was mixed with 18.8 µl formamide Hi-Di (Applied Biosystems), and 0.2 µl standard internal DNA molecular weight marker Genescan 400 HD ROX (Applied Biosystems). The sample mixture was denatured at 95°C for 5 min and immediately cooled on ice, and then loaded onto the instrument. The non-denaturing polymer consisted of 5.6% POP conformational analysis polymer (Applied Biosystems), 10% glycerol, EDTA buffer 10x (Applied Biosystems) and water. The migration time was set to 2000 seconds, the voltage to 15 kV and the temperature was 32°C. Samples were co-migrated with the fluorescent size standard (GeneScan-400 ROX) to allow comparison of migration profiles between samples. Patterns were then aligned with StatFingerprints (version 2.0) and were gathered in a single numerical database before being statistically described by a global Principal Component Analysis (PCA) with R (version 2.14.2). Each sampling point was calculated by analyzing 250 variables corresponding to the SSCP profile scans of each sample. PCA was performed using the correlation coefficient of Pearson. Variables with a cos2≥0.5 on one of the first third principal components  were estimated as sufficiently well represented by the principal plan generated by this PCA.−1. Four hundred µl of CTAB (1x) were added to each sample. After an incubation at 65°C during one hour, 400 µl of chloroform-isoamyl alcohol  were added and the samples centrifuged for 30 min at 3700 rpm. The aqueous phase was transferred to a new tube, and 200 µl of isopropanol were added. Samples were then kept at −20°C over night for DNA precipitation. After 20 min centrifugation at 3700 rpm, the supernatant was discarded and 500 µl of ethanol 70% were added to wash the DNA. Once the ethanol was discarded, the pellets were air dried and then resuspended in 50 µl of sterile distilled water.Fungal genomic DNA was isolated from fresh mycelium scraped at the surface of a Malt Agar plate with a sterile tip. Samples were freeze-dried overnight  and then ground with a little glass ball for 1 min at a frequency of 29.9 sDNA samples were sent to GATC Biotech AG  for sequencing of the ITS region with the primers ITS1f and ITS4 The results of the microbiology study were analyzed using Correspondence Analysis (CA). The data were quantitatively analyzed using the package ade4 of R (version 3.0.1).Species diversity was then calculated using various indexes: species richness , the abundance (number of isolates), the Shannon diversity index, H’ Examination of longitudinal sections of vines showed that there were significantly more necroses in the cordons of the esca-symptomatic plants than in the asymptomatic ones . SpecifiThe mitochondrial 28S rDNA gene was studied to compare the genetic structure and the dynamics of the fungal communities colonizing different types of wood tissue of esca-foliar symptomatic and asymptomatic vines collected over a growing season.The barks of rootstocks (B) and trunks (T), the non-necrotic wood tissues of rootstocks (R), inner trunks (I), outer trunks (O) and arms (A), and the white rot (W) yielded 208 SSCP profiles, which were compared in 3360 pieces of wood were cultured from the 56 vines sampled, 574 fungal strains were isolated from asymptomatic plants, and 612 fungal strains were isolated from esca-symptomatic plants. Depending on the piece of wood, either no fungus, or upto a maximum of three fungi, were isolated. The highest values of fungi isolated were obtained in April and January, with 205 and 163 isolates, respectively. The lowest values were in June, with around 100 isolates taken from the plants. When the numbers of isolates were compared between symptomatic and asymptomatic vines, differences were observed (p = 0.01049 with Chi-square test) (Unpublished data).For each sampling time and for each type of plant, the isolates were ranked in descending taxonomic fashion: division, class, order, family and genus. The Ascomycota were the predominant division, with 92.4% of the total of isolates, followed by the Zygomycota and the Basidiomycota, which were isolated at respectively 7.2% and 0.4%. Trichoderma, Penicillium and for the Botryosphaeriaceae: Botryosphaeria, Diplodia and Neofusicoccum. The fungi isolated from the 50 genera represented 5 classes, 14 orders, 27 families belonging to various lineages of Ascomycota, Basidiomycota and basal fungal lineages.The most frequently isolated genera belong to the class of Sordariomycetes and Dothideomycetes, with a dominance of Sordariomycetes . Altogether, the two classes represented 77% of all the isolates of symptomatic and asymptomatic plants.(i) Botryosphaeriaceae species  with 168 isolates for asymptomatic, and 154 isolates for esca-foliar symptomatic vines , (ii) Trichoderma spp., with 132 isolates for asymptomatic and 163 for symptomatic (no significant difference with Chi-square test), (iii) Penicillium spp., with 45 isolates for asymptomatic plants, and 61 for symptomatic plants  Bionectria spp. with 16 isolates for asymptomatic plants, and 58 for asymptomatic plants (no significant difference with Chi-square test) and (v) Fusarium spp., with 34 isolates for asymptomatic plants, and 20 for symptomatic plants (no significant differences with Chi-square test). These 7 genera accounted for 68.8% of all the strains isolated from the wood of asymptomatic plants, and for 74.5% from the samples of symptomatic plants.The most abundant isolates collected were: Trichoderma spp. For the other species, there were differences between the genera and the number of plants colonized, except for Penicillium spp., Aspergillus spp. and Cladosporium spp., where respectively 50%, 42% and 29% of the both type of plants were inhabited by these fungi. Sixty nine percent of the asymptomatic plants, and 42% of symptomatic ones, were colonized by Fusarium spp. It should be noted that Alternaria spp., Arthrinium spp. and Epicoccum spp. colonize more asymptomatic plants than the symptomatic ones.70% of esca-foliar symptomatic and asymptomatic plants were colonized by Botryosphaeriaceae species and Diplodia seriata is the most abundant species. 58% of the symptomatic vines were inhabited by Diplodia seriata, and 75% for the asymptomatic ones. Fusarium oxysporum (61%/29% vines inhabited = asymptomatic/symptomatic), Epicoccum nigrum (43%/29% = asymptomatic/symptomatic), T. gamsii (43%/36% = asymptomatic/symptomatic) and Arthrinium sacchari (29%/7% = asymptomatic/symptomatic) were more isolated in asymptomatic plants than in the other type of vine. In contrast, more symptomatic plants were colonized by Bionectria ochroleuca (32%/57% = asymptomatic/symptomatic) and N. parvum (29%/46% = asymptomatic/symptomatic) than the asymptomatic plants.Diplodia sp., Neofusicoccum sp., Bionectria spp., Fusarium spp., Aspergillus spp., Microdiplodia spp. were isolated more in June from symptomatic and asymptomatic plants. Rhizopus spp. and Pestalotiospsis spp. were isolated more from symptomatic plants in September and January. Trichoderma spp., Aspergillus spp. and Cladosporium spp. were isolated principally in the inner trunk of asymptomatic plants. Penicillium spp., Rhizopus spp., Bionectria spp., Arthrinium spp. and unknown fungi were isolated, in general, from the inner or outer of the rootstock and from the inner trunk of symptomatic plants. The genera Neofusicoccum, Fusarium and Botryosphaeria were isolated principally from the inner rootstock of symptomatic plants from the outer trunk of asymptomatic plants.H’) were the same. Depending on sampling time, fungal biodiversity in April (H’ sympto = 2.05 and H’ asympto = 2.14) was higher than in January (H’ sympto = 2.87 and H’ asympto = 2.98). Also, dominance was not shown for a relatively large number of species because of the low value of the evenness component (J’). The values varied from 0.18 to 0.21  followed by F. oxysporum (42 sequences), B. ochroleuca (33 sequences), N. parvum (32 sequences), T. gamsii (31 sequences) and T. atroviride (19 sequences). These 6 contigs contained 1 to 4 haplotypes. Some haplotypes were detected in the two types of plants for each sampling time; for instance, for H1 and H3 of D. seriata and H14 of T. gamsii. For the other haplotypes, for which the number of sequences is also generally low, haplotypes were detected only at certain sampling dates.After preliminary analyses using Codon Code Software and Mega 5, 487 sequences were selected for phylogenic and statistical analyses. The 487 sequences were assembled in 43 contigs, with one of them grouping the unassembled sequences. Generally, each contig represented one genus or one species. One hundred and four haplotypes were defined from the 43 contigs. When the sequences of one contig were aligned, only a few nucleotides changed for each species. The objective of this study was to analyze the fungal communities colonizing the healthy wood tissues that were predominant within relatively young (10 year-old) grapevines. This could help in determining the initial factors that make a healthy wood become necrotic, usually a few years later.The results, based on the use of SSCP, clearly indicate that the fungal microflora changed over the year. In the rhizosphere of many plants, shifts in the genetic structures of the fungal communities over time were also observed F. mediterranea is usually the predominant fungus in this zone F. mediterranea is predominant within the white rot of the grapevines we sampled. So as a specific microflora, presumably dominated by F. mediterranea, colonizes white rot, which may explain why that microflora differs from the healthy-wood tissues, as we show by means of SSCP analyses.When the non-necrotic wood of trunks and rootstocks of both esca-foliar symptomatic and asymptomatic plants were studied, their fungal microflora tended to be similar. As expected, the mycoflora were different in the white rot and the non-necrotic tissue for the symptomatic plants. It should be noted that in our study, white rot, a necrosis typical of esca et al.et al.et al.Vitis Vinifera. With the exception of a few samples from Gonzalez and Tello Fomitiporia spp.) or the Stereaceae (Stereum spp.). The main reason is certainly because Basidiomycota, such as F. mediterranea or Stereum hirsutum, were usually found in the necrotic tissues of grapevine plants. Stone et al.The results of the SSCP showed that there were no differences of fungal microflora between the non-necrotic tissues of symptomatic and asymptomatic plants and the results of the cultivable method seemed to show numerous similarities. The number of cultivable fungi we isolated for each sampling date varied for symptomatic and asymptomatic plants from 115 to 280. Ascomycetous fungi represented 92% of the total number of the species obtained. This result is in agreement with reports that have analyzed the microflora colonizing various parts of the vines. For instance, Casieri et al.At the fungal order level, the high proportion of Hypocreales, Botryosphaeriales, Eurotiales and Pleosporales in the trunk and rootstocks of esca-foliar symptomatic and asymptomatic grapevines suggests they were the main components of the endophytic mycota. Our results differ from those of Hofstetter Botryosphaeria, Diplodia, Neofusicoccum, Trichoderma, Penicillium, Bionectria, Fusarium, Mucor, Rhizopus, Aspergillus, Cladosporium, Alternaria, we observed no significant differences between the healthy wood of esca-symptomatic and asymptomatic vines. Most of these genera were detected in the wood of at least 20% of all the plants. The genera Botryosphaeria, Diplodia, Neofusicoccum, have some potentially plant pathogenic species, but the majority of the other genera were non-pathogenic for plants, with some of them being potentially biocontrol agents.For the 12 most abundant genera: Botryosphaeria, Diplodia, Neofusicoccum, they can cause Bot canker on grapevines D. seriata (57% in symptomatic and 43% in asymptomatic plants). In the literature, contradictory results have been reported: depending on the age of the grapevines and cultivar, Botryosphaeriaceae species could be isolated or not. For instance, Hofstetter et al.D. seriata from nurseries and adult vines, and N. parvum from nursery plants. Krol As regards Botryosphaeriaceae species from the genera Trichoderma, is well known for its antagonistic abilities to parasite plant pathogenic fungi and to protect many plants, including grapevine Trichoderma strains were isolated from 75% of esca-foliar asymptomatic and 93% of symptomatic grapevines. The two most frequently isolated species were T. gamsii (in 42/35% of asymptomatic/symptomatic plants) and T. atroviride (in 25/15% of asymptomatic/symptomatic plants). The other 6 species of Trichoderma were isolated to a lesser extent.The second, most abundant genus, D. seriata, and of one haplotype of T. gamsii, were detected at each sampling time, in both esca-foliar symptomatic and asymptomatic plants. This suggests that these two genera, in addition to being abundant, have specific haplotypes that were able to strongly colonize and persist in the wood of grapevines. Also, the data of the four sampling date show that only two haplotypes of Diplodia seriata have been defined, two haplotypes for Trichoderma gamsii and one for Trichoderma atroviride. There is no difference between the haplotypes of symptomatic and asymptomatic plants. Interestingly, Linaldeddu et al.in vitro strains of Trichoderma and Botryosphaeriaceae species, the mycoparasite was predominant over the development of the pathogen. This finding suggests that such competition occurs in the wood of plants; however, to our knowledge, no investigations have been made to determine if it shapes the two fungal communities at the vineyard.In the present study, strains of two haplotypes of E. nigrum, Alternaria alternata, and F. oxysporum were known as useful or promising biocontrol agents. However, it was not possible to postulate that non-pathogenic fungi were always more numerous in asymptomatic grapevines than in esca-foliar symptomatic ones. For instance, the pathogen D. seratia was detected in 57% of asymptomatic and 43% of symptomatic grapevines. Furthermore, as the pathogenic status of F. oxysporum strains is not known, some may be plant pathogenic. So, when the cultivable mycoflora colonizing the healthy woods of the two types of plants were compared, they could not be statistically differentiated.We found that many other fungi, colonizing at least 20% of plants, may have antagonistic activities. For instance, some strains of Trichoderma spp. and Botryosphaeriaceae species. The relative abundance of each endophyte genus reflects an unequal distribution of isolate richness among genera. In fact, in January, the number of different species of isolates was greatest. Also, the low values of evenness underline the fact that few species are abundant in the wood tissue. Unlike the Shannon diversity index (H’) obtained by Gonzalez and Tello i.e. 3.17, our indexes were lower. But the diversity we estimated was based on one single grapevine variety (Cabernet-Sauvignon), and on healthy wood samples only.In our study, 54% and 51% of total isolates identified from, respectively, asymptomatic and symptomatic plants, represented plural genera. Gonzalez and Tello Nowadays, the scenarios that consist in describing why the healthy wood of grapevines usually becomes necrotic still remains to be written and the whole process demonstrated. In the present experiment, as we observed shifts over a year in the mycoflora colonizing the vine wood, it could be speculated that, over a longer period, fungal community structures could also change. Provided that environmental factors are favourable, pathogenic fungi that have already colonized some parts of the wood of the trunks can multiply and subsequently cause necroses.e.g. bacteria, and abiotic, e.g. weather changes, cultural practices and other factors need to be studied in order to understand how those abiotic and biotic factors favor the onset of the pathogenic process.In conclusion, this study demonstrates that the mycoflora colonizing the apparently healthy-trunk wood tissues of grapevines changed over the yearly cycle. However, we have not observed that the equilibrium between the potentially plant pathogenic and potentially plant protective fungi colonizing the healthy wood breaks down over time, leading to the development of necroses. Experiments over a longer period, certainly a few years, would certainly be required to observe such changes. We can assume that biotic,


Both gray-matter (GM) atrophy and lesions occur from the earliest stages of Multiple Sclerosis (MS) and are one of the major determinants of long-term clinical outcomes. Nevertheless, the relationship between focal and diffuse GM damage has not been clarified yet. Here we investigate the regional distribution and temporal evolution of cortical thinning and how it is influenced by the local appearance of new GM lesions at different stages of the disease in different populations of MS patients.We studied twenty MS patients with clinically isolated syndrome (CIS), 27 with early relapsing-remitting MS , 29 with late RRMS (disease duration ≥ 5 years) and 20 with secondary-progressive MS (SPMS). The distribution and evolution of regional cortical thickness and GM lesions were assessed during 5-year follow-up.2 = 50.0, p<0.001) and in early RRMS , compared to late RRMS  and SPMS .The results showed that new lesions appeared more frequently in hippocampus and parahippocampal gyri (9.1%), insula (8.9%), cingulate cortex (8.3%), superior frontal gyrus (8.1%), and cerebellum (6.5%). The aforementioned regions showed the greatest reduction in thickness/volume, although  differences were observed across subgroups. The correlation between the appearance of new cortical lesions and cortical thinning was stronger in CIS (rWe conclude that GM atrophy and lesions appear to be different signatures of cortical disease in MS having in common overlapping spatio-temporal distribution patterns. However, the correlation between focal and diffuse damage is only moderate and more evident in the early phase of the disease. Multiple Sclerosis (MS) is an autoimmune , chronicUnderstanding the mechanisms underlying cortical atrophy is challenging . GM and Although a correlation between CL load and the severity of GM atrophy was previously found , a conclIn this context, we set out to investigate longitudinally, and at different disease stages, the regional distribution and temporal evolution of cortical lesions and cortical thinning in MS patients. In addition, we explored whether the local appearance of new CLs may influence the development of cortical atrophy in the same region.Ninety-six consecutive patients, currently followed at the Multiple Sclerosis Centre of the Neurology Section, University Hospital of Verona , and having at least 5-year of longitudinal MRI follow up performed with the same MRI scan and the same MRI protocol at the Neuroradiology Unit of Euganea Medica , have been included in this retrospective study .At the beginning of the follow-up , according to the MS diagnostic criteria , 20 patiBy the end of the follow-up 5 years later , 13 CIS had a transition to early RRMS, while 3 late RRMS entered the SP phase. Nevertheless, during the data analysis each patient has been considered belonging to his/her original group despite the switching to one of the other groups. Fifty-one patients  had at least 1 relapse during the observation period: among these, 32  had an increase in the EDSS score  related to the relapse and confirmed at 6 months after the relapse. Sixty-four RRMS were treated with IFN beta 1a, IFN beta 1b or Glatiramer Acetate, 6 RRMS were treated with Natalizumab and 4 with Fingolimod, 3 SPMS were treated with Cyclophosphamide and 4 SPM±S were treated with Azathioprine while the remaining 10 patients were untreated.The Ethic Committee of the University Hospital of Verona  approved the study and written informed consent was obtained from all patients before the data analysis.3D Double Inversion Recovery (DIR): 3D sequence without any interpolation techniques, repetition time (TR) 6.500 msec, inversion time 2.800 msec, delay 500 ms, echo time (TE) 265 msec, slice thickness 1.5 mm, number of averages 2, matrix 256 x 256); 2) 3D Fluid-Attenuated Inversion Recovery (3D FLAIR): TR = 10000 msec, TE = 120 msec, TI = 2500 msec, ETL = 23, slice thickness = 1.5 mm, a matrix size = 172 x 288, and a FOV = 250 x 200 mm2; 3) Three volumetric fast-field echo sequence: 120 contiguous axial slices, TR = 25 msec, TE = 4.6 msec, flip angle = 30°, slice thickness = 1.0 mm, matrix size = 256 x 256, and a FOV = 250 x 250 mm2 were acquired. At follow-up, subjects were carefully repositioned according to published guidelines for serial MRI studies of MS [Each patient underwent the same MR protocol at T0 and at T5 (range = 62 ± 2 months). All images were acquired at the Neuroradiology Unit of Euganea Medica , using the same 1.5 T Philips Achieva scanner with 33 mT/m power gradient, and a 16-channel head coil. No major hardware upgrades of the scanner occurred during the study period. The following images were acquired from each subject: 1) es of MS .All images were evaluated by a neurologist (MC) and a neuroradiologist (AM) both with large experience on neuroimaging of MS patients.longitudinal stream included in the Freesurfer image analysis suite (release v5.3.0), available online (http://surfer.nmr.mgh.harvard.edu/). The technical details of these procedures have been described previously [Cortical reconstruction and volumetric segmentation was performed at T0 and at T5 on a volumetric T1-weighted data set by means of the eviously . Topologeviously ,12.The cortical parcellation  was performed on the base of the Talairach Atlas, included in Freesurfer .At T0 and T5, the number of new and pre-existing CLs was assessed region by region on DIR images by consensus following the recent recommendations for CL scoring in patients with MS . Since npost hoc Tukey HSD procedure to account for multiple comparisons. Also differences between CTh changes in region with new CLs compared to regions without new CLs were assessed through analysis of variance (ANOVA). Since CLs were not homogeneously distributed, the Mann-Whitney test was used to compare populations with respect to their CL number. Pearson Chi Square was applied to test the difference between patients. Univariate correlation using the Pearson coefficient has been applied to test the correlation between the baseline number of CLs and the entity of the global CTh change and also between the number of new CLs and the global CTh change.Differences among MS subtypes, between patients having more or less than 5 years of disease duration, and patients developing or not new CLs during the study, were assessed through analysis of variance (ANCOVA), including treatment as covariate (this considering the possible effect of disease modifying drugs on grey matter atrophy)  and postA minimal, anonymized dataset underlying the results of the present study is available . At basep < 0.001) and in CIS group . However, when the number of new CLs was calculated only in those CIS that converted to definite MS during the following 5 years , no significant difference was observed compared to RRMS group (p = n.s.).After 5 years, 331 new CLs  were identified . No significant differences were observed in the number of new CLs between early RRMS  and late RRMS group  while the number of new CLs was significantly lower in SPMS group , the insula (8.9%), the cingulate cortex (8.3%), the superior frontal gyrus (8.1%), and the cerebellum (6.5%). Importantly, significant differences were observed between different disease subtypes  and accop < 0.001; SPMS vs. early RRMS: p = 0.002; late RRMS vs. CIS: p = 0.004). As expected, a moderate correlation was observed between global CTh and disease duration .Global CTh at T0 even after age correction, was significantly lower in SPMS  and in late RRMS  compared to early RRMS  and CIS , (SPMS vs. CIS: p < 0.001 vs. SPMS and p = 0.041 vs. late RRMS) and CIS , indicating increasing loss of cortical GM volume with increasing disease duration.After 5 years follow-up, the mean CTh change was higher in SPMS  and in late RRMS  compared to early RRMS , the cerebellum (5.2%), the hippocampus and the parahippocampal gyrus (5.2%), and the cingulate cortex (5.0%) showed the greatest reduction in thickness/volume .The development of regional cortical thinning was not homogeneous across different MS groups. The reduction of CTh and volume of the hippocampus and the parahippocampal gyrus, the insula, and the cingulate cortex were particularly severe in CIS and early RRMS patients whereas in late RRMS and SPMS cortical thinning and volume loss were significantly greater in the precentral gyrus, the postcentral gyrus, and the cerebellum .2 = 0.26, p < 0.001) in the following 5 years; however, such correlation was stronger in CIS  and early RRMS  compared to RRMS  and SPMS .The mean volume of CLs at baseline moderately correlated with global CTh change (rp < 0.001). The total number of new CLs moderately correlated with the global CTh change in the whole group . However, again, such a correlation was stronger in CIS  and in early RRMS , compared to late RRMS  and SPMS  compared to patients with no new CLs , while no correlation was observed between the appearance of new WM lesions and the CTh change in the whole group nor in the 4 subtypes (p = n.s.).Finally, a modest correlation was also observed between T2-WMLV at baseline and the CTh change  and diffuse  damage than other regions. Our data are in line with previous MRI studies , includiTaken together, these results strengthen the hypothesis that a higher susceptibility to neurodegenerative processes in key brain regions, known to be related to specific clinical (cognitive) functions, is likely to underlie the clinical manifestations of at least a subgroup of MS patients . NeverthAlthough understanding the origin of cortical damage in MS is still challenging, some considerations can be done on the basis of this longitudinal study.First, we observed that the distribution of GM damage is not homogeneous across different disease subtypes and, in turn, different disease durations. Both focal and diffuse GM damage seem to affect in the earliest phases of the disease (CIS and early RRMS) the fronto-temporal regions, especially the hippocampus and the parahyppocampal gyrus, the insula and the cingulate cortex, while they become more widespread, involving also the precentral gyrus, the postcentral gyrus and the cerebellum, later in the disease course (late RRMS and SPMS).Only in CIS and early RRMS we have found a strong correlation between the appearance of CLs and the CTh change suggesting that, at least at the beginning of the disease, the early focal cortical pathology plays a relevant role in the development of brain atrophy. This is in line with natural history studies, demonstrating that the outcome severity is primarily determined during the early phase ,20. The It is worth to underline that such correlation, and even the partial overlap between focal and diffuse damage, do not imply that CLs are the main cause of cortical thinning. Indeed, the relationship between new CLs and cortical thinning does not exist at the level of single cortical areas but only, in the whole brain, between the total number of new lesions and the global cortical thickness change. This means that, at least at the beginning of the disease, those patients with the highest accumulation of new CLs showed the greatest global cortical thinning. In the advanced disease phases, it seems that other factors may influence the development of cortical atrophy as suggested by the high cortical thinning in some regions, such as the calcarine fissure, that show low frequency of CLs presence. Whether this is the consequence of tissue destruction in the subcortical WM, involving axonal transection and retrograde neurodegeneration , it is hA second hypothesis is that cortical thinning in these areas might be more dependent on diffuse subpial CLs , which aA third hypothesis suggests that, in addition to the role of demyelination in cortical thinning, there is a diffuse loss of neurons, axons, and synapses in the non-demyelinated normal appearing gray matter ,39,40, wThis retrospective study is not free from limitations, mainly related to the low MRI field applied and to the low sensitivity of DIR sequence for GM damage when compared to the neuropathological approach. Moreover, the study do not provide any MRI data about the pathology of the normal appearing WM that may significantly contribute to cortical atrophy progression in MS .However, this work has also several strengths: the longitudinal approach, the high number of patients included in the analysis, and the fact that, for the first time, a comparison between the appearance of CLs and cortical thinning has been done region-by-region and in different MS populations.Concluding, from the clinical point of view, considering the potential effect of some new disease-modifying drugs on the cerebro-spinal fluid (CSF) proteome and on the accumulation of CLs ,44, the S1 Dataset(XLSX)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Tablep < 0.001 compared to Patients with DD >5 years (RRMS and SPMS). Regions with more than 0.5% of cortical lesions are shown in the Table.The asterisk (*) indicates (PDF)Click here for additional data file.


Objective: The present study aimed to clarify dissociative symptoms in patients with schizophrenia and its association with negative and positive symptom of schizophrenia.Methods: Based on the 4th edition of Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria, 80 schizophrenic subjects were randomly selected from patients who referred to the clinics of psychiatry hospital in Kerman, Iran. Eighty non-schizophrenic patients were chosen as the control group. Both groups were evaluated for dissociation symptom using the Dissociative Experience Scale (DES). Positive and Negative Syndrome Scale (PANSS) score was also used in the case group for determination of positive and negative symptom of schizophrenia. Results: The prevalence of dissociation symptom was 13% and 4% among schizophrenic and control groups, respectively (p = 0.02). In addition, there was a statistical significant association between DES score and positive symptom in schizophrenia (p = 0.02).Conclusion: The association between dissociative symptom and schizophrenia was significant and dissociative symptoms were associated with positive symptoms of schizophrenia. Declaration of interest: None. Schizophrenia is a clinical syndrome with involvement of thought, perception, emotion, movement and behavior. Its prevalence has been reported from 1 to 1.5%. The ratio of suffering is the same for both males and females. The cause of this disease is obscure. However, there are some assumptions regarding its etiology such as genetic factors, neuro-immuno-virology, and complications during pregnancy and delivery   -4.The classification of schizophrenia into two types has considerably influenced the psychiatric research. Positive symptoms included hallucination and delusion, while the negative symptoms included superficial emotion, difficulty in finding words, obstruction in speech, lack of adornment, lack of motivation, and lack of feeling of pleasure or satisfaction and unsociability. Positive and Negative Syndrome Scale (PANSS) test is a standard method in examining schizophrenic patients , 5. PlusThe term "dissociation" refers to a disorder in normal functioning of perception, memory, identity or consciousness . AlthougThe study of dissociation in schizophrenic patients is mainly due to high traumatic confrontation in patients with severe mental breakdown . It was Although the association between trauma and dissociation has been well-known in patients with dissociative disorders and post trauma stress disorders, and even though patients with severe mental breakdown have higher traumatic confrontations, there are a few studies available regarding the association between trauma and dissociation in those patients.Some studies indicated that there is an association between people’s personal reports of neglect or abuse in childhood and dissociative symptom in adulthood schizophrenia. Emotional abuse has high positive correlation with observed dissociation in schizophrenic population. Such findings describe some genetic predispositions to schizophrenia that may be in connection with disorder of correspondence mechanism. Such process exposes children with pre-schizophrenia to higher impacts of stress . Thus, cThe only way for discovering facilitator events in such patients is re-examination, and checking diagnosis and prior to treatments. Thus, identification of dissociative symptoms in patient with schizophrenia and their association with the negative and positive symptoms of schizophrenia is important.Study subjectsIn this descriptive study, the patients were chosen in a nine-month period in 2002. Two groups of subjects  were recruited from Shahid Beheshti Hospital, Kerman, Iran. Eighty schizophrenic patients aged 18-74 years were included, whose diagnosis was confirmed by clinical interview according to the DSM-IV criteria. Exclusive criteria were serious psychotic disorders other than schizophrenia, mental retardation, substance and/or alcohol abuse, and antiepileptic treatment. Eighty volunteers were selected from workers and employees of hospital as control group who had neither schizophrenic nor other major physiological or physical disorders (matched for gender and age). They were reassessed by a psychiatrist to confirm their physiological status. After explanation the study objectives and obtaining informed consent, the participants were re-interviewed on the basis of the DSM-IV scale.InstrumentsThe patients were assessed by the PANSS  for their positive and negative symptoms by a trained psychiatrist. PANSS is a 30-item scale with 1 to 7 points  for each item and sub-scores for 7 positive (P), 7 negative (N), and 16 global psychopathological symptoms i.e. delusions, formal thought disorders, hallucinations, agitation, grandiosity, suspiciousness/persecution, and hostility(P1 to P7); blunted affect, emotional withdrawal, poor rapport, social passivity and apathy, difficulty in abstract thinking, lack of spontaneity and flow of conversation, and stereotyped thinking (N1 to N7); and general (G) items such as health concerns, anxiety, guilt, tension, mannerisms and posturing, depression, motor retardation, uncooperative behavior, unusual thought contents, disorientation, poor attention, lack of judgment and insight, a volition, poor impulse control, self-centeredness, and active social avoidance (G1 to G16). . PANSS qIn PANSS theory, and in order to estimate positive and negative symptoms in schizophrenia, a classification was introduced by Crow TJ , 3. AccoType I is positive-dominant and defined as three or more positive symptoms and less than three negative symptom. Type II is negative-dominant, and type III is the mixed type. Then, the patients were evaluated through the DES . DES II questionnaire has been used since 1993 after being introduced by Putnam and Carlson as a highly valid and reliable test for screening dissociative experiences with 28 questions covering 4 subscales, which reflects constructs of dissociation symptoms: amnesia, depersonalization/derealization, absorption and imaginative involvement and passive influence. The test was translated to the Persian by the authors and proved by three psychiatrists for its fluency in Persian and English. Its content validity was also approved by the same psychiatrists. Reliability of the questionnaire was examined by split test and Cronbach’s alpha  -19. The collected data were analyzed using the SPSS for Windows 11.5  and Software Minitab. Such statistical procedures as craft-tab tables, spearman coefficient of correlation, ANOVA  and regression were used accordingly.Two groups of schizophrenic and control were matched for age and sex. In schizophrenic group, mean age of men and women were 37.6 ± 31.2 and 33.9 ± 8.68 years old, respectively. Most of the subjects (41.9%) were between 20 and 30 years old and 68 % of them were males. Mean age of the control group was 36 ± 10.9 and 34.1 ± 6.8 among 41% males and 59% females, respectively.In general, the results showed that DES score in 8.1% was above 30, while score in 147 (91.9%) was below, and 30.58 cases (%36.3) received scores below 10 and 89 (%55.6) received scores between 10 and 30. Data analysis revealed that DES score in 23 (29%), 47 (58%), and 10 (13%) of the schizophrenic subjects was below 10, between 10 and 30, and above 30, respectively, whereas in the control group, it was 35 (44%), 42 (52%), and 3 (4%), respectively. This result showed that there was a statistical significant association between schizophrenia and dissociation (p = 0.03) .Distributions of positive, negative, and mixed symptoms according to the gender of the schizophrenic patients are illustrated in Our results demonstrated that DES score was not changed significantly within two genders  or age groups comparison . Furthermore, an analysis of the subdivision of PANSS showed a statistical significant association of supplemental, general psychopathology, depression and activation with DES score.Our results indicated that there was a statistical significant association between dissociative symptom and schizophrenia. The prevalence of dissociative symptom in schizophrenic patients, on the basis of DES test criteria, was significantly higher than the control group. Findings of this research are accordance with those of Spitzer et al.  and someThere is empirical evidence, primarily from the DES studies, that schizophrenic patients meet dissociative phenomena more often and to a higher degree than normal individuals, but with lower intensity and frequency than patients with borderline personality disorder (BPD), post-traumatic stress disorder (PTSD) and dissociative identity disorder(DID) .The association between dissociation and psychosis may also be somewhat more important than recently thought. Dissociative symptom maybe is similar to psychotic symptom in many instances. More severe forms of dissociation may even be misdiagnosed as schizophrenia. In fact, it is not uncommon for patients with PTSD or DID to receive a misdiagnosis of schizophrenia , 21. OnlThe association between the emotional trauma and dissociation has been shown in several studies. In majority of these studies, dissociation was significantly associated with emotional abuse and physical abuse in childhood , 22, 23.We found a statistically significant association between positive symptoms and dissociative experience; whereas there were not any association between negative and dissociative symptoms and dissociative experience. Similar to our results which indicated significant association between DES scores and positive and negative symptoms in schizophrenia, Spitzer et al.  found a The results of the present study provided supportive evidence that dissociative symptoms are also strongly associated with a schizophrenic disorder without gender or age preference and this significant association was observed with positive symptoms in schizophrenia.Therefore, future investigations are required to define the etiologies of dissociation and the exact time of assessment of dissociative symptoms.


Advances in medicine and health policy have resulted in growing of older population, with a concurrent rise in multimorbidity, particularly in Iran, as a country transitioning to a western lifestyle, and in which the percent of the population over the age of 60 years is increasing. This study aims to assess multimorbidity and the associated risk factors in Iran.We used data from 50,045 participants (age 40–75 y) in the Golestan Cohort Study, including data on demographics, lifestyle habits, socioeconomic status, and anthropometric indices. Multimorbidity was defined as the presence of 2 or more out of 8 self-reported chronic conditions, including cardiovascular diseases, diabetes, chronic obstructive pulmonary disease, chronic kidney disease, liver disease, gastroesophageal reflux disease, tuberculosis, and cancer. Multivariate logistic regression models were used to examine the associations between multiple different factors and the risk factors.P < 0.001), and 2.11-fold higher in women than in men (P < 0.001). Other factors associated with higher risk of multimorbidity included non-Turkmen ethnicity, low education, unemployment, low socioeconomic status, physical inactivity, overweight, obesity, former smoking, opium and alcohol use, and poor oral health.Multimorbidity prevalence was 19.4%, with the most common chronic diseases being gastroesophageal reflux disease (76.7%), cardiovascular diseases (72.7%), diabetes (25.3%), and chronic obstructive pulmonary disease (21.9%). The odds of multimorbidity was 2.56-fold higher at the age of >60 years compared with that at <50 years (Apart from advanced age and female sex, the most important potentially modifiable lifestyle factors, including excess body weight and opium use, and opium user, are associated with multimorbidity. Policies aiming at controlling multimorbidity will require a multidimensional approach to reduce modifiable risk factors in the younger population in developing countries alongside adopting efficient strategies to improve life quality in the older population. On the basis of estimates of the World Health Organization (WHO), by 2050, Iran, as a developing country, will have a greater proportion of older individuals (8.4%–29.4%) than the United States (20.1%–27%).6In most countries throughout the world, particularly in the developing countries, the proportion of the older people in the overall population is growing rapidly. This is a result of advances in medical science, technology, and health policy, resulting in a longer life expectancy and a decline in fertility rates.4 The advances in health status of the Iranian population have transformed the most common diseases, from fatal acute diseases into survivable chronic conditions.7 The aging of the population will increase the absolute number of chronic conditions, causing people to suffer from multimorbidity, defined as the simultaneous occurrence of 2 or more chronic health disorders in the same person at one point in time.10 A systematic review of 21 studies showed that the estimated prevalence of multimorbidity varies widely in developed countries; it has been estimated that 1 in 4 adults in the developed countries experiences multimorbidity, and 50% of the older people have 2 or more chronic diseases.13 Data on multimorbidity from low and middle-income countries are sparse, and information about the health of older individuals in these regions is greatly needed. Studies predict that in 2050, 21.1% of world residents will be over 60 years old, and 80% of this group will live in low and middle-income countries.14In Iran, the aging population may be regarded as an indicator of successful population, and public health policies and socioeconomic development.16 Because of the increasing importance of multimorbidity, we conducted a cross-sectional analysis of baseline data from the Golestan Cohort Study (GCS)—a large-scale study in a high incidence area of esophageal cancer in northern Iran17—to identify the prevalence and risk factors of multimorbidity in Iran as a middle-income country that may differ from those observed in developed countries.Multimorbidity is associated with higher mortality risk, functional disabilities, deterioration of quality of life, greater use of multiple medications with associated adverse effects, more frequent and longer hospitalization, and higher healthcare utilization and expenses.18 Baseline data were collected during 2004 to 2008, and total enrollment included 50,045 adults aged 40 to 75 years residing in the Golestan Province, in northern Iran. Of the total participants, 49,946 (99.8%) were of Turkmen, Persian, Turkish, Sistani, Baluch, or Kurdish ethnicities; the other 99 enrollees were foreign nationals, who were excluded from this study.The study was carried out as a cross-sectional analysis of baseline information from the GCS. Details of the cohort enrollment are described elsewhere.Trained physician and nonphysician interviewers administered different parts of a lifestyle questionnaire to each participant in face-to-face interviews to collect information on age, sex, ethnicity, marital status, years of education, employment status, ownership of several appliances, physical activity, body mass index (BMI), smoking, opium and hookah (water pipe) use, alcohol consumption, and Decayed, Missing, Filled Teeth (DMFT) index. For each interviewee, a short physical examination was performed, and blood pressure, height, and weight were measured by trained general physicians.2), normal (BMI 18.5–24.9 kg/m2), overweight (BMI 25–29.9 kg/m2), and obese (BMI >30 kg/m2). In this study, age at the time of interview was clustered as ≤49, 50 to 60, and >61 years. On the basis of the 2-step cluster analysis with the use of similarities of family asset, ethnicity, sex, employment status, age at starting the first job, size (surface area) and the status of house, age, and the status of house, we categorized the socioeconomic status (SES) of participants as low, middle, and high.20In this mainly rural population, most of the physical activities performed by the participants were related to their jobs. Therefore, the physical activity was defined based on occupational activity, and coded as yes (heavy and intense activity) or no . BMI was calculated using the WHO-recommended classification: underweight , including hypertension, coronary heart disease and stroke, diabetes, chronic obstructive pulmonary disease (COPD), chronic kidney disease (CKD), liver disease, gastroesophageal reflux disease (GERD), tuberculosis, and cancers. We chose these diseases because they are frequently observed and have a major impact on health status, quality of life, and mortality in this population.30Multimorbidity in this study refers to the presence of 2 or more chronic diseases.The study was approved by the ethics committee of the Digestive Diseases Research Institute, Tehran University of Medical Sciences (OHRP-IRB-00001641). All participants signed an informed consent during original cohort study allowing investigators to use their anonymized data for further analysis.t tests, Mann–Whitney or chi-square tests, whenever appropriate. To examine the simultaneous effects of different factors and to calculate adjusted prevalence odds ratios (ORs) and 95% confidence intervals (CIs), we used multivariate logistic regression models; cluster effects were evaluated using the generalized estimating equation (GEE) method. All statistical analyses were performed using SPSS . Two-sided P values below 0.05 were considered statistically significant.We estimated the age and sex-standardized proportion of multimorbidity among participants by several sociodemographic and lifestyle factors. To evaluate the differences in distribution of multimorbidity by continuous or categorical factors, we used 2 or higher in 59.4% of the individuals, and 17% had used opium , rural residents (76.8%), and of Turkmen ethnicity (74.6%). BMI was 25 kg/mP < 0.05) . The most common chronic diseases reported by those with multimorbidity were GERD (76.7%), CVD 72.7%), diabetes mellitus (25.3%), and COPD (21.9%). In multivariate models, older people, women, non-Turkmens, unemployed people, opium users, alcohol users, and those with low education, low physical activity, low SES, high BMI, and high DMFT  were at a higher risk of multimorbidity  and 2.11-fold higher in women than in men (P < 0.001). The odds of multimorbidity also increased significantly with higher BMI. For obese participants, the odds was 2.33-fold higher than those in the normal BMI group. Also, the risk of multimorbidity was 1.73-fold higher in opium users than in nonusers (P < 0.001).The multivariate logistic regression models also showed that older age groups and women were at a higher risk of multimorbidity. The odds of multimorbidity was 2.56-fold higher in age groups above 60 years compared with those below 50 years , and the applied statistics procedures seem to be responsible for the majority of this variation.32 and Phaswana-Mafuya et al (2008) reported a prevalence of 22.5% in individuals aged 50 years or above in South Africa.34 On the other hand, prevalence of multimorbidity in the general adult population reported by Loza et al (30%),35 Agborsangaya et al (31%)36 and Foguet-Boreu et al (46.8%)8 in developed countries were considerably higher than the prevalence that we found. Also, in a study by Nagel et al (2008), the prevalence of multimorbidity was 67.3% in the 50–75-year-old population in a prospective cohort study in Germany,37 which is considerably higher than our findings.The prevalence and risk factors of multimorbidity have usually been studied in high-income countries. To our knowledge, this is the first study of this subject conducted in Iran as a developing country. Our findings reveal that multimorbidity is a common phenomenon, with a prevalence of 19.4%. Similar to our study, Agborsangaya et al (2012) showed that the prevalence of multimorbidity was 19.0% among adults in the general population in Canada,38 The prevalence of GERD has been increasing in Iran in recent years.39 Our study showed that GERD as a possible risk factor of esophageal carcinoma was common in Golestan province, a high incidence area of esophageal carcinoma, which is in line with significant correlations of multimorbidity with lifestyle factors and SES. Lifestyle and sociodemographic factors such as BMI, intensity of physical activity, smoking, opium use, irregular dietary habits and low SES are all associated with GERD.38 CVD, diabetes mellitus, and COPD were also common among participants in the GCS. Many of the above lifestyle and sociodemographic factors are associated with these conditions. To some extent the coincidence of GERD and CVD may be due to the high frequency of both of these conditions in the area. Also, some evidences indicate that common inflammation pathway may be involved in the pathogenesis of GERD and following cardiovascular events.42GERD is a common upper gastrointestinal disorder worldwide.44 Some studies showed that obesity was associated with at least a two-fold increased likelihood of having multimorbidity.36 This association may be explained by the fact that obese and overweight individuals are at increased risk of health problems and diseases such as coronary heart disease, certain cancers, diabetes, and pulmonary diseases.44Our findings are consistent with previous studies, which have indicated that older age, obesity and female gender are associated with a higher prevalence of multimorbidity. The association between mulimorbidity and age is an expected finding, as most biological pathways leading to chronic diseases need a long time to affect individuals.46In our study population, opium consumption was common (17%) and significantly increased the risk of multimorbidity. Other studies showed that opium users had an increased risk of death from multiple causes, including circulatory diseases, cancer, asthma, tuberculosis, and COPD.47 This may be because people stop smoking after the symptoms of the related diseases appear.48Former smoking was a risk factor for multimorbidity in this study (OR of 1.36 compared with never smokers), while heavy smokers had a lower relative odd (0.89) for multimorbidity. Similar to our study, Chung et al (2015) showed that being past daily smoker was a significant risk factor for multimorbidity.16 The result in our study can be explained by the fact that the majority of the study participants were rural residents with relatively comparable SES. In addition, the number of people with a lower SES who knew about their nonsymptomatic medical conditions might be lower than those with a higher SES who might have a higher access to care.Regarding the indicators of SES in this study, after adjusting for other lifestyle and socio-demographic factors, the odds of multimorbidity was only 12% higher in the low SES group compared with the high SES group. This is contrary to some studies which found that multimorbidity was much more common in individuals of lower SES.49 Demographic studies indicate that this population will rise to over 14% in the near future.50 A similar pattern is observed in other developing countries.51 Women have a longer life expectancy than men,52 and prevalence of multimorbidity increases with advancing age, especially in women, so the effects of population aging on multimorbidity should be more pronounced in women living in developing countries. GERD, CVD, diabetes and COPD are the most common diseases in this region, so it is important to plan for high burden of these diseases, which will be soon imposed on the healthcare system.During recent years, the population of Iranian adults above 60 years of age has increased to approximately 8%, similar to many developing countries.53 This may be related to some extent to diabetes care in these countries. For instance Glycated hemoglobin testing as an index for diabetes surveillance was 72% in USA in comparison to 6.3% in Iran.53As a limitation, in interpreting our results, the differences in the quality of disease and death registration systems between developed and developing countries (i.e. Iran) should be taken into consideration. The lower rates of mortality and DALY (disability-adjusted life years) related to diabetes in eastern Mediterranean countries including Iran may be related to the lower quality of death and disease registration. Despite the almost similar diabetes mellitus prevalence in developing and western countries, the complications like renal disease were lower.55 This study also has certain limitations, including its cross-sectional design and the potential for residual confounding and validation of self-report of diseases.The major strengths of this study are the use of data from a large, population-based cohort study running in a developing country including different races. As other strengths are the high participation rate and the availability of detailed data on sociodemographic and lifestyle factors. Also, biomarker studies, including comparison of opium intake data against codeine and morphine levels in urine samples, had shown the validity of the questionnaire for measuring exposure to some major risk factors.Our study highlights the importance of multimorbidity in the Iranian population who experience an epidemiologic transition similar to many developing countries, and the importance of recognizing its risk and protective factors, especially in adults and the elderly. The findings of this study indicate that in addition to advanced age and female gender, some lifestyle factors, including being overweight, physical inactivity, and opium and tobacco use, are associated with multimorbidity. Policies aiming to control multimorbidity will require a multidimensional approach, especially in developing countries, to reduce modifiable risk factors in the younger population and adopt efficient strategies to improve quality of life in the adult and elderly population.


Statins are commonly used against arteriosclerotic disease, but recent retrospective analyses have suggested that statins also prevent cancer. The aim of this systematic review is to verify the vitro anti-tumor effects of statins on head and neck squamous cell carcinoma.Studies were gathered by searching Cochrane, MEDLINE, EMBASE, LILACS, and PubMed, up until May 9, 2015, with no time or language restrictions. Only in vitro studies that discuss the effect of statins on head and neck carcinoma were selected.Of 153 identified papers, 14 studies met the inclusion criteria. These studies demonstrated that statins had a significant effect on head and neck squamous cell carcinoma cell lines and influenced cell viability, cell cycle, cell death, and protein expression levels involved in pathways of carcinogenesis, which corroborates with the potential in vitro anti-tumor effects. It provides highlights about the biological mechanisms of statins used alone or associated with traditional therapy for cancer.Though there are few studies on the topic, currently available evidence suggests that statins shows that preclinical experiments supports the potentiality of statin as an adjuvant agent in chemotherapy and/or radiotherapy approaches routinely used in the management of HNSCC and should undergo further clinical assessment. The statin family of drugs is known worldwide as a safe and effective therapeutic agent for the treatment of arteriosclerotic cardiovascular disease . StatinsStatins have demonstrated an ability to enable different tumor induction pathways, mediated by metabolic stress that regulates tumor cell apoptosis. By inhibiting the mevalonate pathway, statins can inhibit the function of epidermal growth receptor (EGFR), which inhibits the mammalian target of rapamycin (mTOR) cascade and the phosphoinositide 3-kinase P13K/AKT) pathway ,11. AddiK/AKT patThe Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) Checklist was followed in this systematic review . We did We selected only articles that compared the effect of statins to control substances in the context of squamous cell carcinoma treatment. The cell lines used should be from head and neck squamous cell carcinoma (HNSCC), such as cells from lip and/or oral cavity, pharynx, larynx, nasal cavity, and paranasal sinuses . All of Population: Cells or animal.Intervention: Statin use for prevention or treatment of HNSCC.Comparison: Cells or animals that did not receive statin treatment but have received a control treatment.Outcome: Cell viability, apoptosis, cell cycle arrest, and regulation of protein expression levels.Study Design: Randomized or non-randomized controlled trials  or studies with comparable or no comparable baseline (in vitro studies).We have excluded: 1) Studies with different targets, such as ones that did not use statins to treat cancer or did not verify the association between statins and HNSCC; 2) Reviews of the literature, letters, case reports, personal opinions, conference abstracts, and book chapters; 3) Clinical studies.A criteria search was performed using the following electronic bibliographic databases: Cochrane, MEDLINE, EMBASE, LILACS and PubMed. More information regarding the search strategies is provided in We selected articles in two phases. In phase one, two authors  independently reviewed the titles and abstracts of all the references. The articles that appeared to meet the inclusion criteria based on their abstract were selected and collected. In phase two, two authors (L.M.C.P and E.N.S.G) read all the full-text articles and excluded those which were not in agreement with the inclusion criteria. The same two authors independently reviewed all full-text articles. Any disagreement between the authors in the first and second phases was resolved by means of discussion and mutual agreement. When they did not reach a consensus, a third author (S.T.E) intervened to make a final decision.One author (L.M.C.P) collected the required information from the selected articles such as authors, year of publication, country, study design, assays, treatment used, results, and main conclusions . A seconThe authors methodically appraised all of the selected studies according to the GRADE method to judge the quality of evidence . Two autCell viability, apoptosis, and cell cycle arrest after statin treatment and regulation of protein expression level in HNSCC were the main evaluated outcomes.A meta-analysis was planned, since the data from the included studies was considered relatively homogeneous.Only to be applied if meta-analysis was possible.In phase one, 153 papers were selected from five electronic databases. After the duplicates were removed, only 119 different citations remained. Subsequently, we conducted a comprehensive evaluation of the abstracts and excluded 101, resulting in 18 articles at the end of phase one. We did not identify any additional studies from the reference lists of these studies. Subsequently, we retrieved the 18 articles, and following a review of the texts, four more articles were discarded . A flow The studies were published from 2001 to 2014, and all of them were in English. They were conducted in five different countries: Canada , 20–23, We used the GRADE method  to assesWe also evaluated the evidence regarding the possible clinical application of statins based on the in vitro studies found in this search and classified each paper as either: (1) showing a potential effect following HNSCC treatment; (2) inconclusive and (3) evidence not supportive of using statins to treat HNSCC .The cytotoxicity of statins in HNSCC cells lines was evaluated using MTT , 27, 28 Some studies , 25, 27 Islam et al.  in 2013 +/+ murine embryonic fibroblasts (MEFs) as a control, which was compared to LKB1-/- treated with a range of lovastatin concentrations of up to 25 μM for 48 and 72 hours. Their data showed that LKB1+/+ cells (20% cell viability after 25 μM dose) were significantly more sensitive than LKB1-/- cells (90% cell viability in 25 μM) after 48 hours. After 72 hours, there was no appreciable difference in the cytotoxicity in these two sets of MEFs (20% cell viability after 25 μM), and both cell lines responded in a dose dependent manner. Lovastatin-induced apoptosis is regulated in part by the inhibition of the EGFR. Western blot analysis of SCC25 cells treated for 24 hours showed that lovastatin regulates downstream targets of EGFR (pAKT), the Integrated Stress Response (ISR), and cell cycle regulators, cyclin D1 and p21, and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1).Ma et al.  in 2012 -/- and CHOP +/+ MEFs after 0–25 μM doses and 48 hours. Statistically significant differences were found according to a one-tailed t-test (p < 0.05). A significant difference was observed in the populations of sub G1 apoptotic cells in CHOP-/- and CHOP +/+ MEFs that were treated with 10 μM doses for 24 and 48 hours (p < 0.05).In 2007, Niknejad, Morley, and Dimitroulakos  used a cThe work of Takeda et al. , in 2006Llobet, et al, in 2014  demonstrDayekh et al,  used monThe studies selected for this analysis were considered heterogenous, and they did not have compatible data that would allow a meta-analysis. In addition to the non-comparability of the results of each study, a meta-analysis could not be conducted due to lack of clinical studies on this subject.The incidence of HNSCC has been on the increase and has become an important issue . Oral caThe statins are a remarkably effective class of drugs that lower cholesterol levels in blood and reduce the frequency of heart attacks . No majoThere has been a growing interest in statins because of their anticancer effects . A systeFurthermore, several studies have tested statin-derived drugs  on HNSCC cell lines , 22, 27.Llobet et al., in 2014,  showed tde novo cholesterol and isoprenoids [Derivatives of the statin drugs are potential inhibitors of HMG-CoA reductase, an enzyme derived from the mevalonate pathway , 11, 36.prenoids , 6, 36. prenoids . Simvastprenoids . In vitrprenoids . In contprenoids .Statins have been shown to inhibit the mevalonate, PI3K/AKT, and mTOR pathways in oral cancer , 8, 29. The tumor suppressor protein p21 has been shown to inhibit tumor proliferation and cell-cycle progression, which may contribute to tumor suppression . One stuIn summary, this is the first systematic review of the effects of statins on HNSCC treatment and the first review to present evidence of a positive association between decrease of HNSCC cell viability and statin using in vitro assays. Although, in 2006, Dale et al.  performeThese in vitro studies , 20–27 cSome methodological limitations of this review should be considered. We found only one clinical study that used statins in patients with HNSCC. Of 153 papers, we selected 14 studies using our inclusion criteria and they were in vitro and in vivo animal studies. For the quality assessment of in vitro studies, we did not use any standard assessment for basic studies; however, we defined a method to assess the quality of all articles. Thereby, we have classified in vitro studies using comparable baselines, comparing them to the RCT studies published by Xiao et al. in 2013  using thThis study demonstrated that statins have a significant effect on HNSCC cell lines with respect to cell viability, cell cycle, cell death, and the regulation of protein expression levels involved in pathways of carcinogenesis, which corroborates with the potential in vitro anti-tumor effects. Though there are few studies on the topic, currently available evidence suggests that statins are potentially useful for HNSCC treatment, and their use requires further clinical analysis.S1 PRISMA Checklist(DOC)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.