L-ascorbic acid (AsA) content of edible crops. One main objective is to establish whether in sink organs such as fruits and tubers, AsA is synthesised in situ from imported photoassimilates or synthesised in source tissues and translocated via the phloem. In the current work we test the hypothesis that long-distance transport is involved in AsA accumulation within the potato tuber, the most significant source of AsA in the European diet.Following on from recent advances in plant AsA biosynthesis there is increasing interest in elucidating the factors contributing to the 3 staining is suggestive of symplastic unloading of AsA in developing tubers. This hypothesis was further supported by the changes in AsA distribution during tuber development which closely resembled those of imported photoassimilates. Manipulation of leaf AsA content by supply of precursors to source leaves resulted in increased AsA content of developing tubers.Using the EDTA exudation technique we confirm the presence of AsA in the phloem of potato plants and demonstrate a correlation between changes in the AsA content of source leaves and that of phloem exudates. Comparison of carboxyflourescein and AgNOOur data provide strong support to the hypothesis that long-distance transport of AsA occurs in potato. We also show that phloem AsA content and AsA accumulation in sink organs can be directly increased via manipulation of AsA content in the foliage. We are now attempting to establish the quantitative contribution of imported AsA to overall AsA accumulation in developing potato tubers via transgenic approaches. L-Ascorbic acid (AsA), the reduced form of vitamin C, is an essential antioxidant for many biological systems and must be obtained via the diet by humans, primates and a few other animals which are unable to synthesise AsA endogenously [Arabidopsis thaliana AsA-deficient mutants (vtc1) which were defective in the activity of a pathway enzyme [A. thaliana mutants have been identified which do not seem to be affected in any of the known pathway genes [genously . The maigenously . The oriy enzyme . Since tay genes ,5. Additay genes -9. This ay genes ,9,10.Arabidopsis thaliana [Mirciaria dubia) [in situ or import from the foliage. Recently Franceschi and Tarlyn [14C-AsA from leaves to flowers and root tips in model systems such as A. thaliana and Medicago sativa. Research in our laboratory demonstrated the occurrence of AsA in the phloem of a number of crop plants and we also observed that the plant phloem was capable of supporting active AsA biosynthesis from a number of precursors [Many of the recent advances in plant AsA biosynthesis have been obtained through investigations of model systems such as tobacco or thaliana ,3,9,11. thaliana  remains thaliana . We havea dubia)  or less a dubia) . Large va dubia)  whilst da dubia) ]. These d Tarlyn  observedecursors . We haveecursors  and potaecursors  when sinecursors . Taken tIn this work we have extended our investigation on AsA accumulation in potato tubers, the main source of vitamin C in the European diet . We pres2. Total AsA  appeared as the largest peak of absorbance at 245 nm retained by the column in both cases. When exudates were collected in the presence of CaCl2 instead of EDTA, all exudate derived peaks were strongly reduced due to the reduction in exudation caused by callose gelation [2 exudation buffers less than 5% oxidation was observed in either case over 90 min (data not shown). AsA localisation to the vascular tissue was confirmed histochemically in sections of potato stems and tubers incubated with ethanolic AgNO3 at 3°C  [Fig. t shown) .t distribution along the axis of stolons and developing tubers. In non-swelling stolons the AsAt content was maximal in the apical section with a sharp basipetal decline such that the more basal sections contained approximately 10% of the AsAt content found in the apex. Tuberising stolons showed a 50% reduction in the AsAt content of the apical section. This was accompanied by increases in the subapical 3–10 mm sections. In developing tubers there was a very substantial increase in the AsAt content in the sub-apical region, corresponding to the swelling area.Fig. t content of source leaves of potato plants acclimatised for 14 days in cabinets with artificial diurnal dark/light cycles. The cabinets were off-phased so that plants at different stages in their lighting regime were available at any given time. The AsAt content of source leaves was monitored for 24 h in plants sampled simultaneously from the two cabinets. At the end of the dark phase, leaf AsAt content was approximately 12 mg/100 gFW and it progressively increased following artificial sunrise. After 10 h of light, the leaf AsAt content increased to approximately 30 mg/100 gFW after which it leveled off. Following artificial sunset the AsAt level gradually fell back to approximately 12 mg/100 gFW within 6–10 h.Figure t content of source leaves and tuberising stolons collected at 12.00 h from the light-phase or dark-phase plants (after collection of phloem exudate for 90 min in a prehumidified atmosphere in leaves). In the table are also reported the chromatographic AsAt peak areas from phloem exudates collected from source leaves and tuberising stolons of the same plants. The exudate data are reported as peak areas as the exact volume of exudate collected could not be established. Whilst the leaves from light-phase plants contained over twice the AsAt level of leaves from dark-phase plants, no significant difference was found in the AsAt content of tuberising stolons from the two sets of plants. The AsAt peak area in chromatograms of light-phase leaf exudates was 1.8-fold larger than that obtained from dark-phase leaves. In tuberising stolons the increase in exudate peak area was more pronounced (4.6-fold).Table t content and the AsAt levels of phloem exudates was also investigated in glasshouse-grown plants following the supply of a range of AsA biosynthetic precursors using the flap technique [t peak area of exudates from leaves pre-treated for 24 h with 20 mM MES pH 5.5, 2 mM CaCl2 alone (control) or containing precursors at a final concentration of 25 mM. Incubation with D-glucose (D-Glc) resulted in a slight (10%) reduction in leaf AsAt content compared with the control but no significant change in AsAt was detected in the exudates. By contrast, supply of L-galactose  or L-galactono-1,4-lactone  increased the AsAt content of leaves (4.9 and 6.2-fold respectively) and, more substantially, of exudates (10.8 and 11.2-fold respectively). With all treatments, the replacement of EDTA with CaCl2 in collection wells resulted in a significant reduction of AsAt peak area in the exudate chromatograms.The correlation between leaf AsAechnique . Figure t content in the totality of source foliage of whole plants was obtained through the supply of the direct AsA precursor L-GalL via the flap technique for 24 h to all terminal leaflets of the four lowermost nodes. At the end of the incubation period the AsAt content of source leaves was 2.2-fold higher than in control plants  and, more substantially in developing tubers (80%) compared with the control. No changes in AsAt levels were observed in petioles, stems or non-tuberising stolons.An artificial increase of AsAnts Fig. . AsAt co3 at low temperature with ensuing formation of metallic silver deposits [14CO2 [t distribution along the stolon axis following tuber induction which closely resemble the changes in sucrose content and radiolabelled assimilate distribution observed in plants labelled with 14CO2 [t in the apices of tuberising stolons accompanied by its accumulation in the subapical region may thus reflect the induction of symplastic phloem unloading in this region resulting in a distal migration of sink activity from the apex. However, this pattern also correlates with changes in the mitotic index along the axis as a result of tuber induction with cessation of cell division in the apical region of the stolon [We have previously shown that AsA represented the largest peak of absorption at 245 nm retained by the column when phloem sap of potato source leaves obtained by aphid stylectomy was analysed by HPLC . In the deposits . In partdeposits  and reses [14CO2 . This was [14CO2 , thus thth 14CO2 . The dece stolon  and its e stolon . This mae stolon .t content of mature source leaves of potato. Circadian or diurnal oscillations of AsA content in photosynthetic tissues are known [e.g. [t content occurred. Leaf AsAt content showed diurnal changes with the maximal levels (observed during the light-phase) 2-fold higher than the lowest levels observed in the dark. Phloem exudates obtained from source and sink organs of light phase plants always showed significantly higher AsAt levels than exudates obtained from dark phase plants. Indeed, the AsAt content of exudates from tuberising stolons of light phase plants was over 4-fold higher that of dark-phase plants. Our findings indicate that changes in source leaf AsA biosynthesis rapidly impact on the phloem AsAt content resulting in transport of de novo synthesised AsA directly to developing sinks. This was also confirmed in experiments where exogenous AsA precursors such as L-GalL or L-Gal were supplied to source leaves via the flap technique, a treatment which resulted in substantial AsAt enrichment in phloem exudates. We were also able to more than double the AsAt content of the source foliage of whole plants by "bulk" supply of exogenous L-GalL to the majority of source leaves for 24 h. This resulted in significant AsAt increases in sink organs such as flowers and developing tubers.In preliminary experiments, we observed diurnal changes in the AsAwn [e.g. ] as wellwn [e.g. ] and sinwn [e.g. ] as wellwn [e.g. . By growArabidopsis genome. NATs belong to a superfamily of integral membrane transporters, which move purines, pyrimidines or ascorbic acid across biological membranes and which also comprises mammalian transporters specific for ascorbate [L-Gal across membranes. We show here that exogenous supply of L-Gal or L-GalL to source leaves leads to AsA enrichment of phloem exudates and AsA accumulation in tuberising stolons and developing tubers. Furthermore, we have observed uptake of L-[1-14C]Gal by source phloem of potato (data not shown), as already demonstrated in N. benthamiana [To our knowledge this is the first demonstration that AsA accumulation in the foliage results in AsA increases in storage organs and it may explain the positive effect of light irradiation on AsA content of fruits and vegetables . The rapscorbate . Functioscorbate . As the scorbate , we havethamiana .A. thaliana and M. sativa [in vitro from nodal cuttings cultured in the dark with sucrose as the sole carbon source contain similar amounts of AsA as field-grown tubers [The evidence we provide here for long-distance transport of AsA in potato plants, corroborates earlier findings in . sativa  and suggn tubers . These l2 s-1 at the top of the canopy. Relative humidity was maintained at a constant 70% and plants were watered daily. In all cases experiments were performed on tuberising plants after 40–60 days from planting. Throughout the text stolons are defined as non-swelling  or tuberising (swelling 2–5 mm diameter). Swellings between 5–10 mm diameter are defined as developing tubers.Potato plants cv. Desiree were grown in 40 cm pots in unheated glasshouses under natural light in compost. In order to subject plants to artificial light/dark cycles, plants were transferred after 35 days to Sanyo Fitotron 1700 controlled environment cabinets and maintained for a further 14 days on 14 h–10 h light-dark cycles with day and night temperatures of 22°C and 15°C respectively. Light was provided by 60 W incandescent lamps to provide a photon flux of 900 μmol mt) i.e. reduced L-ascorbic acid + dehydroascorbic acid. Samples were then centrifuged at 16000 g for 5 min at 1°C and AsAt in the supernatant quantified by HPLC according to the method of Hancock et al [+ guard cartridge (Phenomenex, Macclesfield, UK) maintained at 50°C. Mobile phase was 8 mM H2SO4 at 0.6 ml min-1 and AsAt was detected at 245 nm using a Gynkotech UVD 340S diode array detector (Dionex, Camberley, UK).Tissue was extracted in a mortar and pestle with ice-cold 5% metaphosphoric acid (MPA) containing 5 mM tris(2-carboxyethyl)phosphine hydrochloride TCEP (9:1 v/w). Samples were then held on ice for 60 min to allow reduction of dehydroascorbic acid to AsA therefore, all data are reported as total AsA pool  aqueous ethanol containing 5% (v/v) glacial acetic acid at 3°C in the dark for up to 24 h. The reaction was stopped by washing the tissue twice for 15 min in ethanolic ammonium hydroxide (95% (v/v) 70% ethanol, 5% (v/v) NH4OH ACS reagent, Sigma-Aldrich, Dorset, UK) [Phloem exudates were collected from the petiole of source leaves or tuberising stolons using an adaptation of the method developed by King and Zeevart . Followixudation . At the escribed . Brieflyset, UK) . Finally-1) introduced via open stomata on the abaxial leaf surface using a plastic syringe. The acetylated compound is able to diffuse across cell membranes unlike its deacetylated derivative CF which is produced in vivo by endogenous esterases and is used as a marker for phloem strands and symplastic unloading from the phloem [Phloem transport through potato stems into developing tubers was visualised using the fluorescent transportable molecule carboxyflourescein (CF) as previously described . Plants e phloem . Plants t levels were manipulated using an adaptation of the 'flap' technique [2 alone or with the addition of various intermediates at a final concentration of 25 mM for 24 h. At the end of the incubation period, leaflets were ground in liquid nitrogen and extracted in 5% MPA containing 5 mM TCEP (9:1 v/w) and the AsAt content measured by HPLC. For measurement of phloem exudates, treated leaflets were excised under water and placed in eppendorf tubes for collection of phloem exudates as described previously. In some experiments the terminal leaflets of the four lower nodes on all stems (between 8 and 10 per plant) were simultaneously supplied with 500 μl 20 mM MES pH 5.5, 2 mM CaCl2 alone or containing 25 mM L-GalL for 24 h. Four independent plants were used for each treatment. At the end of the incubation individual plants were separated into flowers, source leaves, leaf petioles, plant stems, non-tuberising stolons , or tuberising stolons (swelling portion). Tissues were immediately frozen in liquid nitrogen and lyophilised. Lyophilised tissue was ground to a powder and 3 × 1 g fractions of each tissue were extracted in 5% MPA, 5 mM TCEP (19:1 v/w) and the AsAt content of each extract analysed by HPLC to give an average value for each tissue. No developing tubers larger than 5 mm diameter were present in the plants.Leaf AsAechnique . An inciLT undertook most of the physiological and biochemical experiments. RDH participated in the design and coordination of the study, the biochemical experimentation and the writing of the manuscript. SA participated in the physiological, biochemical and histochemical experiments. SH undertook the CFDA labeling and silver staining of potato plants and participated in production of the figures. RV conceived the study, participated in its design and coordination and drafted the manuscript.


This study describes the influence of educational level on bone mineral density (BMD) and investigating the relationship between educational level and bone mineral density in postmenopausal women.A total of 569 postmenopausal women, from 45 to 86 years of age (mean age of 60.43 ± 7.19 years) were included in this study. A standardized interview was used at the follow-up visit to obtain information on demographic, life-style, reproductive and menstrual histories such as age at menarche, age at menopause, number of pregnancies, number of abortions, duration of menopause, duration of fertility, and duration of lactation. Patients were separated into four groups according to the level of education, namely no education (Group 1 with 209 patients), elementary (Group 2 with 222 patients), high school (Group 3 with 79 patients), and university (Group 4 with 59 patients).The mean ages of groups were 59.75 ± 7.29, 61.42 ± 7.50, 60.23 ± 7.49, and 58.72 ± 7.46, respectively. Spine BMD was significant lower in Group 1 than that of other groups (p < 0.05). Trochanter and ward's triangle BMD were the highest in Group 4 and there was a significant difference between Group 1 and 4 (p < 0.05). The prevalence of osteoporosis showed an inverse relationship with level of education, ranging from 18.6% for the most educated to 34.4% for the no educated women (p < 0.05). Additionally, there was a significant correlation between educational level and spine BMD (r = 0.20, p < 0.01), trochanter BMD (r = 0.13, p < 0.01), and ward's BMD (r = 0.14, p < 0.01).The results of the study suggest that there is a significant correlation between educational level and BMD. Losses in BMD for women of lower educational level tend to be relatively high, and losses in spine and femur BMD showed a decrease with increasing educational level. Osteoporosis has recently been recognized as a major public health problem by some governments and health care providers. In the European community, the number of men and women aged 65 years of older will increase steadily and the most dramatic changes will occur in the very elderly, in whom the incidence of osteoporotic fracture is greatest . As the Peak bone mass is achieved soon after puberty, and bone is lost with various "insults", including ageing and postmenopausal changes. Factors influencing peak bone mass and loss range from nutrition, to lifestyle, to certain medical disorders. Educational level may also have an effect on bone mineral density since there is relationship between educational level and reproductive factors such as pregnancy and lactation and other lifestyle factors -7.In developed countries a higher prevalence of most chronic diseases has been recognized among lower socio economic levels and in less educated subjects -14; howeSince many risk factors for osteoporosis, such as diet, deficiency of trace minerals, reproductive factors, inactivity and tobacco use, are lifestyle variables related to social and cultural background -25, the Patients were compared according to years of formal education. We used formal education because it may be regarded as a composite or surrogate variable for overall socioeconomic status , and levPurpose of this study was to evaluate the influence of formal education on BMD and investigating the relationship between educational level and bone mineral density in the postmenopausal women.In Department of Physical Medicine and Rehabilitation, of the total 701 consecutive women screened, 132 were excluded. This study was undertaken in Dicle University, Diyarbakir, Turkey. The study protocol was reviewed and approved by the Dicle University Ethics Committee, and informed consent was obtained from all participants. A detailed history was taken from each woman including relevant life-style parameters and risk factors, and their weight and height measurements were recorded. The following exclusion criteria were applied for further analyses: (1) fractures after the age of 25; (2) menopause before the age of 40; (3) amenorrhoea greater than 6 months; (4) chronic conditions affecting bone density; (5) any use of corticosteroids.2 [A total of 569 postmenopausal women, at 45 to 86 years of age (mean age of 60.43 ± 7.19 years) were considered. BMD of the spine and hip (neck, trochanter, and ward's triangle) were measured by dual-energy x-ray absorptiometry (NORLAND, 6938CE, New York, USA). According to the WHO  osteopor2 . The varIn order to standardize the procedure, the patients all answered the same specially developed questionnaire supervised by the doctor (revised from the MEDOS Form) . A stand2) was obtained through height and weight measurements by using a wall-mounted ruler and a digital scale.The level of education is categorized in four groups according to the number of school years and the highest qualification received; no education (Group 1 = 209 patients), elementary (8 years or less, Group 2 = 222 patients), high school (9–11 years, Group 3 = 79 patients), and university (12 years or more, Group 4 = 59 patients). Body mass index  was assessed using standardized food models to estimate portion sizes . DietaryThe statistical analyses were carried out with the SPSS/ PC-program. Differences in proportions for categorical variables were tested by chi-square test. The data are expressed as means ± SD. Statistical significance was tested using one way-ANOVA test and post-hok Bonferroni test for comparison of different groups. Pearson correlation test were computed to measure the association between the variables studied. The statistical significant set if the p-value was less than 0.05.Their reproductive and demographic characteristics are shown in Table Number of abortions was higher in group 1 and 2 than those of group 3 and 4 (p < 0.05). There was no significant difference with respect to number of pregnancies and duration of lactation between group 3 and 4 while there was a significant difference among other groups (p < 0.05), and number of pregnancies and duration of lactation were found to be the highest in Group 1 and 2. Number of pregnancies and duration of lactation in Group 1 were 7.11 ± 3.38 and 133.23 ± 54.34 months and in Group 2 were 4.93 ± 3.61 and 93.62 ± 50.66 months (Table Spine BMD was significant lower in Group 1 than that of other groups (p < 0.05). Trochanter and ward's BMD were the highest in Group 4 and there was a significant difference between Group 1 and 4 (p < 0.05). The prevalence of osteoporosis showed an inverse relationship with level of education, ranging from 18.6% for the most educated to 34.4% for the no educated women (p < 0.05) , trochanter BMD (r = 0.13, p < 0.01), and ward's BMD (r = 0.14, p < 0.01) but wasn't neck BMD (r = -0.02, p > 0.05).The health care costs, morbidity and mortality excess related to osteoporotic fractures are a major health problem in western countries ,30. In oAlthough mechanisms of association between education and osteoporosis remain partly unexplained, most of the risk factors examined have shown distinct trends according to educational level. Although educational level may be an imperfect measure for socio economic status, many studies have clearly established that this marker acts as a good predictor not only for most chronic diseases ,11,14 buIn a study by La Vecchia et al., they found that education is a strong determinant of several chronic conditions, and the pattern of health care utilization also varied extensively according to education .Varenna et al. evaluated 6160 postmenopausal women referred for their first densitometric evaluation and they found age at menarche, past exposure to oral contraceptives, prevalence of chronic diseases, physical activity, overweight and smoking showed significant trends according to years of education . Also, aThe present study showed that there was no significant difference among all groups with respect to age, BMI, age at menarche, age at menopause, and years since menopause. But there were statistically significant differences among groups in respect to number of pregnancies, duration of lactation, bone mineral density and percentage of osteoporosis.The comparison with studies performed in other countries can be misleading since eating habits are strongly influenced by ethnic and geographical backgrounds . The meaMagnus et al. undertook a random sample of 1514 Norwegian women and men to investigate knowledge of osteoporosis and attitudes towards methods for preventing this disease, and they concluded that in both men and women, increased knowledge of osteoporosis was correlated to a high level of education .In several studies, authors have found that reproductive history has an inverse relation to bone density -6,34-41.Though the effect of formal educational level on bone mineral density has not yet been well established, the above findings may suggest some hypothetical comments. The findings of this study imply that osteoporosis which is related to bone mineral density, may be related to the educational level and the risks due to higher birth rate, excessive breast-feeding and insufficient calcium intake, and may be controlled through an improvement in educational level.Because of several limitations, caution must be exercised in interpreting the results of our study. Except for densitometric assessment, the results depend on self-reports. Even though self-report diagnoses have been shown to be valid , the levSimilar studies are recommended to be carried out in different communities in an effort to confirm whether these findings can be generalized or yield a more complete insight into pathogenetic mechanisms. The knowledge of which population strata may be at greater risk of osteoporosis should be considered carefully for the purpose of health care planning and preventive strategies, making it possible to design tailored and culturally appropriate public health intervention programs.The protective role played by educational level, which increases with the years of formal education, could be due to other overall determinants can be indirectly inferred from our data, such as a better health status, a more positive attitude to taking drugs and a more efficient use of health care resources. All these determinants can be considered in the light of a greater concern by the women about their own health status, probably related to a different impact of health promotion messages.In conclusion, the results of the study suggest that there is a significant correlation between educational level and BMD, and shows differences in the prevalence of osteoporosis among educational classes and the protective role played by increases in formal education. Losses in BMD for women of lower educational level tend to be relatively high, and losses in spine and femur BMD showed a decrease with increasing educational level. Although mechanisms of association between education and low bone mineral density remain partly unexplained, most of the risk factors examined have shown distinct trends according to educational level.None declared.None declared.AG participated in the design of the study and performed the statistical analyses.AJS conceived of the study, and participated in its design and coordination.KN and RC participated in the sequence alignment and screened of subjects.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The objective of this article is to present the frequency of cancer in Mexican children who were treated in the hospitals of the Instituto Mexicano del Seguro Social in Mexico City (IMSS-MC) in the period 1996–2001.The Registry of Cancer in Children, started in 1996 in the IMSS-MC, is an on-going, prospective register. The data from 1996 through 2001 were analyzed and the different types of cancer were grouped according to the International Classification for Cancer in Children (ICCC). From this analysis, the general and specific frequencies by age and by sex were obtained for the different groups of neoplasms. Also, the frequency of the stage of the disease that had been diagnosed in cases of children with solid tumors was obtained.A total of 1,702 new cases of children with cancer were registered, with the male/female ratio at 1.1/1. Leukemias had the highest frequency with 784 cases (46.1%) and, of these, acute lymphoblastic leukemias were the most prevalent with 614 cases (78.3%). Thereafter, in descending order of frequency, were tumors of the central nervous system (CNST) with 197 cases (11.6%), lymphomas with 194 cases (11.4%), germinal cell tumors with 110 cases (6.5%), and bone tumors with 97 cases (5.7%). The highest frequency of cancer was found in the group of one to four year-olds that had 627 cases (36.8%). In all the age groups, leukemias were the most frequent. In the present work, the frequency of Hodgkin's disease (~4%) was found to be lower than that (~10%) in previous studies and the frequency of tumors of the sympathetic nervous system was low (2.3%). Of those cases of solid tumors for which the stage of the disease had been determined, 66.9% were diagnosed as being Stage III or IV.The principal cancers in the children treated in the IMSS-MC were leukemias, CNST, and lymphomas, consistent with those reported by developed countries. A 2.5-fold reduction in the frequency of Hodgkin's disease was found. Of the children, the stage of whose disease had been determined, two thirds were diagnosed as having advanced stages of the disease. The frequency of malignant neoplasms in children has been found to vary among countries. For example, in children in Canada, the United States, and Europe, the three most common cancers are leukemias, tumors of the central nervous system (CNST), and lymphomas -3, whereThe percentage of cases of each type of neoplasm in relation to the total number of cancers is also different. In the developed countries, the percentages for leukemias range between 30 and 37%; for CNST, between 18 and 27%; and for lymphomas, between 7 and 12% -6. In AfThe study of the frequency of cancer in children not only is of interest to the clinical physician because it helps him/her to establish the pre-testing probability for a child suspected of having cancer , but alsIn Mexico, there existed data only from retrospective studies on the epidemiology of cancer in children and those studies had been carried out prior to 1993 . TherefoThe objective of this paper is to present the frequency of malignant neoplasias in the child population residing in the area served by the Instituto Mexicano del Seguro Social (IMSS-MC). The data were obtained from the Registry of Cancer in Children which was started in 1996 at, and is maintained by the Hospital de Pediatría del Centro Médico Nacional "Siglo XXI" of the Instituto Mexicano del Seguro Social in Mexico City (IMSS-MC).Observational, descriptive, and prospective hospital inquiry.The Instituto Mexicano del Seguro Social (IMSS) offers medical attention to the population of workers and their families, which comprises 50% of the Mexican population . The IMSNewly diagnosed cases of malignant neoplasias in children less than 15 years of age treated in the IMSS-MC were included in this study. In 100% of these cases, the diagnosis was confirmed by histological tests and/or by aspiration of the bone marrow.The IMSS-MC has two hospitals that provide medical attention to children with cancer. The Departments of Hematology and of Pediatric Oncology of both the Hospital de Pediatría of the Centro Médico Nacional "Siglo XXI" (HP) and the Hospital General del Centro Médico Nacional "La Raza" (HR) participated in this study. Both facilities have the infrastructure (competent personnel and suitable technology) needed for the precise diagnosis of a cancer.Included in this study were the cases attended from 1 January 1996 to 31 December 2001.Prior to carrying out the study, a form for recording the variables of interest was designed. For this article, the variables analyzed were the following: type of neoplasia, sex of the patient, age at the time of diagnosis, and the stage of the cancer for those children with solid tumors.At each hospital, a fulltime nurse was assigned to register all new cases of cancer. Prior to collecting the data, each nurse was instructed in procedure necessary for obtaining all the different variable of the study. The nurse interviewed the parents and reviewed the clinical record of each child in order to obtain all the necessary information.The nurse was also taught how to encode and to determine the stage of cases of solid tumors. The standardization of the coding and determination of the stage of the solid tumors was done by all personnel (three physicians and two nurses) concerned with the registry; an excellent concordance was obtained (unweighted Kappa of 0.85) .In addition to other duties related to the Registry, each nurse spent three days a week in the oncology and hematology departments of the hospital (HP or HR), searching for cases of children who, being suspected of having cancer, had been registered in a specific file and boarded at the hospital. After reviewing the clinical record in each file, the nurse either included the case in the study (encoding the data if the diagnosis of cancer had been confirmed) or eliminated the case if the diagnosis was not confirmed. If for any reason, the patient was discharged from the hospital and the diagnosis was undetermined, the nurse reviewed the clinical record in the clinical archive of the hospital in order to learn what the final diagnosis was.To encode the different cases of cancer, topographical and morphological coding was used. The second edition of the "International Classification of Diseases for Oncology" (ICD-O-2) was used for the cases collected from 1996 through 1999; the third edition (ICD-O-3), for the cases collected from 2000 through 2001 ,13. For The Child-Check Program developed by the International Agency for Research on Cancer (IARC)  was usedCases were grouped according to the ICCC  that hasA total of 1,702 cases of malignant neoplasias were analyzed. Of these, in the order of the most frequently found, were the following types of tumors: leukemias, 784 cases (46.1%); CNST, 197 cases (11.6%); lymphomas, 194 cases (11.4%); germinal cell tumors (GCT), 110 cases (6.5%); and bone tumors (BT), 97 cases (5.7%); and the remainder of the neoplasias were found in low percentages ; lymphomas ; and CNST . In females, 73.6% of the cases consisted of leukemias ; CNST ; GCT ; and lymphomas . Overall, the ratio of males to females was 1.1; however, this ratio varied for the different groups of neoplasias, most notably a high of 2.5 for lymphomas and a low of 0.8 for both hepatic tumors, BT and GCT , although retinoblastoma was only the fourth most frequent there, its frequency (8.9%) was one of the highest Table .In regard to the spread of the disease at the time of diagnosis for those children the stage of whose solid tumors had been determined, 89 (13.5%) were Stage I; 129 (19.6%), Stage II; 242 (36.8%), Stage III; and 198 (30.1%), Stage IV or higher.This is the first report of data, covering the six year period from 1996–2001, taken from the on-going Registry of Cancer in Children that was started in 1996 in Mexico City by the Instituto Mexicano del Seguro Social (IMSS-MC). The strategy of having placed a nurse in each of the two hospitals involved with the Registry of Children with Cancer resulted in the great majority (more than 90%) of the new cases that were treated by the two hospitals being identified and duly registered. We therefore concluded that this Registry of Cancer in Children was one of the most complete of its kind undertaken in Mexico City.Having access to the clinical records of the patients served to improve the quality of the data that was registered because, since cases were registered as soon as they were diagnosed, few cases were overlooked.The quality of the data was also enhanced by the use of the Child-Check Program, with which the possible errors, not only of registry but also of capture, were reviewed and were eliminated upon rechecking the respective clinical record in the hospital files. As mentioned in Methods, in 100% of the cases, the histopathology reports for children with solid tumors, or the reports on the aspirated bone marrow for children with leukemia, were obtained. Therefore, the data that was obtained for the children with cancer who were residents in the area covered by IMSS-MC and treated in Mexico City were the most precise that have been gathered to date in Mexico.The frequency of a disease in a population is a method of obtaining the pre-test probability that a patient has before a diagnostic test is performed . It is iAs has been mentioned, knowing the frequency of diseases serves in the estimation of administrative needs, not only with respect to personnel but also to the equipment and supplies necessary for diagnosis and treatment, and for providing medical attention in general and in particular for children with cancer . Given tA more clinical aspect, particular to children with cancer, that indicated the spread of their disease was the stage of the disease at the time of diagnosis. It was established that 66.9% were in stages III or IV, a finding that generally means the prognosis for the patient is not good. This datum was consistent with, and more precise than the result of the previously reported retrospective study, in which it was found that 56.4% had been diagnosed as having an advanced stage (III or IV) of the disease . This faAlthough the results obtained for the different groups of cancer in Mexican children in this study showed general agreement with data previously reported, there were some differences. Consistent with data obtained in other studies ,10, it wIn contrast to previous studies in Mexico, the frequency of Hodgkin's disease was notably lower, thus reducing the overall frequency of lymphoma to slightly lower than that of CNST and putting it in the range reported for developed countries (11.4% vs. 7–12%) [The previous studies, being retrospective, had a greater tendency to under-register the number of cases and therefore result in an lower frequency than the actual value; yet, here, with a more accurate study, the frequency obtained was lower still 10% a 3.8%) Table  9,10]. ,10. 9,10% a 3.8% In this and the two prior studies in Mexico, the frequency of CNST was found to be less than that in developed countries (18–27%). However, the lowered frequency of lymphomas found in the present study changed the hierarchical pattern of tumors, with CNST now having just edged out lymphomas for second place. We do not consider that this population has, strictly speaking, the pattern of U.S.-Canada-Europe, nor that of Latin America. We can say that the pattern of neoplasias in Mexican children was found to be a phenomenon in transition. It was interesting that the pattern of presentation found in children in Mexico City was different to that of the children that live in the northern part of the country (data not shown). The pattern in children from the north was similar to that of children in the U.S. and Canada, in that the principal neoplasias were leukemias, CNST, and lymphomas and had very similar frequencies ,24. ThisData for children under one year of age showed that, whereas neuroblastoma was the principal tumor for this age group in developed countries ,3, in ouAnother interesting aspect of the present study was that one of the highest frequencies of GCT (6.5%) in the world was found, a frequency similar to that for some Asiatic countries, such as Japan and Singapore (6.8%) . We do nFinally, it should be mentioned that one state of the Mexican Republic, Chiapas, was found to have one of the highest frequencies of retinoblastoma (8.9%), a frequency very similar to that of countries of Africa (Zimbabwe, 9.6%) and in India  . ChiapasIt may be concluded that, in the children residing in Mexico City that were included in this study, the principal neoplasias were leukemias, CNST, and lymphomas, findings that were consistent with previously published data. It was found that, in comparison to previous studies in Mexico City, there was a reduction in the frequency of lymphomas and especially of Hodgkin's disease, and that, of the children with solid tumors, two thirds were diagnosed as having advanced stages (III-IV) of the disease.None declared.S J-O analyzed the data and wrote the first draft of the manuscript. G G-M registered, recorded, and analyzed the data. JM M-A, ME R-M, and MC M-G conceived and the designed the study and analyzed the data. A F-G conceived and designed the study, analyzed the data, and provided guidance to all aspects of this project. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


DC derived-exosomes are nanomeric vesicles harboring functional MHC/peptide complexes capable of promoting T cell immune responses and tumor rejection. Here we report the feasability and safety of the first Phase I clinical trial using autologous exosomes pulsed with MAGE 3 peptides for the immunization of stage III/IV melanoma patients. Secondary endpoints were the monitoring of T cell responses and the clinical outcome.+, or -B35+ and HLA-DPO4+ leukocyte phenotype, tumor expressing MAGE3 antigen) were enrolled from 2000 to 2002 and received four exosome vaccinations. Two dose levels of either MHC class II molecules (0.13 versus 0.40 × 1014 molecules) or peptides (10 versus 100 μg/ml) were tested. Evaluations were performed before and 2 weeks after immunization. A continuation treatment was performed in 4 cases of non progression.Exosomes were purified from day 7 autologous monocyte derived-DC cultures. Fifteen patients fullfilling the inclusion criteria (stage IIIB and IV, HLA-A114 exosomal MHC class II molecules allowing inclusion of all 15 patients. There was no grade II toxicity and the maximal tolerated dose was not achieved. One patient exhibited a partial response according to the RECIST criteria. This HLA-B35+/A2+ patient vaccinated with A1/B35 defined CTL epitopes developed halo of depigmentation around naevi, a MART1-specific HLA-A2 restricted T cell response in the tumor bed associated with progressive loss of HLA-A2 and HLA-BC molecules on tumor cells during therapy with exosomes. In addition, one minor, two stable and one mixed responses were observed in skin and lymph node sites. MAGE3 specific CD4+ and CD8+ T cell responses could not be detected in peripheral blood.The GMP process allowed to harvest about 5 × 10The first exosome Phase I trial highlighted the feasibility of large scale exosome production and the safety of exosome administration. In preclinical studies, few vaccination strategies were shown to counteract tumor induced-tolerance and to promote immunity to cancer leading to tumor eradication [+ T cells [+ Th1 lymphocytes [+ Th1 activation have been rarely reported [Immunization protocols rely on the capacity of the vaccine design to elicit long term protective, peptide specific, MHC restricted-CD4dication . One of dication ,3. Dhoda T cells ,6 and hephocytes , correlareported . Thereforeported ,12 might+ and CD8+ T cell responses restricted by exosomal MHC class II and I molecules respectively. Up to 41 ± 6.7 (9–115) id/sc vaccinations/patient at the lowest dosage  could be generated for all patients from one leukapheresis. One partial response and some other tumor regressions at skin and lymph node sites even in tumors that did not respond to other vaccine formulations were observed in the absence of toxicity. A case report of MART1 antigen spreading and MHC class I loss variant suggested that exosomes mediated bioactivity in vivo, supporting to conduct Phase II clinical trials.DC process exogenous antigens in endosomal compartments such as multivesicular endosomes  which ca+, or -B35+ and HLA-DPO4+ phenotype , tumor expressing MAGE3 antigen  and subcutaneously (9/10th) every week for a total of 4 vaccinations , incront see . Patienteously 9/0th everyeously 9/0th everyBased on size and density, we could rapidly purify exosomes from monocyte derived-DC (MD-DC) culture supernatants according to a good manufacturing process already described . BrieflyExosomes secreted in the supernatant of MD-DC cultures (1–4 liters) were purified to 175 fold the starting volume according to a GMP method previously described .168–176.A1/B35; EVDPIGHLY) and MHC class II peptides (Mage3247–258.DP04, KKLLTQHFVQENYLEY) in pharmaceutical quality (Multiple Peptide Systems, San Diego, CA) at 10 μg/ml at day 6 of the MD-DC culture and the exosomes were purified from day 7 culture supernatant as described above. The direct loading of MHC class I peptides at pH 4.2 has been reported [This method has been previously described ,22. Brie14 MHC class II molecules in immunocapture assays) and included a functional assay (bioactivity in the superantigen SEE test of potency).Insurance quality criteria allowing exosome release in patients were qualitative (expression of CD81 tetraspanins), quantitative , after 2 (week 4, 50 ml heparinized blood sample) and 4 vaccines (week 7, by leukapheresis), isolated by Ficoll density gradient and conditioned either for immediat testing or kept frozen for further analyses. In patient #12, single cell suspensions from dissociated cervical lymph nodes resected at the end of the induction and retreatment phases were also collected.Determination of peripheral lymphocytes subsets were performed by four color immunostainings using combinations of FITC-, PE-, Cychrome-, APC-labeled mAbs directed against CD2, CD3, CD4, CD5, CD8, CD19, CD25, CD27, CD45RA, CD56, CD69, CD122, HLA-DR . Controls included isotype-matched immunoglobulins. Thawed PBMCs were stained for 15 min at 4°C, washed twice and fixed in 1X PBS with 0.1% paraformaldhehyde. Ten thousand viable PBMCs were acquired on a FACSCalibur cytometer (Becton Dickinson, BDIS, San Jose, CA, USA) according to standard FSC/SSC criteria and analyzed with the Cellquest software.168–176.A1/B35, MAGE3247–258.DP04 (Multiple Peptide System), and control viral peptides (HIV/nef113–128.A1, HIV/nef73–82.B35, FLU/pb1591–599.A1, FLU/np265–274.A2, EBV/bzlf154–64.B35, EBV/bmlf1259–267.A2, CMV/pp65495–503.A2 TT/p2830–844, Eurogentec, Seraing, BEL) used according to the HLA classe I phenotype of patients, as well as, against tetanus anatoxin (at 100, 10 and 1 μg/ml), tuberculin (at 50, 5 and 0.5 IU/ml), and phytohemaglutinin (at 2.5, 0.5, and 0.1 μg/ml, PHA HA16, Murex Biotech, Dartford, UK).Reactivity of PBMCs was assessed in response to MAGE35 cells/well) were cultured in triplicates in presence of graded doses of peptides (at 50, 5, and 0.5 μg/ml) or control antigens or medium alone. Proliferative capacity was determined after overnight pulsing with [3H]thymidine (1μCi/well, NEN, Paris, France) at day 5 of the coculture (earlier at day 3 for PHA). Cells were harvested onto 96-well Unifilter microplates, dried overnight and radioactivity counted on a microplate scintillation counter (TopCount-NXT, Packard, CA, USA). All determinations were made in triplicate wells and data were calculated as means ± SEM of cpm.Fresh PBMCs (2 × 105cells/well) were cultured in triplicate in nitrocellulose-bottomed 96-well plates (MAHA S4510, Millipore, Saint Quentin- en-Yvelines, FR) precoated with 2 μg/ml of a primary anti-IFNγ mAb (1-D1K, Mabtech, Hamburg, GER) in presence of peptides (at 5 μg/ml) or control antigens in RPMI1640 medium supplemented with 8% human AB serum. After incubation for 48 hrs, wells were washed five times and incubated with a secondary biotinylated anti-IFNγ mAb (7-B6-1, Mabtech) for 2 h, washed and stained using an extravidine-alkaline phosphatase conjugate substrate kit (Biorad, Hercules, CA, USA). Spots were evaluated and counted using a computer-assisted video imaging analyser (Bioreader 2000, Biosys, Karben, GER).ELISPOT assay for the detection of antigen-specific IFNγ-producing T cells was performed as described previously . Briefly5 peptide-pulsed PBMC (20 μg/ml) were cultured in Iscove's medium with 10% human AB serum supplemented with IL-2 (20 IU/ml), IL-4 (10 ng/ml) and IL-7 (10 ng/ml) and restimulated at day 7 with replacement of 50% fresh medium with addition of MAGE3.A1/B35 peptide (10 μg/ml). On day 15, microcultures were screened for specific CD8+ T cells using the relevant MAGE3.A1 or B3501 tetramers coupled to PE together with a control APC labeled-HLA-A1 containing an influenza peptide . This microculture procedure was also applied on patient #12's tumor infiltrating LN to highlight CD8+ T cells specific for HLA-A2/MART126–35, and HLA-B3501/MAGE3168–176. Flow cytometry acquisition and analysis of tetramer positive cells were performed as recommanded by P. Coulie [The microculture method developed by Coulie et al.  was used. Coulie .Immunostainings were performed on sections obtained from formalin-fixed and paraffin-embedded lymph node samples. Sections were deparaffinized, placed in 10 mmol/L Na-citrate buffer (pH.7), and heated in a microwave during 20 min. Endogenous peroxidase was blocked with 1% hydrogen peroxide in methanol for 30 min. Slides were stained using anti-CD3 and anti-CD57 mAb (Pharmingen, France) and secondary antibodies with appropriate controls.9 ± 0.8 PBMC (range: 4.4–15) containing 20.7% ± 1.8 CD14+ cells (range: 12.4–33.0). These monocytes differentiated into immature MD-DC in rhu GM-CSF and rhu IL-4 (means: 313 × 106 ± 100, range: 50–1200) as assessed at day 7 in flow cytometry highlighting loss of CD14 molecules, acquisition of CD1a, poor cell surface expression of CD83. In 3 patients out of 15, the first leukapheresis did not allow CD14+ cells to adhere and consequently, a second leukapheresis was required to harvest exosomes. The GMP process allowed to purify a mean of about 5.22 × 1014 ± 0.9 (range: 1.2–15.0) exosomal MHC class II molecules from supernatants of day 6 to day 7 MD-DC required for up to 41 ± 6.7 (9–115) id/sc vaccinations/patient at the lowest dosage . As for the patients who benefited from continuation treatment requiring a second leukapheresis (pt #3, #12 and #14), the second exosome recovery was not significantly different from the first one. The quality control parameters required for GMP exosome batch release are reminded in M&M.Leukaphereses of 1.5 blood mass performed in the 15 metastatic melanoma patients enrolled in the study allowed the recovery of 9.7 × 10GMP exosomes were successfully produced from DC cultures derived from all melanoma patients enrolled in this phase I study, allowing at least 4 exosome inoculations.No major (>grade II) toxicity was observed. A grade 1 fever was recorded in 5 patients. We noted slight inflammatory reactions at vaccine sites without outward delayed type hypersensitivity reactions. Some patients (#4, #8, #11, #12 and #15) reported a transient swelling and sensitivity of cutaneous and lymph nodes metastases 48 hrs after each exosome inoculation.Among the six patients vaccinated in the first part of the study, only one patient #3) presenting with a stage III disease exhibited a minor response (disappearance of one sc lesion out of 3). This patient benefited from a continuation therapy with exosomes every other 3 weeks for 21 months and remained stable for up to24 months. It is noteworthy that this patient was progressing despite vaccination with MAGE3 protein prior to enrollment in the Phase I exosome trial. In the second part of the study («direct loading»), responses were only observed in the second group of patients receiving the highest dosages of peptides (100 μg/ml) pulsed onto exosomes. One female patient (#12) presenting with five supraclavicular invaded lymph nodes after conventional chemotherapy (DTIC, 2 cycles) exhibited a partial response (PR) after the induction therapy. Interestingly, disappearance of arterial neovasculature concomittant with tumor shrinkage and necrosis as assessed by doppler pulsed ultrasonography could be demonstrated . These studies did not allow to conclude for significant anti-MAGE3A1/B35 CTL responses induced by exosome therapy nor to suggest the clonal expansion of discrete T cell specificities (not shown) in patients' peripheral blood.The phenotypic analyses of lymphocyte subsets did not reveal any significant changes in the percentages nor absolute counts following exosome therapy (not shown). Interestingly, the CD122 molecule i.e IL-2Rβ chain was upregulated in both CD4 and CD8 T cell subsets after exosome therapy (p < 0.05 using Student t'test, week 7 versus week 1 for CD4+ T cells). Serial ex vivo ELISPOT analyses of PBMC before W1, Fig.  and afte26–35, -/gp100209M-217 -/Tyrosinase239–251 and HLA-B3501/MAGE3168–176 -specific CD8+ lymphocytes using soluble fluorescent tetramers were performed directly ex_vivo and after microculture restimulation assays using MART126–35 or MAGE3 peptides.Only high frequencies of MART1 specific CD8+ T cells were detected in TILN from site 1 and 6 (1.22 and 0.85 % respectively) and in peripheral blood (0.53% in May 2002 versus 0.15% in Oct. 2001) that were confirmed by expansion in all microculture assays  and secondary (July 2002) tumor specimen revealed quantitative modifications of tumor infiltrating CD3+CD57+ T cells. While activated T cells were mostly surrounding the tumor bed initially, a two fold expansion or recruitment of activated T cells invade tumor areas after exosome vaccines Fig. .14-1015 exosomal MHC class II molecules could be purified in GMP conditions for all patients, allowing 9–123 sc/id vaccinations with 0.13 × 1014 exosomal MHC class II molecules. The optimal dosages of exosomal MHC class II molecules required to trigger an efficient peptide-specific CTL response leading to tumor rejection was about 1010-1011 molecules in preclinical studies in the HLA-A2 transgenic mouse model [This work is reporting for the first time the feasability and safety of DC derived-exosome-based vaccination in melanoma patients. From a single leukapheresis, up to 10se model . The optse model ,22. A dose model .Only one objective response (PR) according to the RECIST criteria was recorded. However, one minor response and two stabilisations prompted the continuation of treatment as well. Tumor regression was exclusively observed in skin and lymph node lesions, as already reported for peptide-based vaccines . It is nb harboring exosomes pulsed with H-Y epitopes could promote expansion of peptide specific CD4+ transgenic T cells. Nevertheless, MHC class I restricted-CTL responses elicited by exosomes pulsed with Mart1 peptides in HLA-A2 transgenic mice were dramatically boosted by adjuvants [No DTH responses to iterative inoculations of exosomes or MHC class II restricted-direct or recall responses (to TT epitopes (3 pts tested), or Mage DP04 epitopes (12 patients tested)) were detected at 4 and 6 weeks of exosome vaccination. It is noteworthy that peptides and not whole proteins (such as TT or KLH) were used to pulse DC in vitro, in contrast to what has been reported in most DC trials. However, in the transgenic Marilyn mouse model , id inocdjuvants . Therefodim NK cells) were markedly infiltrating the tumor after therapy (N. Chaput, manuscript in preparation). Therefore, it is conceivable that DC derived-exosomes from melanoma patients are specifically endowed with NK cell stimulatory capacity in vivo. This hypothesis deserves further investigations and should be pursued in the next clinical studies. Moreover, since our preclinical data in tumor bearing mice highlighted a critical role of suppressor T cells in restricting exosome-mediated specific T cell responses, T regulatory cells should be monitored and combination of cyclophosphamide together with exosomes could be invisioned (N. Chaput, in preparation). Phase II/III trials will address the potential of exosomes to enhance time to progression in advanced non small cell lung carcinoma patients.Since no specific CD4+ or CD8+ T cells generated by the exosome vaccines could be detected and could account for the tumor regression in patient#12, what could have been the primary effectors accounting for antigen spreading? We were able to show enhanced NK cell effector functions following exosome administration in peripheral blood of 8/13 patients including patient #12 for whom CD3-CD57+ cells expressing PEN5 (mostly expressed on CD56DC: Dendritic cells; MD-DC: monocyte-derived DC; Dex: Dendritic cell -derived exosomes; CTL: Cytotoxic T Lymphocytes; GMP: good manufacturing processes; MHC: Major Histocompatibility Complex; LN: lymph nodes.1• was the principal investigator,Bernard Escudier2•, Sophie Piperno2, Caroline Robert3,9 were the clinicians ensuring patients' enrolment, follow up and clinical care.Thierry Dorval• BE and TD equally contributed to this work4, Sophie Novault3, Christophe Leboulaire3, Mojgan Movassagh3 and Christophe Leboulaire3 were monitoring T cell responses corrdinated by Olivier Lantz4 and Eric Angevin1Marie-Pierre Cabi3, Fabrice André3 and Caroline Flament3monitored NK cell responsesNathalie Chaput5Catherine Boccaccio was in charge of the cell therapy unit at the Institut Gustave Roussy4, Christian Bonnerot4 and Laurence Zitvogel1 were the scientists at the source of preclinical data orienting the study design.Sebastian Amigorena1 was heading the Host and Tumor Development Programm of the Institut Gustave Roussy, initiating the clinical study.Thomas Tursz6, Nancy Valente7, Olivier Dhellin6 were the Director of Anosys SA, the medical director of Anosys Inc. and the Pharmacist in charge of production, regulatories and quality insurance in Anosys SA respectively. Jean-Bernard Le Pecq6,7was the Chief and Scientific Officer of Anosys leading the research and development of the exosome programm.Vincent Serra8, Christophe Borg3 were in charge of the immunohistochemitry analyses on tumor specimen.Alain SpatzDosing, demographic, base line data and clinical outcomeClick here for file


In many eukaryotic cells, double-stranded RNA (dsRNA) triggers RNA interference (RNAi), the specific degradation of RNA of homologous sequence. RNAi is now a major tool for reverse-genetics projects, including large-scale high-throughput screens. Recent reports have questioned the specificity of RNAi, raising problems in interpretation of RNAi-based experiments.Trypanosoma brucei as a model, we designed a functional complementation assay to ascertain that phenotypic effect(s) observed upon RNAi were due to specific silencing of the targeted gene. This was applied to a cytoskeletal gene encoding the paraflagellar rod protein 2 (TbPFR2), whose product is essential for flagellar motility. We demonstrate the complementation of TbPFR2, silenced via dsRNA targeting its UTRs, through the expression of a tagged RNAi-resistant TbPFR2 encoding a protein that could be immunolocalized in the flagellum. Next, we performed a functional complementation of TbPFR2, silenced via dsRNA targeting its coding sequence, through heterologous expression of the TbPFR2 orthologue gene from Trypanosoma cruzi: the flagellum regained its motility.Using the protozoan This work shows that functional complementation experiments can be readily performed in order to ascertain that phenotypic effects observed upon RNAi experiments are indeed due to the specific silencing of the targetted gene. Further, the results described here are of particular interest when reverse genetics studies cannot be easily achieved in organisms not amenable to RNAi. In addition, our strategy should constitute a firm basis to elaborate functional-dissection studies of genes from other organisms. RNA interference (RNAi) can be triggered by introduction of long double-stranded RNA molecules (dsRNAs) in cells , and proRNA-silencing processes have been described in a variety of organisms: post-transcriptional gene silencing in plants ,8, quell>50 base pairs) triggers the activation of sequence-unspecific interferon-related pathways [in vivo synthesis of small hairpin RNAs, which were demonstrated to produce gene-specific silencing [While RNAi and associated phenomena constitute exceptional recent basic science findings, they also provided a basis for the elaboration of powerful research tools. RNAi methodologies have been set up to perform reverse-genetics studies in a number of organisms. RNAi potency and flexibility have allowed to perform high-throughput genetic screens in several organisms -26. In mpathways -29. To cpathways  or in viilencing ,30,31; rilencing ,33.However, an siRNA might trigger a number of potential unspecific events such as the degradation of partially complementary mRNA due to cross-hybridization, leading to unspecific RNAi, or the translational arrest due to a micro RNA-like effect where an siRNA hybridizes to a mRNA with one or few mismatches. It is thus of paramount importance to ensure that the phenotypic effects observed as a result of siRNA presence in cells are due to silencing of the target gene only. Two large-scale studies show that siRNA-induced gene silencing of transiently- or stably-expressed mRNA is highly gene-specific and does not produce secondary effects detectable by genome-wide expression profiling ,35. In cTbPFR2 silencing leads to flagellar paralysis and trypanosomes do not swim anymore [Trypanosomes are protozoan parasites belonging to the Kinetoplastida order. These unicellular flagellated organisms diverged very early in eukaryotic evolution, and exhibit a number of original features -40. Tryp anymore ,47. Duri anymore . This asTbPFR2 gene as a model system, involved the silencing of the TbPFR2 target via its UTRs and the expression of a RNAi-resistant copy of the targetted gene. The RNAi-resistant gene was either a copy of TbPFR2 with different UTRs or its Trypanosoma cruzi orthologue: TcPFR2. We found that inter-species complementation experiments were straight forward. This strategy opens a venue for functional gene dissection experiments where modified gene sequences can be tested for their ability to encode functional protein that can complement the RNAi-based loss-of-function phenotype.We previously established the degree of identity between the gene sequences capable of leading to cross-RNAi ,49. HoweTbPFR2 and FLA1; TbPFR2 encodes one of the two major components of the paraflagellar rod and is necessary for flagellum motility [FLA1 encodes a protein required for flagellum attachment to the cell body [GFP dsRNA.We wanted to establish if the co-transfection of two distinct dsRNAs, targeting two different genes, could trigger their simultaneous silencing. The genes selected were motility ; FLA1 enell body . These dFLA1 gene silencing was analyzed by differential interference contrast microscopy, as it results in the visible detachement of the flagellum from the cell body . The trypanosomes shown in Figure FLA1 silencing is no longer visible in the new flagellum while that same flagellum still exhibits the phenotype due to the TbPFR2 silencing, clearly indicating different turn-over for TbPFR2 and FLA1 proteins.The transfection of The RNAi machinery could cope with two different dsRNA populations, without – in our conditions – any visible saturation effect. These results show the feasibility of experiments involving the use of multiple dsRNAs, thus allowing studies on complex processes in the cell physiology. However, such complex experiments can only be envisaged after ensuring that the phenotypes resulting from RNAi are specifically due to silencing of the target gene. In order to address that specific problem, we elaborated a method that involves RNAi experiments on trypanosomes that were engineered to possess an extra RNAi-resistant copy of the targeted gene, leading to functional complementation.TbPFR2 gene, which is present in four copies in the WT trypanosome genome . For each experiment, the presence or absence of TbPFR2 in the new flagellum of bi-nucleated/bi-flagellated cells was monitored by immunofluorescence 14 h after the transfection . This observation is probably explained by the organization of the TbPFR2 locus: the polycistronic transcript is rapidly spliced into three different types of mRNA, each encoding one of the four copies of TbPFR2 [TbPFR2 RNA is destroyed, the three remaining ones would likely provide enough RNA to synthesize TbPFR2 levels compatible with normal PFR formation.When WT trypanosomes were transfected with f TbPFR2 -53. ThusTbPFR2, we used two cell lines expressing a supplementary tagged TbPFR2 gene copy. The TbPFR2tag cell line expresses the TbPFR2-TAG protein which correctly localizes to the flagellum.To demonstrate that the silencing observed upon transfection of WT trypanosomes with the UTRs MIX dsRNAs was due to the actual targeting of TbPFR2tag-ΔHLA cell line expresses TbPFR2-TAG-ΔHLA, lacking the HLA tripeptide, which prevents its localization to the flagellum. To determine both the cellular localization of the tagged TbPFR2 proteins (TbPFR2-TAG and TbPFR2-TAG-ΔHLA) and the completeness of the PFR assembly, immunofluorescence experiments were carried out with the BB2 and ROD-1 antibodies; the former recognizes the Ty-1 epitope tag present on the two tagged TbPFR2 proteins [The proteins , while tproteins ,56.TbPFR2 silencing: TbPFR2 was decorated in both the old and new flagella by the anti-TbPFR2 antibody, and the PFR could be assembled fully, as evidenced by its staining with the ROD-1 antibody  decorated with the anti-TbPFR2 antibody, demonstrating that both the WT and the recombinant TbPFR2 gene copies were effciently silenced . TbPFR2tag-ΔHLA does not access the flagellar compartment and thus cannot be functional [TbPFR2 copies were silenced, thus leading to an incomplete PFR edification  has been implicated as one possible step in the formation of siRNA in fungi , plants anosomes -64.TbPFR2i cell line – that expresses TbPFR2 dsRNA under the control of a tetracycline-inducible promoter [Trypanosoma cruzi TbPFR2 orthologue (TcPFR2) was established using stable transfection procedures. TbPFR2 and TcPFR2 proteins share 90 % identity (both of them are recognized by the anti-TbPFR2 L8C4 antibody), but their gene sequences have diverged enough for us to envisage that the RNAi-silencing of TbPFR2 would not affect significantly the introduced TcPFR2 gene . We thus created two new cell lines based on the previously described TbPFR2i cells [We next asked if an RNAi-mediated loss of function could be complemented by the expression of a gene orthologue to the silenced one. The system used to answer that question involved the promoter  – into w2i cells  (see MetPCGFP cells constitutively expressed GFP, as detected by microscope observation of living cells (data not shown). Both the PCGFP and PCTcPFR2 cell lines were induced to express TbPFR2 dsRNA for 48 hours. Immunofluorescence revealed that non-induced PCGFP cells exhibit a WT-like phenotype . When these cells were induced with tetracycline, expected TbPFR2 silencing occurred . When these same cells were tetracycline-induced, the flagellar staining was still perfectly visible, at a level comparable to the one previously observed in the non-induced PCGFP control cells ? To address this question, we performed a sedimentation assay [PCGFP and PCTcPFR2 trypanosomes , is roughly 80 % ([The complementation described above shows the robustness of our strategy, because TbPFR2 and TcPFR2 are highly similar (they share 82 % identity at the nucleotide level ) and arely 80 %  T. brucei cells are engineered to ensure constitutive heterologous expression of the gene of interest, still allowing RNAi-mediated silencing of the T. brucei gene; 3) function of the investigated gene is assessed by checking if the loss-of-function phenotype observed in the first place gets complemented. Additionally, one application of the strategy described herein is genetic functional dissection, which is of interest when protein domains are to be characterized with respect to their function .In this report, we demonstrated that RNAi-mediated silencing of a gene by targeting its UTRs is useful in studies where the loss of function resulting from this silencing must be complemented with the expression of an RNAi-resistant copy of the silenced gene, in order to demonstrate that the phenotype is indeed due to silencing of that gene, and not to inactivation of another one. The results obtained in this work are of particular interest when reverse-genetics studies cannot be easily achieved in organisms not amenable to RNAi, like ishmania  or Trypama cruzi , or wherComplementation had previously been demonstrated following transformation of mammalian cells with EGFP siRNA and expression of a codon-modified, but functional, EGFP version . Our strT. brucei brucei strain 427 (or its derivatives) was used throughout this work. Cells were cultured at 27°C in semi-defined medium 79 (SDM 79) containing 10% foetal calf serum. PFRAi cells were described in [TbPFR2i trypanosomes can be tetracycline-induced to express TbPFR2 dsRNA, thus eliciting an RNAi response against that gene. Note that this cell line is referred to as TbPFR2i in this article because of a change in the gene nomenclature [The procyclic ribed in . The TbPnclature .in vitro with T3 and Sp6 polymerases using PCR products as templates [RNA was synthesized emplates . The folGFP , AATTAACCCTCACTAAAGGGAGAAG AACGGCATCAAGGTGAAC  and ATTTAGGTGACACTATAGAAG AGTGATCCCGGCGGCGGTCACG ;for FLA1, AATTAACCCTCACTAAAGGGAGA CCAAACCGTGGGCACCAAGG  and ATTTAGGTGAACTATAGAAGAG GTGGGATGATTAAAACGAGC ;for TbPFR2 5' untranslated region , AATTAACCCTCACTAAAGGGAGA (T3 promoter) and ATTTAGGTGACACT-ATAGAAGAG (Sp6 promoter);for the TbPFR2 intergenic untranslated region (igUTR), AATTAACCCTCACTAAAGGGAGA CGCTGCGCTTAAATGTCTT  and ATTTAGGTGACACTATAGAAGA GTGATGCTTTATTGCTTTCT ;for the TbPFR2 3'untranslated region , AATTAACCCTCACTAAAGGGAGA  and ATTTAGGTGACACTATAGAAGAG ;for the TbPFR2 coding sequence , ATTTAGGTGACACTATAGA GAGGTGAAGCGCCGTATTGAGGA  and AATTAACCCTCACTAAAGGGAGA GTTTTGTACAGGCGACGGAA ;for the TbPFR2 locus and the position of the two dsRNA populations that were used, and their homology to either the coding sequence (labelled "CDS dsRNA") or the different 5'UTR, igUTR and 3'UTR all together (labelled "UTRs MIX dsRNAs"). A third dsRNA, homologous to the GFP gene is labelled "GFP dsRNA" throughout this work and was used as a control dsRNA. dsRNA was introduced into trypanosomes by electroporation, as described [Figure escribed .Hind III and Eco RI to remove the GFP gene. Oligonucleotides AGCT GTCTAGCGATATCGGATCCG (forward) and AATT CGGATCCGATATCGCTAGCA (reverse) were annealed  and the resulting double-strand oligonucleotide was ligated into the pSk1-GFP plasmid, resulting in the insertion of a poly-linker containing restriction sites Cla I, Hind III, Nhe I, Eco RV, Bam HI and Eco RI . Plas-mid pPCTcPFR2 was generated as follows: amplification of the TcPFR2 gene was performed using Trypanosoma cruzi genomic DNA  and the two primers TcPFR2H (GAGTCTAAGCTTATGAGCTACAAGGAGGCATC) and TcPFR2ER (GCGTGGAATTCTTACTGTGTGATCTGCTGCAC). Both the amplified DNA fragment and the pPC plasmid were digested with Eco RI and Hind III. The fragment was ligated into pPC so as to yield the plasmid pPCTcPFR2  for selection.The  plasmid  was tran plasmid ,72. In c plasmid  Fig 4G)TbPFR2tagporation , cells wTbPFR2i cells [PCGFP and PCTcPFR2 new cell lines. The PCGFP cell line was established by transfecting TbPFR2i cells with plasmid pPCGFP after linearization with BstX I. For establishing the PCTcPFR2 cell line, the pPCTcPFR2 plasmid was linearized with BstX I and transfected into TbPFR2i cells. Recombinant cells were selected by addition of puromycin (1 μg/mL), phleomycin (2 μg/mL), G418 (15 μg/mL) and hygromycin (20 μg/mL) to the culture medium.2i cells  constituT. brucei TbPFR2 and cross-reacting with T. cruzi orthologue TcPFR2 [Three different monoclonal antibodies were used as hybridoma supernatants: L8C4, IgG recognizing e TcPFR2 ; BB2, Ige TcPFR2 ; and RODe TcPFR2 . For imme TcPFR2 . Experime TcPFR2 .6 cells/mL in normal culture medium, with or without 48 hour tetracycline induction. 1 mL of these cultures was dispensed to 5 plastic spectrophotometry cuvettes, for time points 0, 2, 4, 6, 8 hours, and left still. At each time point, the optical density at 600 nm was measured twice: first without mixing (O.D.no mix) and second after mixing the cuvette (O.D.mix). Data were plotted as a function of time.The trypanosome sedimentation assay was performed as described in . BrieflyF.R. carried out most of the experiments reported and wrote the manuscript, M.D.-D. performed the double transfection reported at Table


Transfection involved a combination of electrical parameters and a specific solution to deliver plasmid directly to the cell nucleus by using the Nucleofector® electroporation system. Nucleofection resulted in the production of IL-2 with a mean of 478.5 pg/106 cells (range of 107.6–1079.3 pg /106 cells/24 h) compared to mock transfected CIK cells (31 pg/106 cells) (P = 0.05). After co-culturing with DC their functional ability was assessed in vitro by a cytotoxicity assay. On comparison with non-transfected CIK cells co-cultured with DCs (36.5 ± 5.3 %), transfected CIK cells co-cultured with DC had a significantly higher lytic activity of 58.5 ± 3.2% (P = 0.03) against Dan G cells, a human pancreatic carcinoma cell line.Modulation of the immune system by genetically modified immunological effector cells is of potential therapeutic value in the treatment of malignancies. Interleukin-2 (IL-2) is a crucial cytokine which induces potent antitumor response. Cytokine-induced killer cells (CIK) have been described as highly efficient cytotoxic effector cells capable of lysing tumor cell targets and are capable of recognizing these cells in a non-MHC restricted fashion. Dendritic cells (DC) are the major antigen presenting cells. This study evaluated the antitumor effect of CIK cells which were non-virally transfected with IL-2 and co-cultured with pulsed and unpulsed DC. Human CIK cells generated from peripheral blood were transfected  Advances in the characterization of cytokines and tumor antigens, coupled with our increasing ability to manipulate gene expression, have fostered a new era of tumor immunotherapy . InterleDendritic cells (DC) are specialized antigen-presenting cells located throughout the human body. They represent heterogeneous cell population, residing in most peripheral tissues where they represent 1–2% of the cell numbers. In the absence of ongoing inflammatory and immune responses, dendritic cells constitutively patrol through the blood, peripheral tissues, lymph and secondary lymphoid organs . MorpholExocrine pancreatic carcinomas have a very poor prognosis and a resistance to conventional therapy. This is mainly induced by lack of immuno-competent cells. Therefore, it might be beneficial for patients with pancreatic cancer to induce an immune attack against the tumor by inserting a cytokine gene into the immunological effector cells. The aim of this study was to evaluate the antitumor immune responses of a cytokine immunotherapy using gene transfer to provide continuous and local cytokine production and therefore showing an improved cytotoxic effect against pancreatic cancer cells.6 cells/ml for one hour at 37°C in complete RPMI 1640 with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin. The non-adherent cells were collected for generating CIK cells. The adherent cells were cultured in 2 ml RPMI 1640 with autologous, heat-inactivated serum, 750 IU GM-CSF and 500 IU IL-4 (Essex Pharma, Nürnberg, Germany), 100 U/ml penicillin and 100 μg/ml streptomycin per well for seven days for generating DC. The media along with the necessary cytokines were changed every third day.DC were generated as described before ,7. Blood2 and sub-cultured every third day in fresh complete medium with 300 U/ml IL-2 at 3 × 106 cells/ml. CIK cells were harvested on day +7 and were co-cultured for seven days with autologous DC at a stimulator (DC) to responder (CIK) ratio of 1:5.CIK cells were generated as described previously . In brie2.The human pancreatic carcinoma cell line DAN-G was purchased from DSMZ (Deutsche Sammlung für Zellkultur, Braunschweig, Germany). The cells were maintained in RPMI 1640 supplemented with 10% fetal calf serum (FCS, PAA) 100 U/ml penicillin and 100 μg/ml streptomycin (Seromed, Jülich, Germany) and grown at 37°C in a humidified atmosphere of 5% COcDNA of human IL-2 was cloned in the plasmid pMTV.05 (Invitrogen, Karlsruhe, Germany). The recombinant pMTv-hIL-2 (referred to as pIL-2) was transformed and the plasmid was eluted using a mini-prep column (Qiagen GmbH, Hilden Germany) according to the manufacturer's protocol.DCs were pulsed with tumor lysate of Dan-G cells on day +5 .6 CIK cells were nucleofected in an electroporation cuvette along with pre-warmed nucleofector solution and 3 μg of pMTV-hIL-2 using the programme U-14. Once nucleofected, CIK cells were transferred into fresh pre-warmed media with the necessary cytokines and serum.CIK cells were subjected to a combination of electrical parameters and specific solution to deliver the DNA directly to the cell nucleus under mild conditions by using a commercially available nucleofection system on day 10 according to manufacturer's protocol (Nucleofector Amaxa Biosystems GmbH, Cologne, Germany). Five times 10Cell culture supernatants from the nucleofected and non-nucleofected CIK cells were sampled at 24 hrs and 48 hrs, respectively. An enzyme linked immunosorbent assay for IL-2 with matched antibody pairs was performed according to the manufacturer's instructions (R and D Systems, Wiesbaden, Germany).® non-radioactive cytotoxicity assay was used as a fluorometric alternative to the 51Cr release assay . The assay is based on loading target cells with a fluorescence enhancing ligand. After cytolysis the ligand is released and introduced to the DELFIA® Europium solution. The measured signal correlates directly with the amount of lysed cells [A DELFIA EuTDAed cells . Each exP value < 0.05 was considered significant.Students't' test was applied. A + monocytes using GM-CSF and IL-4. Adherent cells showed cytoplasmic processes typical for DC. After co-culture with CIK cells they formed typical cluster. Flow cytometry showed CD14 negative populations, expressing markers typical for DC (CD80+, CD83+, CD86+ and HLA-DR expressing cells). The CIK cells were phenotyped with antibodies against CD3, CD8, CD16, CD40L, CD56, HLA-ABC and HLA-DR. Data was similar to other studies by our group (data not shown) [DC were generated from CD14t shown) ,14.6 cells (range of 107.6–1079.3 pg /106 cells/24 h) compared to irrelevantly transfected (containing eGFP) CIK cells (31 pg/106 cells) (P = 0.05). CIK cells secreting IL-2 were co-cultured from days +7 to +14 with DC, 10 days of age. Cytotoxicity of effector cells was analyzed. Co-culture of effector cells with DC led to an increase in cytotoxic activity as measured in a Eu-release assay using Dan-G. Eu-release in co-cultured CIK cells transfected with pIL-2 and DC was 58.5 ± 3.2% at an effector:target ratio of 1:40 . In order to further enhance cytotoxic activity DC were pulsed with tumor lysate of Dan-G cells on day +5. However, lytic activity (50.3%) was not significantly enhanced (P = 0.33) when compared to non-transfected cells . Nucleofection resulted in the production of IL-2 with a mean of 478.5 pg /10in vitro against pancreatic carcinoma cell lines via secreting significant amounts of IL-2.In this report, transfection of CIK cells with IL-2 demonstrated a prominent augmentation of antitumor immunity in vitro in a non MHC restricted fashion [Ductal pancreatic adenocarcinoma is the fourth leading cause of cancer death in the Western world. Unfortunately, recent advances in diagnostics, staging, and therapy have not resulted in significant improvements. Thus, new approaches are necessary to improve the outcome of patients with exocrine pancreatic cancer. CIK cells are the most potent mediators of the lyses of autologous and allogeneic cancer cells  fashion , have a  fashion ,6,15 and fashion . Therefo fashion . Transge fashion  it can b fashion  by stimuP = 0.05). This results matches perfect to a previously reported result by our group [The use of gene transfected lymphocytes were hampered by a poor efficiency of gene transfer in lymphocytes and a down regulation of cytokine expression . In contur group . To the This effect is due to increased amounts of CIK cells during co-cultivation of IL-2 secreting CIK cells with DC. IL-2 secretion by the CIK cells enhances the NK cell antitumor activity . NK cell