A developing organism captured on time-lapse video is a wonder to behold. If you're watching a chick embryo, by day 3, you'll see millions of cells engaged in a frenzy of activity, as rapidly dividing cells migrate to new positions, acquire the characteristics of specialized cells, and craft well-defined tissues, organs, and limbs in just under two weeks. In addition to the cells destined for specialization is another important group, stem cells, whose progeny have two very different fates. They can either “self renew”—that is, make identical copies of themselves—or generate intermediate progenitor cells that give rise to mature, differentiated cells.Both differentiation and self renewal are guided by an elaborately regulated genetic program, which transforms embryonic stem cells into the many different cell types that make up the body. Adult stem cells share the hallmark trait of self renewal, but are relatively rare: in bone marrow, the source of hematopoietic, or blood-forming, only an estimated one in 10,000–15,000 cells is an adult hematopoietic stem cell (HSC).Studies that have compared the gene expression profiles of different types of stem cells to identify genetic signatures of “stemness” have found only a limited number of signature genes. And the molecular mechanisms that regulate this so-called potency and the self renewal process have remained obscure. Now, focusing on HSCs, Margaret Goodell and colleagues have undertaken a systematic evaluation of HSC renewal. The study identifies molecular signatures associated with discrete stages of the HSC self renewal cycle and proposes a molecular model of the process.HSC renewal passes through three stages: quiescence, activation and proliferation, and a return to the dormant state. HSCs give rise to both red blood cells, which carry oxygen and carbon dioxide, and white blood cells, which fight infection. Certain stressors—including blood-cell-inhibiting chemotherapy and bone marrow transplants—trigger HSC activation, which induces rapid proliferation, generating both progenitors to deal with the threat and new stem cells that return to quiescence. Once activated by a trigger, dormant HSCs engage a regulatory program that rapidly churns out billions of cells, then puts the brakes on cell division, prompting the return to a nondividing, quiescent state.To understand the genetic programs underlying this process, Goodell and colleagues induced proliferation in HSCs , then allowed the cells to return to quiescence, so they could characterize the changes in gene expression that occurred during each stage. They compared these time-specific patterns to the gene expression profiles of naturally proliferating fetal mouse HSCs (which undergo massive proliferation) and quiescent adult mouse HSCs  to find genes associated with the two different states.Genes were grouped into proliferating or quiescent groups based on when they were expressed after 5FU treatment, and these groupings were refined based on comparisons to previously published HSC gene expression data. Functional analysis of these genes found a bias toward genes involved in cell division processes in the proliferation stage and toward cell division inhibitors in the quiescent stage, supporting the logic of the groupings. To understand the activation process at a global level, the authors employed some novel analysis strategies, including the “Gene Ontology” (GO) system for classifying genes.With these results, Goodell and colleagues constructed a model of the HSC self renewal cycle: quiescent HSCs maintain a “state of readiness,” molecularly speaking, that allows a quick response to environmental triggers. A stressor (like the chemotherapy mentioned above) triggers a “prepare to proliferate” state—a kind of pregnant pause—and then the proliferation machinery kicks in, going through an early and late phase before quiescence returns. By shedding light on the molecular mechanisms of stem cell renewal, this study will aid efforts to develop stem-cell-based clinical therapies, which depend on replicating the HSC self renewal cycle to replenish diseased or damaged tissue, and will ultimately guide efforts to grow stem cell colonies outside the body, a long-standing goal that would have many clinical applications. The authors suggest their findings may also be relevant to studies of cancer stem cells, tumor cells with self renewal properties.


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 . 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  as wellwn [e.g. ] and sinwn [e.g. ] as wellwn 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  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 .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 , trochanter BMD , and ward's BMD .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  were measured by dual-energy x-ray absorptiometry . 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 , high school , and university . 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 , and ward's BMD  but wasn't neck BMD .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%) . ,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  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 thymidine  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 . 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  precoated with 2 μg/ml of a primary anti-IFNγ mAb  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  for 2 h, washed and stained using an extravidine-alkaline phosphatase conjugate substrate kit . Spots were evaluated and counted using a computer-assisted video imaging analyser .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  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  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 , 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  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  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 . 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 . 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 . 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  . 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 %  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 , 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 . The cells were maintained in RPMI 1640 supplemented with 10% fetal calf serum  100 U/ml penicillin and 100 μg/ml streptomycin  and grown at 37°C in a humidified atmosphere of 5% COcDNA of human IL-2 was cloned in the plasmid pMTV.05 . The recombinant pMTv-hIL-2 (referred to as pIL-2) was transformed and the plasmid was eluted using a mini-prep column  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 . 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 .® 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 . 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


Injection of APL resulted in positive selection of CD8 T cells expressing the relevant viral specificity and in the export of those virus-specific CTL to lymph nodes without inducing T cell proliferation. Thus, exogenous APL administration offers the potential of expanding repertoires in vivo in a manner useful to the organism. To efficiently peripheralize antigen-specific T cells, concomitant enhancement of mechanisms promoting thymocyte migration appears to be required. This commentary describes the rationale for thymic vaccination and addresses the potential prophylactic and therapeutic applications of this approach for treatment of infectious diseases and cancer. Thymic vaccination-induced peptide-specific T cells might generate effective immune protection against disease-causing agents, including those for which no effective natural protection exists.Conventional vaccines afford protection against infectious diseases by expanding existing pathogen-specific peripheral lymphocytes, both CD8 cytotoxic effector (CTL) and CD4 helper T cells. The latter induce B cell maturation and antibody production. As a consequence, lymphocytes within the memory pool are poised to rapidly proliferate at the time of a subsequent infection. The "thymic vaccination" concept offers a novel way to alter the primary T cell repertoire through exposure of thymocytes to altered peptide ligands (APL) with reduced T cell receptor (TCR) affinity relative to cognate antigens recognized by those same TCRs. Thymocyte maturation (i.e. positive selection) is enhanced by low affinity interaction between a TCR and an MHC-bound peptide in the thymus and subsequent emigration of mature cells into the peripheral T lymphocyte pool follows. In principal, such variants of antigens derived from infectious agents could be utilized for peptide-driven maturation of thymocytes bearing pathogen-specific TCRs. To test this idea, APLs of gp Vaccination has improved healthcare by providing the most cost effective means to prevent disease on a global basis ,2. SinceTo date, the fundamental principles of vaccination have remained unchanged. The overriding concept for each vaccine has been the establishment of protective immunity largely due to antigen-specific T cell expansion, facilitating subsequent proliferation and differentiation of CD8 cytotoxic effector T cells and CD4 helper T cells capable of producing antiviral cytokines and chemokines Fig. . CD4 T cHowever, conventional vaccines have their pitfalls. Microorganisms including HIV and malaria, among others, may alter their antigenic proteins through rapid mutagenesis, thereby hindering cytotoxic T lymphocyte (CTL)-based immunity, exploiting holes in the T cell repertoire, and/or misdirecting both cellular and humoral responses away from key cell-binding receptors to pathogen components which cannot provide epitopes for neutralizing antibodies -13.in vivo, controlling the generation of naive T cells and hence, subsequent memory development within the peripheral lymphoid compartment. Through repertoire manipulation, it should be possible to sculpt the specificity and diversity of disease-fighting cells. This thymic vaccination approach aims to deliver, by parenteral administration, positively selecting APL of cognate antigens into the thymus, eliciting maturation of thymocytes with desired TCR specificities at the level of thymic repertoire development . APL derived from infectious agents or tumor antigens, with low affinity to the TCR could, in principle, mediate positive selection and export of specific T cells from the thymus . As suchent Fig. . Note hoExpanding T cell repertoires has enormous potential in aiding the organism's fight against infections or in affording tumor immunity. This strategy is principally different from conventional "peripheral" vaccination, which leads to proliferation of pre-existing mature T cells but does not alter the repertoire through creation of T lymphocytes with new T cell specificities. Thymic vaccination, by contrast, will alter the thymic repertoire to create desired T cell specificities. Moreover, a key feature of thymic vaccination is that it should be capable of directing the immune response towards those non-mutable components of proteins derived from infectious agents and tumors and away from misguiding cues that are part of pathogen or cancer chicanery. The rationale for this approach and the advantages over traditional vaccines are described below.-CD8- double negative) → DP ] is depicted in Fig. T cells bearing a highly diverse αβ T cell receptor (TCR) repertoire develop in the thymus from stem cells originating in the hemopoietic tissues -17. On eThe avidity of pMHC/TCR interactions plays a major role in T cell recognition . Crystal52–59 (VSV8), VSV8 triggers negative selection of DP thymocytes in the context of H-2Kb. In contrast, a weak agonist peptide variant, identical to the VSV8 peptide except for substitution of leucine for valine at the p4 peptide residue, termed L4, induces positive selection [b-restricted immunodominant LCMV epitope gp33–41 [33–41 peptide causes negative selection due to high affinity pMHC/TCR interactions. However, certain mutations of amino acid residues in the gp33–41 peptide affects the fate of thymocyte development in fetal thymic organ culture (FTOC) leading to positive selection P14-bearing thymocytes [366–379 in the context of H-2Db, a peptide antagonist mediates positive selection in FTOC [TCR-transgenic mice provide useful tools for studies of peptide-based thymocyte selection. For example, in N15 transgenic mice carrying a TCR specific for the vesicular stomatitis virus nucleoprotein octapeptide Nelection . Similar gp33–41 , the cogymocytes . In yet  in FTOC ,36, wher33–41 and VSV8 peptides with substitutions at residues interacting with the TCR aimed at reducing TCR-pMHC affinity via diminution of the number of atomic contacts between the peptide and the TCR. Subsequently, we examined the effects of these APL on thymocyte maturation and emigration in vivo in two well-defined TCR-transgenic mouse systems. In the case of gp33–41 cognate peptide, amino acids at the peptide positions p4  and p6  were modified to Ser (S) and Ala (A), respectively, based on the crystal structure of the gp33–41/H-2Db complex showing exposure of the side chains of these amino acid residues to the solvent and hence, TCR accessibility [b, thus ensuring proper peptide presentation in the context of MHC. In the other less extreme peptide variant of gp33–41, Ala (A) at p7 was substituted with Glu (E). For the VSV8 peptide, the weak L4 agonist with the substitution of Leu (L) for Val (V) at the p4 peptide residue has been employed. The crystal structure of the N15 TCR-VSV8/Kb complex as well as the space-filling models of Kb in complex with VSV8 and L4 peptides are shown in Fig. b as compared to L4/Kb, these focal changes (p4 and Lys 66 on the α1 helix of H-2Kb) determine the outcome of thymic selection [b/ gp33–41 vs. APL in which amino acid residues at p4 and p6 are altered, differentially affect development of thymocytes expressing the P14 TCR (data not shown). Experimental data using these APL (Y4S/F6A and A7E) for studies of thymocyte selection and emigration as applied to the thymic vaccination approach are summarized below .-/- lymph node and spleen showed a response to the high TCR affinity cognate peptide gp33–41, but not to the Y4S/F6A variant peptide. In support of this observation, tetramers of H-2Db in complex with the Y4S/F6A peptide did not bind to SP CD8 thymocytes from P14 Rag2-/- mice at any tetramer dilution, as judged by immunofluorescence analysis, whereas high fluorescence intensity staining was detected using tetramers of H-2Db in complex with the gp33–41 peptide. The A7E/H-2Db tetramer gave intermediate staining. These data suggested that the Y4S/F6A mutant must interact with the P14 TCR with extremely weak affinity, if at all.The binding of the APL to the MHC class I molecules using RMA-S cells confirmed that amino acid substitutions at peptide residues interacting with the TCR did not affect peptide binding and, by extension, peptide presentation to T cells. A series of experiments was next performed to evaluate the functional potential of the APL to stimulate peripheral T cells in mice injected with the variant peptides. Results of both proliferation and cytokine secretion assays using mature T cells from P14Rag233–41 and VSV8 leads to negative selection of P14- and N15TCR-bearing thymocytes, respectively, due to relatively high affinity pMHC/TCR interactions [In vivo administration of gp33–41 in P14Rag2-/- mice and VSV8 injection into N15Rag2-/- mice resulted in pronounced depletion of DP thymocytes. Surprisingly, injection of P14 Rag2-/- mice with the Y4S/F6A peptide mutant resulted in a significant increase in the total number of thymocytes as well as the DP thymocyte subpopulation, while the A7E variant had no effect on thymocyte counts. As with Y4S/F6A in the P14Rag2-/- system, injection of L4 in the N15Rag2-/- mouse preserved the DP thymocytes and led to an increase in total thymocyte counts. The unusual increase in the number of DP thymocytes following exposure to Y4S/F6A peptide was not due to cellular proliferation and attendant DNA synthesis as examined by in vivo BrdU incorporation assay. Rather, Y4S/F6A peptide administration prevented apoptosis as confirmed by staining of thymocytes with anti-Annexin V mAb. To more directly test this hypothesis, we injected P14 Rag2-/- mice with mixtures of the negatively selecting cognate peptide gp33–41 plus the Y4S/F6A APL. Increasing the amount of Y4S/F6A peptide in the injection mixture resulted in a higher number of total and DP thymocytes. Thus, we infer that the Y4S/F6A variant may compete with other endogenous negatively-selecting peptides for binding to H-2Db molecules expressed on thymic stroma either by binding to "empty" surface MHC class I molecules or, perhaps, by a cross-presentation mechanism [b/A7E tetramer binding assays suggest that this APL does not reduce TCR binding affinity sufficiently to stimulate positive selection.Injection of the cognate viral peptides gpractions ,34. In v-/- mice (donor) were injected into irradiated congenic B6 mice (recipient) and the development of donor-type cells was monitored weekly by immunofluorescence staining and multicolor FACS analysis [The numerically small population of antigen-specific recent thymic emigrants (RTE) makes thymic selection/emigration studies difficult even with the use of TCR transgenic mice. To resolve this issue, we employed irradiation chimeras of congenic mouse strains (expressing the CD45.1 marker in B6 and CD45.2 in P14 and N15 transgenic mice) to determine whether interactions between the low affinity ligands, Y4S/F6A and L4, and their specific TCRs would result in thymic positive selection and subsequent emigration from the thymus. For this purpose, lineage-minus BM precursors of P14 – or N15- TCR transgenic Rag2analysis . Followi+ thymocytes in irradiation chimeras injected with Y4S/F6A were greatest, suggesting that this ligand mediated positive selection of P14 Rag2-/--specific T cells. Similarly, in N15 Rag2-/--B6 irradiation chimeras injected with L4, the numbers of both DP and SP CD8 donor thymocytes were highest, consistent with positive selection. In contrast, injection of either gp33–41 or VSV8 cognate viral peptides into irradiation chimeras led to thymocyte depletion by negative selection. Analysis of the peripheral lymphoid organs in these chimeras by triple color immunofluorescence with anti-CD45.2, anti-CD8α and anti-TCR-specific mAbs showed the greatest number of donor-type CD45.2+CD8+Vα2+ T cells in the lymph nodes of Y4S/F6A -injected chimeras (2–3 fold over PBS-injected control mice), suggesting that donor-type thymocytes expressing the P14 TCR had developed in the presence of Y4S/F6A, matured and emigrated to the lymph nodes. A similar increase in the donor cell numbers were observed up to 9 weeks after injection of Y4S/F6A peptide. Positive selection was also evident in N15 Rag2-/- -B6 irradiation chimeras injected with the L4 variant. In this case, higher CD8+Vβ5.2+ N15 TCR transgene donor-type T cell numbers were observed both in lymph nodes and spleens. The functional analysis of donor-type CD8+ lymph node T cells in irradiation chimeras injected with the positively selecting Y4S/F6A or L4 peptides showed approximately two-fold higher proliferation levels in response to the cognate peptides gp33–41 and VSV8, respectively, in vitro, compared to cells from PBS control-injected chimeric mice, reflecting the two-fold difference in the number of donor-type CD8+ T cells in lymph nodes of chimeras injected with the APL. However, importantly, these mature donor-type T cells did not proliferate in response to Y4S/F6A or L4 variant peptides in vivo. Injection of the viral peptides and their APLs in vivo led to reduction of CFSE+ staining in the case of gp33–41 and VSV8, suggestive of proliferation and/or activation-induced cell death (AICD). In contrast, no change in CFSE+ staining was observed upon injection of Y4S/F6A or L4 peptides, implying that these APLs do not facilitate T cell expansion per se. In sum, the cognate peptide ligands gp33–41 and VSV8 which interact with the TCR with relatively high affinity compared to their respective APL, induce activation of peripheral T cells, whereas peptide variants Y4S/F6A and L4, which bind TCR with low affinity and mediate positive selection, do not stimulate mature T cell divisions.The numbers of donor DP and SP CD8in vivo. In addition, we show that thymocyte emigration is dependent on the affinity/avidity of pMHC/TCR interactions. These results suggest that although the low affinity pMHC/TCR interactions are insufficient to trigger cell divisions in mature cells, differentiation of immature thymocytes nevertheless follows. Affinity measurements support the idea that positively selecting peptide ligand affinities are lower than those of negatively selecting ligands for TCRs, but additionally linked to their MHC binding/stability properties [The data described above and previously  represenoperties . Our repoperties . Two recoperties ,44.in vivo in a manner useful to the organism. Whether these peptide-specific T cells generate stronger defense mechanisms to fight viral infection or tumors in normal, non-transgenic mice remains to be investigated. The magnitude of the APL-driven increase in thymocytes and subsequent egress is only 2–3 fold, however. This level of change likely reflects the tightly regulated thymocyte egress process. In this respect, exploring peptide-based means of enhancing differentiation of thymocytes bearing desired TCRs together with the modulation of mechanisms controlling thymocyte emigration to the periphery would be of a great importance. To this end, various pathways regulating egress from the thymus are described below and should be considered as potential targets for such manipulation in conjunction with APL administration. Although not discussed further here, thymic vaccination followed by conventional cognate antigen immunization may be the best way to insure a robust memory T cell response.Collectively, our data show that cognate peptides can be modified at key TCR recognition positions to create variants that result in selection, directly or indirectly, of desired TCR specificities at the level of thymic development. This exogenous peptide administration offers a potential of expanding repertoire generation Lymphocyte migration plays an important role in regulating the localization and orchestration of immune responses. As thymocytes progress through the developmental stages, they migrate from the cortico-medullary junction, the site of entry of T cell progenitors from the BM, to the subcapsular region of the thymus, then to the cortex and to the medulla ,19. Finaiα protein-coupled seven-transmembrane receptors (GPCR), leading to activation of intracellular secondary mediators which control directional cell migration. To date, 43 human chemokines have been identified, acting via binding to 19 different GPCR.Chemokines are basic polypeptides of about 100 amino acids, usually containing four Cys residues linked by disulphide bonds, which are produced by certain thymic stroma cells and are abundantly expressed in the thymus. Specifically, thymic epithelial, medullary epithelial and dendritic cells have been shown to secrete various chemokines. Growing evidence suggests that chemokines and their receptors, expressed differentially on thymocytes during discrete maturational stages, control homing of T cell progenitors to the thymus, their intrathymic migration, and exit to the periphery. Chemokines deliver signals for lymphocyte proliferation and survival, and regulate thymocyte trafficking by functioning in concert with other adhesion molecules such as selectins and integrins. Chemokines stimulate responding cells by activating pertussis toxin-sensitive GSome chemokine receptors are expressed in DP and SP thymocytes, e.g. CCR9, with its ligand CCL25 secreted by TEC and DC. Others, e.g. CCR5 and CCR8, expressed on mature SP thymocytes, have been suggested to play a role in mediating thymocyte emigration. In particular, CCR7 has been demonstrated to mediate homing of naïve T cells to peripheral lymphoid organs via ligands CCL19 and CCL21.+ macrophages in the thymus. Other ECM proteins including nidogen, associated with laminin, and galectins -1, -3, and -5 as well as glycosaminoglycans are produced by thymic epithelium. ECM proteins form molecular bridges between thymocytes and the thymic microenvironment, mediating adhesion of thymocytes via their ECM receptors VLA-4, -5 and -6, and their disassembly from the cell complexes. In the absence of ECM proteins, normal thymocyte development and migration are severely perturbed, both in in vitro cultures of TEC and in in vivo knockout mouse models, suggesting a crucial role of the ECM protein network in the thymic function.Extracellular matrix (ECM) proteins laminin and fibronectin are produced by TECs, fibroblasts and MHC class II1–5/Edg) with up to nanomolar affinity, triggering diverse effects, including proliferation, survival, migration, morphogenesis, adhesion molecule expression, and cytoskeletal changes. S1P receptors are widely expressed during embryonic development and in the adult. The tissue distribution shows that lymphoid organs express high levels of S1P1 and S1P4. Thus, these receptors may be potential targets for pharmacological drug design aimed at effecting thymocyte migration.Sphingosine 1-phosphate (S1P), a member of sphingolipid family, is an important signaling molecule present in high concentrations in body fluids. SIP binds to members of a family of G protein-coupled receptors .Thymic vaccination might offer promising clinical applications as a way of immunization against these infectious diseases and cancers, enabling "designer" thymic development to produce suitable and long-lasting protective T cell immune specificities. In a converse role, deletion of unwanted T cell specificities in the case of autoimmunity by agonist administration early in life could be considered. Assuming thymic vaccination proves clinically viable, immune responses against invariant components of infectious agents, such as HIV and malaria, which otherwise utilize their intrinsic mutational capacity to evade human immune recognition, can be targeted. Design of novel vaccines based on the thymic vaccination approaches will benefit from the information gained in the recently completed Human Genome Project, particularly as genetic polymorphism associated with high risk of developing certain diseases later in life including cancers, infectious disease susceptibility and autoimmunity are uncovered. For example, the ability to manipulate the T cell repertoire to elicit anti-tumor responses early in life may prevent clinical disease evolution later. Powerful bioinformatic tools such as computer-based identification of HLA-allele specific binding epitopes and structural insight into TCR-pMHC interactions will aid in the epitope-based APL design process .in vivo mouse models using irradiation chimeras reconstituted with bone marrow progenitors from TCR-transgenic animals. Increasing emigration of antigen-specific T cells from the thymus to the periphery is a challenging goal. In the future, a combined approach of exposing the subject to a positively selecting APL plus a thymic export-enhancing agent might generate practical and efficient protective repertoire manipulations. Potential applications may include design and administration of APL against cancer, infectious and autoimmune diseases.A thymic vaccination strategy has been conceived based on the current knowledge of thymocyte differentiation and repertoire generation. This approach differs substantially from conventional vaccination since it aims to shape T cell responses through thymic repertoire manipulation, exposing developing thymocytes to positively selecting APL derived from infectious agents or tumors. Experimental data to date suggest that this strategy is possible in APL, altered peptide ligand; BM, bone marrow; CMJ, cortico-medullary junction; DC, dendritic cells; DN, double negative; DP, double positive; GPCR, G-protein-coupled receptors; HEV, high endothelial venule; MMP, matrix metalloproteinases; RTE, recent thymic emigrants; SP, single positive; S1P, sphingosine 1-phosphate; TCR, T cell receptor; TEC, thymic epithelial cells.The author(s) declare that they have no competing interests.MFH carried out the study, including experimental design and data acquisition, drafted and revised the manuscript. ELR conceived of the study, participated in its design and coordination, and helped to draft and revise the manuscript. The authors read and approved the final manuscript.