There is an increasing prevalence of asthma in the Caribbean and patients remain non-compliant to therapy despite the development of guidelines for management and prevention. Some patients may self-medicate with medicinal herbs for symptomatic relief, as there is a long tradition of use for a variety of ailments. The study assessed the prevalence of use and the factors affecting the decision to use herbs in asthmatic patients attending a public specialty care clinic in Trinidad.de novo, pilot-tested, researcher-administered questionnaire between June and July 2003.A descriptive, cross-sectional study was conducted at the Chest Clinic in Trinidad using a Fifty-eight out of 191 patients (30.4%) reported using herbal remedies for symptomatic relief. Gender, age, ethnicity, and asthma severity did not influence the decision to use herbs; however, 62.5% of patients with tertiary level schooling used herbs, p = 0.025. Thirty-four of these 58 patients (58.6%) obtained herbs from their backyards or the supermarket; only 14 patients (24.1%) obtained herbs from an herbalist, herbal shop or pharmacy. Relatives and friends were the sole source of information for most patients (70.7%), and only 10.3% consulted an herbalist. Ginger, garlic, aloes, shandileer, wild onion, pepper and black sage were the most commonly used herbs.Among patients attending the Chest Clinic in Trinidad the use of herbal remedies in asthma is relatively common on the advice of relatives and friends. It is therefore becoming imperative for healthcare providers to become more knowledgeable on this modality and to keep abreast with the latest developments. Recent reports from the Caribbean suggest that the incidence of asthma is following the global trend of increasing prevalence. In Jamaica, a prevalence of 20.8% for exercise-induced asthma was estimated in a cross-sectional study in schoolchildren . About oInhaled corticosteroids as prophylaxis and 'as required' bronchodilator for symptomatic relief are established modalities for asthma management and prevention and the Commonwealth Caribbean Medical Research Council/Global Initiative for Asthma guidelines were adopted in the Caribbean in 1997 . It has Over the last few decades, a global resurgence in the use of herbal remedies has fuelled the growing multi-billion dollar international trade of botanical products. Many patients, dissatisfied with conventional medicines because they expect permanent cures, believe that herbal remedies are 'natural' and sometimes self-medicate without informing their attending physician.Although there is a long history of traditional use of medicinal herbs throughout the Caribbean ,8 few stThis study was undertaken to assess the extent of use of herbal remedies by asthmatic patients attending a specialty chest clinic in Trinidad for symptomatic relief and to determine the factors influencing the patient's decision to use herbs.The study was approved by the Ethics Committee of the Faculty of Medical Sciences, University of the West Indies, St. Augustine campus and permission to interview patients was granted by the Director of the Chest Clinic of the Ministry of Health, Trinidad and Tobago. The study was conducted over the two-month period June to July 2003.de novo, pilot-tested, researcher-administered questionnaire.The Chest Clinic was chosen as the source of subjects as this is the only national tertiary level health facility specializing in the management of respiratory diseases. Patients entering the study were physician-diagnosed asthmatics based on self-reporting symptoms of wheezing, chest tightness and nocturnal coughing in the previous year. Patients were recruited by consecutive sampling and the nature and purpose of the study were explained on an individual basis. Those confirming their willingness to participate signed their informed consent and were interviewed using a The questionnaire assessed demographic data such as age, gender, ethnicity, residential district, education, employment and socioeconomic status. Subjects reported their disease severity as intermittent, moderate or severe as determined by the Global Initiative for Asthma (GINA) guidelines with respect to symptom frequency . Patient2 tests were performed to determine whether there were statistically significant associations between the use of herbs and these variables. The p value was set at <0.05 for statistical significance. The data was analyzed using SPSS for Windows .The sample size was calculated as 185 patients assuming a prevalence of 86%  with a cDuring the study period one hundred and ninety one patients consented to participate. The demographic details of the sample are given in Table 2-agonists (relievers) were prescribed at very high rates, Table 2-agonists. This high level of prescription and use of β2-agonists suggest a lack of symptomatic control in our sample population. Theophylline and anticholinergics were prescribed in both categories of patients, but to a lesser extent.The GINA guidelines were recently adopted in the Caribbean and asthmatic patients are currently treated according to their symptom severity. In our sample population, particularly in patients with moderate and severe symptoms, corticosteroids (controllers) and βGender, age, ethnicity, residential district, employment status, income and asthma severity had no statistically significant effect on the use of herbal remedies within the sample population, Table Most patients (70.7%) using herbs were advised by a relative or friend and only 10.3% sought the advice of an herbalist, Table Most patients (58.6%) obtained their herbs or medicinal plants from either their backyards or the supermarket. Only fourteen (24.1%) obtained their herbal supplies from an herbalist, herbal shop or pharmacy. Seventeen (29.3%) of these patients reported using herbs within the last week and most these patients (60.3%) used herbs within the last six months.Many of these patients were using both physician-prescribed antiasthmatic drugs and herbal remedies, Table Allium sativum) or ginger  for symptomatic relief of asthma, Table Aloe vera) shandileer (Leonotis nepetifolia), wild onion , pepper (Capsicum spp.) tulsi (Ocimum gratissimum), black sage (Cordia curassavica), shadon beni (Eryngium foetidium), lemongrass (Cymbopogon citratus) and nutmeg (Myristica fragrans) were the more popular traditional indigenous West Indian medicinal plants used. Two patients reported using marijuana (leaves and roots). Herbs of European and North American origin, identified as Echinacea (Echinacea purpurea), Golden Seal (Hydrastis canadensis) and Chamomile (Matricaria chamomilla) were less frequently used. Five patients reported using trade name imported tablets for asthma.Most patients in the sample used more than one medicinal herb simultaneously, which were usually prepared and administered as mixtures in teas. Almost one in four patients using medicinal herbs (22.5%) used either garlic  and aloes (Aloe vera), and traditional indigenous medicinal herbs such as shandileer (Leonotis nepetifolia) and tulsi (Ocimum gratissimum) were more likely to be earning less than US$12,000, Table Echinacea purpurea and Matricaria chamomilla) were more likely to be used by patients earning in excess of US$12,000 per annum. Income did not affect the use of either garlic or cocoa onion.Patients using easily accessible herbs such as ginger (Aloe vera), tulsi (Ocimum gratissimum) and golden seal were preferred in patients with at least twelve years of formal education, Table Leonotis nepetifolia), wild onion  or ginger .Aloes  were more expensive than conventional medicines. We assumed that an additional expense would have only been incurred by those patients purchasing processed, imported herbs obtained from a herbalist, herbal shop or pharmacy (24.1%) and who actually consulted a herbalist (10.3%). We also reasoned that since all the other herbs used were inexpensive and available from either the backyard garden or supermarket (58.6%) that the cost to patients selecting these remedies was minimal.The findings of this study are important in that local medicinal plants in Trinidad have been identified in the self-management of asthma in a significant number of patients attending the specialty clinic. These identified herbs can now be targeted for scientific investigation to determine whether their pharmacological efficacy will assist in the development of viable healthcare alternatives in a developing country. These findings are also important for policymakers in the health sector who are given the mandate to regulate issues pertaining to the public's health. We are also becoming more aware of the potential for critical interplay between herbs and drugs when taken concomitantly to produce life-threatening interactions. Since herbs are here to stay and patients will continue to self-medicate with increasing frequency, it is imperative that healthcare providers become more knowledgeable on this modality and keep abreast with the latest developments in herbal therapy.The author(s) declare that they have no competing interests.YNC was the P.I. in this study. He was responsible for the study concept, development of methodology, coordinating the research activities, analyzing the data, and writing the manuscript. AFW was responsible for data input and analysis. DA was involved in methodological development, data collection, data input and analysis and presentation at regional conference. RC was involved in methodological development, data collection, data input and analysis. NW was involved in methodological development, data collection and input. RM was involved in methodological development, data collection and input. OS was involved in methodological development, data collection and input. DW was involved in methodological development, data collection and input. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Escherichia coli sequences display the period close to 11 base pairs.Sequence periodicity with a period close to the DNA helical repeat is a very basic genomic property. This genomic feature was demonstrated for many prokaryotic genomes. The Escherichia coli. The noncoding sequences reveal this periodicity much more prominently compared to protein-coding sequences. The sequence periodicity of ApC/GpT, ApT and GpC dinucleotides along the Escherichia coli K-12 is found to be located as well mainly within the intergenic regions.Here we demonstrate that practically only ApA/TpT dinucleotides contribute to overall dinucleotide periodicity in E. coli suggests that the periodicity is a typical property of prokaryotic intergenic regions. We suppose that this preferential distribution of dinucleotide periodicity serves many biological functions; first of all, the regulation of transcription.The observed concentration of the dinucleotide sequence periodicity in the intergenic regions of  E. coli as well, is distributed in a non-uniform way, in scattered segments of the size 100–150 bases. It was also known for a long time that quite a few DNA promoter regions of E. coli possess the sequence periodicity of AA and TT dinucleotides [DNA sequence periodicity with the period about 10–11 base pairs (bp) has been long known in eukaryotic DNA sequences. It was discovered recently in prokaryotic sequences as well . The perE. coli promoters have upstream curved sequences [E. coli DNA curvature peaks are frequently located downstream of the CDS.The sequence periodicity of AA/TT dinucleotides is frequently associated with sequence-dependent DNA curvature, which is known to play an important role in the initiation of transcription of many genes . Usiequences ,17. Pedeequences  showed tSince the dinucleotide periodicity with the period close to the helical repeat is associated with DNA intrinsic curvature -23, the E. coli and its distribution along the genome are systematically analyzed. A strong preference of intergenic regions to express the sequence periodicity of AA, AC, GC, and TT dinucleotides is discovered.In this work, the sequence dinucleotide periodicity in E. coli, as well as its coding and noncoding regions, was subjected to this procedure. Resulting autocorrelation profiles for all 16 dinucleotides (data not shown) were further analyzed by Fourier transform. In Fig. Positional autocorrelation analysis of the nucleotide sequences is an appropriate tool to detect all major characteristic distances in the sequences, the periodicities in particular. The complete genome of E. coli and find out where the periodical regions are located, we chose the period 11.2 bp [E. coli genome sequence and to detect the periodical sites. This calculation shows that the periodicity is not evenly distributed along the E. coli genome.To screen the genome of  11.2 bp  and this 11.2 bp ,26. We uE. coli genome are shown. The periodicity is distinctly located in certain regions. Many of the peaks observed are found to correspond to the intergenic regions (indicated by the black bars at the top). For example, two such peaks of periodicity in Fig. In Fig. To verify the apparent strong correlation between the intergenic regions and AA/TT periodicity, we split intergenic regions in several families by size and analyzed the subsets separately by aligning (centering) the regions and summing up the respective local periodicity distributions. The combined maps for intergenic regions with a size from 50 to 150 bp, from 150 to 250 bp, from 250 to 350 bp, from 350 to 450 bp, and from 450 to 550 bp are shown in Fig. E. coli DNA sequence periodicity.To verify the choice of the period 11.2 bases, we calculated the periodicity maps for highly populated group of the regions of the size 200 ± 50 bp, by assuming different periods in the range 10.5–12.5 bases. The resonance 3D plot in Fig. The spectral analysis Fig.  and examThe observed concentration of the sequence periodicity in the intergenic regions corroborates earlier results and suggests that the periodicity is a typical property of the intergenic regions.Escherichia coli K-12 MG1655, locus U00096, 4639221 base pairs, was taken from the National Center of Biotechnology Information . Intergenic regions were identified in accordance with the annotation to this genome of E. coli and gathered in a separate dataset.The sequence of the whole genome of X was calculated for each dinucleotide separately. For the calculation of ApA autocorrelation, for example, we calculated the number of occurrences of pairs ApA – ApA in a distance k, and designated it by Xk. Spectral analysis of autocorrelation profile X was obtained using the following formulae:Autocorrelation profile fp is normalized wave-function amplitude of period p, X is an autocorrelation profile for one chosen dinucleotide, Xi is its value in position i,  is its average value, and W is a maximal considered autocorrelation distance (in our case 100 bp).where T were taken to describe idealized periodical distribution of AA and TT dinucleotides within window W. The probes were correlated with E. coli sequences by moving the probes along the sequences and calculating the value C for every position.As a probe of periodicity the sine waves with period i is an index of a dinucleotide position in the window W andwhere Cmax is introduced for the normalization purposes. It is calculated as follows:The value i is a position in the window W andwhere C = 1, while segments with no periodicity would correspond to C = 0. The results of these calculations are presented as maps of the sequence periodicity. The four sample maps are shown in Fig. Ideally periodical sequence segments would be, therefore, described by The maps around intergenic regions were combined (summed) separately for the groups of similar sizes of the intergenic regions. Five such groups were analyzed: 100 ± 50 bp, 200 ± 50 bp, 300 ± 50 bp, 400 ± 50 bp, and 500 ± 50 bp. For each group the maps were synchronized at the respective intergenic centers and the sums of the maps were calculated and smoothed by a running average within 51 bp. The standard deviations for the combined plots were estimated by generating random sequences of the same size and dinucleotides composition for each group separately and averaging the respective periodicity maps.T in the interval 10–12.5 bp. One-third (202) of the most periodic maps of this group was taken for the calculation. The maps for different periods T were smoothed five times by a running average over 51 bp. The baselines were set to 0. The surface of 3D plot was smoothed 3 times by a running average over 9 point square elements, on the grid with separations 0.1 bp for T, and 20 bp for sequence position.The resonance 3D plot for the intergenic regions of length 200 ± 50 bp was built from calculations with different periods None declared.SH carried out all graphics. ENT and AB participated in the design of the study and analysis of results. All authors drafted the manuscript. All authors read and approved the final manuscript.


Fasciola hepatica primarily involves the liver, however in some exceptional situations other organs have been reported to be involved. The ectopic involvement is either a result of Parasite migration or perhaps eosinophilic reaction.Here we report a known case of multiple myeloma who was under treatment with prednisolone and melphalan. He was infected by Fasciola hepatica, which involved many organs and the lesions were mistaken with metastatic ones.Presented here is a very unusual case of the disease, likely the first case involving the pancreas, spleen, and kidney, as well as the liver. Human fascioliasis, a commonplace infection caused by a leaf-shaped Trematode Fasciolahepatica affects a human host by chance -4. It seIn Dec 2001, a 52 year old man, who was a known case of Multiple Myeloma (MM), was presented to one of our affiliated hospitals with persistent right upper quadrant and epigastric pain, and anorexia for a period of 1 month. At the time of admission, the patient had been receiving prednisolone and melphalan for his MM, which was currently in remission. His recent condition began with tongue and facial edema two weeks before appearance of the abdominal pain.Upon physical examination, mild epigastric tenderness and a palpable liver were found. Neither icterus nor any positive sign of cardiopulmonary abnormalities were noted. Additionally, the patient did not have a fever and his peripheral lymph nodes were not enlarged. Initial laboratory findings were as follows: a hemoglobin of 10.6 g/dL, white blood cell count of 10,800/mm3 with 18% eosinophils, and a sedimentation rate of 90 mm at the end of the first hour. The total bilirubin was 0.5 mg/dL (0.2–0.8 mg/dl), alanine aminotranferase (ALT) 48 IU/L (0–40 IU/L), aspartate aminotransferase (AST) 46 IU/L (0–40 IU/L), and alkaline phosphatase (Alk P) 651 IU/L (60–140 IU/L). The eosinophilia fluctuated between 12 to 55 percent in various tests performed during the time period in question, with no unique patterns noted. Serum electrophoresis showed a monoclonal spike in the gamma region. Specific enzyme-linked immunosorbant assay (ELISA) produced a positive result for Fasciola hepatica, while the test was negative for Toxocara canis. Serologic tests for the presence of hepatitis A, B, and C viruses were negative. Blood and urine cultures were found to be sterile. Other laboratory studies, including repeated stool examinations for ova and parasites, showed no abnormalities. Chest x-rays did not demonstrate any parenchymal or pleural abnormality. Abdominal ultrasonography showed a mild hepatomegaly with multiple hypoechoic lesions in the liver. A CT scan revealed multiple but poorly defined, hypodense lesions in the liver, and a completely enlarged pancreas with mild bilateral pleural reaction, suggesting metastatic cancer . In the search for a potential malignancy, diagnostic laprascopy was performed, which revealed the presence of white-colored lesions ranging from 1 to 3 cm in diameter on the surface of both lobes of the liver with mild ascites. Multiple liver and peritoneal biopsy specimens revealed fibrinoid necrosis, associated with granulomatous reaction and a high concentration of eosinophils in the liver,  accompanied by markedly inflamed peritoneal tissue with eosniphilic infiltration. No malignant cells were identified and no evidence of extramedulary plasmocytoma was found. Specific staining for fungal organisms and acid-fast bacilli were negative. The ascitic fluid also had a high level of eosinophils. Endoscopic retrograde cholangiopancreatography (ERCP) failed to show any filling defect within the biliary tree. Furthermore, the patient underwent bone marrow aspiration that only indicated high eosniophilic infiltration. The patient was placed on albendazole (400 mg twice daily for 1 week). The treatment was well tolerated and the abdominal pain was improved rapidly. At the time of discharge, the patient was in good clinical condition. During a follow-up visit two months later, a second CT unexpectedly showed not only an increase in the number and size of the hypodense lesions in the liver, but also the extension of lesions into the pancreas, the spleen and both kidneys . No evidence of peripheral enhancement of the hepatic lesions or ascites was documented. The patient was still experiencing upper quadrant pain on the right side. Laboratory investigations produced a white blood cell count of 7200/mm3 with 16% eosinophilia. Repeated stool examinations failed to identify ova and parasites. The patient was given triclabendazole . As recommended, the patient had another follow-up CT scan three months later. At that time all of his symptoms were resolved. Follow-up CT scans revealed a considerable improvement in the number and size of the lesions. At the time of the CT scan, all of his symptoms were resolved . At this time the WBC was 6000/mm3 with 6% eosinophils. After 5 months and in the last CT, the lesions had almost disappeared completely .While fascioliasis is a well-known human parasite, it sometimes produces unusual characteristics that may influence a clinician to misdiagnose the condition.In the vast majority of cases, the diagnosis is difficult in both acute and chronic phases and some important conditions such as liver abscesses and metastasis cannot be easily differentiated from fascioliasis ,13. InteAmong imaging tools, ultrasonography is of little diagnostic value during the acute phase, while a contrast-enhanced CT scan can be very useful for diagnosis ,17. In CAs clinical and laboratory findings of fascioliasis may easily be confused with many other conditions, a high index of suspicion is required to establish a correct diagnosis ,17. BothThe ingested metacercariae of Fasciola hepatica penetrate the intestinal wall and migrate through the peritoneal cavity to reach the liver. However, ectopic migration to other locations is one of the strangest manifestations of the infection ,9. The pThe pre-publication history for this paper can be accessed here:


The endometrium prepares for implantation under the control of steroid hormones. It has been suggested that there are complicated interactions between the epithelium and stroma in the endometrium during menstrual cycle. In this study, we demonstrate a difference in gene expression between the epithelial and stromal areas of the secretory human endometrium using microdissection and macroarray technique.The epithelial and stromal areas were microdissected from the human endometrium during the secretory phase. RNA was extracted and amplified by PCR. Macroarray analysis of nearly 1000 human genes was carried out in this study. Some genes identified by macroarray analysis were verified using real-time PCR.In this study, changes in expression <2.5-fold in three samples were excluded. A total of 28 genes displayed changes in expression from array data. Fifteen genes were strongly expressed in the epithelial areas, while 13 genes were strongly expressed in the stromal areas. The strongly expressed genes in the epithelial areas with a changes >5-fold were WAP four-disulfide core domain 2 (44.1 fold), matrix metalloproteinase 7 (40.1 fold), homeo box B5 (19.8 fold), msh homeo box homolog (18.8 fold), homeo box B7 (12.7 fold) and protein kinase C, theta (6.4 fold). On the other hand, decorin (55.6 fold), discoidin domain receptor member 2 (17.3 fold), tissue inhibitor of metalloproteinase 1 (9 fold), ribosomal protein S3A (6.3 fold), and tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (5.2 fold) were strongly expressed in the stromal areas. WAP four-disulfide core domain 2 (19.4 fold), matrix metalloproteinase 7 (9.7-fold), decorin (16.3-fold) and tissue inhibitor of metalloproteinase 1 (7.2-fold) were verified by real-time PCR.Some of the genes we identified with differential expression are related to the immune system. These results are telling us the new information for understanding the secretory human endometrium. Many studies have sought to understand the mechanism of implantation. Recently, the rate of pregnancy in the in vitro fertilization and embryo transfer (IVF-ET) cycle has declined, and this has been attributed to a decrease in the rate of implantation. The recently developed laser microdissection method has gained widespread use throughout the research field. Information about cells can be determined without contamination by using this method. Moreover, with the macroarray technique, which was already widely used for this purpose, the profiling of the gene expression of specific cells types has become possible. Torres et al. have already reported differences in gene expression between cell types or regions within the monkey endometrium using laser microdissection and differential display ,2. Identification of cell-specific proteins, which are expressed in the endometrium during the secretory phase, has been performed using a multi-disciplinary approach in the same trial. IGF-II mRNA is expressed in the mid-to-late secretory phase and in early pregnancy . During In this study, we demonstrate differential in gene expression between the epithelial and stromal areas obtained from secretory human endometrium using laser microdissection and the macroarray method. Confirmation of differential expression of candidate genes was performed by real-time PCR. Human endometrium was obtained from 8 patients (25–38 years old) with normal menstrual cycles (28~30 days) during the mid secretory phase. These patients had had at least one intrauterine pregnancy in the past . Part of the endometrial biopsy was obtained with a curetting technique. The day of the menstrual cycle was determined by the patient's history, plasma progesterone levels (9.8~17.3 ng/ml) and the histological criteria of Noyes et al . These pThe endometrium was embedded in OCT compound and frozen immediately in isopentane that had been cooled in the liquid nitrogen. This freezing block was sliced by a cryomicrotome at 8 μm thickness. Frozen sections were fixed in 100% methanol for 3 min and stained with 1% toluidine blue. The section was laser-microdissected by the PALM MicroBeam system (PALM Microlaser. Technologies A.G.) for epithelial and stromal areas and collected in a small tube Fig . Approxi32P dCTP (3000 Ci/mmol) using a randam primer. Labeled probes were hybridized to a nylon array ) in ExpressHyb solution at 68°C overnight. After hybridization, the nylon membrane was washed with 2 × standard saline citrate (SSC) + 1% sodium dodecyl sulphate (SDS)  once, twice with 1.0 × SSC + 0.5% SDS at 68°C [The RNAs obtained were synthesized from cDNA using a modified oligo (dT) primer and the BD SMART™ PCR cDNA Synthesis Kit . cDNA was PCR amplified for 24–29 cycles according to the user manual. ). 550 ng of cDNA sample was labeled with α- at 68°C ,13. The  at 68°C ,15. In tRNA was reverse transcribed using oligo (dT) primers by TaKaRa RNA PCR Kit (AMV) Ver 2.1  according to the manufacturer's instructions. PCR was performed using the ABI PRISM 7700 Sequence Detection System. TaqMan Universal PCR MasterMix and Assays-on-Demand Gene Expression probes (Applied Biosystems) were used for the PCR step . Primer sequences are not publicly available, although their validity has been established by the manufacturer. The expression values obtained were normalized against those from the control human GAPDH [Secretory endometrium was collected from 8 patients . Each sample was carefully dissected by laser microdissection for epithelial and stromal areas Fig  to 1d.Total RNA was extracted and subjected to macroarray with nearly 1000 genes on the nylon membrane. Fifteen genes were strongly expressed in the epithelial areas Table . Mean vaReal-time PCR  was used to verify the changes in expression of certain candidate genes that are highly expressed in the array. Five samples were used for this study. WFDC2 and MMP7, which are both strongly expressed in the epithelial areas and decorin and TIMP1, which are both strongly expressed in the stromal areas by the cDNA array were chosen for verification. Each value was corrected for differences in loading relative to GAPDH mRNA expression. WFDC2 and MMP7 mRNA expression increased by 9.4- and 9.7-fold, respectively, compared to that of stromal cells. Decorin and TIMP1 mRNA expression increased by 16.3-and 7.2-fold, respectively, in stromal cells compared to that of epithelial cells. Statistically significant changes in expression of these genes were observed (p < 0.05) Fig  and 2D.in vivo. It has recently become possible to acquire the information about the cell by the microdissection method. To date, various methods have been used for understanding the function of the endometrium. It is a well-known fact that epithelial cells and stromal cells in the endometrium play specific roles and are influenced by steroid hormones. However, it is very difficult to understand the molecular composition of each cell type as a function of time during the menstrual cycle. One of the problems of a cell culture experiment is that separation cultivation makes changes the composition of the cells. This is especially true as these cells are influenced by neighboring cells In this study, laser microdissection was used to isolate epithelial and stromal areas from the human endometrium. RNA was amplified by PCR and global gene expression was demonstrated by cDNA macroarray. Twenty-eight genes were identified in this study. These constitute only 2.8% of the 1000 genes on the array. Although this seems to be a small number, these genes were expressed at least 2.5-fold greater in all three samples and normalized to two house keeping genes. A similar percentage (1.2–5.8%) of genes with differential expression were reported using array analysis -19. HoweRecently, some papers focused on endometrial gene expression have been reported. However, lots of them were compared between phases in the menstrual cycle. While Okulicz et al. demonstrated a difference in the gene expression between cell compartments in the monkey endometrium, the genes they identified are not the same as ours ,2. One oFifteen of 1000 genes were strongly expressed in the epithelial areas compared to the stromal areas. Of these, WFDC2 and MMP 7 were strongly expressed in the epithelial areas as confirmed by real-time PCR. WFDC2 was originally described as an epididymis-specific protein is expressed in a number of normal human tissues. A possible role for this gene in sperm maturation is indicated by amino acid similarities to extracellular proteinase inhibitors of genital tract mucous secretions . AlthougHOXB gene induction is related to the immune system, and is specifically associated with IL-2-induced NK cell proliferation ,28. AlthOf the strongly expressed genes in the stromal aeas, decorin and TIMP1 gene expression were verified by real-time PCR. San Martin et al. reported the expression of decorin which is a leucine-rich proteoglycan in the mouse uterine and suggested it localized in the undifferentiated interimplantation site stroma . Some re


Previously we have shown that there are significant differences in empA expression in two strains of V. anguillarum, M93Sm and NB10. It is hypothesized that differences in empA regulation are due to differences in binding of regulatory elements.The induction of metalloprotease encoded by V. anguillarum, M93Sm and NB10, were examined and compared for the presence of DNA regulatory proteins that bind to and control the empA promoter region. Gel mobility shift assays, using a digoxigenin (DIG)-labeled oligomer containing a lux box-like element and the promoter for empA, were done to demonstrate the presence of a DNA-binding protein. Protein extracts from NB10 cells incubated in Luria Bertani broth + 2% NaCl (LB20), nine salts solution + 200 μg/ml mucus (NSSM), 3M , or NSS resulted in a gel mobility shift. No gel mobility shift was seen when protein extracts from either LB20- or NSSM-grown M93Sm cells were mixed with the DIG-labeled empA oligomer. The azocasein assay detected protease activity in all incubation conditions for NB10 culture supernatants. In contrast, protease activity was detected in M93Sm culture supernatants only when incubated in NSSM. Since the luxR homologue in V. anguillarum, vanT, has been cloned, sequenced, and shown to be required for protease activity, we wanted to determine if vanT mutants of NB10 exhibit the same gel shift observed in the wild-type. Site-directed mutagenesis was used to create vanT mutants in V. anguillarum M93Sm and NB10 to test whether VanT is involved with the gel mobility shift. Both vanT mutants, M02 and NB02, did not produce protease activity in any conditions. However, protein extracts from NB02 incubated in each condition still exhibited a gel shift when mixed with the DIG-labeled empA oligomer.Two strains of V. anguillarum NB10 cells contain a protein that binds to a 50 bp oligomer containing the empA promoter-lux box-like region. NB10 cells express empA during stationary phase in all growth conditions. The DNA binding protein is not present in M93Sm extracts. M93Sm cells express protease activity only when incubated at high cell density in fish gastrointestinal mucus. The gel shift observed with NB10 cells is not due to VanT binding. The data also suggest that the DNA binding protein is responsible for the less restrictive expression of empA in NB10 compared to M93Sm.The data demonstrate that protein extracts of  Vibrio anguillarum is the causative agent of vibriosis, one of the major bacterial diseases affecting fish, bivalves, and crustaceans  × (1 × 109)]  .V. anguillarum cells as previously described [vanT-F2 and vanT-R2 . Sequencing was performed on a Beckman-Coulter CEQ 8000. The Dye Terminator Cycle Sequencing (DTCS) quick start kit was used for the sequence reactions that were prepared according to the manufacturer's specifications and run in a thermal cycling program. DNA samples were mixed with the appropriate primer Table  and thenS.M.D. carried out the experimental part of the study and drafted the manuscript. D.R.N. conceived of the study, participated in its design and coordination, and edited the manuscript. P.S. participated in the experiment design and assisted with gel shift analysis. All authors have read and approved the final manuscript.


The correct final concentrations for this buffer are as follows:After the publication of this work  it was bHEPES 50 mMNaCl 100 mMNaF 10 mMEDTA 5 mMNa3VO4 0.5 mMNEM 2 mMTriton 0.1%Complete protease inhibitors (Roche).We regret any inconvenience that this inaccuracy may have caused, and thank Dr. Bräuer for bringing it to our attention.


Women report fear of pain in childbirth and often lack complete information on analgesic options prior to labour. Preferences for pain relief should be discussed before labour begins. A woman's antepartum decision to use pain relief is likely influenced by her cultural background, friends, family, the media, literature and her antenatal caregivers. Pregnant women report that information about analgesia was most commonly derived from hearsay and least commonly from health professionals. Decision aids are emerging as a promising tool to assist practitioners and their patients in evidence-based decision making.Decision aids are designed to assist patients and their doctors in making informed decisions using information that is unbiased and based on high quality research evidence. Decision aids are non-directive in the sense that they do not aim to steer the user towards any one option, but rather to support decision making which is informed and consistent with personal values.We aim to evaluate the effectiveness of a Pain Relief for Labour decision aid, with and without an audio-component, compared to a pamphlet in a three-arm randomised controlled trial. Approximately 600 women expecting their first baby and planning a vaginal birth will be recruited for the trial.The primary outcomes of the study are decisional conflict (uncertainty about a course of action), knowledge, anxiety and satisfaction with decision-making and will be assessed using self-administered questionnaires. The decision aid is not intended to influence the type of analgesia used during labour, however we will monitor health service utilisation rates and maternal and perinatal outcomes. This study is funded by a competitive peer-reviewed grant from the Australian National Health and Medical Research Council (No. 253635).The Pain Relief for Labour decision aid was developed using the Ottawa Decision Support Framework and systematic reviews of the evidence about the benefits and risks of the non-pharmacological and pharmacological methods of pain relief for labour. It comprises a workbook and worksheet and has been developed in two forms – with and without an audio-component (compact disc). The format allows women to take the decision aid home and discuss it with their partner. Making evidence-based decisions in clinical practice is not always straightforward: patients and their healthcare providers may need to weigh up the evidence between several comparable options, the evidence for some treatments may be inconclusive, and the information needs to be tailored to each patient's clinical context and personal preferences ,2. Good To assist patients and their doctors in making informed decisions, information must be unbiased and based on current, high quality, quantitative research evidence. However, patient information materials are often outdated, inaccurate, omit relevant data, fail to give a balanced view and ignore uncertainties and scientific controversies ,5. It isDecision aids are "interventions designed to help people make specific and deliberative choices among options by providing (at minimum) information on the options and outcomes relevant to the person's health status" . AdditioInternationally decision aids have been evaluated in a variety of health and clinical settings. Although their use in pregnancy and birth has only just begun to be explored, this is an area in which consumers are known to want to participate actively in decision making . A surveThe pain of labour is a central part of women's experience of childbirth and is a constant feature of antenatal discussion groups . Most woHowever satisfaction with childbirth is not necessarily contingent upon the absence of pain . Many wo• Continuous caregiver support for a single individual should be available to women in labour• Midwives must involve women in decisions about analgesia and recognise the value of promoting personal control• Maternity services should ensure access to written and verbal information on pain relief and should support women in their choices for pain relief• Maternity services should respect women's wishes to have some control over their pain relief• Improved public information and data on pain and analgesiaIn Australia over 250,000 women give birth annually and the increasing use of epidural analgesia means some 75,000 women have an epidural in labour each year . Among pRandomised controlled trials have shown epidural analgesia provides the most efficacious pain relief for labour, but the adverse consequences include prolonged labour, restricted mobility, use of oxytocin augmentation and an increased incidence of instrumental delivery ,18. ConsAlthough not as effective as epidural, randomised trials show inhalational analgesia (e.g. 50% nitrous oxide in oxygen) and systemic opioid analgesics (e.g. pethidine) can provide modest benefit to some patients during labour or supplement an unsatisfactory epidural . Both thA number of women prefer to avoid pharmacological analgesia if possible . The wisWomen report fear of pain in childbirth and often lack complete information on analgesic options prior to labour . For exaDickerson stresses the importance of discussing preferences for pain relief before labour begins . A womanThe management of pain in labour is a clinical decision that fulfils Eddy's criteria for a decision in which patients' values and preferences should be included . The outDuring 2003 and 2004, we developed an evidence-based decision aid about the management of pain in labour for women having their first baby. This followed a needs assessment that collected data on the attitudes, preferences and knowledge of nulliparous women who were making plans about pain relief for labour and childbirth. The needs assessment found that women's knowledge of pain relief options was limited and these women would benefit from a decision aid for labour analgesia.In developing the decision aid we utilised the NHMRC guideline "How to prepare and present information for consumers of health services"  and the The decision aid was designed for women to use at home or in the clinical setting, and takes about 30 minutes to complete. After working through the decision aid, women should take the completed worksheet to their next antenatal appointment to discuss their preferences with their health care provider. The worksheet is also useful for the practitioner, who can see rapidly from it what evidence the patient has considered, what her values and preferences are and which way she is leaning in her preferences for analgesia during labour.The decision aid was developed, pilot tested and revised with extensive consumer involvement, as outlined in the NHMRC guideline on preparing information for consumers . The conA number of draft decision aids , were developed and each subjected to pilot testing and revision as we obtained feedback. The process of testing and revising started with the study project group. The next phase included a review by a group of national and international content experts, including decision aid experts, obstetricians, midwives, perinatal epidemiologists, parent educators and psychologists. Once we were convinced that the content was accurate the decision aid was pilot-tested amongst consumers. There were several rounds of consumer review and refinement.Initially we aimed to compare the Decision Aid (workbook and audio-component) with usual care and counselling however preliminary work led us to alter our original study design. We could find no studies that compared Decision Aids with and without an audio-component. As the audio-component adds considerable complexity to the development and cost of the Decision Aid we decided to have two intervention arms: a Decision Aid with an audio-component and a Decision Aid without an audio-component. Further in pilot testing we found that women in the usual care arm were disappointed to not receive any information. Thus, to minimise refusals and losses to follow-up we decided to issue the women in the control group with a pamphlet called "Pain relief during childbirth – A guide for women" This pamphlet is published by the Royal Australian and New Zealand College of Obstetricians and Gynaecologists, is publicly available and includes information about methods of pain relief during labour . These cTo compare the relative effectiveness of the Pain Relief for Labour Decision Aid with a pamphlet on women's decisional conflict, knowledge, expectations, satisfaction with decision making and anxiety, and examine its impact on service utilisation and perinatal outcomes (as secondary outcomes).The primary study hypotheses are:Use of the Pain Relief for Labour Decision Aid by women expecting their first baby:1. Reduces decisional conflict (uncertainty about the course of action)2. Increases knowledge of labour analgesia3. Increases satisfaction with their decision making4. Reduces anxiety.The secondary hypotheses of the study are:Use of the Pain Relief for Labour Decision Aid by women expecting their first baby will not influence:1. The type of analgesia women use for labour2. Maternal and infant outcomes.We will conduct a randomised trial with the following study groups to assess the impact of the decision aid:Group 1: The pamphlet, "Pain relief during childbirth – A guide for women" Group 2: Decision aid with an audio-componentGroup 3: Decision aid without an audio-componentAn Australian tertiary obstetric hospital with a full range of non-drug and anaesthetic options for pain relief in labour. Epidurals are available 24 hours a day from anaesthetic staff designated to labour ward. All forms of antenatal care  will be included in the study.Primiparous women in late pregnancy (≥36 weeks gestation) who are expecting to have a vaginal birth of a single infant will be eligible for the study. Primiparous women were selected because previous pregnancies have a strong impact on decision making and analgesia use in labour ,16. ExclThe study procedure draws on the usual schedule of weekly antenatal visits in late pregnancy Figure . We planBrief baseline data will be collected to assess comparability of the study groups. The baseline assessment will include age, brief socio-demographic data, highest level of education achieved, anxiety as assessed by the state component of the short Spielberger anxiety scale , and infThe aim of the decision aid is to assist preference elicitation, and not to influence the direction of the decision taken. Women in each study group will be given the opportunity to review the intervention they are allocated (decision aid or pamphlet) while in the antenatal clinic and/or to take home, which ever is most convenient. Many women will also want to discuss their preferences with their partner. At the next antenatal visit, women will be contacted by the research nurse to discuss the information materials and any questions they may have had.All participants will be given a follow-up questionnaire prior to their next antenatal consultation. (See Outcome Measure details below).After a study participant delivers, the midwife who provided the labour care will complete a brief questionnaire to assess the impact of the decision aid on the management of labour analgesia. Information will also be collected on caregiver support in labour, birthplace (delivery suite or birth centre), use of non-drug analgesic options and stage of labour at admission.At 12–16 weeks postpartum all participants will be mailed a second follow-up questionnaire. This will assess women's satisfaction with the decisions made and the decision-making processes. (See Outcome Measures below). Questionnaires will be mailed with reply paid envelopes, with up to two reminder telephone prompts to non-responders.We will conduct in-depth interviews to explore the impact of the decision aid on women's experiences in labour and childbirth. A sub-sample of 30 women will be purposively selected, to reflect heterogeneity of experience of labour. The interviews will provide an understanding of the complexities of analgesic preferences, management, expectations, satisfaction, and psychological health following delivery. This data will enable examination of unpredicted and subtle effects of the decision aid on psychosocial outcomes that may not be captured using quantitative methods. Interviews will be face-to-face and conducted in women's homes or at a clinic, according to participants' preferences. Interviews will be recorded and transcribed. Data will be analysed using thematic analysis.As with many obstetric interventions blinding is virtually impossible. The main outcomes of this study are self-reported and the women are clearly not blinded to their treatment allocation. However, we will institute a number of measures aimed at keeping antenatal staff blind to the treatment allocation and preventing contamination of the control group:• Women will review the decision aid with the research nurse and complete the first questionnaire (primary outcome measures) prior to their next antenatal consultation• Usual antenatal care providers will be blinded to the exact content and format of the decision aid• Regular in-service  for the antenatal care providers to explain the trial protocol and to make clear the potential effect of unmasking or contamination.• Monitoring decision aid distribution and keeping them locked up and only accessible by the research nurse• Asking participants not to reveal their treatment allocation, or share their decision aid material with antenatal staff or other women. If participants do not want to keep their decision aid they will be asked to return it.The primary outcomes of this study will be:Decisional conflict (uncertainty about which preference to choose) will be assessed by the Decisional Conflict Scale which has established reliability, good psychometric properties and is short (16 items) [6 items) . It has is short 6 items [Measures of knowledge and realistic expectations about labour analgesia options and the benefits and risks of these options will be specific to this project. Thus we will need to develop, and test these measures as part of the project.Anxiety will be measured by the state component of the short Spielberger anxiety scale which has been extensively used and validated [alidated ,32. We dSatisfaction with analgesia decisions will be assessed using the Satisfaction with Decision Scale – a very brief six item scale with high reliability was developed specifically to assess satisfaction with health care decisions [ecisions .Satisfaction with the decision and anxiety will be measured again at 12–16 weeks postpartum. This interval was chosen to avoid the potential bias arising from questioning women still in the hospital who may feel a disloyalty to their caregivers by a critical appraisal and whose opinions have been shown to be more positive and short-lived than those obtained further out from the birth itself . At thatThe aim of the decision aid is to assist preference elicitation, and not to influence the direction of the decisions taken. Nevertheless, it is important to collect service utilisation and pregnancy outcome data so we will record and compare the pain relief methods used by women in all arms of the study, as well as recording and comparing rates of pregnancy complications and perinatal outcomes. The latter will be obtained (with informed consent) from the existing computerised obstetric database and include: medical or obstetric complications, induction or augmentation of labour, mode of delivery , enrolment to delivery interval, gestational age, birthweight, Apgar scores, perinatal deaths, Neonatal Intensive Care Unit admission and length of stay.The planned sample size is 600 women, with approximately 200 women to be recruited to each arm of the trial. Based on data for 2001 from the tertiary obstetric hospital where the study will be conducted, about 1500 primiparous women give birth to singleton infants after 36 weeks gestation and 92% use some form of analgesia. We anticipate that at least 50% of women will be both eligible and willing to participate.The sample size calculations for the trial  are based on the mean difference in the decisional conflict scale between any two arms of the trial. The effect of decision aids on this scale is documented and effect size data are available . Meta-anApproximately 20% of primiparous women have a caesarean section (6% before labour and 14% after labour has commenced) . Some ofIf there are no significant differences in outcome for the two decision aid groups (with or without the audio-component), the decision aid groups will be pooled giving two women with the intervention for each woman in the pamphlet group thereby increasing the power to detect differences between the decision aid and the pamphlet.Analyses will be by intention to treat, including withdrawals and losses to follow-up firstly of all women randomised and then excluding women who lose their options for analgesia. Study groups will be compared in terms of baseline characteristics. As this is a randomised trial, we would anticipate minimal differences in baseline characteristics. If however, important differences are found, these potential confounders will be adjusted for in the analysis of outcomes. For the primary outcomes, the mean score for each measure for each group will be compared using t-tests. If adjustment for confounders is needed a multiple linear regression model will be used. The secondary outcomes will be compared using chi-squared tests of significance for categorical data and t-tests for continuous data. If adjustment for confounding is necessary logistic regression and multiple linear regression will be used respectively.This work involves the development of a decision aid for the management of pain in labour and childbirth. Women must decide between a range of non-pharmacological and pharmacologic methods of pain relief. However this decision must be made in the context of the likely analgesic effects of each option, the risk of complications and adverse obstetric effects, and maternal preference for relief of pain. There are currently no evidenced based materials available. We therefore expect this project to be beneficial for participating women. A systematic review of decision aids found they improved knowledge without increasing anxiety. Nevertheless we will measure anxiety levels at baseline and follow-up to document any adverse effects. A trained research nurse will interview all women and obtain written informed consent. Women will be encouraged to discuss any concerns/anxiety with the research nurse and/or with their usual antenatal care provider. Women will be reassured that they are able to withdraw from the study at any time with no adverse effects on their pregnancy management. Participation will require women to complete self-report questionnaires during and after pregnancy. Working through the decision aid will take approximately 30 minutes and review of their preferences or outstanding questions will be at a routine antenatal visit. Therefore we do not consider this to be an excessive burden on their time.The study has been approved by the Central Sydney Area Health Service Ethics Review Committee (Protocol no. X02-0247) and the University of Sydney Human Ethics Committee (Ref No. 3419). This project is funded by a nationally competitive peer-reviewed grant from the Australian National Health and Medical Research Council (No. 253635).Participants in the trial will be identified by a study number only, with a master code sheet linking names with numbers being held securely and separately from the study data. To ensure that all information is secure, data records will be kept in a secure location at the University of Sydney and accessible only to research staff. As soon as all follow-up is completed the data records will be de-identified. De-identified data will be used for the statistical analysis and all publications will include only aggregated data. The electronic version of the data will be maintained on a computer protected by password. All hard copy patient identifiable data and electronic backup files will be kept in locked cabinets, which are held in a locked room accessed only by security code and limited staff. Data files will be stored for seven years after completion of the project as recommended by the NHMRC. Disposal of identifiable information will be done through the use of designated bags and/or a shredding machine.The author(s) declare that they have no competing interests.CR, CRG, LT and KM were involved in the conception and design of the study. CR, NN and CRG were responsible for the drafting of the protocol. All authors have read and given final approval of the final manuscript.The pre-publication history for this paper can be accessed here:


Chaperones (CH) play an important role in tumor biology but no systematic work on expressional patterns has been reported so far. The aim of the study was therefore to present an analytical method for the concomitant determination of several CH in human tumor cell lines, to generate expressional patterns in the individual cell lines and to search for tumor and non-tumor cell line specific CH expression.Human tumor cell lines of neuroblastoma, colorectal and adenocarcinoma of the ovary, osteosarcoma, rhabdomyosarcoma, malignant melanoma, lung, cervical and breast cancer, promyelocytic leukaemia were homogenised, proteins were separated on two-dimensional gel electrophoresis with in-gel digestion of proteins and MALDI-TOF/TOF analysis was carried out for the identification of CH.A series of CH was identified including the main CH groups as HSP90/HATPas_C, HSP70, Cpn60_TCP1, DnaJ, Thioredoxin, TPR, Pro_isomerase, HSP20, ERP29_C, KE2, Prefoldin, DUF704, BAG, GrpE and DcpS.The ten individual tumor cell lines showed different expression patterns, which are important for the design of CH studies in tumor cell lines. The results can serve as a reference map and form the basis of a concomitant determination of CH by a protein chemical rather than an immunochemical method, independent of antibody availability or specificity. Drosophila busckii. Since then, efforts from a large number of investigators have shown that the heat shock response is ubiquitous and highly conserved. It is observed in all organisms from bacteria to plants and animals. CH form an essential defense mechanism for protection of cells from a variety of harmful conditions, including temperature elevation or heat shock, decrease in pH, hypersalinity, alcohols, heavy metals, oxidative stress, inhibitors of energy metabolism, fever or inflammation -1-propane-sulfonate) , 65 mM 1,4-dithioerythritol , 1 mM EDTA (ethylenediaminetetraacetic acid) , protease inhibitors complete  and 1 mM phenylmethylsulfonyl fluoride (PMSF). The suspension was sonicated for approximately 30 sec in an ice bath. After homogenisation samples were left at room temperature for 1 h and centrifuged at 14,000 rpm for 1 h. The supernatant was transferred into Ultrafree-4 centrifugal filter units , for desalting and concentrating proteins. Protein content of the supernatant was quantified by the Bradford protein assay system . The staI values were used as given by the supplier of the immobilised pH gradient strips . Excess of dye was washed out from the gels with distilled water and gels were scanned with an ImageScanner (Amersham Bioscience). Electronic images of the gels were recorded using Adobe Photoshop and Microsoft Power Point softwares.Samples prepared from each cell line were subjected to 2-DE as described elsewhere . 1 mg pr+ ions of angiotensin I, angiotensin II, substance P, bombesin, and adrenocorticotropic hormones (clip 1–17 and clip 18–39). Each spectrum was produced by accumulating data from 200 consecutive laser shots. Those samples which were analysed by PMF from MALDI-TOF were additionally analysed using LIFT-TOF/TOF MS/MS from the same target. A maximum of three precursor ions per sample were chosen for MS/MS analysis. In the TOF1 stage, all ions were accelerated to 8 kV under conditions promoting metastable fragmentation. After selection of jointly migrating parent and fragment ions in a timed ion gate, ions were lifted by 19 kV to high potential energy in the LIFT cell. After further acceleration of the fragment ions in the second ion source, their masses could be simultaneously analysed in the reflector with high sensitivity. PMF and LIFT spectra were interpreted with the Mascot software . Database searches, through Mascot, using combined PMF and MS/MS datasets were performed via BioTools 2.2 software (Bruker). A mass tolerance of 100 ppm and 1 missing cleavage site for PMF and MS/MS tolerance of 0.5 Da and 1 missing cleavage site for MS/MS search were allowed and oxidation of methionine residues was considered. The probability score calculated by the software was used as criterion for correct identification.Spots visualised by Colloidal Coomassie Blue staining were excised with a spot picker , placed into 96-well microtiter plates and in-gel digestion and sample preparation for MALDI analysis were performed by an automated procedure  ,47. BrieThe algorithm used for determining the probability of a false positive match with a given mass spectrum is described elsewhere .CH chaperoneHSP heat shock protein2-DE two dimensional gel electrophoresisMALDI-MS matrix-assisted laser desorption ionisation mass spectrometryOGP octyl-β-D-glucopyranosideCHCA α-cyano-4-hydroxy-cinnamic acidPMF peptide mass fingerprintGRP glucose regulated proteinTCP t-complex proteinThe author(s) declare that they have no competing interests.JKM did data mining and contributed to the preparation of the manuscript. LAS performed protein extraction, two-dimensional electrophoresis and data handling. MFC carried out MALDI-TOF-TOF analyses. IS developed methodology for studying proteins from cell lines. GL initiated and planned the study developing the concept, supervised 2-DE, mass spectrometry, creating data and the manuscript. All authors have read and approved the final manuscript.Table 1. Identified proteins in different human tumor cell lines: Saos-2, SK-N-SH, HCT 116, CaOv-3, A549, HL-60, A-375, A-673, MCF-7 and Hela.Click here for fileTable 1-1. Identified proteins in different normal cell lines: Kidney, Lymphocyte, FibroblastClick here for fileI, observed pI, total score and peptide matched of molecular chaperones in tumor cell linesTable 2. Theoretical molecular weight, theoretical pClick here for fileTable 3. The list of tumor cell linesClick here for file


Ischemia within the optic nerve head (ONH) may contribute to retinal ganglion cell (RGC) loss in primary open angle glaucoma (POAG). Ischemia has been reported to increase neurotrophin and high affinity Trk receptor expression by CNS neurons and glial cells. We have previously demonstrated neurotrophin and Trk expression within the lamina cribrosa (LC) region of the ONH. To determine if ischemia alters neurotrophin and Trk protein expression in cells from the human LC, cultured LC cells and ONH astrocytes were exposed to 48 hours of oxygen-glucose deprivation (OGD). Also cells were exposed to 48 hours of OGD followed by 24 hours of recovery in normal growth conditions. Cell number, neurotrophin and Trk receptor protein expression, neurotrophin secretion, and Trk receptor activation were examined.Cell number was estimated using an assay for cell metabolism following 24, 48 and 72 hours of OGD. A statistically significant decrease in LC and ONH astrocyte cell number did not occur until 72 hours of OGD, therefore cellular protein and conditioned media were collected at 48 hours OGD. Protein expression of NGF, BDNF and NT-3 by LC cells and ONH astrocytes increased following OGD, as did NGF secretion. Recovery from OGD increased BDNF protein expression in LC cells. In ONH astrocytes, recovery from OGD increased NGF protein expression, and decreased BDNF secretion. Trk A expression and activation in LC cells was increased following OGD while expression and activation of all other Trk receptors was decreased. A similar increase in Trk A expression and activation was observed in ONH astrocytes following recovery from OGD.In vitro conditions that mimic ischemia increase the expression and secretion of neurotrophins by cells from the ONH. Increased Trk A expression and activation in LC cells following OGD and in ONH astrocytes following recovery from OGD suggest autocrine/paracrine neurotrophin signaling could be a response to ONH ischemia in POAG. Also, the increase in NGF, BDNF and NT-3 protein expression and NGF secretion following OGD also suggest LC cells and ONH astrocytes may be a paracrine source of neurotrophins for RGCs. Glaucoma is an optic neuropathy defined by characteristic optic nerve head and associated visual field changes. Nearly 67 million people worldwide are believed to have glaucoma, including an estimated 2.2 million in the USA ,2. PrimaNeurotrophins are polypeptide growth factors involved in the development and maintenance of neurons, as well as non-neuronal cells. Included in this family of trophic factors are nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin 4 (NT-4). Neurotrophin signaling occurs via two types of receptors including (a) tyrosine kinase high affinity Trk receptors and (b) the low affinity p75 receptor . The TrkIn addition to their localization at axon terminals, Trk receptors have been localized at neuronal cell bodies, at dendritic projections, and along axons -27. DiscThe administration of exogenous NTs has been shown to protect neurons from ischemic damage ,39 suggeIn POAG, the laminar plates of the LC are compressed and bow backward from the sclera producing an excavated and exaggerated optic cup . BecauseGiven that ischemia can be a component of ocular hypertension, we used oxygen-glucose deprivation (OGD) as an acute model of in vitro ischemia and examined the expression of NTs and their receptors by cultured LC cells and ONH astrocytes following OGD. We are aware that the model used (oxygen and glucose deprivation) is not a perfect model for what occurs in the glaucomatous optic nerve head. However, this acute model was an attempt to mimic the end results of a chronic condition, and as a "first step" we felt this model was adequate for examining the response to injury in these cell types.The LC and ONH astrocyte cell lines used in this study have been previously characterized and described . PrelimiTo determine an exposure of OGD that would result in cellular changes while allowing a majority of cells to remain viable, we examined cell number at various time points of OGD using an assay that estimates cell number based on cell metabolism. The oxygen level within the anoxic incubator was determined to be below detectable levels as measured using an Oxygen Test Kit . Lamina cribrosa and ONH astrocyte cell metabolism/cell number following OGD is shown in Figure The expression of NT protein in LC cells and ONH astrocytes following OGD is shown in Figures As seen in Figure Following 48 hours of OGD, ONH astrocytes Figure  demonstrFigure The expression of Trk receptor protein by ONH astrocytes following OGD and recovery from OGD is shown in Figure The expression of phosphorylated Trk receptor protein following OGD and recovery from OGD is shown in Figure Overall, exposure to OGD resulted in decreased phospho-Trk protein expression by LC cells and ONH astrocytes. Expression of the 148 kDa phospho-trk isoform was decreased to a statistically significant level in both LC cells and ONH astrocytes, as was the 120 kDa and 80 kDa isoforms in ONH astrocytes. The only increase in phospho-Trk receptor protein expression following OGD was observed in LC cells. Protein expression of the 120 kDa phospho-Trk receptor isoform was increased 60% over the controls in LC cells, which was statistically significant.Recovery from OGD resulted in increased protein expression of phospho-Trk receptor isoforms in LC cells and ONH astrocytes when compared to OGD alone, with the exception of the 120 kDa isoform in LC cells, which decreased toward control levels. Although its expression increased compared to OGD alone, expression of the 148 kDa isoform was still decreased to a significant level. Interestingly, the 120 kDa phospho-trk isoform was again the only isoform whose expression was increased above control levels. This 100% increase was observed in ONH astrocytes following recovery from OGD and was statistically significant.The secretion of NGF and BDNF following OGD and recovery from OGD is shown in Figure In this study we examined the protein expression of NTs and Trk receptors by LC cells and ONH astrocytes following OGD, an in vitro model of ischemia. Lamina cribrosa cell and ONH astrocyte responses to OGD and recovery from OGD are summarized in Tables Ischemia due to elevated IOP during POAG may cause changes within the ONH and contribute to RGC loss -11. As aNeurons, including RGCs, express Trk receptors not only at the axon terminal and cell body, but also along their axons -27 suggeThe expression of Trk receptors (both full length and truncated) by LC cells, ONH astrocytes or other cells within the LC could limit NT availability to RGCs -24. TherThere is evidence that reperfusion following ischemia is actually more detrimental to cells than ischemia itself ,46. To dIn conclusion, we have demonstrated that LC cells and ONH astrocytes increase the expression of NGF, BDNF and NT-3 protein following OGD, which may be neuroprotective for RGCs. Neurotrophins expressed by LC cells and ONH astrocytes following OGD or recovery from OGD bind and activate Trk receptors expressed by these cells. Increased NT signaling within LC cells and ONH astrocytes could increase cell survival following ischemic injury, but may compromise RGC survival during reperfusion. Further studies examining the expression of NTs and Trk receptors following hypoxia or transient ischemic insults would provide a better model for ischemic injury in POAG. Using this model, RGCs could be co-cultured with LC cells or ONH astrocytes to determine the neuroprotective effects of NTs during ischemic injury. By better understanding NT signaling within the LC under normal and injurious conditions, new strategies involving these factors could be developed to better treat patients with POAG.Lamina cribrosa cells and ONH astrocytes respond to conditions that mimic ONH ischemia by increasing NGF, BDNF and NT-3 protein expression and NGF secretion.Increased protein expression of Trk receptors and phosphorylated Trk receptors by LC cells and ONH astrocytes following OGD and recovery from OGD respectively suggest paracrine and/or autocrine NT signaling occurs within the ONH following ischemic injury.Lamina cribrosa cells and ONH astrocytes may be a paracrine source of NGF, BDNF and/or NT-3 for RGCs, especially during ischemic injury within the ONH throughout POAG progression.® Aqueous Non-Radioactive Cell Proliferation Assays and Emax™ ImmunoAssay Systems specific for NGF, BDNF, NT-3 and NT-4 were purchased from Promega Corporation, Madison, WI.DMEM and fetal bovine serum (FBS) were purchased from HyClone Labs, Logan, UT. The following materials were purchased from Gibco BRL Life Technologies, Grand Island, NY; glucose free DMEM, L-glutamine, penicillin/streptomycin and fungizone (amphotericin B). Costar 96-well plates and Nunc ELISA/EIA 96 well Maxisorp plates were purchased from Fisher Scientific, Pittsburgh, PA. Polyclonal antibodies to Trk A, Trk B, Trk C, truncated Trk B (Trk B.T) and phosphorylated Trk were purchased from Santa Cruz Biotechnology Inc, Santa Cruz, CA. CellTiter 962/95% air at 37°C and media was changed every 2 to 3 days. Characterization of these cells was performed as described previously [Lamina cribrosa and ONH astrocyte cell lines were obtained from human LC explants from separate donors as described previously . Cells weviously . Cells eeviously ,33,36. Ceviously -36. Botheviously ,33,36. A2/5% CO2) for 48 hours. The oxygen level within the anoxic incubator was measured using a Oxygen Test Kit  and was determined to be below detectable levels. Recovery following OGD was achieved by placing cells in OGD conditions for 48 hours and then allowing them to recover in growth media (Ham's F-10 Media or DMEM plus 10% FBS) and an aerobic environment (95% air/5% CO2) for 24 hours. Cells cultured in growth media and an aerobic environment for served as controls.Preliminary studies examining cell survival following anoxia, hypoxia, hypoglycemia or serum withdrawl demonstrated LC cells and ONH astrocytes were resistant to hypoxia and serum withdrawl alone (data not shown). To approach what is occurring in vivo, we used the more acute oxygen-glucose deprivation (OGD) model to examine NT and trk expression and NT signaling in cells from the ONH. Preconfluent, age-matched adult LC cells and ONH astrocytes were treated with serum free media for 24 hours. Oxygen-glucose deprivation (OGD) was achieved by culturing LC cells and ONH astrocytes in glucose free serum free DMEM in an anoxic incubator (95% N2) for 24 hours. Cells cultured in growth media and an aerobic environment for served as controls.Adult LC cells and ONH astrocytes were trypsinized, counted using a hemacytometer and plated into Costar 96-well plates at a density of 1,000 cells/well. Cells were allowed to attach overnight and were then placed in serum free media for 24 hours. Lamina cribrosa cells and ONH astrocyte were exposed to OGD as described above for 24, 48 or 72 hours. Recovery following OGD was achieved by placing cells in OGD conditions for 24 or 48 hours and then allowing them to recover in growth media and an aerobic environment . Metabolically active cells convert MTS into formazan, which is soluble in aqueous solutions. The quantity of the formazan product measured by the amount of absorbance at 490 nm is therefore directly proportional to the number of living cells. Cell metabolism/cell number per well was calculated from a standard curve generated using known amounts of cells per well. A standard curve was generated for each cell line assayed. Three LC cell lines and three ONH astrocyte cell lines were assayed. The entire experiment, including standard curves, was repeated twice. Changes in cell metabolism/cell number following OGD and recovery from OGD were reported as a percent of the control ± SEM.Cell number was estimated using the CellTiter 96c Protein Assay System . Cellular lysate (50 μg) was separated on denaturing polyacrylamide gels and then transferred by electrophoresis to nitrocellulose membranes. Blots were processed using primary antibodies and the Western Breeze Chemiluminescent Immunodetection System . Blots were then exposed to Hyperfilm-ECL  for various times depending on the amount of target protein present. The density (O.D. × mm2) of unsaturated bands was measured using the Discovery Series scanner and the Diversity One program from pdi  and a digital Venturis FP466 computer . Western blots for NTs and trks were stripped and re-probed for β-actin to ensure equal loading.Cellular protein was collected in lysis buffer modified from Watson et al.  [20 mM TConditioned media was collected and concentrated using Millipore Centriplus YM-3 Centrifugal Filter Devices . Emax™ ImmunoAssay Systems specific for NGF, BDNF, NT-3 and NT-4 (Promega) were performed according to manufacturer's instructions. Conditioned media was added to Nunc ELISA/EIA 96 well Maxisorp plates coated with anti-NT polyclonal antibodies. Secreted NT was detected by treating the plates with the respective NT monoclonal antibody followed by a horseradish peroxidase conjugated secondary antibody. Enzyme substrate was added to generate a color product whose absorbance was read at 450 nm. A NT standard included in each assay was used to generate a standard curve that was used to calculate the amount of secreted NT per well. The amount of secreted NT per sample was normalized to total protein per sample. Samples were assayed in triplicate. Conditioned media from three LC cell lines and three ONH astrocyte cell lines were assayed. Each immunoassay was repeated twice. Changes in NT secretion following OGD and recovery from OGD were reported as a percent of the control ± SEM.® statistical package, version 7.4.41 [Cell metabolism/cell number, western blot densitometry and immunoassay data were analyzed using one way analysis of variance (ANOVA) followed by validation using Student-Newman-Keuls tests. The MedCalcn 7.4.41  was usedn 7.4.41 .WL carried out tissue culture, cell proliferation assays, Western blotting, and immunoassays. WL, AC, and RW participated in design of the study, interpretation of the results, and in the writing and revision of the manuscript. All authors read and approved the final manuscript. This study is taken in part from a dissertation submitted to the UNT Health Science Center in partial fulfillment of the requirements for the degree Doctor of Philosophy for WL.


In particular, we compared the migration activity of mature Dendritic Cells (mDC) with that of immature Dendritic Cells (iDC) and also assessed intradermal versus subcutaneous administration.Dendritic Cell (DC) vaccination is a very promising therapeutic strategy in cancer patients. The immunizing ability of DC is critically influenced by their migration activity to lymphatic tissues, where they have the task of priming naïve T-cells. In the present study 99mTc-HMPAO or 111In-Oxine, and the presence of labelled DC in regional lymph nodes was evaluated at pre-set times up to a maximum of 72 h after inoculation. Determinations were carried out in 8 patients .DC were labelled with It was verified that intradermal administration resulted in about a threefold higher migration to lymph nodes than subcutaneous administration, while mDC showed, on average, a six-to eightfold higher migration than iDC. The first DC were detected in lymph nodes 20–60 min after inoculation and the maximum concentration was reached after 48–72 h.in vivo provide preliminary basic information on DC with respect to their antitumor immunization activity. Further research is needed to optimize the therapeutic potential of vaccination with DC.These data obtained  These iDC possess functional characteristics typical of this maturation status, such as phagocytosis, macropinocytosis, receptor-mediated endocytosis and antigen processing [2 may considerably increase migration, inducing CCR7 expression on the surface of DC. Penetration may be limited to the peripheral zones of lymphoid tissue when the DC are still immature, or may reach the deeper T-cell zones, where a greater number of naïve T-cells are present, when DC are mature and activated.Dendritic Cell (DC) vaccination is one of the most promising tools of immunological therapy for cancer. Administration of DC, generated and loaded with tumor antigens olerance ,2. A larocessing ,4. Afterocessing . Followiocessing ,7. The mSurface antigen CCR7, present on the cell membrane of DC, strongly influences migratory capacity through its interaction with transporter molecules, TREM-2, LTC4, LTD4, etc. -10. The in vitro-generated vaccines have shown that mature, but not immature DC, induce an effective antitumor response [Recent studies on cancer patients evaluating the efficacy of response -18. Animresponse . Similarresponse -23.in vivo migration ability of DC by labelling them with 99mTc-HMPAO or 111In-Oxine. In particular, migratory activity was assessed in iDC and mDC in terms of time required for migration to lymph nodes, duration of activity, and number of cells that migrated. Migratory capacity was further evaluated by comparing subcutaneous and intradermal administration.In the course of a vaccination trial using DC pulsed with autologous tumor lysate (ATL) in cancer patients, we evaluated the In vivo migration was assessed using a part of the DC obtained for one of the therapy cycles. Three of the 8 patients were evaluated twice.The case series consisted of a subset of the 19 patients enrolled onto a phase I/II vaccination trial for advanced melanoma and renal cell carcinoma in which the first 9 patients were treated with iDC and the remaining 10 received mDC, both pulsed with autologous tumor lysate and keyhole limpet hemocyanin . In the present study 8 patients were analyzed  for a total of 11 treatments. Two melanoma patients were treated with iDC (one of whom twice), while 4 other patients with melanoma and 1 with renal cell carcinoma (treated twice) received mDC. The remaining melanoma patient was treated with iDC and subsequently with mDC.The clinical trial was approved by the Italian Ministry of Health and by the Ethical Committee of Forlì Health and Social Services . All patients gave written informed consent.g) and the supernatant was passed through a 0.2-μm filter. Protein contents were determined and aliquots were stored at -80°C until use.Tumor samples surgically removed from the patients were immediately placed in PBS. Adjacent non malignant tissue was removed by scalpel and tumor cells were dispersed to create a single-cell suspension. Cells were lysed by incubation in sterile distilled water. Lysis was monitored by light microscope. Larger particles were removed by centrifugation . From days 2–6, IL-2  was administered subcutaneously at a dose of 3 million IU/die. This procedure was repeated after two weeks and once a month until progression occurred.DC were prepared from peripheral blood monocytes (PBMC) obtained by leukapheresis without previous mobilization. 5–9 liters of blood were processed in each collection. PBMC were purified on Ficoll-Paque. An aliquot of PBMC was utilized immediately for DC generation and the rest was frozen in bags for use at a later date (4–5 bags/1 collection).6 cells/ml for 2 h. The non-adherent cells were discarded and the adherent cells were incubated in CellGro DC Medium containing 1000 IU/ml rhIL-4 (Cell Genix) and 1000 IU/ml rhGM-CSF  for 7 days to generate a DC-enriched cell population. On day 6 DC were pulsed with autologous tumor lysate (100 mg/ml) and with KLH (50 mg/ml) and incubated overnight. On day 7, they were defined as iDC. After eliminating the previous culture medium, pulsed iDC were cultured for a further 2 days with a cocktail of cytokines . On day 9 they were defined as mDC. iDC or mDc were removed, washed and suspended in sterile saline for therapeutic infusion into the patient.PBMC were incubated in tissue culture flasks with CellGro DC Medium  at 10 × 106) were resuspended in platelet-poor autologous plasma (CFP1) and incubated for 15 min at room temperature with 99mTc-HMPAO (20 mCi)  111In-Oxine (1 mCi) . After two washes to eliminate the unbound isotope, the cells were resuspended in a total volume of 1.5 ml of CFP1. Radiolabelling of the DC and of the culture supernatant was evaluated with a gamma counter, after which DC were inoculated intradermally into the patient near healthy lymph nodes and in the contralateral zone not used for therapeutic vaccination . The patient then underwent serial acquisitions with gamma-camera positioned at the site of inoculation, with a field of view that included all the lymphatic regions of interest. The first acquisition was performed with a dynamic study of 20 min, followed by 10-min static acquisitions carried out every 30 min for the first 4–6 h and from 18 to 28 h. Other static determinations were made at 36, 48 and 72 h. The maximum duration of observation of DC migratory activity, which depended on the half-life of the radioisotope used, was 72 h for 111In-Oxine and 36 h for 99mTc-HMPAO.Labelling of DC was performed according to the methods described for leucocyte radiolabelling -26. A paThe identification of lymph node stations involved in the migratory activity was initially visual, after which we carried out a semiquantitative evaluation of the percentage of DC that migrated to lymph nodes from the inoculation site and an assessment of the speed of DC migration, expressed by activity/time curves obtained through the compartmental mathematical model.99mTc-HMPAO and the other was labelled with 111In-Oxine. The labelled cells were then suspended in CellGro DC Medium, divided into 4–5 culture flasks for each labelling molecule and incubated for 0 h, 4 h, 21 h, 24 h (99mTc-HMPAO) and 0 h, 4 h, 21 h, 24 h, 48 h (111In-Oxine). The DC from one flask were removed and centrifuged. The supernatant containing the free molecule, and the pellet containing the labelled cells, were then measured with a gamma counter.DC obtained from the culture of frozen PBMC were divided into two parts: one was labelled with 5 cells were suspended in 100 μl of buffer  and incubated for 30 min at 4°C with 10 μl of appropriate fluorescein isothiocyanate or phycoerythrin-labelled monoclonal antibodies (mAbs). The cells were then washed twice and resuspended in 500 μl of assay buffer. The fluorescence was analyzed by a FACS Vantage flow cytometer . mAbs specific for human CD1a, CD14, CD80, CD86, (Becton Dickinson) CD83  and CCR7  were used.iDC and mDC phenotypes were determined by single or two-color fluorescence analysis. 3–5·10At each pre-set time the supernatant was collected and stored at -80°C until analysis was carried out using commercially available ELISA Kit  to measure the production of IL-12 + p40 (bioactive heterodimer of IL-12) and IL-10 by DC.Single cell-based measurement of endocytosis was carried out as described 27). Dendritic cells were incubated for 30 min at 37°C with 0.5 mg/ml FITC-Dextran . DX-FITC  was centrifuged before use to remove aggregates. As negative control, cells were incubated with DX-FITC at 4°C. The cells were washed with cold PBS containing 2% FCS and 2 nM sodium azide to exclude dead cells and were then analyzed on a FACS Vantage flow cytometer (Becton Dickinson) [. DendritAll the patients  had advanced disease and all but one had undergone previous treatment. Median age was 49 years (range 46–52 years). Three patients were HLA-A1, 3 were HLA-A3, 1 was HLA-A2 and 1 was HLA-A11  are reported in Table in vitro stability of DC labelled with 99mTc-HMPAO and 111In-Oxine was evaluated using DC cultured from frozen PBMC. 99mTc-HMPAO-labelled DC showed a 75% loss of activity 4–24 h after the beginning of in vitro culture. 111In-Oxine-labelled DC showed a higher labelling stability (50%) that lasted for up to 24 h  was evaluated by comparing radioactive uptake in axillary lymph nodes. The intradermal route showed a threefold higher migration than that observed for the subcutaneous route Table . Evaluat111In-Oxine or 99mTc-HMPAO. A simple numerical analysis shows that the maximum uptake ratio between 99mTc-HMPAO-labelled mDC and iDC varies from 2 to 35, with an average of 8.4. The same ratio for 111In-Oxine-labelled cells varies from 4 to 7, with an average of 6. 99mTc-HMPAO labelling is influenced by the very low iDC uptake due to its greater binding instability and to the short half-life of the radioisotope, which does not permit the acquisition of reliable counts beyond 24–36 h.iDC and mDC migration was evaluated in all 8 patients (4 iDC and 7 mDC treatments) (Table 99mTc-HMPAO and 48–60 h with 111In-Oxine). A curve fitting analysis also seemed to indicate a progressive increase in uptake after the first 60 h, but the number of patients evaluated is too low for any definitive conclusions to be drawn.A lymph node uptake can be observed in all patients within the first two hours of inoculation, reaching a maximum uptake after 12 h in 7 patients Fig. ,5. In th99mTc-HMPAO alone in the same inoculation sites used for the study, no labelled hot spots were observed. This would seem to suggest that pure tracers move through lymphatic vessels without accumulating inside lymph nodes.After injection of DC-based immunotherapy has undergone a remarkable transformation in its development from basic research to clinical application ,28. Howe2 is essential for activating DC chemotaxis through the expression of CCR7 on the cell surface [2 may therefore prove to be important for increasing migration activity and DC efficacy. However, it has also been observed that PGE2 inhibits IL-12 production, resulting in a weaker in vivo activation of T-cells [in vivo experimentation.Recent published data have shown that the choice of maturation stimulus may be crucial for therapeutic success. In particular, it has been seen that PGE surface ,32. This T-cells ,34. Thes99mTc-HMPAO and 111In-Oxine) iDC and mDC [In the present study we aimed to clarify some issues concerning DC migration activity in a clinical vaccination trial utilizing radiolabelled ; it was observed that intradermal administration had a threefold higher migration to lymph nodes than the subcutaneous route. Although it remains to clarify the extent to which this migratory capacity is active or passive, it is clear that DC must be administered intradermally to obtain a higher migration.A crucial phase of the study was the comparison between the migratory activity of iDC  and that of mDC . The result was once again unequivocal, showing a greater progressive concentration of mDC that was, on average, six-eightfold higher than that of iDC, in accordance with data reported by other authors ,23 and i111In-Oxine-and 99mTc-HMPAO-labelled DC in succession and in the same site, to follow their migratory course.Notwithstanding the results obtained from the present study, many issues remain to be clarified. It has yet to be determined whether the increased activity detected in lymph nodes corresponds to an effectively greater migratory capacity or whether it is the result of a more effective adhesion capacity between surface molecules. Both hypotheses could even prove to be correct. We also do not know how long DC remain in lymph nodes. The increase in activity in lymph nodes is high in the first few determinations but tends to diminish or stabilize after around 36 h. It remains to be seen whether this presumed stabilization is the result of a sort of saturation or whether it can be attributed to the attainment of a dynamic equilibrium. The former hypothesis would indicate the need for an optimization of the number of DC to administer, as only a limited number would be functionally active. The latter would highlight the importance of the timing of administration and perhaps also the degree of DC maturation. To further investigate this, we plan to administer in vitro transiently stimulated DC (semimature DC). The therapeutic use of this type of DC, which have already begun the process of maturation and may be capable of reaching lymph nodes before their functional exhaustion, could increase the duration of their activation and stimulation. If these semimature DC prove to be equipped with a good migratory capacity, further improvement in the therapeutic use of DC may be possible.Finally, we aim to assess the migratory capacity of The migration activity of DC to regional lymph nodes is one of the many critical factors that influence the therapeutic result of antitumor vaccination. In the present study we used radioisotope-labelled DC and demonstrated that a better migration activity is obtained using intradermal than subcutaneous administration and that mDC show, on average, a six-to eightfold higher migration than iDC. Numerous other issues on DC functionality have yet to be clarified before antitumor therapeutic efficacy can be improved. The next important step will be to closely monitor the quantity and quality of responses observed in T-cells, and it is hoped that a consensus will be reached on standardized criteria for the definition and validation of clinical results obtained.DC, dendritic cell; iDC, immature dendritic cell; mDC, mature dendritic cell; ATL, autologous tumor lysate; PBMC, peripheral blood monocytes.RR and LR participated in the design of the study and were responsible for the clinical side of the study. AR, MP, LF and MS participated in the design of the study and were responsible for the biological part of the study. GM performed the apheresis collections. RG, AM and GF carried out DC labelling and migration evaluation. GG performed the mathematical and statistical analysis. All authors read and approved the final manuscript.None declared.