BmR1 has been extensively employed in both ELISA and immunochromatographic rapid dipstick (Brugia Rapid) formats for the specific and sensitive detection of IgG4 antibodies against the lymphatic filarial parasites Brugia malayi and Brugia timori. In sera of individuals infected with Wuchereria bancrofti the IgG4 reactivity to BmR1 is variable, and cross-reactivity of sera from individuals infected with Onchocerca volvulus or Loa loa was observed only in single cases. In order to characterize the homologs of the BmR1 antigen in W. bancrofti (Wb-BmR1), O. volvulus (Ov-BmR1) and L. loa (Ll-BmR1) the cDNA sequences were identified, the protein expressed and the antibody reactivity of patients' sera was studied.The recombinant antigen W. bancrofti, O. volvulus and L. loa. The clones obtained were sequenced and compared to the cDNA sequence of BmR1. Ov-BmR1 and Ll-BmR1 were expressed in E. coli and tested using an IgG4-ELISA with 262 serum samples from individuals with or without B. malayi, W. bancrofti, O. volvulus and L. loa infections or various other parasitic infections. BmR1, Ov-BmR1 and Ll-BmR1 were also tested for reactivity with the other three IgG subclasses in patients' sera.PCR methodology was used to identify the cDNA sequences from cDNA libraries and/or genomic DNA of BmR1 was found to be identical to BmR1. Ov-BmR1 and Ll-BmR1 were found to be identical to each other and share 99.7% homology with BmR1. The pattern of IgG4 recognition of all serum samples to BmR1, Ov-BmR1 and Ll-BmR1 were identical. This included weak IgG4 reactivities demonstrated by L. loa- and O. volvulus-infected patients tested with Ov-BmR1 and Ll-BmR1 (or BmR1). With respect to reactivity to other IgG subclasses, sera from O. volvulus- and L. loa-infected patients showed positive reactions  only with IgG1. No reactivity was observed with IgG2 or with IgG3. Similarly, ELISAs to detect reactivity to other anti-filarial IgG subclasses antibodies showed that sera from individuals infected with B. malayi or W. bancrofti (active infections as well as patients with chronic disease) were positive with BmR1 only for IgG1 and were negative when tested with IgG2 and with IgG3 subclasses.Wb-BmR1 antigen are present in W. bancrofti, O. volvulus and L. loa and that these antigens are highly conserved. Recognition of this antigen by patients' sera is similar with regard to IgG1, IgG2 and IgG3, but different for IgG4 antibodies. We conclude that the BmR1 antigen is suitable for detection of IgG4 antibodies in brugian filariasis. However, its homologs are not suitable for IgG4-based diagnosis of other filarial infections.This study demonstrates that homologs of the  Brugia malayi and Brugia timori is endemic in several Asian countries and infects approximately 13 million people. In May 2000, The Global Program for Elimination of Lymphatic Filariasis (GPELF ) was officially formed with the goal of eliminating the disease as a public health problem by the year 2020. To this end, sensitive and specific field-applicable diagnostic tools are required for mapping the distribution of the disease and monitoring the various phases of the program. Many areas endemic for LF are remote and have poor access to well-equipped laboratories, thus a rapid and field-applicable diagnostic test is important to ensure that it can be easily be performed by field workers and reliable, reproducible, results can be obtained. For bancroftian filariasis caused by Wuchereria bancrofti, the ICT antigen card test  is widely used for this purpose. This test is based on the detection of a circulating adult worm antigen of W. bancrofti. Although this antigen is also present in Brugia [B. malayi infection is not available. Therefore, despite its inconvenience and insensitivity, routine diagnosis of brugian filariasis is made by light microscopy of night blood.Lymphatic filariasis (LF) caused by Although PCR assays are highly sensitive, these mainly detect individuals with circulating microfilariae (mf); and they are both time consuming and labour-intensive requiring well-equipped laboratory facilities.Detection of anti-filarial IgG4 antibody provides a good alternative diagnostic tool for brugian filariasis, as this antibody subclass has been shown to be elevated in active infection and decline post-treatment -9. RecomBmR1 recombinant antigen, expressed by gene pPROEXHT/Bm17DIII (GenBank accession no. AF225296), has been shown by us to be a highly specific and sensitive antigen for IgG4 assays to detect exposure to both B. malayi and B. timori infections. The antigen has been used in both ELISA and immunochromatographic rapid dipstick (Brugia Rapid) formats, and evaluation in various laboratories and field trials has revealed a sensitivity of 93%–100% in detecting microfilariaemic individuals [BmR1 antigen is highly specific (99%–100%) with respect to reactivity with sera from non-filarial infections [W. bancrofti, followed by Loa loa, while only one sample of nine patients with Dirofilaria infection was found to be reactive. No cross-reactivity was exhibited in patients infected with O. volvulus or Mansonella [The ividuals . Furtherividuals -15. The fections ,12. The nsonella ,16.BmR1 antigen more closely. The varying degree of BmR1 recognition in other filarial infection raises the question of whether the homologous antigen is also present in W. bancrofti, L. loa and O. volvulus. In the present study we have shown that BmR1 antigen is highly conserved (99–100% amino acid identity), and that almost identical antigens are present in the other human filarial parasites of public health importance. Interestingly, however, the ability of the hosts to mount IgG4 response to BmR1 homologs was found to be highly variable in some infections. In addition, the antibody responses of other IgG subclasses to BmR1 and its homolog were also investigated.Due to its diagnostic significance, it is therefore important to characterize the W. bancrofti microfilaria (mf), adult male and adult female cDNA libraries were obtained from the Filarial Genome Project Resource Centre . Genomic DNA of W. bancrofti mf were prepared from samples provided by Dr. B Ravindran, Division of Immunology, Regional Medical Research Centre, Indian Council of Medical Research, Bhubaneswar, India . The samples were comprised of two from individuals whose serum/blood samples were negative by the Brugia Rapid test and two from individuals who were positive by the Brugia Rapid test. L. loa L3 and O. volvulus mf cDNA libraries were kindly provided by Dr. P Fischer, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany .Bm17DIII gene sequence the following PCR primers were used: RNF (24 mer) 5' ATT ACT GAT TAG TAT TTT ATC GTT 3' and RNR (24 mer) 5'ATG ATA AAA ATG AAT GAG AAA TAT 3'. λ phage plaques were amplified and the DNA was extracted using a λ DNA extraction kit . PCR was then performed in a thermocycler  at the following conditions: 94°C, 5 mins.; 55°C, 5 mins.; 35 cycles for 94°C, 45 sec; 55°C, 45 sec & 72°C, 90 sec; 72°C, 10 mins.To amplify the entire W. bancrofti mf was prepared using Genispin Tissue DNA Kit . PCR amplifications using the above primers were then performed using the following thermocycler conditions: 94°C, 5 mins; 35 cycles for 94°C, 1 min; 55°C, 1 min & 72°C, 1 min; 72°C, 10 mins.Genomic DNA from W. bancrofti, O. volvulus and L. loa, the PCR products were cloned into TOPO-TA vector , then transformed into E. coli TOP10 host . The recombinant plasmids were then amplified, purified using QIAprep® Spin Miniprep Kit , and subsequently sent for sequencing . The results of the DNA sequences were analyzed using vector NTI software .For sequence analysis of the gene homologs in Bm17DIII gene homologs in O. volvulus and L. loa were subcloned into a bacterial expression vector, pPROEX-HT which contain 6-His tag , then transformed into E. coli TOP 10 host cells.The ). The suspension was sonicated at 200 W for 10 minutes, followed by centrifugation at 12 000 g for 30 minutes. The resulting supernatant was purified using Ni-NTA resin  and buffers containing imidazole. The protein-containing fractions were then pooled.The recombinant bacteria were cultured in Terrific broth and placed in a shaker incubator at 37°C until the optical density reached 0.5. The culture was then induced with 1 mM IPTG  for 3 hrs at 30°C. The bacterial pellet was reconstituted with lysis buffer containing 50 mM Tris HCl (pH 8.5), 5 mM 2-mercaptoethanol and a cocktail of protease inhibitors  were coated with 100 μl of either BmR1 (20 μg/ml) or the homologous recombinant antigens  in NaHCO3 buffer (pH 9.6). After a blocking step, serum samples (1:20 or 1:50 or 1:100) were incubated for 2 h, followed by 0.5 h incubation with the secondary antibody HRP conjugated to monoclonal anti-human IgG1 (1:6000), IgG2 , IgG3  or IgG4 (1:4500) . Subsequently ABTS substrate  was added for 30 minutes before the optical densities (OD) were read at 410 nm with an ELISA spectrophotometer (Dynatech (now DYNEX Technologies), USA ).The methodology employed was as previously reported . BrieflyO. volvulus, L. loa, W. bancrofti, B. malayi and other parasitic infections. In addition serum samples from endemic normals  and non-endemic normals  were also tested. The O. volvulus sera were from microfilaremic from western Uganda [L. loa sera were from microfilaremic individuals from the clinical department of the Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany. W. bancrofti sera samples were from India; while sera from B. malayi infections, endemic normals, non-endemic normals and other parasitic infections were from Malaysia. Infections with other parasites comprised of patients from Malaysia:Serum samples were from existing serum banks, collected according to the ethical requirements of each institution. The samples were as follows: n Uganda . L. loa lumbricoides, Trichuris trichiura, hookworm, Strongyloides stercoralis)• whose stool specimens were positive for parasite ova/larva • with BmR1 antigen in W. bancrofti (Wb-BmR1), O. volvulus (Ov-BmR1) and L. loa (LI-BmR1). PCR of W. bancrofti cDNA libraries and W. bancrofti genomic DNA  produced a single band of 618 bp. PCR products were eluted from band 618 and cloned into TOPO vector. A total of 12 recombinant clones from six TOPO reactions  were sequenced. A total of 31 DNA sequencing reactions were analyzed and all obtained sequences were identical. Comparison of the obtained nucleotide sequence showed that it was identical to cDNA sequence of BmR1, irrespective whether the template DNA came from cDNA libraries, or microfilaria originated from individuals positive or negative for Brugia Rapid.In order to explain the pattern of antigen recognition in patients with other filarial infection we identified the homologs of the BmR1 and Ll-BmR1, a total of 5 and 3 recombinant clones were sequenced, respectively . The Ov-BmR1 and Ll-BmR1 were 100% identical to each other, and only two base pairs were different from BmR1 and Wb-BmR1 i.e. at 97 bp and 483 bp. When the amino acid sequences were compared only one amino acid difference was observed: the uncharged polar isoleucine at position 33 was substituted by a neutral threonine  using a panel of 201 sera samples from individuals with various parasitic infections and 61 healthy controls  indicated that the exchange of one amino acid had no influence on the reactivity of IgG4 antibodies. The IgG4-ELISA results showed all recombinant antigens were identical in reactivity with the various categories of sera  readings of ≥0.300 were considered to be positive . The comBmR1 and its homologs (Ov-BmR1 and Ll-BmR1) with serum antibodies of the other three IgG subclasses  using samples from O. volvulus and L. loa infected individuals showed positive reactions with only IgG1. Most IgG1 positive samples had an OD >1.000. Similarly, the reactivities of anti-filarial IgG1, IgG2 and IgG3 antibody subclasses with BmR1 on serum samples from active and chronic cases of W. bancrofti and B. malayi showed positive reactions only with IgG1. It is also noted that sera from non-endemic normals and soil-transmitted infections also showed similar reactivities i.e. IgG1 positive and IgG2- & IgG3-negative , has been consistently shown to be a sensitive and specific antigen for the immunodiagnosis of brugian filariasis in studies employing either ELISA or immunochromatographic rapid test (Brugia Rapid) formats [Bm17DIII cds exhibited 94% homology with the reported EST sequence derived from B. malayi microfilaria cDNA (GenBank AW244981). Southern blot hybridization assays performed on cDNA libraries of L3, L4, mf, adult male and adult female B. malayi showed that it is present in all of the above stages . Bands of the correct molecular weight were observed in a Western blot of B. malayi mf, adult male and adult female soluble antigens probed with monopurified antibody to BmR1 . formats ,11-13,15BmR1 to sera of W. bancrofti-infected patients. Reactivity to sera from Chennai, India was 54.5% (12/22); from Indonesia was 70% (14/20) and from the Cook Islands was 90% (9/10) [W. bancrofti is identical to the cDNA of BmR1 – irrespective of the source of the parasites – whether the mf were isolated from the individuals whose sera showed positive or negative reactivity with the Brugia Rapid test. Thus the observed differences in the reactivity of BmR1 antigen with W. bancrofti sera collected from different geographical regions does not appear to be due to genotypic variability between different isolates of mf. Further studies are currently underway to determine if the variability in the expression of the gene could account for the variability in the Brugia Rapid results with serum samples collected from W. bancrofti infected individuals.Multicenter evaluations performed with Brugia Rapid showed variable reactivity of as 54.5% /22; from% (9/10) . The widW. bancrofti genomic DNA samples to obtain an amplicon with a size greater than 618 bp . However, only one prominent band of 618 bp was obtained . PCR on W. bancrofti genomic DNA to amplify the intron sequence (using primers based on the Bm17DIII intron) produced a sequence that shared ~75% homology to the intron of Bm17DIII. This is believed to be an amplification on another part of W. bancrofti genome, since PCR using a pair of internal primers that flank the possible intron site produced a PCR product of ~300 bp (a size that is expected if there was no intron). Conversely, amplification of B. malayi genomic material produced two kinds of amplicons: 618 bp and 1010 bp. The latter was comprised of an intron (393 bp) and two flanking exons (237 bp and 381 bp), the sequences of which were consistent with B. malayi data at TIGR website . Thus at Universiti Sains Malaysia, genomic DNA of Wb17DIII was found to be intronless, whereas genomic DNA of Bm17DIII was shown to have two variants (i.e one with and one without an intron). These results, though seemingly controversial, were a result of exhaustive efforts with appropriate PCR controls. Data from other laboratories will hopefully confirm these results.PCR experiments were performed on the BmR1 IgG4 was detected in 84.6% (44/52) of L. loa sera but generally not detected in O. volvulus serum samples [BmR1 and Ll-BmR1 were identical to each other and 99.7% similar to BmR1 (and to Wb-BmR1) on the nucleotide level  did not alter their antigenicity. It is interesting to note that although IgG4 has been shown to be elevated in onchocerciasis with assays using other recombinant antigens [BmR1 or Ov-BmR1 in O. volvulus was generally negative. One possible explanation for this is that adult worms mostly express Ov-BmR1 and the immune response to O. volvulus is predominantly due to mf [BmR1 and Ov-BmR1. It is possible that the uptake of antigen from lymphatic filariae by antigen presenting cells is significantly different compared to O. volvulus .Anti- samples ,16. Ov-B4.6% 44/5 of L. loantigens ,19, the ue to mf . This maBmR1, Ov-BmR1 and Ll-BmR1 recombinant antigens were also used to determine if IgG1, IgG2 or IgG3 antibodies in O. volvulus and L. loa serum samples were reactive with the recombinant proteins. In addition, the three IgG subclasses were also tested with BmR1 on assays using sera collected from patients with B. malayi and W. bancrofti infections. In all cases only anti-filarial IgG1 was reactive, while anti-filarial IgG2 and IgG3 assays were consistently negative. It is important to note that IgG1 antibodies to BmR1 and its homologs are unspecific and without any diagnostic value. The BmR1 antigen obviously contains widespread epitopes that are recognized by IgG1 antibodies.The BmR1 and its homologs in W. bancrofti, O. volvulus and L. loa induce IgG antibody responses restricted to IgG1 and IgG 4 subclasses only. Unlike the anti-filarial IgG4 response in B. malayi infection, the IgG4 response to BmR1 in W. bancrofti and L. loa was not consistently detected in all infected individuals, indicating that this recombinant antigen will not be of much use in the diagnosis of these two filarial infections. Although IgG1 response to BmR1 was observed in all the filarial infections tested, it lacks specificity since it was also positive when tested with serum samples from normal individuals and with those infected with other parasites.Thus based on the current study, BmR1 antigen in other filarial parasites. However, they do not seem to induce consistent antibody responses in all infected subjects. Thus the immunogenicity of BmR1 in brugian filariasis appears to be clearly different from that of bancroftian filariasis, onchocerciasis and loiasis.The study demonstrates the presence of identical and almost identical homologs of the diagnostic The author(s) declare that they have no competing interests.RN – was the principle researcher, designed the study, supervised the experiments and result analysis, wrote the first draft of the manuscript.RAAA – performed the experiments and participated in the analysis of the data.BR – provided parasite materials, collected patients' sera, edited the paper.


II and p30II, which are incompletely defined in the virus life cycle or HTLV-1 pathogenesis. Proviral clones of the virus with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. Exogenous expression of p30II differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and represses tax/rex RNA nuclear export.Human T-lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus that causes adult T-cell leukemia/lymphoma and is implicated in a variety of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13II in regulation of cellular gene expression, using stable p30II expression system employing lentiviral vectors to test cellular gene expression with Affymetrix U133A arrays, representing ~33,000 human genes. Reporter assays in Jurkat T cells and RT-PCR in Jurkat and primary CD4+ T-lymphocytes were used to confirm selected gene expression patterns. Our data reveals alterations of interrelated pathways of cell proliferation, T-cell signaling, apoptosis and cell cycle in p30II expressing Jurkat T cells. In all categories, p30II appeared to be an overall repressor of cellular gene expression, while selectively increasing the expression of certain key regulatory genes.Herein, we further characterized the role of p30II, while repressing the expression of many genes, selectively activates key gene pathways involved in T-cell signaling/activation. Collectively, our data suggests that this complex retrovirus, associated with lymphoproliferative diseases, relies upon accessory gene products to modify cellular environment to promote clonal expansion of the virus genome and thus maintain proviral loads in vivo.We are the first to demonstrate that p30 Briefly, 293T cells (5 × 106) were seeded in a 10-cm dish and transfected the following day with 2 μg of pHCMV-G, 10 μg of pCMVΔR8.2 and 10 μg of pWPT-p30IIHA-IRES-GFP or pWPT-IRES-GFP using the calcium phosphate method. Supernatant from 10 to 20 dishes was collected at 24, 48 and 72 h post transfection, cleared of cellular debris by centrifugation at 1000 rpm for 10 min at room temperature and then filtered through a 0.2 μm filter. The resulting supernatant was then centrifuged at 6,500 g for 16 h at 4°C. The viral pellet was suspended in cDMEM (DMEM containing 10% FBS and 10% streptomycin and penicillin) overnight at 4°C and the concentrated virus was aliquoted and stored at -80°C. To determine the virus titer, serial dilutions of the virus stock were used to spin infect 293T cells and 48 h post infection, eGFP expression and p30II expression was measured by flow cytometry and RT-PCT respectively. Briefly, on the day before infection, 293T cells (1 × 105) were seeded in a 6-well plate. The medium was removed the following day and the cells were then incubated with the diluted virus containing 8 μg/ml polybrene . Cells were then spin-infected by centrifugation at 2700 rpm for 1 h at 30°C, supplied with fresh medium and cultured for 48 h. Then cells were treated with trypsin , pelleted and resuspended in D-PBS (Invitrogen) for fluorescence activity cell sorting (FACS) analysis on an ELITE ESP flow cytometer (Beckman Coulter). One × 106 cells were used to perform western blot to detect the expression of p30II HA. Jurkat T lymphocytes  were transduced with recombinant virus at multiplicity of infection of 4 in the presence of 8 μg/ml polybrene (Sigma) and spin-infected at 2700 rpm for 1 h at 25°C. Primary CD4+ T cells were extracted using dynabead CD4 positive isolation kit  according to manufacturer's instructions. Primary CD4+ T cells were stimulated with Phytohemagglutinin (PHA) for 48 h, transduced with recombinant virus at multiplicity of infection of 20 in the presence of 8 μg/ml polybrene (Sigma) and spin-infected at 2700 rpm for 1 h at 25°C. At 10 days post-transduction, GFP expression of controls and samples were verified to be above 90% by FACS analysis and the presence of p30II mRNA expression in samples (and absence in controls) was verified by RT-PCR . Equal amounts of proteins were mixed with Laemmli buffer . After boiling for 5 min, samples were electrophoresed through 12% polyacrylamide gels. The fractionated proteins were transferred to nitrocellulose membranes (Amersham Pharmacia Biotechnology) at 100 V for 1 h at 4°C. Membranes were blocked with 5% non-fat dry milk in PBS with 0.1% Tween for 16 hours, then incubated with mouse anti-HA monoclonal Ab  (clone 16B-12) , for overnight at 4°C, and developed by using horseradish peroxidase-labeled secondary Ab  and enhanced chemiluminescence reagent .Cells were lysed in buffer containing phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS). Cell lysates were prepared by centrifugation at 14,000 rpm (Beckman) for 20 min at 4°C. Protein concentrations were determined by BCA assay (micro-BCA Protein AssayIn vitro transcription was performed on the cDNA to produce biotin-labeled cRNA with ENZO RNA Transcript labeling kit (Affymetrix), according to the manufacturer's instructions. Complimentary RNA (cRNA) cleanup was performed using Genechip Sample Cleanup module (Affymetrix). The quality of total RNA and biotin-labeled cRNA of all the samples and controls were checked by calculating the ratio of absorbance at 260 nm and 280 nm (between 1.9 to 2.1) using a spectrophotometer (Genequant) and agarose gel electrophoresis. The labeled cRNA was fragmented to 50–200 nucleotides, and hybridized to U133A arrays (Affymetrix) using GeneChip® Hybridization Oven (Affymetrix). Arrays were washed and stained using GeneChip® Fluidics Station 400 (Affymetrix) and scanned by GeneArray Scanner (Affymetrix).According to the instructions of manufacturers, total cellular RNA was isolated from transduced Jurkat T lymphocytes using RNAqueous . To test the concentration and purity of the RNA samples, absorbance at 260 nm and 280 nm were measured and the 260/280 ratio was calculated using a spectrophotometer . The 260/280 ratio of all the RNA samples were between the range of 1.9–2.1. The probe preparation for GeneChip was performed according to the Affymetrix GeneChip Expression Analysis Technical Manual . Briefly, cDNA was synthesized using genechip T7-Oligo (dT) promoter primer kit (Affymetrix) and superscript double stranded cDNA synthesis kit (Invitrogen), according to the manufacturers instructions. cDNA cleanup was done using Genechip Sample Cleanup module (Affymetrix). ® (Microarray suite 5) to identify probes with at least 1.5 fold difference in expression between control and p30II and verified for cluster formation by dCHIP software [Quality control criteria evaluations done as part of the basic analysis include (1) The ratios of 3' signal to 5' signal of two housekeeping genes, beta-actin and GAPDH were between 0 and 3. (2) The hybridization controls BioB, BioC, BioD, and Cre were all present and in a linear relationship of intensity. (3) The scale factors between arrays did not vary by 3 fold. (4) The background intensity was not significantly higher than expected. (5) The percent of gene present was monitored and found to be not less than the standard 30%. To determine the quantitative RNA level, the average differences representing the perfectly matched minus the mismatched for each gene-specific probe set was calculated. Differential gene expression and comparative analysis was done using Data Mining Toolsoftware . The bioII were as follows: TAG CAA ACC GTC AAG CAC AG (forward) and CGA ACA TAG TCC CCC AGA GA (reverse). The PCR primers for CHP were as follows: CCC ACA GTC AAA TCA CTC GCC (forward) and ATG GTC CTG TCT GCG ATG CTG (reverse). The PCR primers for JUN-D were as follows: CTC TCA GTG CTT CTT ACT ATT AAG CAG (forward) and TTA TCT AGG AAT TGT CAA AGA GAA GATT (reverse). The PCR primers for NFATc were as follows: TTG GGA GAG ACA TGT CCC AGA TT (forward) and TCA TTT CCC CAA AGC TCA AAC A (reverse). The results were expressed as a graph. Statistical analysis was performed using Student's t test, P < 0.05.One μg of RNA was converted to cDNA  as described by the manufacturer. cDNA from 100 ng of total RNA was amplified with AmpliTaq DNA polymerase , PCR products were separated by agarose gel electrophoresis, normalized to GAPDH and quantified using alpha imager spot densitometry . DNA contamination was tested by performing a control with no reverse transcriptase. The PCR primers for p30II-transfected Jurkat T lymphocytes was performed as described previously [7 cells in cRPMI  and 10% streptomycin and penicillin) at 350 V and 975 μF using Bio-Rad Gene Pulser II  with 30 μg of pME-p30 or pME empty plasmid, 10 μg of reporter plasmid , and 1 μg of pRSV-Gal plasmid or 1 ug pWPT-IRES-GFP plasmid. The transfected cells were seeded in six-well plates at a density of 5 × 105/ml and were either left untreated or stimulated with 20 ng/ml of phorbol myristate acetate (PMA) (Sigma) or with 2 μM ionomycin (Sigma), or both at 6 h post-transfection, followed by incubation for 18 h prior to lysis for analysis of luciferase activity. Stimulations with anti-CD3 and/or anti-CD28 antibodies (each at 3 μg/ml)  were carried out 18 h post-transfection. Following 8 h of stimulation, to measure luciferase activity, the cells were lysed with Cell Culture Lysis Reagent (Promega), and the cell lysates were tested for luciferase activity according to the manufacturer's protocol. Transfection efficiency was normalized by staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside  (Sigma) and counting β-Gal expressing cells. Transfection efficiency was also normalized by counting GFP positive cells under the fluorescence microscope. Results were expressed as mean of optimized luciferase activity  in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed using Student's t test, P < 0.05.Analysis of AP-1, NF-κB, and NFAT transcriptional activity in pME- and pME-p30eviously . BrieflyArbitrary light units, ALUPhorbol myristate acetate, PMAFetal bovine serum, FBSHuman T-lymphotropic virus type 1, HTLV-1Adult T cell leukemia/lymphoma, ATLThe author(s) declare that they have no competing interests.Bindhu Michael, Amrithraj M. Nair, Hajime Hiraragi, Lei Shen, Gerold Feuer, Kathleen Boris-Lawrie and Michael D. Lairmore have all met the definition of author as outlined by the Retrovirology journal. Each has made substantive intellectual contributions to a published study. Bindhu Michael, Amrithraj Nair, Hajime Hiraragi Gerold Feuer, Kathleen Boris-Lawrie and Michael D. Lairmore have made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data. Lei Shen performed drafting the article or revising it critically for important intellectual content in particular sections related to biostatistical analysis. Each author has given final approval of the version to be published. Each author have participated sufficiently in the work to take public responsibility for appropriate portions of the content.II.Listing of genes modulated by HTLV-1 p30Click here for file


The human immunodeficiency virus type I (HIV-1) transactivator protein Tat is an unusual transcriptional activator that is thought to act solely by promoting RNA polymerase II processivity. Here we study the mechanism of Tat action by analyzing transcription complex (TC) assembly in vivo using chromatin immunoprecipitation assays. We find, unexpectedly, that like typical activators Tat dramatically stimulates TC assembly. Surprisingly, however, the TC formed on the HIV-1 long terminal repeat is atypical and containsTATA-box-binding protein (TBP) but not TBP-associated factors (TAFs). Tat function involves direct interaction with the cellular cofactor positive transcription elongation factor b (P-TEFb). Artificial tethering of P-TEFb subunits to HIV-1 promoter DNA or nascent RNA indicates that P-TEFb is responsible for directing assembly of a TC containing TBP but not TAFs. On the basis of this finding, we identify P-TEFb-dependent cellular promoters that also recruit TBP in the absence of TAFs. Thus, in mammalian cells transcription of protein-coding genes involves alternative TCs that differ by the presence or absence of TAFs. The transcriptional activator, HIV-1 Tat, not only acts by promoting RNA polymerase processivity, but it is able to promote transcription complex assembly in the absence ofTATA- box-binding protein-associated factors In eukaryotes, gene regulation is largely controlled at the transcriptional level. Factors involved in the accurate transcription of eukaryotic structural genes by RNA polymerase II (class II genes) can be classified into two groups. First, general (or basic) transcription factors (GTFs) are necessary and can be sufficient for accurate transcription initiation in vitro . Such fTranscriptional activity is greatly stimulated by the second class of factors, promoter-specific activator proteins (activators). In general, cellular activators are sequence-specific DNA-binding proteins whose recognition sites are usually present in sequences upstream of the core promoter . In aThe Tat protein of human immunodeficiency virus type I (HIV-1) is a potent activator of the viral long terminal repeat (LTR) and is required for viral replication . The chromatin immunoprecipitation (ChIP) assay has provided a powerful approach to study in vivo the mechanism by which activators stimulate transcription and to delineate the composition of transcription complexes (TCs). In yeast, ChIP analysis has been used to show that activators function, at least in part, by stimulating TC assembly. These studies have also revealed that at certain promoters TBP is recruited in the absence of TAFs ,17, indiTo analyze transcription stimulation by Tat and other activators we performed ChIP experiments in transiently transfected mammalian tissue culture cells. Plasmids expressing a reporter construct, containing a core promoter and various combinations of activator-binding sites, were transiently transfected into HeLa or 293T cells. In some experiments, a second plasmid expressing an activator was co-transfected. Reporter activity or primer extension was used to quantitate transcription levels. TC assembly was analyzed by a ChIP assay using antibodies directed against components of four representative GTFs involved in distinct stages of TC assembly: TFIID , TFIIB, mediator (CDK8 and hMed6), and RNA polymerase II (RBP1). In most experiments the analysis was performed using two sets of primers: one set encompassed the core promoter, transcription start site, and immediate downstream region, and a second set was located far downstream of the transcription start site, within the open reading frame (ORF).In yeast, it has been shown that activators stimulate TC assembly, which is evident at the earliest step of this process, interaction of TBP with theTATA box ,19. To cThe artificial activator Gal4-VP16 contains an unusually potent acidic activation domain and can stimulate transcription in many species and cell types including mammalian tissue culture cells . Figure cis-acting enhancer element that is often located at a distance from the transcription start site . The encription . Figure Finally, we analyzed transcription directed by the Ad E1a protein. Efficient transcription from Ad early promoters requires the viral E1a protein as well as the participation of cellular transcription factors that also bind to the promoter (reviewed in ). For exIn summary, the results of We next investigated Tat-mediated transcription activation and TC assembly on the HIV-1 LTR promoter. We note that recruitment of Tat was not observed in the ChIP assay, which detects proteins bound either directly or indirectly to DNA (reviewed in ). UnlikeTo determine whether the lack of TAF recruitment was a general feature of Tat-directed transcription, we analyzed a chronically HIV-1-infected cell line, 8E5/LAV, which harbors an integrated provirus that is constitutively transcribed . Figure We also analyzed a second chronically HIV-1-infected cell line, U1, which has been used as a model to study viral latency. U1 cells harbor an integrated viral genome, but, unlike 8E5/LAV cells, the constitutive level of viral expression is extremely low. Viral expression can be induced by phorbol esters, which stimulate transcription through upstream NF-κB-binding sites in the HIV-1 LTR, leading to Tat synthesis and a subsequent substantial Tat-mediated transcriptional increase ,28. ThusTo investigate further the composition of the TC formed on the HIV-1 LTR, we obtained a panel of antibodies directed against nine additional human TAFs , as well as antibodies against four subunits of the STAGA complex  and the TBP-interacting protein Mot1. The results A show thThe results of the ChIP experiments strongly suggested that transcription activation by Tat did not require TAFs. To confirm this supposition, we examined the ability of Tat to stimulate transcription following TAF inactivation. First, we examined the ability of Tat to activate transcription in ts13 cells, which harbor a temperature-sensitive mutation in TAF1 ,30. FiguThe results described above indicate that on the HIV-1 LTR transcription activation involves assembly of an atypical TC that contains TBP but not TAFs. However, these experiments do not distinguish whether the critical determinant for recruitment of TBP and not TFIID is Tat or the promoter, the HIV-1 LTR. To address this issue we performed a series of artificial tethering experiments.Previous studies have shown that Tat can activate transcription when directed to the HIV-1 LTR through a heterologous DNA-binding domain ,32,33. WAs discussed above, Tat interacts directly with CycT1, a subunit of the transcription elongation factor P-TEFb. Thus, Tat is an adaptor whose function is to recruit P-TEFb. We therefore considered that P-TEFb was ultimately responsible for the selective recruitment of TBP in the absence of TAFs. To address this possibility, we analyzed transcription activation by the two P-TEFb subunits, CycT1 and CDK9. Consistent with previous results ,35, bothIn the experiments shown in P-TEFb is also a cofactor for several cellular activators, the best studied of which is CIITA, a transcription factor involved in the expression of major histocompatibility complex (MHC) class II genes (reviewed in ). In facHLA-DM and HLA-DR, and, as a negative control, GAPDH. RT-PCR analysis confirmed, as expected, that both HLA-DM and HLA-DR were expressed in 293T cells (data not shown). The associated ChIP experiment , We have shown that transcription from the HIV-1 LTR involves TBP but not TFIID. This result was particularly unexpected because in mammalian cells TBP is bound to TAFs extremely tightly and numerous biochemical studies have failed to find free TBP ,3,4. Nonβ-globin, E4CAT, G6(−83)HIV LTRΔTARCAT, and GAPDH). Third, when TC assembly was directed by Tat or P-TEFb the ChIP assay detected all GTFs analyzed, including TBP, but not TAFs on six different promoters  or lacking (pB/E) the SV40 enhancer were provided by Walter Schaffner; pBS contains a 196-bp SV40 enhancer–containing fragment inserted into a plasmid derived from pB6 ), and twice with TE (10 mM Tris-HCl [pH 8.0] and 1 mM EDTA). Chromatin was eluted with freshly prepared elution buffer (1% SDS and 0.1 M CHNaO3). After reversal of the cross-links, the samples were treated with proteinase K for 1 h at 45 °C, extracted by phenol/chloroform, and ethanol precipitated. The pellet was resuspended in TE, and PCR was performed. Autoradiograms were scanned and quantitated by the National Institutes of Health ImageJ program. IP DNA was quantitated and presented as the ratio of IP to input. Primer sequences are available upon request.HeLa and 293T cells were transfected with 10 μg of effector plasmids and 5 μg of reporter plasmid using standard CaCl methods . Forty-eFor TAF1 inactivation, ts13 cells  were inihttp://www.ncbi.nlm.nih.gov/LocusLink/) accession numbers for the genes and gene products discussed in this paper are CDK8 (1024), CDK9 (1025), CIITA (4261), CycT1 (904), GAPDH (2597), hGCN5 (2648), HLA-DM (3109), HLA-DR (3122), hSpt3 (8464), MED6 (10001), Mot1 (9044), PAF65β (27097), RBP1 (5430), TAF1 (6872), TAF11 (6882), TAF12 (6883), TAF13 (6884), TAF2 (6873), TAF4 (6874), TAF5 (6877), TAF6 (6878), TAF7 (6979), TAF8 (129685), TAF9 (6880), Tat (155871), TBP (6908), TFIIB (2959), and TRRAP (8295).The LocusLink (


In our work, we have introduced an ant colony optimisation (ACO) algorithm to address the non-deterministic polynomial-time hard (NP-hard) combinatorial problem of predicting a protein's conformation from its amino acid sequence under a widely studied, conceptually simple model – the 2-dimensional (2D) and 3-dimensional (3D) hydrophobic-polar (HP) model.The protein folding problem is a fundamental problems in computational molecular biology and biochemical physics. Various optimisation methods have been applied to formulations of the We present an improvement of our previous ACO algorithm for the 2D HP model and its extension to the 3D HP model. We show that this new algorithm, dubbed ACO-HPPFP-3, performs better than previous state-of-the-art algorithms on sequences whose native conformations do not contain structural nuclei  at the ends, but rather in the middle of the sequence, and that it generally finds a more diverse set of native conformations.The application of ACO to this bioinformatics problem compares favourably with specialised, state-of-the-art methods for the 2D and 3D HP protein folding problem; our empirical results indicate that our rather simple ACO algorithm scales worse with sequence length but usually finds a more diverse ensemble of native states. Therefore the development of ACO algorithms for more complex and realistic models of protein structure holds significant promise. An example of stigmergy is the communication of ants during the foraging process: ants indirectly communicate with each other by depositing pheromone trails on the ground and thereby influencing the decision processes of other ants. This simple form of communication between individual ants gives rise to complex behaviours and capabilities of the colony as a whole.Ant Colony Optimisation (ACO) is a population-based stochastic search method for solving a wide range of combinatorial optimisation problems. ACO is based on the concept of et al.  is the probability of choosing direction  as the relative direction di at sequence position i. Unlike in our previous implementation [c' improves over the current solution quality (energy) of c. This search process is greedy in the sense that it does not allow worsening steps, and it is terminated when no improving steps have been found after a specific number of scans through the chain . Since this local search procedure has a relatively high time-complexity, in each iteration of ACO-HPPFP-3 it is only applied to a certain fraction of the highest-quality conformations constructed by the ants in the preceding construction phase.where entation , the locAfter each construction and local search phase pheromones are updated according toτi,d:= ρ·τi,d,     (4)ρ ≤ 1 is the pheromone persistence, a parameter that determines how much of the information gathered in previous iterations is retained. Subsequently, selected ants with low-energy conformations update the pheromone values according towhere 0 <τi,d:= τi,d + Δi,d,c,     (5)i,d,c is the relative solution quality of the given ant's candidate conformation c if that conformation contains local structure motif d at sequence position i, and zero otherwise.where ΔMAX - MIN Ant System [τ values are normalised such that ∑d∈{S,L,R,U,D} τi,d = 1 for every residue i; additionally, whenever for a given sequence position i the minimal normalised pheromone value / falls below a threshold θ , the minimal τi,d value is set to θ, while the maximal τi,d value is decreased by θ - mind∈{S,L,R,U,D} τi,d.  This guarantees that the probability of selecting an arbitrary local structure motif for the corresponding sequence position does not become arbitrarily small, and hence ensures the probabilistic approximate completeness of our algorithm ..ACO-HPPFP-3 has been implemented in C++ and compiled using gcc (version 3.3.3) for the Linux operating system; a Linux executable is available from Both authors contributed to the development of ideas, design of experiments, analysis and interpretation of results, and the writing of the paper. AS implemented the proposed method and performed the computational experiments.Additional information on biological and randomly generated HP sequences. This file (in .pdf format) contains tables providing additional information on our new test sets of biological and randomly generated HP sequences and the results from our computational experiment with ACO and PERM.Click here for file


It's a common runner's complaint. Just when you've built up enough strength and endurance to make running fun, those niggling aches and pains won't go away. Every time your foot hits the ground, a force equal to about twice your weight shoots through your body, eventually chipping away at bones, cartilage, muscles, tendons, ligaments, and joints. For those lucky souls who can take the pounding, the main limitation to running performance stems from muscle fatigue. Now, Randall Johnson and colleagues report that a protein found in skeletal muscle profoundly influences muscle endurance.Running, like any sustained skeletal muscle activity, consumes large quantities of adenosine triphosphate (ATP), a molecule that fuels many essential cell processes. A number of metabolic pathways supply muscle tissue with the ATP needed to power muscle contraction and sustain ongoing exercise. Which pathway predominates depends on factors like speed, duration, and type of activity, as well as on the availability of oxygen, which fluctuates during activity. Say you start a half-hour run with a sprint. Within a few seconds, your body uses up the oxygen in its muscles and has to switch to anaerobic pathways, which metabolize sugars and fats to regenerate ATP. Aerobic pathways operate inside mitochondria, the cell's major power generators. Anaerobic pathways like glycolysis function in the cytoplasm.Hypoxia  governs how ATP is recycled and which energy-producing substrates  are used; it also generates metabolic by-products, like lactic acid, during strenuous exercise. (Runners know the “lactic acid burn” associated with reduced blood pH.) Glycolysis—the primary source of anaerobic energy in animals—uses glucose, stored as glycogen in muscle cells, to produce ATP. When blood oxygen levels drop, the gene transcription factor hypoxia-inducible factor 1α (HIF-1α) triggers the glycolytic pathway.To understand how HIF-1α regulates skeletal muscle function, Johnson's team generated mice that couldn't express HIF-1α in skeletal muscle. Normal and mutant mice went through exercise routines that included swimming and running on treadmills. After exercise, the normal mice had increased levels of gene transcripts and enzymes involved in glucose transport and metabolism. In the mutant mice, expression of these glycolysis-associated genes and enzymes was significantly lower. The mutants' ATP levels, however, were normal. Without the molecular machinery to engage anaerobic metabolism, their muscles switched to aerobic pathways. The presence of enzymes that respond to reduced ATP levels by increasing mitochondrial ATP production, combined with low levels of lactic acid, confirmed the switch.During endurance tests, the mutants could swim and run uphill (on treadmills tilted upward) longer than the normal mice, but when it came to running downhill, the normal mice prevailed. Downhill running, it turns out, favors glycolytic metabolism; uphill running and swimming favor oxidative pathways, which the mutants were predisposed toward. But their inappropriate use of this pathway came at a cost. By the final day of a four-day exercise routine, the mutants' run time was significantly shorter and their muscles were clearly damaged.The mutants displayed a number of the trademark muscle defects seen in human patients with glycolytic processing disorders. These patients often have reduced lactate levels and elevated levels of mitochondrial enzymes, which apparently can cause a second wind and enhance endurance. This inappropriate use of oxidative pathways—which compensates for the inability to trigger glycolysis—may account for the exercise-induced muscle damage associated with these diseases.These results demonstrate that losing the molecular wherewithal to engage hypoxia response pathways has serious consequences for muscle function during exercise; it can give increased endurance, but at a high price. The mouse model presented here will help researchers explore how muscles normally function in response to low oxygen and how metabolic deficiencies cause debilitating muscle disease.


The function of esophagus is to move food by peristaltic motion which is the result of the interaction of the tissue forces in the esophageal wall and the hydrodynamic forces in the food bolus. The structure of the esophagus is layered. In this paper, the esophagus is treated as a two-layered structure consisting of an inner collagen-rich submucosa layer and an outer muscle layer. We developed a model and experimental setup for determination of elastic moduli in the two layers in circumferential direction and related the measured elastic modulus of the intact esophagus to the elastic modulus computed from the elastic moduli of the two layers.in vivo length and pressure-diameters relations were recorded for the rat esophagus. Furthermore, the zero-stress state was taken into consideration.Inflation experiments were done at 2O the muscle layer had a larger radius and strain than the mucosa-submucosa layer. The strain for the intact esophagus and for the muscle layer was negative at low pressures indicating the presence of residual strains in the tissue. The stress-strain curve for the submucosa-mucosa layer was shifted to the left of the curves for the muscle layer and for the intact esophagus at strains higher than 0.3. The tangent modulus was highest in the submucosa-mucosa layer, indicating that the submucosa-mucosa has the highest stiffness. A good agreement was found between the measured elastic modulus of the intact esophagus and the elastic modulus computed from the elastic moduli of the two separated layers.The radius and the strain increased as function of pressure in the intact as well as in the individual layers of the esophagus. At pressures higher than 1.5 cmH The majority of previous mechanical studies on visceral organs, including the blood vessels, have considered them as homogenous tubes; i.e., a single layer structure. Most visceral organs are, however, multilayered, e.g. the arteries consist of intima, media and adventitia and the gastrointestinal tract has circumferential and longitudinal muscle layers, submucosa and mucosa layers.The esophagus represents a very interesting biomechanical model since it is the only organ that can be separated into two layers without damage to either layer. Hence, the muscle layers can be separated from the mucosa-submucosa layer by dissection, leaving two intact tubes. Separation experiments of the esophagus in guinea pigs and rabbits showed that the submucosa-mucosa layer had larger residual strains and opening angles than the muscle layer -3. ConsiIn this study we recognize that the esophagus consists of mucosa-submucosa and muscle layers. We analyze these layers as elastic shells. Each layer has its own zero-stress state, and its own elastic constants. We will determine the material properties of each layer separately. Specifically, the material properties in the individual layers will be computed from the pressure-diameter relation and zero-stress state with the method of analysis presented below. We will then propose a simple model to combine the two layers to predict the overall behavior of the esophagus under certain hypotheses. The limitations and implications of the model will be discussed.-1 ip). Papaverine (15 mg) was injected into the tail vein to relax the visceral muscles and to euthanize the rat. The cervical segment of the esophagus was dissected free from its adjacent tissue. Next, the thoracic and abdominal cavities were opened. After pouring cold Krebs solution into the thoracic cavity, the esophagus was quickly dissected free from adjacent tissues and its in situ length was measured. A 2-cm-long segment from the middle part, intended for the distension test, was marked. The length of this segment and that of the entire esophagus was measured. The entire esophagus was then cut at the proximal and distal ends including the very first part of the stomach, and immediately placed in calcium-free Krebs solution containing 6% dextran and 0.25% ethylene glycol-bis (β-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA). The solution was aerated with a gas mixture of 95% O2-5% CO2 at pH of 7.4. After careful removal of all extra-esophageal tissue, the length was measured in vitro.Eight male Wistar rats, weighing 380–420 grams, were used in the study. Approval of the protocol was obtained from the Danish Animal Experiment Committee. The animals were anesthetized with sodium pentobarbital  monitored the changes in diameter and length during the distension and images were grabbed by a PC.The middle part of the intact esophagus was mounted in an organ bath containing the Ca2O.After the test of the intact esophagus, the muscle and submucosa-mucosa layers were gently separated into two tubes. The tubes were studied separately using the same procedure outlined above. The only difference was that the maximum pressure was set to 6 cmHThe zero-stress state of the esophagus was obtained in accordance with the method used for blood vessels . Briefly2O increment. The outer diameter was measured at each pressure level and averaged over three locations. At the no-load and zero-stress states, the inner and outer circumferential lengths were measured along with the thickness and area of the wall and layers . The opening angle was defined as the angle subtended between two radii drawn from the midpoint of the inner wall to the tips of the inner wall of the open sector.The morphometric measurements were made using SigmaScan Pro image analysis software . The data were obtained from the images of the tubes in the distended state, rings in the no-load state and sectors in the zero-stress state. In the distended state for both the intact esophagus and the separated tubes, images were analyzed for each 0.5 cmHi-p), the wall thickness (Hp), the mid-wall circumference (Cm-p) at a given pressure were computed as ri-p = [(r2o-p - An/πλ1)]1/2, Hp = ro-p - ri-p, and Cm-p = 2π(ri-p + Hp/2). The outer radius (ro-p) of the intestine was computed according to the outer diameter (Do). The circumferential Green's strains and Kirchhoff's stress were computed according to the equations:The stresses and strains of the esophagus and its sublayers in the pressurized state were determined under the assumption that the geometric configuration of the lumen is cylindrical, the wall of the esophagus is incompressible, and the material in each layer is homogenous. Based on the above measurements and assumptions, parameters of the esophagus such as the luminal radius  corresponds to Young's modulus in the linear stress-strain regime.We assume that the circumferential stress-strain relationships for the inflation experiment obey Hooke's law for each layerσθθsm) (= Eθθsm)(eθθsm) (σθθm) (= Eθθm)(eθθm) (    (4)σ, E and e indicate the Cauchy stress, Tangent modulus and Green strain, respectively. θθ, sm and m indicate the circumferential direction and the submucosal and muscle layers, respectively.where θ the circumferential axis and z the radial coordinate. Nθ denotes the tensile membrane stress resultant in θ direction. The displacement in the x, θ, and z directions of a point on the neutral axis surface are denoted by u, v, and w, respectively. The displacements of any point, A, is denoted by uA, vA, wA as follows: 1) uA = displacement along the generator, positive in the direction of increasing x; 2) vA = displacement along a circle of radius a + z, positive in the direction of increasing θ and 3) wA = radial displacement, positive outward.In general, the circumferential stress is a function of circumferential and longitudinal strain. In the present analysis, we assume that the cross-modulus is small such that the longitudinal term is negligible in comparison with the circumferential term and hence eq. (4). We further assume that the esophagus is a circular cylinder. The basic equations of equilibrium and deformation are given in Flugge . Let x dAccording to the Bernoulli-Kirchhoff hypothesis , we havewhere a indicate the neutral axis.The circumferential strain which is assumed to be small is given byθ is given byThe circumferential membrane stress resultant Nθ and w does not change with x, we obtainBy substitution of Eqs. (4), (5), and (6) into Eq. (7) and noting that in the inflation experiments u, v, and w do not change with Eq. (8) can be integrated to yieldI, asm and am are the neutral axes for the submucosa (1.35) and muscle (1.15). Equation (9) can be solved in terms of EI aswhere I stands for intact esophagus; aI from Eq. (3) with EI computed from Esm and Em as given by Eq. (9b).Hence, we can compare measured EThe data were assumed to be representative of a normal distribution. The results are expressed as means ± SE. Student's t test and analysis of variance were used to detect possible differences between curves obtained from the intact esophagus and the two sublayers. The results were regarded as significant if P < 0.05.in vivo length before the mechanical tests. The length was fixed during the distension protocol.The esophagi shortened by approximately 30% after excision. However, the segments were stretched to the 2O the muscle layer had a higher radius and strain than the mucosa-submucosa layer. The strain for the intact esophagus and for the muscle layer was negative at low pressures indicating the presence of residual strains in the tissue. The thickness of the intact wall and the separated layers decreased as function of pressure. The mucosa-submucosa was the thinnest layer.The outer radius, wall thickness, and circumferential Green's strain as function of pressure is shown in figure Cross-sectional views of the esophagus and its two layers were obtained at the no-load state and zero-stress state. Upon reducing the no-load state to the zero-stress state by cutting the ring radially, the opened ring expanded itself into a sector with an opening angle of about 140° for the intact esophagus figure , top. SeThe stress-strain data are depicted in figure 2O) where the assumption of linear stress-strain relationship is most justified.Figure The peristaltic transport of swallowed material by the esophagus to the stomach is a neuromuscular function affected by a number of neuromuscular factors -14. The The unique structure of the esophagus as a composite of submucosa and muscle layers allows the separation of the two layers and an experimental determination of the constitutive properties of each layer. This is difficult to carry out in the blood vessels or in other organs. The coronary arteries can be separated at the external elastic laminae but only by tearing the adventitial layer . The resA major difference between the structures of the esophagus and blood vessels is the ease with which the wall can be separated into layers. This fact allowed us to obtain the stress-strain relation and the zero-stress state of the esophageal tissue layers, as reported above. In contrast, in spite of the extensive effort on theoretical and experimental bilayer models of arteries by Berry et al , DemirayThe opening angle of the inner submucosa layer is larger than that of the outer muscle layer which agrees with our previous report ,3 and laα, can be used as a measure of stiffness [The stress-strain curve for the submucosa-mucosa layer was shifted to the left of the curves for the muscle layer and for the intact esophagus at strains higher than 0.3, indicating that the submucosa-mucosa has the highest stiffness figure . This cotiffness . The dattiffness ,23,25,26A limitation of the study is that only uni-axial data were obtained in this study. Intuitively the circumferential direction seems to be the most important for cylindrical organs. Since longitudinal changes may also be important for esophageal function, future studies should implement bi- or tri-axial data. Another limitation is that the analytical model is restricted to the linear stress-strain regime. Furthermore, the esophagus and its layers are assumed to be cylindrical tubes, and that the esophageal tissue is incompressible. The last assumption is possibly true in the pressure range studied and it is also known from yet unpublished studies that the esophagus attains circular geometry both at the inner and outer surfaces even at low pressures. The linearity assumption needs to be generalized. We also assumed that each layer was homogeneous, though it is well known that the muscle layer is composed of longitudinal and circumferential muscle bundles. Hence, the muscle layer can be modeled into further sublayers.We have developed an analytical tool that can be used to analyze the mechanics of bilayered organs. The model was used to study the esophagus. The model may be useful for studying the mechanical properties of other organs that can be separated into layers. There are two immediate implications of the results in this study for the understanding of esophageal function and for clinical practice. It is well known that pain may arise from the esophagus and that the receptors involved in the mechanotransduction are located at different positions in the wall. Detailed information about the stress and strain distributions in the layers is therefore important for the interpretation of receptor-mediated responses. Furthermore, the stress reduction during loading  may serve as a mechanism to reduce damage to the esophagus during excessive loadings caused by swallowing of large objects or by acute esophageal obstruction.For the esophagus the model may be applied to the study of remodeling of the individual layers in response to disease. For example, in systemic sclerosis the muscle layers in esophagus are slowly replaced by fibrotic tissue, creating a passive conduit . It is already known that the esophageal stiffness increase in patients with systemic sclerosis ,28 but wYanhua Fan carried out the experimental work, the measurements, and read and approved the final manuscript. Hans Gregersen designed the study, partly analyzed the data, and drafted the manuscript. Ghassan S. Kassab suggested the analysis, analyzed the data, drafted and approved the final manuscript.


Design and test the reliability of a web-based system for multicenter, real-time collection of data in the emergency department (ED), under waiver of authorization, in compliance with HIPAA.This was a phase I, two-hospital study of patients undergoing evaluation for possible pulmonary embolism. Data were collected by on-duty clinicians on an HTML data collection form (prospective e-form), populated using either a personal digital assistant (PDA) or personal computer (PC). Data forms were uploaded to a central, offsite server using secure socket protocol transfer. Each form was assigned a unique identifier, and all PHI data were encrypted, but were password-accessible by authorized research personnel to complete a follow-up e-form.From April 15, 2003-April 15 2004, 1022 prospective e-forms and 605 follow-up e-forms were uploaded. Complexities of PDA use compelled clinicians to use PCs in the ED for data entry for most forms. No data were lost and server log query revealed no unauthorized entry. Prospectively obtained PHI data, encrypted upon server upload, were successfully decrypted using password-protected access to allow follow-up without difficulty in 605 cases. Non-PHI data from prospective and follow-up forms were available to the study investigators via standard file transfer protocol.Data can be accurately collected from on-duty clinicians in the ED using real-time, PC-Internet data entry in compliance with the Privacy Rule. Deidentification-reidentification of PHI was successfully accomplished by a password-protected encryption-deencryption mechanism to permit follow-up by approved research personnel. The ability of medical researchers to obtain and store electronic clinical data was complicated by requirements of the Patient Privacy Rule of the Health Insurance Portability and Accountability Act ("HIPAA") of 1996 in title 45 of the Federal Register, parts 160, subparts A and E of part 164 . The HIP® do not mandate user identification, do not partition and encrypt sensitive data, and do not maintain a record and audit trail of use. Accordingly, we developed a comprehensive electronic system for clinicians to capture clinical research data from the bedside using commercially available hardware and data upload over the Internet. The system was programmed with multiple security steps and authentication procedures to maintain data security and privacy.Existing electronic data collection methods are limited in their ability to centralize data in a fashion that expedites data sharing while remaining in compliance with HIPAA. For example, commercial spreadsheets that run on WindowsWe tested the hypothesis that real-time clinical data can be obtained from clinicians in multiple hospitals using electronic data collection stored in an off-site server, under waiver of Authorization, while remaining in compliance with the Privacy Rule. This study represents the development and implementation phase of an ongoing multicenter study to collect prospective and follow-up clinical data from patients undergoing evaluation for pulmonary embolism in the emergency department. The specific aims of this study were to: 1. Test the feasibility of real-time, electronic data collection on personal digital assistants and personal computers in the emergency department setting in two hospitals. 2. Test if the system would correctly upload protected health information (PHI) in a secure and encrypted fashion, but allow follow-up to be performed by selected individuals using password-protected access to PHI.Patients were enrolled from two hospitals in Charlotte, NC: Carolinas Medical Center Main and Carolinas Medical Center University. The clinical protocol was approved under waiver of informed consent and waiver of authorization by the Carolinas HealthCare Institutional Review Board and the Institutional Privacy Board in accordance with the published guidelines of the Department of Health and Human Services (DHHS) , which wBecause of institutional sensitivity about maintaining compliance with the privacy rule, this project required intensive planning and due diligence. Over a 6-month period, the authors scheduled several meetings with the Director of Privacy in Clinical Research, the hospital's Assistant Vice President of Privacy, and the Director of Information Security to discuss the protocol and methods. These individuals had oversight for privacy issues for both hospitals. Then, to facilitate the process of gaining assistance and approval from the Information Systems Department in implementing the technical aspects  at both hospitals, we obtained a letter of approval from each of these individuals to physically show to technical support personnel.All patients in this study underwent evaluation for pulmonary embolism. The method of selection and diagnosis have been described previously . For eacThis system was designed to allow data to be transferred from multiple sites and stored on one server using technical requirements described in part 160 and 164 of the Privacy Rule. Figure The trigger for data entry was the decision to order a diagnostic test to rule out pulmonary embolism in a symptomatic emergency department patient. Patient data were entered on the prospective e-form. The e-forms were programmed using hypertext markup language (HTML) in conjunction with active server pages (ASP) and Standard Query Language (SQL). The prospective and follow-up e-forms are shown in Figures To test for data validity in the prospective forms uploaded by clinicians, two authors independently examined each of 70 fields for all patients uploaded. We evaluated for blank cells, nonsensical character entry, or numeric entry that fell outside the predefined ranges.® operating system followed by synchronized upload to the hosted server. All clinicians owned a compatible PDA. The authors and staff assistants provided technical support to assist clinicians in the process of downloading the prospective e-form to their PDAs and uploading completed e-forms to the study's central server using commercial software (Microsoft ActiveSynch® v. 3.5). The clinicians were shown that prospective data entry forms could also be accessed through a URL hyperlink that was posted on the desktop of all Internet-connected computers in both emergency departments. When the user clicked the hyperlink, this action routed the user through the firewall directly to the hosted server for this study. All computers ran Windows 95 or higher, with ethernet connection to a T3 44.736 Mbps channel.Forms were completed by attending physicians (N = 22), resident physicians (N = 20), and physician assistants (N = 6) in two emergency departments while the patient of interest was still in the emergency department. Prior to study deployment, each clinician was individually trained in a 10-minute session by the study principal investigator using a pre-defined protocol, and each clinician received a follow-up letter that summarized the training session. Data forms could be accessed in the emergency department using designated Internet-connected personal computers within patient care areas, or could be completed using individually owned personal digital assistants using the Pocket PCUpon opening the first web page, the user viewed a list of clinician names Figure . The cli®, Seattle WA). However, all PHI data fields were remained encrypted.Multiple methods were used to ensure that protected health information would not be subject to unauthorized access, viewing or hijacking. When clinicians entered data in the emergency department, the server polled the form for inactivity exceeding 30 minutes, at which time the page would automatically close without being uploaded. We anticipated scenarios where a clinician would enter data on a prospective form that would need to be revised as a result of updated information . To allow for such editing, the clinician could re-access any prospective form for a period of 60 minutes after initial upload, provided that the Internet provider address of the computer was the same as the computer from which the form originated. After 60 minutes, the prospective e-form could be altered only by a study administrator. All data were transferred using secure socket link (SSL) protocol. The central server (Win2000 OS) was located off-site at a large commercial web hosting facility (NTT/Verio Inc). Upon upload, all fields containing any of the 18 elements of that constitute PHI were subjected to 128 key-bit encryption. Data were stored in relational tables. To allow data analysis for research purposes, the study PI could access stored by file transfer protocol and exported into a format compatible with commercial software  to determine if the patient had any of the outcomes of interest in the follow-up form. If no follow-up data were available within the hospital system to prove the patient was alive at 45 days, then the follow-up protocol required query of the public social security master death index to determine if a death certificate had been filed for the patient. Finally, if no valid follow-up were documented by electronic database search, we then attempted to contact the patient through a previously described, stepwise procedure, consisting of a mailed questionnaire, followed by a telephone call, if necessary .When all of the data required for the follow-up form were obtained and input into the form, the research coordinator would complete the form and press the "check form for completeness" button. This would activate a system to ensure that all necessary follow-up data were entered. For example, every patient had to have a valid 45-day follow-up, either in the form of a documented follow-up to a clinic, telephone follow-up with the patient, or confirmation of patient death within 45 days.The system was fully implemented on April 15, 2003. As of April 15, 2004, prospective data forms were uploaded from 1022 patients evaluated for acute pulmonary embolism. Prospective data have been entered by 42 clinicians and 6 physician assistants from two hospitals in Charlotte, NC. The primary technical barrier to implementation was the process of loading and using the prospective e-forms on a personal digital assistant. All 48 clinicians required individual help and training, of over one hour each to show them how to install the e-form on their PDAs. This impedance was compounded by real-time difficulties associated with stylus use on a small PDA screen, followed by difficulties with uploading to the website from the PDA led to abandonment of this method of data entry. Out of 48 clinicians, only 6 successfully uploaded more than one e-form from the PDA. Only 12 of 1022 uploaded e-forms originated from a PDA.The primary technical barrier to implementation on the personal computers included maintaining the URL icon on desktops in the ED . This problem was solved by the permanent link on the hospital's intranet home page. In two separate instances, clinicians reported that they had populated the e-form, attempted to submit, and for unknown reasons, were unable to upload the e-form, and they had to reenter the data and resubmit the e-form. The server has maintained a log of all successive e-forms uploaded by each clinician. No uploaded prospective forms have been lost or deleted. The side-server system was designed to prevent e-form upload with missing or erroneous data.To examine if this system properly, two observers reviewed the eight parametric field entries  that were key-entered by clinicians for 1022 patients. Validity required that both observers agree that the entry was a real number within the prespecified range of the parameter. In 44/8176 fields of 12 patients (0.6%), two observers deemed the parametric field entry to be useless for statistical analysis. Stated another way, these data would be coded as missing after data cleaning were completed. However, no categorical data were missing or erroneous. As a result, 1010/1022 (98.8%) of prospective e-forms had usable data in all 70 fields.Ninety-four percent of all 1022 patients have reported the site hospital to be their hospital of choice. Follow-up forms Figure  have beeFollow-up has revealed that all prospective e-forms were authentic, and each was completed on an emergency department patient who underwent at least one clinical test for pulmonary embolism. No bogus forms were detected during follow-up to date. This demonstrated a low likelihood of an unauthorized person generating a spoofed form on one of the designated computers in the ED treatment area, given that the automatic control system would not allow a form to be uploaded until all 70 fields are completed.All uploaded prospective and follow-up data were obtained by the central PI using file transfer protocol and were inputted into a spreadsheet without difficulty. Figure ® Server 2003 operating system. The only means of electronic access to the server is via hypertext transfer protocol (HTTP) and file transfer protocol (FTP). Both of these system services log all requests made to their ports. An example log entry is shown in the appendix.Query of the server log revealed no evidence of website hijacking or other intrusion. The server computer which houses the study database and runs the web application uses the WindowsThe step of obtaining written Authorization to comply with HIPAA can impart a selection bias in registries intended to study acute disease processes . In sectThe first research objective was to determine if PDAs would represent an efficient and secure mechanism for clinicians to record real-time data at the bedside in the emergency department setting. Our experience in this phase I, two-hospital study demonstrated that PDAs presented unexpected complexities that eroded our enthusiasm. The clients were clinicians with variable levels of technical sophistication. Despite our use of a relatively standard process, clinicians found it difficult to download the e-form from the website onto their PDAs, and many needed help from the study authors. Clinicians frequently forgot to bring their PDA devices to work, and during the one-year course of this study, 10 of 48 clinicians bought new PDA devices. Clinicians consistently reported difficulty with the small screen size and data entry with a stylus. Unfortunately, we did not quantify this opinion using a structured survey. We believe this represents the first published experience at using PDAs to collect research data in the emergency department setting. Our results are somewhat less positive than other studies that have reported the use of hand-held computers to maintain clinical databases . HoweverThe second objective was a relatively complex task intended to determine if the system would allow prospective and follow-up data collection over the Internet in compliance with the requirements of the Privacy Rule. From a functional standpoint, we sought to determine if the system would allow us to protect the data fields that needed to be protected, but allow the non-sensitive data to be accessed by study personnel who did not have local IRB approval. This was accomplished while maintaining strict security standards at each step of data transfer . Accordingly, the data collection instrument must accurately capture the information that the clinician uses to motivate his or her test ordering behavior, rather than to collect data after the test results have returned, and more complete medical records may have arrived. This report represents a phase I study. For the second phase, we will deploy this system to 10 US hospitals to allow collection of data from 5000 patients. The ultimate goal of this project is to collect a large, multicenter database, as the substrate for a mathematical model to generate a pretest probability of pulmonary embolism based upon beliefs of many clinicians.Research data can be successfully collected, entered and uploaded to a hosted server by emergency physicians working in different emergency departments, and in compliance with the Privacy Rule. Use of server side controls to test for data validity ensured that 98.8% of uploaded forms contained complete data usable for statistical analysis. The PHI data were successfully encrypted and deencrypted using password access to allow follow-up at a later date. Server log query demonstrated no evidence of intrusion or data loss, suggesting that data were securely stored.ASP – Active server pagesED – Emergency departmentFTP – File transfer protocolDHHS – Department of Health and Human ServicesHIPAA – Health Insurance Portability and Accountablity ActHTML – Hypertext markup languageHTTP – Hypertext transfer protocolIRB – Institutional Review BoardPDA – Personal digital assistantPHI – Protected health identifiersPI – Principal investigatorSQL – Standard query languageRUL – Uniform resource locatorJeffrey A. Kline is primary author on a US copyright certificate "Electronic Form ("e-form") For Secure Collection of Clinical Data Via the Internet" and inventor on a US patent (pending) that describes the present work. Jeffrey A. Kline and Charles L. Johnson own stock in BreathQuant Medical Systems.Example of a log entry for the HTTP system service" "Mozilla/4.0 "208.10.156.69 – [02/Mar/2004:23:55:55 +0000] "POST /pestudy/peadmin.asp HTTP/1.1" 302 1349 "208.10.156.69 and the resource requested is the ASP file . For the FTP system service this would be a typical log entryThe IP address of the HTTP requesting browser is 208.10.156.69 – generic [06/Aug/2003:17:00:14 +0000] " [22786]USER generic FTP" 331 0 "-" "-"208.10.156.69 and the USER identity that was logged in was "generic". Both these log files are scanned once a week to look for any suspicious requests. To date there have been no identified intrusion or hijack attempts on the specific study database. As an HTTP responding server on the public Internet, the web (HTTP) server program does receive many well identified "virus spreading" requests which it denies and whose denials are logged. One such "virus related" request common to all web logs today isThe IP address of the HTTP requesting browser is 404 380661.100.6.181 – [01/May/2004:11:30:52 +0000] "GET /scripts/nsiislog.dll HTTP/1.1" 404 code denotes that the request was denied. As security updates from Microsoft become available the study web server is updated. At present there are no known HTTP vulnerabilities which would allow an unauthorized user to gain access to files on the server.This example virus was developed to attack a vulnerability which existed in Microsoft Web Servers but was eliminated by a security update for their web (HTTP) server. The highlighted JAK conceived the study, obtained funding and participated in the system design, data collection and drafted the manuscript. CLJ carried out all technical programming, participated in system design and drafting the manuscript. WBW participated in idea conception, system design, data collection and drafting the manuscript. MSR participated in data collection and drafting the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Human metastatic cutaneous melanoma has gained a well deserved reputation for its immune responsiveness. The reason(s) remain(s) unknown. We attempted previously to characterize several variables that may affect the relationship between tumor and host immune cells but, taken one at the time, none yielded a convincing explanation. With explorative purposes, high-throughput technology was applied here to portray transcriptional characteristics unique to metastatic cutaneous melanoma that may or may not be relevant to its immunogenic potential. Several functional signatures could be identified descriptive of immune or other biological functions. In addition, the transcriptional profile of metastatic melanoma was compared with that of primary renal cell cancers (RCC) identifying several genes co-coordinately expressed by the two tumor types. Since RCC is another immune responsive tumor, commonalities between RCC and melanoma may help untangle the enigma of their potential immune responsiveness. This purely descriptive study provides, therefore, a map for the investigation of metastatic melanoma in future clinical trials and at the same time may invite consideration of novel therapeutic targets. Human metastatic cutaneous melanoma relative to other common solid tumors shares with renal cell cancer (RCC) the well deserved reputation of being responsive to immune manipulation ,2. HowevWe have previously shown that the microenvironment of a subgroup of melanoma metastases expresses at the transcriptional level an array of biologically active factors that may influence both the innate and the adaptive arm of the immune system . We have2-value ≤ 0.001 (unpaired two-tailed Student t test). This test identified 4,658 cDNA clones differentially expressed between melanoma metastases and tumors of other histology  which is a previously described serum marker of RCC also associated with renal carcinogenesis [-7 and 1 × 10-6 for two clones representing this gene . Overall, this cluster was enriched of genes associated with active cellular metabolism and included only few genes of previous known relevance to melanoma with the exception of a member of the melanoma antigen family D  predominantly associated with growth regulation and apoptosis. Among the genes included in this cluster was BNIP3L  that we have previously reported to be associated with the immune responsiveness of melanoma metastases [cluster c and d) included 262 and 613 clones, respectively (112 and 296 named corresponding respectively to 110 and 289 genes). These clusters were characterized by a high density of unnamed clones and by relatively low Cy5/Cy3 ratios. However, it should be noted that these clusters may be of particular interest because the gene expression profile was similar between melanoma and RCC tumors suggesting that some of these genes may conceal the enigma of immune responsiveness. In particular, a relatively sizable sub-cluster was noted with genes predominantly up-regulated in RCC but also expressed by melanoma lesions compared with other tumors  that was previously also reported in association with melanoma immune responsiveness to interleukin-2-based immunotherapy [JAK-1 was recently linked to the apoptotic role that interleukin-24 (melanoma differentiation associated gene-7: MDA 7) may exert on melanoma cells [cluster e) included 208 clones (151 named representing 143 different genes) predominantly associated with immune function. This immune signature was underlined by the high prevalence of expression of these genes in samples obtained from lymph node metastases whether from melanoma or colorectal primaries. Cluster f integrated 129 clones (91 named representing 87 distinct genes) including a mixture of genes with disparate functions difficult to categorize into a predominant pattern. This group also included APPBP1  which was previously reported in association with melanoma immune responsiveness [APPBP1 is a recently discovered epidermal growth factor that regulates dendrite motility and melanin release in epidermal melanocytes and melanoma cells [Cluster g included genes preferentially up-regulated in subcutaneous melanoma metastases known to be more responsive to immunotherapy with interleukin-2 [PRAME and tyrosine-related protein-1; TRP-1). In a small proportion, this cluster included a group of genes only over-expressed in fine needle aspirates (FNA) and generally expressed by circulating cells revealing blood contamination of FNA material . Cluster I included 222 clones (171 named identifying 155 genes) with most of the melanoma differentiation antigens (MDA) clustering in close proximity with the exception of the TRP-1 already discussed in cluster g. Interestingly, this cluster was also highly enriched of genes associated with ribosomal function and active translation. Furthermore, it included the melanocyte master regulator MITF  which has been shown to modulate lineage survival and melanoma cell viability through interaction with the anti-apoptotic protein BCL2 [MITF was coordinately expressed with several genes associated with calcium and other solute metabolism including cytochrome p450  solute carrier family 7 , G protein coupled receptor 56  and calpain 3 , a calcium-regulated gene found to be highly expressed in melanoma cells [cluster J included 88 clones of which the 62 named identified 58 genes.Several signatures representing genes preferentially expressed by melanomas were identified that could be partially linked to specific gene functions. Those signatures were segregated according to unsupervised gene rearrangement based on the Eisen's clustering method. The first cluster  and the tyrosine-related protein-1 (TRP-1). When the melanoma associated genes were studied alone, PRAME clustered close to the other MDA believe to be involved in the pigmentation process . This is of particular interest because PRAME has been also reported to be highly expressed in other cancers of ectodermic origin such as medulloblastoma and neuroblastoma suggesting a link between ectoderm and pigmentation [SILV) in melanoma metastases while genes of the MAGE family manifested an independent behavior [Genes previously described to be preferentially expressed by melanoma lesions were confirmed to be so at a very high level of significance Figure . Excepti d Table . Clusterentation ,20. The entation -25, we ncluster e. These genes appeared up-regulated in lymph node metastases of melanoma as well as those from colorectal primaries suggesting that their expression results from lymphoid cell infiltration  natural killer cell-related genes suggests potent chemo-attraction toward natural killer cells by the tumor micro-environment of subcutaneous and cutaneous melanoma metastases. This is also emphasized by the high expression of interleukin-21 receptor which is usually expressed by natural killer cells and stimulates their cytolytic activity upon ligation with interleukin-21 produced by activated T cells [IRF)-7 implicated in the amplification of the innate immune response [The large majority of genes associated with immune function were included in n Figure . The samer cells , was coner cells  and to bin vitro ,29. Of itivation ,31. The d CXC3L1  may be ad CXC3L1 . Overall T cells ,35. The response  through response  may leadresponse . More puresponse  and CD4+response ; a relatclusters f to j. These genes had an expression profile opposite to the immune-related genes seen included in cluster e and appeared over-expressed in subcutaneous compared to lymph node metastases. Two granzyme-related genes were found strongly up-regulated in cluster e including granzyme A and M. This observation contrasted with the increased expression of cathepsin F and L in cluster f to j suggesting an opposite regulation of these genes involved in cell death or survival.A second and smaller group of immunologically-related genes was identified and included genes that segregated separately in clusters g - j appeared to demonstrate a preferential expression of genes in subcutaneous metastases independent of the technique used for biopsy (excision versus FNA). In particular, cluster g contained a small node of 47 clones highly expressed in subcutaneous metastases that included PRAME and TRP-1. This cluster also included the renal tumor antigen RAGE which has been previously shown to be highly expressed by melanomas [PRAME was the pattern of expression of the serine/threonine-specific protein kinase B-RAF. This gene is mutated in approximately 70 % of melanomas and it is often over-expressed [It has been reported that subcutaneous metastases of melanoma are more responsive to immunotherapy with interleukin-2 than lymph nodal and visceral metastases . Therefoelanomas  and melaelanomas . Interesxpressed . Althougt test and identified 4,221 genes at a cut off p2-value of ≤ 0.001. The data set was then filtered using the Cluster Program  selecting genes that were expressed in at least 80 % of the experiments and for which a Cy5/Cy3 log2 ratio ≥ 2 was present in at least one experiment. Two-thousand eight hundred and forty-three genes resulted from this filter. Because of the predominance of melanoma lesions (69 melanoma lesions compared to 14 RCC) the genes identified strongly represent differences between melanomas and other tumors. Therefore, we identified among them those that were not differentially expressed between melanoma and RCC samples to identify those genes that are truly uniquely expressed by the two immune responsive cancers. Two-thousand three hundred and fifty-eight genes were expressed similarly between the two types of cancer . A significant number of genes commonly expressed by melanoma and RCC and not by other tumors had no known function (681 genes). The remaining 1, 677 genes were further analyzed by separating those up-regulated from those down-regulated in melanoma and RCC compared with other tumors. The genes up-regulated in RCC and melanoma were considered those with a median LogRatio above 0.3 in either RCC or melanomas  which is a modulator of cell cycle progression and angiogenesis in melanoma [MIF has modulatory properties on natural killer cell mediated lysis of cancer cells contributing, therefore, to an immune privileged microenvironment in uveal melanoma [LCAM) and melanoma cell adhesion molecule (MCAM) were also up-regulated in both lesions and may play an important role in mediating migration of immune cells to the tumor deposits [We then pooled together melanoma and RCC samples data to identify genes commonly expressed by these tumors and not by tumors of other histology. Significance was assessed by a two-tailed unpaired student's ure MITF ,44. thature MITF  was founure MITF . It is pure MITF . In addimelanoma ,48. was melanoma . Two gendeposits . FinallyThis is a descriptive study where genes specifically expressed by melanoma metastases were identified comparing a large collection of samples from patients with metastatic cutaneous melanoma with other primary tumors and lymph nodal metastases. A limitation of the study is the lack of other samples including visceral metastases of melanoma and metastases from tumors of other histology. Nevertheless, we considered useful to compile a list of genes characteristically expressed by subcutaneous and lymph nodal lesions of melanoma for reference purposes and we are willing to provide full information about these genes upon request. In spite of the limitations of this study, few general conclusions could be drawn.Fourteen primary renal cell carcinoma (RCC) specimens were collected at the Department of Urology of The Johannes Gutenberg-University, Mainz, Germany; one primary melanoma, three in transit metastases, seven cutaneous metastases, thirty-five lymph nodal metastases and two visceral metastases of cutaneous melanoma were collected at the Department of Surgical Sciences, University of Padua, Italy; twenty-one fine needle aspirates of cutaneous melanoma metastases were obtained at the Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD; seventeen primary epithelial ovarian cancer (EOC) specimens were obtained at the Department of Gynecologic Oncology, MD Anderson Cancer Center, TX; three primary sarcoma, one primary endometrial cancer, one primary laryngeal cancer, two primary breast cancers and one primary colon adeno-carcinoma were obtained from the Tissue Network ; twelve primary carcinomas of the esophageal junction were obtained from the NCI (Division of Cancer Treatment and Diagnosis); thirty-five primary, 16 lymph node metastases and one hepatic metastasis from colorectal adeno-carcinomas were obtained from the Department of Pathology of the University of Pisa, Italy. Specimens were collected as the result of routine operative procedures and portions were frozen for subsequent analysis while the remnant tissue was used for pathological confirmation. Tissue procurement followed standard ethical procedure according to institutional policy. A summary of the specimens studied is presented in Table Total RNA was extracted from frozen material using Trizol reagent according to manufacturer's instructions  and amplified into anti-sense RNA (aRNA) as previously described ,27,51,52. Micro-arrays were printed at the Immunogenetics Section, DTM, CC, NIH with a configuration of 32 × 24 × 23 and contained 17,500 elements. Clones used for printing included a combination of the Research Genetics RG_HsKG_031901 8 k clone set and 9,000 clones selected from the RG_Hs_seq_ver_070700 40 k clone set. The 17,500 spots included 12,072 uniquely named genes, 875 duplicated genes and about 4,000 expression sequence tags.Total RNA from peripheral blood mononuclear cells pooled from six normal donor was extracted and amplified to serve as constant reference as previously described ,27,51,522-based ratios normalizing the medial log2 ratio value across the array equal to zero. Validation and reproducibility were performed using our internal reference concordance system as previously described [t test. The same analyses were performed using un-paired Wilcoxon's non-parametric assessment and provided the same conclusions (not shown). Details of each analysis are presented in the results section.All statistical analyses were performed using the logescribed . Unsuperescribed  and visuescribed . DetailsAVE Ratio = average Log2 CY5/Cy3 ratio between test and reference sample. The t test p2-value refers to a two-tailed unpaired analysis between the samples mentioned below. RCC = renal cell cancer; MEL = melanoma; Other = tumors other than RCC and melanoma.Click here for file