EYFP reporter as a model system to quantitatively analyze HTLV-1 particles released from producer cells.Human T-lymphotropic virus type 1 (HTLV-1) is an important human retrovirus that is a cause of adult T-cell leukemia/lymphoma. While an important human pathogen, the details regarding virus replication cycle, including the nature of HTLV-1 particles, remain largely unknown due to the difficulties in propagating the virus in tissue culture. In this study, we created a codon-optimized HTLV-1 Gag fused to an The codon-optimized Gag led to a dramatic and highly robust level of Gag expression as well as virus-like particle (VLP) production. The robust level of particle production overcomes previous technical difficulties with authentic particles and allowed for detailed analysis of particle architecture using two novel methodologies. We quantitatively measured the diameter and morphology of HTLV-1 VLPs in their native, hydrated state using cryo-transmission electron microscopy (cryo-TEM). Furthermore, we were able to determine HTLV-1 Gag stoichiometry as well as particle size with the novel biophysical technique of fluorescence fluctuation spectroscopy (FFS). The average HTLV-1 particle diameter determined by cryo-TEM and FFS was 71 ± 20 nm and 75 ± 4 nm, respectively. These values are significantly smaller than previous estimates made of HTLV-1 particles by negative staining TEM. Furthermore, cryo-TEM reveals that the majority of HTLV-1 VLPs lacks an ordered structure of the Gag lattice, suggesting that the HTLV-1 Gag shell is very likely to be organized differently compared to that observed with HIV-1 Gag in immature particles. This conclusion is supported by our observation that the average copy number of HTLV-1 Gag per particle is estimated to be 510 based on FFS, which is significantly lower than that found for HIV-1 immature virions.In summary, our studies represent the first quantitative biophysical analysis of HTLV-1-like particles and reveal novel insights into particle morphology and Gag stochiometry. There are approximately 15-20 million people infected by human T-lymphotropic virus type 1 (HTLV-1) worldwide . DespiteThe Gag polyprotein is the main retroviral structural protein and is sufficient, in the absence of other viral proteins, for the production and release of immature VLPs . The GagStudies with many retroviruses, including human immunodeficiency virus type 1 (HIV-1), have shown that retroviral assembly is initiated by binding the myristoyl moiety of MA with lipid rafts at the plasma membrane -11. The Cryo-electron tomography (cryo-ET) combined with three-dimensional (3D) reconstructions have provided highly detailed structural information for HIV-1. Structural studies have revealed that HIV-1 Gag proteins form an incomplete paracrystalline lattice in immature particles ,15. ThisDespite limited amino acid sequence homology among different retroviruses, the atomic tertiary structures of individual Gag domains exhibit high similarity -18. Thercis-elements on the RNA transcript required for efficient nuclear export.Previous molecular analyses of HTLV-1 replication have been severely hampered by the fragility of HTLV-1 proviral sequences as well as the low levels of viral replication in tissue culture. Given the technical and experimental limitations of working with HTLV-1, we first sought to create an experimental model system that would be amenable to successfully and efficiently analyze HTLV-1 Gag trafficking and virus particle assembly and release. It is well-established that retroviral Gag polyprotein is sufficient for the assembly and release of VLPs [reviewed by ]. Our prEYFP is expressed from a CMV promoter, and a Kozak consensus sequence was engineered upstream of the start codon to facilitate translation initiation as well as an in-frame insertion of the EYFP gene sequence just prior to the HTLV-1 Gag gene stop codon. The plasmid is quite stable and readily amplified in E. coli (data not shown).In order to create a tractable and robust system for Gag expression and virus-like particle production, we designed and created a codon-optimized HTLV-1 Gag construct to improve HTLV-1 Gag expression. In order to readily detect Gag expression, trafficking, and incorporation into VLPs, we fused the EYFP to the C-terminal end of the Gag protein. Figure To confirm expression of the fusion construct, 293T cells were transiently transfected with three independent clones of pEYFP-N3 HTLV-1 Gag in parallel experiments. Thirty-six hours post-transfection, HTLV-1 Gag-EYFP protein expression was examined from both cell culture supernatants  on independently prepared samples resulted in a mean diameter of 75 ± 4 nm for the VLPs.FFS provides information about the size of a particle through the autocorrelation function and the brightness and concentration of the particles through the photon counting histogram (PCH). Recent advances have expanded this technique to allow for the examination of protein oligomerization of larger complexes, including our recent analysis of HIV-1 particles . In the 2 ≥ 10). Including a second VLP brightness species into the fit model was required to reproduce the experimental data. A fit of the photon counting histogram to a 2-species model (reduced χ2 = 1.5) is shown in Figure i is characterized by its normalized brightness bi and average particle number Ni in the observation volume. Note that the normalized brightness is the same as the Gag copy number of the VLP. It is illustrative to briefly ignore the brightness heterogeneity by calculating the average Gag copy number bavg of the VLP sample according to [The same raw data was analyzed with PCH analysis to determine the average copy number and concentration of VLP samples. A model assuming a single VLP brightness species leads to poor fits of the experimental PCH data (reduced χrding to .n = 5) we determined an average Gag copy number per VLP of 510 ± 50  remained approximately constant for all measured samples, f2 = 19 ± 7%. Thus, a population of ~19% of the VLPs is associated with the higher Gag copy number. Note that a similar heterogeneity in Gag copy numbers has also been reported for HIV-1 VLPs [The average Gag copy number was determined from the two brightness species identified by PCH analysis. Repeated measurements of multiple independent sample preparations confirmed the presence of the two species. Their brightness values, which typically varied very little across experiments, correspond to Gag copy numbers of V-1 VLPs .Recent progress in cryo-TEM, cryo-ET and 3 D reconstruction has led to many major breakthroughs in our understanding of virus structure. For instance, the architecture of immature and mature HIV-1 ,23,24, mWhile our model system does not express Gag in the context of a proviral sequence , our results indicate that the VLPs produced have the morphology of the authentic HTLV-1 immature particles. Furthermore, while the Gag trafficking pathways used by the HTLV-1 Gag in this model system may be different from that of Gag expressed from the provirus, the production of VLPs argues that the trafficking pathways are biological relevant since VLP production is the result of expression of the codon-optimized Gag-EYFP fusion. The altered Gag trafficking pathways could influence envelope incorporation into VLPs, though our cryo-TEM data revealed an abundance of VLPs with VSVG. The VLPs characterized in our study resemble immature HTLV-1 and can be readily observed in ultrathin sections of 293Ts transfected with pEYFP-N3 HTLV-1 Gag Figure . In addiWe found that the intracellular localization of HTLV-1 Gag-EYFP was comparable to that of authentic Gag in HeLa cells Figure . This imOur cryo-TEM and FFS analysis determined that the average VLP diameter was 71 ± 20 nm and 75 ± 4 nm, respectively. As observed in other retroviruses, the size of HTLV-1-like particles varies greatly, ranging from 30 to 237 nm. According to the size distribution Figure , over 25We used FFS to also investigate Gag stoichiometry in the VLPs by performing brightness analysis of the FFS data. We determined that the average Gag copy number per VLP is ~510, which implies that only half of the available membrane surface is covered by Gag. Brightness analysis further revealed heterogeneity of the Gag copy number by identifying two brightness species. The presence of heterogeneity in the Gag copy number has also been observed for HIV-1 Gag-based VLPs . Since FAmong the thousands of cryo-TEM images of VLPs examined in our study, we commonly observed particles that did not have electron density consistent with a Gag shell covering the entire membrane surface Figure . These rIn vitro studies suggest that the HTLV-1 Gag shell is very likely to be organized differently compared to that of HIV-1 Gag [IV-1 Gag -18. WhenIn summary, we have developed the first efficient and robust model system for the analysis of HTLV-1 Gag cellular trafficking, virus particle assembly, release and particle morphology. This system will allow for significant advancements in understanding of the basic mechanisms of HTLV-1 replication - which has been severely hampered due to the limitations in studying HTLV-1 in tissue culture. Our study also represents the first description of immature HTLV-1 particles as well as quantitative measurements of particle size, Gag copy number, and an initial analysis of the HTLV-1 Gag lattice. Future application of cryo-electron tomography will aid in gaining greater insight into HTLV-1 particle morphology. A deeper understanding of the basic mechanisms involved in HTLV-1 particle assembly and morphology should help to enhance our global understanding of the basis of HTLV-1 particle infectivity, transmission and pathogenesis.gag contains an optimal Kozak consensus sequence [ATGG (start codon in bold). Two restriction enzyme sites, Hind III and Bam HI, were also engineered into the 5' and 3' end of the gene, respectively, for sub-cloning purposes. For reporter gene construction, the artificial HTLV-1 gag was cloned into a pEYFP-N3 vector using the HindIII and BamHI restriction sites, creating pEYFP-N3 HTLV-1 Gag.A codon-optimized HTLV-1 Gag gene was designed using the UpGene program  and syntsequence ,39 at th293T cells were transiently transfected with the pEYFP-N3 HTLV-1 Gag construct using GenJet  according to the manufacturer's instructions. Thirty-six hours post-transfection, cell pellets and supernatant were collected and lysates were prepared as previously described . LysatesHeLa cells were grown on Lab-Tek II chamber slides (Fisher Scientific) and transfected with either the pEYFP-N3 HTLV-1 Gag construct or a HTLV-1 proviral clone (a kind gift from Dr. Marie-Christine Dokhelar) . Thirty-G) protein (10:1) expression construct using GeneJet. Twenty-four hours post-transfection, the cell culture media was changed to a serum-free media and incubated for an additional 12 hr. In order to harvest VLPs, tissue culture supernatant was centrifuged at 3000 × g for 5 min to remove large cellular debris, then the supernatant was passed through an Amicon Ultra- 15 Centrifugal Filter Unit (100 KDa)  to concentrate samples. The concentrated samples were then subjected to a 10-40% linear sucrose gradient prepared with a Gradient Master . Samples were then ultracentrifuged at 35,000 rpm for 30 min at 4°C using a SW55 Ti rotor. The VLP fraction was extracted and pelleted at 35,000 rpm, 4°C for 1.5 hr using a SW55 Ti rotor (Beckman). After centrifugation, the pellet was resuspended in 1× STE buffer  at 4°C for 4 hr and then analyzed by cryo-TEM.293T cells were co-transfected with pEYFP-N3 HTLV-1 and a vesicular stomatitis virus G  were acquired and stained with uranyl acetate and lead citrate, then examined by electron microscopy using a JEOL 1200EX transmission electron microscope.293T cells were transfected with either pEYFP-N3 HTLV-1 or a HTLV-1 proviral clone as described above. Thirty-six hours post-transfection, cells were harvested and washed twice with 1× PBS followed by an additional wash in 0.1 M sodium cacodylate. To prepare thin sections, cell pellets were first fixed with 2.5% glutaraldehyde for 40 min and then washed three times with 0.1 M sodium cacodylate. After washing, the samples were post-fixed with 1% OsOA 3ul aliquot of the purified and concentrated HTLV-1 VLP sample preparation was applied to a glow-discharged c-flat holey carbon grid  and used for plunge freezing into liquid ethane  with a Fhttp://cryoem.ucsd.edu/programDocs/runRobem.txt. The center of each particle was determined using the program EMCORORG that calculates cross-correlation of each image with its 180° rotational image http://cryoem.ucsd.edu/programDocs. The radial profile for each particle was then calculated by computing the rotationally averaged density relative to the center of the particle. A group of 14 VLP images with a diameter in the range of 70-80 nm was used for calculation of the averaged radial profile. Each pixel in the image corresponds to a 3.0-Å spacing in the VLP. The defocus levels of these images were between 1.5-3.7 μm, which allows for visualization of both membrane leaflets of the viral membrane. The radial profile of each particle was first calculated to obtain the highest density position of the outer membrane leaflet. The radial profile of each particle was then linearly interpreted to match the position of the outer membrane to the averaged position (367-Å radius). The average radial profile and standard deviation were then calculated.In order to calculate the radial density profile, images of VLPs with spherical morphology were boxed using RobEM Cryo-TEM images were analyzed using ImageJ software . For each VLP, two perpendicular diameters were used to calculate the average diameter. The histogram was generated using GraphPad Prism 5 software .293T cells were co-transfected with pEYFP-N3 HTLV-1 and a VSV-G expression construct (10:1) as described earlier. Aliquots of the cell culture supernatants used for subsequent cryo-TEM analysis were removed for parallel analysis by FFS. Thirty-six hours post-transfection, VLPs were harvested and clarified of cellular debris by low-speed centrifugation at 3000 × g for 5 min as well as passing through a 0.22 μm filter. The resulting clarified supernatants were then used for FFS measurements.A mode-locked Ti:sapphire laser  pumped by an intracavity doubled Nd:YVO4 laser  is the source of two-photon excitation. Experiments were performed on a modified Zeiss Axiovert 200 microscope  as previously described . Each FFD1/τD2 = r1/r2. The measured diffusion time of fluorescent spheres with a known radius of 50 nm serves as a reference to calculate the average diameter of the VLPs [ε and the average number N of particles in the optical observation volume. The particle number N is converted into a concentration after the observation volume is calibrated with a dye sample. One of the three species is required to take the auto-fluorescent background of the solution into account as discussed in a recent paper on HIV-1 VLPs [A brief description of the analysis method is provided here. A detailed discussion of FFS analysis of VLP samples can be found elsewhere . The difthe VLPs . The FFSthe VLPs . Each inV-1 VLPs . This baV-1 VLPs . PCH fitV-1 VLPs .N remains constant, which establishes a constant optical observation volume. The brightness of a protein complex scales with the number of YFP-labels it contains [b = ε/εYFP, where εYFP is the brightness of the YFP monomer and ε is the brightness of the complex. A calibration measurement of YFP brightness, εYFP, is necessary to determine copy number. Because YFP brightness is difficult to determine at the low powers that the VLPs must be measured at to avoid saturation of the detector, the YFP brightness, εhigh, is measured at a higher excitation power, Phigh. Conversion to the YFP brightness εlow for the low power Plow of VLP experiments is achieved by using the relationship of power to brightness εlow = εhigh(Plow2/Phigh2). The data acquisition time for the VLP measurements was chosen such that at least 1000 VLPs passed through the observation volume, which is sufficient for statistical analysis of the data. All VLP measurements were performed at excitation powers that are free from saturation and bleaching artifacts [bavg of the two species is determined by a non-linear relationship [To avoid unwanted optical effects, all experiments are conducted in a power range where the fluorescence intensity of YFP scales quadratically with excitation power. We also confirmed that within this power range the average occupation number contains . The YFPrtifacts . The FFStionship ,bi and Ni are the normalized brightness and the number of particles in the observation volume of each species.where The authors declare that they have no competing interests.IFG, WZ, JLJ, KF, YC and JR carried out the experimental work, participated in the data analysis and interpretation, and contributed in the writing of the manuscript. WZ, JDM, YC and LMM conceived of the study, oversaw experimental design, data analysis, and interpretation as well as edited the manuscript. All authors read and approved the final manuscript.Supplemental Figure 1. Low magnification cryo-TEM image of VLPs produced from 293T cells. Image provides another example of the types of particles observed by cryo TEM. Scale bar = 100 nm.Click here for file


Strongyloides stercoralis infection by augmenting regulatory T cell numbers, which in turn down regulate the immune response to the parasite.Human strongyloidiasis varies from a chronic but limited infection in normal hosts to hyperinfection in patients treated with corticosteroids or with HTLV-1 co-infection. Regulatory T cells dampen immune responses to infections. How human strongyloidiasis is controlled and how HTLV-1 infection affects this control are not clear. We hypothesize that HTLV-1 leads to dissemination of Strongyloides stercoralis larval antigen-specific cytokine responses in strongyloidiasis patients with or without HTLV-1 co-infection.To measure peripheral blood T regulatory cells and Strongyloides larval antigens. Supernatants were analyzed for IL-5 production.Peripheral blood mononuclear cells (PBMCs) were isolated from newly diagnosed strongyloidiasis patients with or without HTLV-1 co-infection. Regulatory T cells were characterized by flow cytometry using intracellular staining for CD4, CD25 and FoxP3. PBMCs were also cultured with and without Strongyloides co-infection had higher parasite burdens. Eosinophil counts were decreased in the HTLV-1 and Strongyloides co-infected subjects compared to strongyloidiasis-only patients . The proportion of regulatory T cells was increased in HTLV-1 positive subjects co-infected with strongyloidiasis compared to patients with only strongyloidiasis or asymptomatic HTLV-1 carriers . Strongyloides antigen-specific IL-5 responses were reduced in strongyloidiasis/HTLV-1 co-infected patients . Reduced IL-5 responses and eosinophil counts were inversely correlated to the number of CD4+CD25+FoxP3+ cells.Patients with HTLV-1 and Strongyloides stercoralis co-infection and correlate with both low circulating eosinophil counts and reduced antigen-driven IL-5 production. These findings suggest a role for regulatory T cells in susceptibility to Strongyloides hyperinfection.Regulatory T cell counts are increased in patients with HTLV-1 and  Strongyloides stercoralis by augmenting regulatory T cell numbers, which in turn down regulate the immune response to the parasite. In our study, patients with HTLV-1 and Strongyloides co-infection had higher parasite burdens than patients with only strongyloidiasis. Eosinophils play an essential role in control of strongyloidiasis in animal models, and eosinophil counts were decreased in the HTLV-1 and Strongyloides stercoralis co-infected subjects compared to patients with only strongyloidiasis. The proportion of T cells with a regulatory cell phenotype was increased in HTLV-1 positive subjects co-infected with strongyloidiasis compared to patients with only strongyloidiasis. IL-5 is a key host molecule in stimulating eosinophil production and activation, and Strongyloides stercoralis antigen-specific IL-5 responses were reduced in strongyloidiasis/HTLV-1 co-infected patients. Reduced IL-5 responses and eosinophil counts were inversely correlated to the number of regulatory T cells. These findings suggest a role for regulatory T cells in susceptibility to Strongyloides hyperinfection.Human strongyloidiasis varies from a mild, controlled infection to a severe frequently fatal disseminated infection depending on the hosts. Patients infected with the retrovirus HTLV-1 have more frequent and more severe forms of strongyloidiasis. It is not clear how human strongyloidiasis is controlled by the immune system and how HTLV-1 infection affects this control. We hypothesize that HTLV-1 leads to dissemination of  Strongyloides stercoralis infects 60 million individuals in tropical and subtropical areas of the world Strongyloides hyperinfection has a high fatality rate S. stercoralis, because the parasite cannot complete the lifecycle in mice Animal models have only provided limited understanding of how the host controls S. stercoralis infection S. stercoralisThere are few studies on the human immune response to S. stercoralis and HTLV-1 frequently develop S. stercoralis hyperinfection HTLV-1 is another human retrovirus endemic in Japan, Africa, the Caribbean and South America Leishmania major infection, regulatory T cells prevent complete elimination of the parasite, yet parasite persistence is required for maintaining the protective immune response S stercoralis infected patients. We hypothesize that HTLV-I leads to dissemination of Strongyloides stercoralis by augmenting regulatory T cells (Treg), which in turn down regulate the immune response against this parasite, allowing for the hyperinfection syndrome.Regulatory T cells are increasingly recognized as playing a key role in reducing injurious host inflammatory and immune responses S. stercoralis in stool were tested for HTLV-1 infection by Enzyme-Linked Immuno Sorbent Assay  with confirmatory western blot analysis . Consenting patients underwent further clinical and laboratory evaluation, including complete blood count (CBC) with differential, flow cytometry analysis and immunological studies of peripheral blood mononuclear cells (PBMCs). Demographic and symptoms data were obtained from patient interviews and reviews of clinical records. Stool and blood samples were collected at the outpatient clinic at the time of enrollment. In addition, asymptomatic HTLV-1 infected patients and healthy HTLV-1 negative subjects were enrolled as controls.The Instituto de Medicina Tropical ‘Alexander von Humboldt’ at the Universidad Peruana Cayetano Heredia (IMT AvH - UPCH) in Lima, Peru is a national referral center for the study of HTLV-1 and its associated diseases. Patients with strongyloidiasis are routinely tested for HTLV-1 infection. Between November 2005 and August 2007, all newly diagnosed subjects with strongyloidiasis were invited to participate. Strongyloidiasis diagnosis was confirmed by stool examination. All those with All participants signed a written informed consent form prior to enrollment in the study. The Institutional Review Board  of the Universidad Peruana Cayetano Heredia in Lima, Perú approved the study protocol and consent forms.S. stercoralis using the Baermann technique modified by Lumbreras S. stercoralis parasite load was reported in a semi-quantitative scale as negative, 1+, 2+, 3+ or 4+ according to the number of larvae observed under microscopy by personnel who were blinded to the HTLV-1 status of the participant.Routine stool evaluation for ova and parasites included direct examination, and spontaneous and fast sedimentation techniques. Stools were examined for PBMCs were isolated from heparinized blood by density gradient centrifugation . Regulatory cells were defined by staining for CD4, CD25, and FoxP3. PBMCs were first stained using Cy-5 conjugated anti-CD4 and phycoerythrine (PE)-conjugated anti-CD25 monoclonal antibodies . After fixing and permeabilizing, the cells were then stained for intracellular FoxP3 using a fluorescein-isothiocyanate (FITC)-conjugated anti-FoxP3 monoclonal antibody . Cells were analyzed using a FACScalibur flow cytometer . Regulatory T cells were identified as CD25+ and FoxP3+ cells among CD4+ cells within the lymphocyte gate. Absolute CD4+ cell counts were performed using a 4 color single platform staining of whole blood cells . Flow cytometry analysis used FlowJo software .S. stercoralis third-stage larvae (L3) were obtained and cleaned as previously described 2) for 72 hours in the presence or absence of 2-ug/ml crude infective stage S. stercoralis larvae (L3) antigen. PBMCs were adjusted to a final concentration of 1×106 cells/ml. Supernatants were collected and stored at −80°C until cytokine analysis.Interleukin 5 (IL-5) was measured by enzyme-linked immuno sorbent assay on the cell culture supernatant following manufacturer's instructions .HTLV-1 proviral load in PBMCs was quantified in the strongyloidiasis and HTLV-1 co-infected patients. HTLV-1 proviral load was quantified using a real-time SYBR Green PCR method, as described previously We compared symptoms and parasite load between patients with and without HTLV-1 by Chi square test. Blood results including regulatory T cell numbers and proportions were compared by ANOVA. The IL-5 responses to antigen stimulation were compared by Mann-Whitney U test. The correlation between the IL-5 responses and regulatory T cells were compared by Spearman's rank test.S. stercoralis. Among them, 3 were HIV positive and excluded from further analysis. Of the 40 remaining S. stercoralis patients, 27 were infected with S. stercoralis alone, and 13 were co-infected with S. stercoralis and HTLV-1. Controls included 17 healthy subjects and 9 patients with HTLV-1 with stools negative for S. stercoralis.A total of 69 subjects were enrolled in the study. Forty-three patients were identified as infected with S. stercoralis alone (45.0 vs. 39.0 years. p = 0.36). Gender distribution was similar for both groups, with 55% male and 45% female. Patients were originally from Andean, Coastal, and Jungle regions in decreasing order of frequency for both groups. Most patients had visited the jungle within three months of onset of gastrointestinal symptoms.Demographic data are presented in S. stercoralis groups, with over 50% of patients affected. Patients with S. stercoralis and HTLV-1 co-infection reported more symptoms than those only infected with S. stercoralis  was noted in 10 (83%) of twelve of the co-infected patients. Conversely, patients with just S. stercoralis had a low burden of disease (84% were 1+ or 2+) , Trichuris trichiura (10%), hookworms (8%), Giardia lamblia (5%), and Ascaris lumbricoides (2.5%).S. stercoralis/HTLV-1 co-infected group compared to HTLV-1 asymptomatic carriers and S. stercoralis-only patients . Total white blood cell counts and neutrophil counts did not differ significantly among the groups. HTLV-1 asymtpmatic carriers did not show increased eosinophil numbers. Among S. stercoralis infected subjects there was a trend towards lower median eosinophils in patients co-infected with HTLV-1  was significantly increased in the HTLV-1 co-infected patients (median proportion of CD4+ T-cells that were CD25+FoxP3+: 17.9%) when compared to all other groups . These patients had a significantly higher mean proviral load than did asymptomatic carriers  and were similar to HTLV-1-associated myelopathy patients , as reported elsewhere Proviral load data were available for 9 of the 13 HTLV-1 co-infected patients . We also performed an analysis using CD127 expression (low or negative) to define regulatory cells. In all of these analyses, the results were similar. Taken together, these data strongly suggest that Tregs may suppress antigen driven function of T cells, which influences the function of eosinophils.We reasoned that regulatory T cells might be responsible for the reduced IL-5 and eosinophil responses in S. stercoralis co-infection or as a response to the proinflammatory effects of these infections. Further studies are needed to determine whether the expansion is in HTLV-1 infection of the Treg cells.Pro-viral loads were also elevated in the strongyloidiasis and HTLV-1 co-infected patients, similar to levels increased for other HTLV-1 associated diseases like HTLV-1 associated myelopathy. This suggests that this expanded regulatory T-cell population may be induced by HTLV-1-S. stercoralis and HTLV-1 co-infection do not control S. stercoralis infection as well as patients with S. stercoralis only as indicated by the increase in the number of larvae in stool samples. This defect in immune host response is associated with lower eosinophil counts and decreased antigen-driven production of IL-5. This reduced response is inversely correlated with the proportion of CD4 cells, which are CD4+CD25+FoxP3+, suggesting a role for these cells in blunting antigen-driven protective responses. Additional data is needed to determine whether regulatory T cells are increased because they are HTLV-1 infected or as a response to parasite infection, and to explore mechanisms for suppression of eosinophil counts and function.In summary, we have demonstrated that patients with


A fast, specific, accurate and precise reverse phase high performance liquid chromatographic method was developed for the simultaneous determination of butenafine hydrochloride and betamethasone in cream formulation. The determination was carried out on licrocart licrosphere RP-select B  column in isocratic mode, the mobile phase consisting of 50 mM ammonium acetate buffer and acetonitrile in the ratio of 60:40, adjusted to pH 4.5 ± 0.1 with glacial acetic acid. The flow rate was 2.0 ml/min and eluent was monitored at 254 nm. The retention times of butenafine hydrochloride and betamethasone were 4.70 min and 7.76 min, respectively, and the resolution factor was greater than 4.0. Linearity of butenafine hydrochloride and betamethasone were in the range of 100-300 μg/ml and 5-15 μg/ml, respectively. The proposed method is also found to be precise and robust for the simultaneous determination of butenafine hydrochloride and betamethasone in cream formulation. Earlier days the practices of curing the skin diseases with single drug molecule for prolong period, this general practice leads to develop drug resistance for bacterial and fungal strains. Now the practice has been changed and combination of drug molecules implies for simultaneous mechanism of action, this leads to application of drugs for short period, which will exhibit fast curing and minimum development of drug resistance to the microbial strains. There is a simultaneous requirement of fast, potential analytical methods to determine the drugs in the given formulation.The past decade has witnessed a significant increase in the prevalence of resistance to antibacterial and antifungal agents. Resistance to antimicrobial agents has important implications for morbidity, mortality and health care. Antifungal can be grouped into three classes based on their site of action. Azoles,23H27 N.HCl and a molecular weight of 353.93. It inhibits squalene epoxidase, an enzyme which converts squalene to lanosterol and leads to accumulation of squalene and is primarily fungicidal against dermatophytes. The azoles antifungal have been extensively used to treat dermatophytosis since the 1970s. The most commonly used topical agents are clotrimazole and miconazole. The azoles act as fungi-static agents by inhibiting the cytochrome P450-dependent enzyme lanosterol 14-demethylase, which is important for the biosynthesis of ergosterol, a component of fungal cell membrane. It is freely soluble in methanol, ethanol and chloroform and slightly soluble in water.Butenafine hydrochloride  is a new benzylamine derivative with a chemical structure and mode of action similar to allylamine antifungal. It has the empirical formula C28H37FO7 and a molecular weight of 504.6. Betamethasone dipropionate is a synthetic adrenocorticosteroid, for dermatologic use. Betamethasone, an analog of prednisolone has high corticosteroid activity and slight mineralocorticoid activity. Betamethasone dipropionate is a white or almost white, crystalline powder, practically insoluble in water, freely soluble in acetone and in methylene chloride, sparingly soluble in alcohol.Betamethasone dipropionate is designated chemically as 9-fluoro-11b-hydroxy-16b-methyl-3,20 dioxopregna-1,4-diene-17,21-diyl dipropionate. It has the empirical formula CThe developed cream formulation contained 1% butenafine hydrochloride synthetic antifungal agent and 0.05% synthetic corticosteroid betamethasone as betamethasone dipropionate is member of the class of steroids. A survey of literature revealed that few chromatographic and spectrophotometric methods are reported for determination of butenafine hydrochloride and betamethasone individually–7. HowevThe drug sample, butenafine hydrochloride was procured from Hetero Drugs Limited, Hyderabad, India, betamethasone dipropionate and cream sample were used in-house. HPLC grade acetonitrile, ammonium acetate and glacial acetic acid were purchased from Merck India Limited, Mumbai, India.An isocratic high performance liquid chromatography (Jasco system) with intelligent sampler (AS-2057), quaternary gradient pump (PU-2080), intelligent UV/Vis detector (UV-2075), intelligent column oven (CO-2065) was used. The chromatography column used was licrocart licrosphere RP-select B  column.A mixture of 50 mM ammonium acetate buffer (adjusted pH to 4.5 with ten percent solution of acetic acid) and acetonitrile in the ratio 60:40 v/v was filtered through 0.45 μ membrane filter and used as mobile phase. The flow rate of mobile phase was maintained at 2.0 ml/min. For calibration, standard butenafine hydrochloride and betamethasone solutions were prepared at concentration range of 100-300 μg/ml for butenafine hydrochloride and 5-15 μg/ml for betamethasone in mobile phase. These standard solutions were injected in duplicate and the average detector response measured at 254 nm.In-house research and development samples  two tubes each 5 g cream sample was taken in 100 ml beaker and mixed properly. An accurately weighed quantity of cream equivalent to 20 mg of butenafine hydrochloride and 1 mg of betamethasone was taken in 100 ml volumetric flask and dissolved in mobile phase. Volume was made up to mark with mobile phase. Whole solution was transferred in 250 ml volumetric flask and chilled it in ice bath for 10 min. The solution was filtered through Whatman filter paper No. 40. The aliquot portion of the filtrate was further diluted to get final concentration and detection was done at 254 nm. The results obtained in the experiment were tabulated in The method was validated for accuracy, precision, specificity and robustness . The accLinearity experiment was performed by using definite concentrations of butenafine hydrochloride  and betamethasone  solutions were injected and peak areas were recorded. A linear graph was plotted by using the peak areas against concentration in μg/ml.The instrument precision was evaluated by injecting the butenafine hydrochloride (200 μg/ml) and betamethasone (10 μg/ml) solution six times repeatedly and peak areas were measured. The results are reported in terms of relative standard deviation. The intra-day and inter-day precision study of butenafine hydrochloride and betamethasone was carried out by estimating the corresponding responses six times on the same day and six times on the second day. The results obtained were reported in terms of relative standard deviation (RSD). The specificity was estimated by spiking known quantity of drug in to placebo preparation. The chromatography was performed by using appropriate dilutions and quantities of drugs were estimated. Robustness of the method was studied by deliberately changing the experimental conditions like flow rate, percentage of organic phase. Stability of the standard and sample solutions were observed at 25+2° for 24 h. The sample solution was assayed at every 6 h intervals up to 24 h.Optimization of mobile phase was performed based on resolution, total runtime, asymmetric factor and theoretical plates obtained for butenafine hydrochloride and betamethasone. Mobile phase consisting of 50 mM ammonium acetate buffer and acetonitrile in the ratio of 60:40, adjusted pH 4.5±0.1 with glacial acetic acid was selected which gave sharp, well resolved peaks for butenafine hydrochloride and betamethasone . The retThe accuracy of the method was determined by calculating recoveries of butenafine hydrochloride and betamethasone by placebo spiked method. The recoveries obtained were 99.26-101.98 % for butenafine hydrochloride and 98.02-101.88 % for betamethasone. The high values indicate that the method is accurate. Instrument precision was determined by performing injection repeatability test for standard solution, and the RSD values for butenafine hydrochloride and betamethasone were found to be 1.94 and 1.99, respectively.For inter-day study RSD values were found to be 0.81, 0.66 for butenafine hydrochloride and betamethasone respectively. The low RSD values indicate that the method is precise. Robustness of the method was studied by changing the flow rate of the mobile phase from 2.0 ml/min to 1.8 ml/min and 2.2 ml/min. Using 1.8 ml/min flow rate, retention time for butenafine hydrochloride and betamethasone were observed to be 5.2 and 8.5 min, respectively and with 2.2 ml/min flow rate retention time for butenafine hydrochloride and betamethasone were observed to be 4.2 and 6.9 min, respectively without affecting the resolution of the drugs. When mobile phase composition was changed to increase in 2% organic phase, the retention time for butenafine hydrochloride and betamethasone were observed to be 3.9 and 5.9 min, respectively and decrease in 2% organic phase, the retention time for butenafine hydrochloride and betamethasone were observed to be 5.8 and 8.2 min, respectively. The solution stability study revealed that butenafine hydrochloride and betamethasone solutions were stable for 24 h without detectable degradation and the percentage recovery of both the drugs were found to be more than 98.02%.


HTLV-I is the causal agent of adult T cell leukemia (ATLL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Biomarkers are needed to diagnose and/or predict patients who are at risk for HAM/TSP or ATLL. Therefore, we investigated using luciferase immunoprecipitation technology (LIPS) antibody responses to seven HTLV-I proteins in non-infected controls, asymptomatic HTLV-I-carriers, ATLL and HAM/TSP sera samples. Antibody profiles were correlated with viral load and examined in longitudinal samples.P < 0.0001) and ATLL patients (P < 0.0005). Anti-Env antibody titers above 100,000 LU had 75% positive predictive value and 79% negative predictive value for identifying the HAM/TSP sub-type. Anti-Tax antibody titers were also higher (P < 0.0005) in the HAM/TSP compared to the asymptomatic HTLV-I carriers. Proviral load correlated with anti-Env antibodies in asymptomatic carriers (R = 0.76), but not in HAM/TSP.Anti-GAG antibody titers detected by LIPS differentiated HTLV-infected subjects from uninfected controls with 100% sensitivity and 100% specificity, but did not differ between HTLV-I infected subgroups. However, anti-Env antibody titers were over 4-fold higher in HAM/TSP compared to both asymptomatic HTLV-I (These studies indicate that anti-HTLV-I antibody responses detected by LIPS are useful for diagnosis and suggest that elevated anti-Env antibodies are a common feature found in HAM/TSP patients. Human T lymphotropic virus type I (HTLV-I) is a retrovirus that infects 20 million people worldwide . HTLV-I in vitro [HAM/TSP is a chronic progressive neurodegenerative disorder that involves demyelination of the spinal cord and is characterized by CNS perivascular infiltration of inflammatory cells . Epidemiin vitro -19. HAM/in vitro -22. On tin vitro  and showin vitro .Although there are adequate methods for determining if people are infected with HTLV-I, there are no serological diagnostic tests available for discriminating asymptomatic carriers from HAM/TSP patients or ATLL patients. Currently HTLV-I diagnosis is performed by immunoassays for HTLV-I gene products, HTLV-I-specific antibody production, detection of HTLV-I DNA, Southern blotting for ATLL diagnosis and more recently, proteomic approaches . The abiCurrently, most immunoassays measuring anti-HTLV-I antibodies do not quantitatively evaluate multiple antigens and are incapable of detecting conformational epitopes in these antigens. We recently developed a highly sensitive immunoprecipitation technology called Luciferase Immunoprecipitation System (LIPS) that utilizes mammalian cell-produced, recombinant fusion protein antigens for efficiently evaluating antibody responses to multiple viral proteins and even a full virus proteome . Here, LSera samples analyzed in this study were derived from 115 well-characterized participants including healthy volunteers, asymptomatic HTLV carriers, ATLL, and HAM/TSP patients. The gender, race/ethnic group and mean age of sample acquisition are summarized in Table P < 0.0001) from the mean value of 143,250 ± 56,169 LU for the confirmed 73 HTLV-I infected samples including the asymptomatic carriers, ATLL and HAM/TSP samples. Using a value of 875 LU as a cut-off derived from the mean plus 5 SD of the controls revealed 100% sensitivity (73/73) and 100% specificity in detecting positive anti-Gag antibodies in the known HTLV-I positive samples. Some of weak background signals seen in control samples were below the cut-off value and were dramatically lower than any of the HTLV-I positive samples. While the anti-Gag antibody was highly useful for the diagnosis of HTLV-I infection, the mean anti-Gag antibody titers were not statistically different between the 15 asymptomatic HTLV-I carriers, 18 ATLL and 40 HAM/TSP HTLV-I positive patient groups . A cut-off of the mean plus 5 SD of the control subjects revealed 85% sensitivity (62/73) and 100% specificity in detecting positive anti-Env antibodies in the HTLV-I positive samples. The anti-Env antibody responses were less useful than the anti-GAG antibody titers to discriminate HTLV-I infected from uninfected controls and this is reflected by the slightly lower area under the curve (AUC) value of 0.95 for anti-Env antibodies verses 1.00 for the anti-Gag antibody test as determined by analyzing receiver-operating characteristics (ROC).Anti-Env antibodies were also evaluated in the 115 sera samples Figure . While tP < 0.0001) and ATLL patients (P < 0.0005). In contrast, the anti-Env antibody titers were not statistically different between the asymptomatic HTLV-I-infected and ATLL patients (P = 0.1782).As shown in Figure Inspection of patient characteristics did not reveal any obvious differences that explained the 4 ATLL and 4 asymptomatic HTLV-I-infected outliers with relatively high anti-Env antibody titers Figure . For exaP < 0.0001). Using a cut-off of the mean plus 5 SD of the control subjects revealed 85% sensitivity (14/15) in detecting positive anti-Tax antibodies in the HTLV-I asymptomatic carriers and 98% sensitivity (39/40) in detecting positive anti-Tax antibodies in the HAM/TSP samples. In the case of the ATLL samples, 100% (18/18) were found to have anti-Tax positive titers.HTLV-I Tax is a major regulator of gene expression in the human host and several studies have suggested anti-Tax antibodies are involved in the pathogenesis of HAM/TSP ,28. UsinP < 0.0001). In contrast, no statistical difference (P = 0.1815) was found between the mean anti-Tax antibody titer of the HAM/TSP and ATLL samples.Inspection of the mean anti-Tax antibodies among the different HTLV-I infected groups showed that the 40 HAM/TSP patients had a mean anti-Tax antibody titer of 518,849 LU that was close to twice the mean value of 282,900 LU for the asymptomatic HTLV-I carriers. This elevated anti-Tax antibody in the HAM/TSP patients compared to the asymptomatic HTLV-I carriers was highly statistically significant . Comparison by regression analysis of the HTLV-I proviral load with the HTLV-I antibody titer data revealed that there was no significant correlation between the different antibody titers determined by LIPS and the proviral load in the HAM/TSP patients. For example as shown in Figure R = 0.18, P = 0.345). In contrast, anti-Env antibody titers from asymptomatic HTLV-I carriers markedly correlated  with the HTLV-I proviral load  from a number of selected patients were also analyzed for anti-Gag, anti-Env, and-Tax antibodies. Representative results from several of these patients are shown in Figure Several of the HTLV-I antigens showed markedly higher antibody titers in HAM/TSP patients compared to ATLL and asymptomatic HTLV-I carriers. Therefore, ROC analysis was used to evaluate the most useful antibody markers. The anti-Env antibody titer showed the best discriminative power for identifying patients with HAM/TSP versus asymptomatic HTLV-I carriers and ATLL, as shown by the large AUC value of 0.83 from the ROC curve. Anti-Tax and anti-Gag antibodies were still informative but had lower AUC values of 0.72 and 0.61, respectively (data not shown). When a cutoff threshold greater than 100,000 LU was applied to the anti-Env antibody titers, the sensitivity and specificity for identifying HAM/TSP patients versus ATLL and asymptomatic HTLV-I carriers were 81% and 72%, respectively . Of the 7 antigens tested for diagnosing HTLV-I infection, the anti-Gag antibody test was the most informative, achieving 100% sensitivity and 100% specificity. The LIPS assay likely detects more conformational epitopes than alternative immunoassay formats providing high sensitivity, high specificity and robust signals that provide a substantial and clear distinction between positive and negative HTLV-I sera. Additionally, the high-throughput format used here makes this approach highly feasible for screening large numbers of sera samples.R = 0.76; P = .003). These results suggest that anti-Env antibody titers detected by LIPS may have utility in monitoring HTLV-I proviral load in asymptomatic individuals. This correlation between anti-ENV antibody titers and proviral load was unique to HTLV-I seropositive asymptomatic patients and consistent with previous studies demonstrating HTLV-I proviral load associations with anti-HTLV-I antibodies [From our LIPS studies profiling 7 different HTLV-I antigens, anti-Env antibody titers significantly correlated with the proviral load in asymptomatic HTLV-I seropositive samples  and HAM/TSP patients (diagnosed according to WHO guidelines) evaluated under Institutional Review Board-approved protocols of the NCI and NINDS, National Institutes of Health . The gender, race/ethnic group and mean age of sample acquisition are summarized in Table Renilla luciferase (Ruc) expression vector, was used to generate all plasmids [pREN2, a mammalian plasmids . HTLV-I plasmids ,36,37. I9 light units (LU) of Ruc-Gag protein per 100 mm2 plate of Cos1 cells . The LIPS immunoprecipitation assay was performed in a 96-well plate format at room temperature as described [2, 1% Triton X-100) in a 96-well polypropylene microtiter plate. For evaluating antibody titers by LIPS, 40 μl of buffer A, 10 μl of diluted human sera , and 50 μl of the equivalent of 1 × 107 light units (LU) of Ruc-antigen Cos1 cell extract, diluted in buffer A, were added to each well of a polypropylene plate and incubated for 1 hour at room temperature. Next, 7 μl of a 30% suspension of Ultralink protein A/G beads  in PBS were added to the bottom of each well of a 96-well filter HTS plate . To this filter plate, the 100-μl antigen-antibody reaction mixture was transferred and incubated for 1 hour at room temperature on a rotary shaker. The washing steps of the retained protein A/G beads were performed on a BioMek FX work station  using an integrated vacuum manifold. After the final wash, LU were measured in a Berthold LB 960 Centro microplate luminometer  using coelenterazine substrate mix . All LU data were obtained from the average of at least two independent experiments and corrected for background by subtracting the LU values of beads incubated with Cos1 cell extract, but without sera.Following transfection of mammalian expression vectors, crude protein extracts were obtained as described . For exaescribed . First, 6 cells using Puregene DNA Isolation Kit , and 100 ng of the sample DNA solution was analyzed by this system. The HTLV-I proviral DNA load was calculated by the following formula: copy number of HTLV-I (pX) per 100 cells = (copy number of pX)/(copy number of β-actin/2) × 100.Real-time PCR analysis of HTLV-I (Tax) proviral load was performed as previously described . DNA wasP < 0.05. For the calculation of sensitivity and specificity between HTLV-I infected and uninfected samples, the cut-off limit for each antigen was derived from the mean value of the 25 control samples plus 5 standard deviations. The sensitivity, specificity, negative predictive value, positive predictive value and diagnostic odds ratio were calculated by using 2 × 2 contingency tables and calculated using the GraphPad software. The multivariate data which included antibody titers and clinical covariates was also evaluated using the RapidMiner  suite of data mining tools.The GraphPad Prism software  was used for other statistical analysis, including evaluating test performance by area under the curve (AUC). Results for qualitative antibody titers between the controls, seroindeterminates, asymptomatic HTLV-I carriers, HAM/TSP and ATLL are reported as the mean ± SD. Mann-Whitney U tests were used for comparison of antibody titers in different groups. Statistical significance of the regression analysis was evaluated by Pearson correlation coefficient and the level of significance was set at The authors declare that they have no competing interests.FK initially conceived of the study. FK and RM provided a pilot set of sera for proof of concept. PB generated the needed constructs. PB and HL analyzed the sera by LIPS. SJ, EM, JG, KY, UO, and JJ provided the sera samples used in this study, participated in the design of the experiments and analyzed the data. PB analyzed the data and drafted the manuscript. MI funded the study. All authors read and approved the manuscript.


Giardia duodenalis is a ubiquitous protozoan parasite that has emerged as a significant opportunistic human pathogen. G. duodenalis may have a deleterious effect on animal growth and performance, therefore its potential as a production limiting organism should not be discounted. We therefore undertook this study to determine management and environmental factors in feedlots that influence the prevalence and environmental load of G. duodenalis cysts in fecal material deposited by feedlot cattle in the central and western United States.Giardia duodenalis cysts were identified and counted using direct immunofluorescent microscopy. The estimated overall point prevalence of G. duodenalis was 19.1%, representing feedlots from a wide range of climates and management systems. Pen-level prevalence varied from 0 to 63.3%, with pen-level shedding estimates ranging from 0 to 261,000 cysts/g feces. Higher environmental temperatures, increased animal density, and increased time in the feedlot were associated with a lower prevalence of G. duodenalis. Removing manure before placing a new group of cattle in a pen was associated with a decreased prevalence of G. duodenalis in fecal pats. Using coccidiostats as a feed additive was associated with a higher prevalence of Giardia.Twenty two feedlots from 7 states were included in the study, and up to 240 fecal samples were collected from pen floors of up to 6 pens per feedlot. G. duodenalis in their feces and therefore potentially limit contamination of their environment.Management practices could be employed that would limit the probability that feedlot cattle shed  Giardia duodenalis is a ubiquitous protozoan parasite that has emerged as a significant opportunistic human pathogen. At least seven distinct assemblages (A - G) of G. duodenalis have been identified [Giardia infections are usually self-limiting in people with normal immune systems, but can be severe in immuno-compromised individuals [G. duodenalis cysts [entified  however entified . Giardiaividuals -5. Sinceis cysts  it is imG. duodenalis infections in ruminants have been attributed to either assemblage A mono-infection [G. duodenalis infection in dairy cattle had been studied because the confinement practices of dairies has been linked with increased G. duodenalis infection and reinfection rates of calves [Giardia cysts were found in agricultural run-off at an increased concentration during peak calving time [Giardia in cattle coming from pastures into a confinement operation [G. duodenalis cysts than adult cattle. In particular, previous studies have found that the prevalence of G. duodenalis shedding for beef and dairy calves up to 9 months ranges from 17-55% and for adult beef and dairy cattle ranges from 0-17% [G. duodenalis is associated with clinical disease [G. duodenalis can have a deleterious effect on animal growth and performance [While cattle have been found to be primarily infected with the livestock-specific assemblage E genotype, a portion of nfection , to mixenfection ,9, or tonfection . This ranfection ,3. G. duf calves ,12. The ing time . Furtherperation . Young com 0-17% ,15-19. I disease ,12 and s disease . An expeformance , therefoG. duodenalis per animal unit or mass of fecal material is conditional on the underlying age distribution of the herd [Giardia infection was not associated with factors such as calf management, confinement, manure accumulation, farm hygiene, or duration of the calving season [Much of the variation in reports of prevalence is likely due to the differences in populations of cattle studied, which include the type of operation (beef versus dairy), age distribution of cattle, and variation in management practices . It has the herd -17. Otheg season ,19,23.G. duodenalis in cattle in commercial feedlots. Large numbers of cattle are concentrated in feedlots across the United States, leading to high-volume point sources of fecal waste. In order to develop management plans to reduce the potential for waterborne transmission of G. duodenalis, a valid estimate of the prevalence of infection and number of cysts present in the fecal material deposited by feedlot cattle is required. Therefore, we undertook this study to determine management and environmental factors in feedlots that influence the prevalence and environmental load of G. duodenalis cysts in fecal material deposited by feedlot cattle in the central and western United States.Presently, little is known about the prevalence of One to 4 feedlots from selected states that were deemed by collaborators to be representative of feeding operations in those states were identified for inclusion in the study. Factors considered included location, animal capacity, type of cattle fed, and management system. A total of 22 feedlots in 7 states  were eventually enrolled. Feedlots in different states were sampled in sequence so that fecal samples were collected from feedlots with a variety of animal husbandry practices both within and across states and across different climatic conditions and seasons. Approximately 240 fecal samples were collected at each feedlot, from between 2 and 6 pens of cattle. Samples were collected from a randomly selected pen of cattle that had recently arrived to the feedlot, a randomly selected pen of cattle nearing their harvest date, and the remaining pens were randomly selected from across the feedlot. One-hundred-gram fecal samples were collected from freshly-deposited fecal pats, placed on ice packs, and shipped by overnight courier to the Veterinary Medicine Teaching and Research Center, Tulare, CA within 24 hours of collection. A questionnaire related to feedlot and pen management was administered to the feedlot manager at the time of fecal sampling. The following data was recorded: number of animals in the pen, temperature and precipitation during the previous month, animal class , water source, pen size, days in feedlot, coccidiostats in feed, days between lots of cattle, pen cleaning/scraping frequency, presence of a cover for the cattle, percent concentrate in feed, primary concentrate used, source of cattle , and water/feed trough cleaning frequency and method.G. duodenalis cysts as previously described [Cryptosporidium/Giardia detection kit, Meridian Diagnostic, Inc., Cincinnati, OH). The entire slide was scanned at ×200 magnification. The total number of G. duodenalis cysts was recorded for each smear.Direct immunofluorescent microscopy (DFA) was used for detecting and enumerating escribed . BrieflyG. duodenalis and added purified wild-type dairy calf G. duodenalis cysts to a final concentration of either 100, 1000, or 10,000 oocysts/L. Three replicates per concentration per fecal sample were used for a total of 26 fecal samples. The immunofluorescent microscopy procedure was then performed as described above. Percent recovery was estimated by fitting a negative binomial regression model to the observed number of oocysts, with the number of spiked or expected oocysts functioning as the offset variable and fecal identification as a cluster variable due to using three replicates per concentration per fecal sample [G. duodenalis cysts using the immunofluorescent microscopy procedure was 36.2% . The estimated concentration of G. duodenalis was therefore calculated by dividing the number of cysts observed by the average amount of fecal material on each slide (0.0117 grams) multiplied by 0.362, the estimated cyst recovery rate.To determine percent recovery for immunofluorescent microscopy, we collected three fecal samples from beef feedlot cattle that tested negative for l sample . The estG. duodenalis was calculated by type of feedlot pen, management practices, and climate factors. Pen order was categorized into three groups; entry pens (nutrition based primarily on forages), middle pens (feedlot diets transition from high roughage to high concentrates), and exit pens (diets containing a high proportion of concentrates). The concentration of cysts in each positive sample was placed into categories of fecal cyst load. The prevalence of G. duodenalis positive samples was calculated for each category of fecal cyst load by pen order. Differences in proportion G. duodenalis positive between groups were assessed using a univariate logistic regression.The prevalence of ® for Windows, Cytel Software Corporation, Cambridge, MA) to identify cattle characteristics , farm management practices, and climate factors associated with the presence of G. duodenalis cysts in fecal samples from feedlot cattle. Descriptive statistics were performed to determine variable ranges, the extent of outliers, and to check for normality of continuous variables. All potential risk factors were initially screened for their association with the presence of G. duodenalis cysts using univariate logistic regression. Variables with a Pearson's chi-square statistic of p ≤ 0.25 were offered entry into the model building process. Two-way interaction variables were created from this list and offered for entry. A forward stepping approach was used to develop the logistic regression model, with p ≤ 0.05 as determined by a likelihood ratio test set as the criterion for inclusion of the variable in the final model. The variable "pen" was included as a random effect term in the final model because fecal samples from the same pen were not considered to be independent.A logistic regression model was created using a commercial software program ; prevalence within pens varied from 0% to 63.3%, while prevalence within feedlots varied from 5.4% to 43.6% , in mid-duration pens was 17.2% (n = 2488), and in pens nearing harvest was 15.5% (n = 1371). The prevalence for entry pens was significantly higher than in middle and exit pens (p < 0.001).A total of 5,260 fecal samples were collected from 22 feedlots in California, Colorado, Nebraska, Oklahoma, South Dakota, Texas, and Washington. The overall proportion of fecal samples containing detectable G. duodenalis by pen type is shown  were estimated to have more than 106 G. duodenalis cysts per gram of feces. These fecal samples were from 6 different feedlots; four of these high intensity fecal samples were from the same pen of exit cattle.The distribution of fecal concentration of n Figure . There wG. duodenalis was inversely associated with temperature; prevalence was greatest in feedlots which had had the coldest temperatures in the past month, and least in feedlots which had had the hottest temperatures in the past month , fed corn as their primary concentrate, had no cover for the cattle, and used wells as their primary water source; these variables are not included in the logistic regression model due to a lack of variation. The final logistic regression model included number of days in the feedlot, average temperature during the previous month, animal density within pens, whether manure was removed between lots of cattle, and whether a coccidiostat was included in the ration Table . SeveralG. duodenalis by the number of days in the feedlot and by temperature is shown in Figures Giardia prevalence, that feeding a coccidiostat increases the prevalence over not feeding a coccidiostat, and that manure removal prior to placing cattle in a pen is associated with reduced prevalence of infection.The predicted prevalence of G. duodenalis was 19.1%, with 26.1% of samples from newly-arrived cattle and 15.5% of samples from cattle near harvest testing positive. While the cumulative infection rate with Giardia may reach 100% in range beef calves [G. duodenalis in 6.5% and 11% of fecal samples [Giardia infection as compared to pasture settings, especially when cattle first enter the feedlot. As cattle remain in the feedlot, prevalence is reduced. A longitudinal study in calves found a cumulative prevalence of 100%, with 85% prevalence in the first month, decreasing to 21% prevalence in months 6-7 [In this sample of fecal material from feedlots in the central and western United States, the point prevalence of f calves , previou samples ,26. Thisnths 6-7 . While aG. duodenalis in pen fecal deposits; our model predicted that as temperature increased the odds of detecting Giardia cysts decreased by 2.2% per °C. This may result from the desiccation of Giardia cysts in the fecal material by the high temperatures, thereby reducing transmission among cattle within a pen and the probability that a freshly collected fecal pat would contain cysts. Bingham et al. studied the physical factors involved with excystation of Giardia and viability in varying temperatures [Giardia cysts and decreases the potential for infection of penmates. Other researchers have demonstrated that Giardia cyst viability was compromised by shearing forces generated during freeze-thaw cycles, while lower temperatures were protective of cysts [Giardia in feedlots that had an average temperature range of 1 to 10°C, which may reflect potential freeze/thaw conditions, was significantly lower than feedlots with an average temperature range below freezing (Figure Giardia positive cattle (Figure Results from this study indicate that hot climates are associated with reduced prevalence of eratures . Cysts seratures  which fuof cysts . The pree Figure . All of G. duodenalis (Table Increased pen area per animal was found to have a slight positive association with prevalence of is Table . While tEimeria spp. However, the primary reason for including most coccidiostats in feedlot rations is to improve feed to gain conversion. In this study, feeding a coccidiostat in the ration was associated with an increased prevalence of infection with G. duodenalis. These feed additives selectively inhibit certain ruminal microorganisms, thereby altering fermentation efficiency and end products available for absorption and performance [Coccidiostats, which include monensin, lasalocid, and decoquinate, are feed additives commonly incorporated into feedlot rations in order to control infection with various formance . We specG. duodenalis (Table Removing manure prior to placing a new group of cattle in a pen was found to be associated with a decreased prevalence of is Table . PhysicaCryptosporidium parvum [Giardia cysts to compare the results from this research. The current study found that feedlot cattle can shed G. duodenalis in the range of tens of millions to hundreds of millions of cysts per animal per day. Ralston et al. performed a longitudinal study that demonstrated a decline in prevalence of G. duodenalis over time and attributed the higher prevalence in young cattle entering the feedlot to a temporary relaxation of immunity from the stresses of weaning and being shipped to a feedlot [Giardia positive cattle (Figure Giardia in the entry pens, as compared with middle and exit pens. These findings may indicate that many animals stop shedding Giardia cysts as the animal ages and the duration of their stay at a feedlot lengthens.While there are reports examining the environmental load of m parvum ,23,25, t feedlot . The curGiardia "super-shedders". Researchers have noted this same phenomenon of cattle shedding high concentrations of Escherichia coli O157:H7 and Mycobacterium avium spp. paratuberculosis and have put forth the concept of super-shedders and heavy-shedders, respectively [We found a small percentage of fecal pats containing a high concentration of cysts. These cattle were found from all stages of the feeding period, which may provide evidence for the existence of ectively -35. WhilG. duodenalis which limits their role as reservoirs of giardiasis in humans [G duodenalis has any adverse clinical consequences in cattle, knowledge of prevalence and risk factors for shedding is important in order to generate management plans. While grazing on pasture may not concentrate cattle and cysts, a feedlot setting may create a sufficient concentration of fecal deposits on a limited area of land; therefore it is recommended that feedlot fecal waste management plans be implemented in order to minimize the potential for environmental contamination with this and other potential pathogens.Recent evidence indicates that cattle are most commonly infected with the non-zoonotic livestock genotype of n humans ,3,26. NeBH helped conceive the project and assisted in writing initial grant application, provided input and advice on statistical modeling, and revised the manuscript. RP assisted in developing statistical models, drafted the initial manuscript. JS performed initial descriptive statistics, and provided input into the initial manuscript. MP performed all laboratory analyses, provided editing for manuscript. EA wrote final draft of grant, supervised all sampling and data collection, assisted in statistical concepts, and edited the final manuscript. All authors read and approved the final manuscript


The aim of this biennial meeting is to promote discussion and share new findings between researchers and clinicians for the benefit of patients infected by human T-lymphotropic virus (HTLV). HTLV infects approximately 15–20 million individuals worldwide and causes a broad spectrum of diseases including neurodegeneration and leukemia. The scientific program included a breadth of HTLV research topics: epidemiology, host immune response, basic mechanisms of protein function, virology, pathogenesis, clinical aspects and treatment. Exciting new findings were presented in these different fields, and the new advances have led to novel clinical trials. Here, highlights from this conference are summarized.The "14th International Conference on Human Retrovirology: HTLV and Related Retroviruses" was held in Salvador, Bahia, from July 1 In the opening ceremony, Carlos Brites , chair of the conference, underscored the importance of research in preventing HTLV-induced diseases in Brazil as well as throughout the world. Many physicians are not aware of the consequences of HTLV infection. HTLV-1 causes two major types of diseases: adult T-cell leukemia (ATL) and HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Despite improved therapies, ATL still has a very poor prognosis and HAM/TSP has no satisfactory treatment. Graham Taylor, former president of the International Retrovirology Association, highlighted the key questions that each scientist or clinician should remember: "What do we know, what do we think to know and, ... what do our patients want us to know?". The meeting started with memorial lectures remembering three colleagues who departed us too early: John Brady, Ralph Grassmann and Bill Harrington. These three scientists were pillars of retrovirus research and made outstanding contributions to our understanding of HTLV-1 and patient care. The biennial HTLV Retrovirology prize was renamed the "Brady-Grassmann-Harrington prize"  downregulation though inhibition of LAT (Linker for Activation of T Cells) accumulation at the virological synapse. LAT is known to interact with the TCR that binds the MHC during cell conjugation with the antigen presenting cell. Therefore, p8I and p12I have opposite effects on cell proliferation. Franchini showed that p8I was transferred from the initial infected cells to the recipient T cells within minutes, and increased the adhesiveness of cells via LFA-1. The transfer occurred through nanotubes emerging from infected cells (MT-2) to uninfected Jurkat cells. p8I increased tunneling nanotube formation. Non-infected cells were labile until they were touched by these nanotubes. Whether the virus is transferred through these structures is presently unknown. Cytotoxic T cells (CTL) were also recruited by the nanotubes and were anergized by p8I. This mechanism could lead to the inhibition of cytotoxic cell killing.In the keynote lecture, Genoveffa Franchini , newly elected president of the International Retrovirology Association, focused on the role of accessory proteins in the development of HTLV pathogenesis. In addition to the classical structural, enzymatic and regulatory proteins, the HTLV-1 genome encodes a series of viral factors whose functions have been poorly understood [II [II. In the presence of Tax, p13II is stabilized and localizes to the nucleus. Franchini reported that p13II induced Tax degradation and inhibited it's transcriptional activity, thereby decreasing viral replication. In contrast, p13II was stabilized in the presence of Tax through a mechanism that involved ubiquitination. Vincenzo Ciminale  described another role of p13II that included the induction of mitochondria swelling due to insertion into the inner membrane [II induced a dose-dependent depolarization of the mitochondrial membrane and O2 consumption. Reactive oxygen species (ROS) production measured by Amplex red was increased by p13II. Expression of p13II decreased the tumor growth in mice but activated primary PBMCs. This dual regulatory function illustrates the "ROS rheostat" theory that postulates that minimal levels of ROS are required to initiate cell proliferation, but that an excess ROS level induces apoptosis.Another important accessory open reading frame (ORFII) encodes p30II  and p13IA special lecture presented by Becca Asquith  was entitled "HBZ binding to HLA class 1 determines the outcome of HTLV-1". The goal of this project is to test the hypothesis that CD8 efficiency is determined by the binding of HLA class 1 molecules. Asquith synthesized peptides spanning each of the HTLV-1 proteins and tested them for binding affinity. In this manner, she validated the epitope predictions made by an in silico computer program (METASERVER). HLA-alleles that were HTLV-protective appeared selectively to bind HBZ more efficiently when compared to other viral proteins. In addition, HLA class 1 alleles from asymptomatic HTLV carriers (AC) had a better binding to HBZ when compared to alleles from HAM/TSP individuals. Asquith suggested that HLA-binding to HBZ is associated with a reduced proviral load. The most likely mechanism is that HBZ inhibits expression of other HTLV-1 genes allowing escape from the host immune response.On the other hand, HBZ expression drives infected cell proliferation conferring a survival advantage to HBZ-expressing cells . Masao MThese recent data underscore an essential function of HBZ in HTLV-1 pathogenesis and an intricate interplay between HBZ and Tax . HTLV-2 Besides antisense-strand encoded factors, the Tax oncoprotein plays an essential role in viral replication and pathogenesis . Novel pKuan-Teh Jeang  outlined that Tax expression induced micronuclei resulting from trapping of chromosomes by nuclear envelope reformation after telophase. Jeang reported that chromosome missegregation can be seen in HTLV-cells probably due to a defect in their spindle assembly checkpoint (SAC). He discussed the two major checkpoints in mammalian cells: p53 at the G1/S junction and the SAC with its constituent mitotic arrest deficiency proteins (MAD) in the mitotic (M) phase of the cell cycle. He showed that mice knocked out (KO) for 1 allele of MAD1 (+/-) have a significantly higher probability of tumor development, and mice simultaneously with two checkpoint defects (MAD+p53) have highly increased chromosome instability. Jeang suggested that loss of two discrete checkpoints may lead to the emergence of multiple primary tumors in the same patient .In the latter part of his talk, Jeang also addressed the current inability to transform human primary somatic cells using the HTLV-1 Tax oncogene. He showed that while differentiated human cells resist transformation by Tax, human stem cells could be efficiently transformed by Tax to give rise to tumor in immune-deficient mice. He then speculated that the seeds for human cancers may not be tissue somatic cells, but may instead be the tissue stem cells. This view converges with some  of the issues currently debated regarding the "cancer stem cell" hypothesis.Tax activities are regulated by complex post-translational modifications, including phosphorylation  and ubiquitination . In the cytoplasm, Tax activated NFκB by interaction with IKKγ/NEMO. Tax also bound to optineurin, a Golgi resident protein. Journo reported that optineurin stabilized Tax ubiquitination and enhanced Tax-dependent NFκB activation. Another Tax-binding protein, Tax1BP1, an ubiquitin adaptor for A20/Itch/RNF11 is involved in the same pathway.Although increased proliferation of infected cells has clearly been observed in vitro as well as in patients, the mechanisms involved are still unknown. Tax is a major player in this process through interaction with the minichromosome maintenance protein (MCM2–7) complex . Tax interacted and co-localized with MCM proteins in T lymphocytes. Moreover, Tax facilitated MCM3 binding to chromatin and increased the number of active replication origins during the synthesis phase of the cell cycle, thereby accelerating DNA replication. Silencing of MCM3 with shRNAs abrogated Tax-stimulation of replication origins. Tax also triggered re-replication, generating cells with > 4N DNA content. Replicative lesions activated the DNA damage response pathway, as revealed by phosphorylation of H2AX in cell lines established from ATL patients. These lesions can be converted into fatal replication lesions and aberrant mitosis (mitotic catastrophe) using DNA repair inhibitors, a strategy that may be useful for the treatment of ATL.CD4+ lymphocytes and to a lesser extent CD8+ T cells are considered as the main targets of HTLV-1. During his presentation, Francis Ruscetti  demonstrated that plasmacytoid dendritic cells (pDC) were highly infected by HTLV-1 in patients. In fact, all types of DC  have been shown to be easily infected by HTLV-1 in vitro and efficiently transmit HTLV-1 to T cells . InteresKathy Jones  in collaboration with Claudine Pique  reviewed the consecutive steps of virus infection involving heparan sulfate proteoglycans (HSPG), neuropilin 1  and the glucose transporter (GLUT1) . She poiUsing EM tomography, infection of T cells has previously been shown to occur through a virological synapse . This prAndrea Kress  reported that increased levels of cAMP are present in long lived murine T cells and in ATL cell lines. In TESI cells derived from primary lymphocytes transduced with a Tax-expressing recombinant rhadinovirus vector, downregulation of Tax expression decreased the levels of cAMP. Elevated cAMP levels are due to downregulation of phosphodiesterase 3B (PDE3B) mRNA through epigenetic silencing. Whether higher levels of cAMP exert an immunosuppressive function remains an open question.Ricardo Khouri  presented that HAM/TSP cells have decreased level of SOD1, which is involved in regulation of reactive oxygen species (ROS). He found that the SOD1 inhibitor D1 synergized with IFN-α but not AZT to induce apoptosis of infected cells. SOD1 may explain the efficacy of compounds such as vitamin C. ROS also appeared to be important mediators of BLV persistence . Indeed, spontaneous expression of ROS in short term cultures inversely correlated with proviral loads.The host immune response is believed to exert a tight control over the virus that continuously attempts to replicate . SeveralThe group of Mari Kannagi  provided evidence for the suppression of HTLV expression by stromal cells through a type I IFN response. The process was reversible since viral expression in infected cells was restored by their separation from stromal cells. The mechanism was reported to involve an interferon response since (i) an antibody directed against IFNβ blocked suppression by stromal cells and (ii) HTLV expression was suppressed in wild type but not in IRF-7 KO mice.Invariant NKT (iNKT) cells were known to have anti-HTLV-1 activity . These cells, which recognize and are activated by α-galactosylceramide  at the cell surface, connect innate and adaptive immune responses. The frequencies of iNKT were shown to be reduced in PBMCs from HAM/TSP and ATL patients compared to AC subjects. In PBMC cultures from AC but not ATL, stimulation of iNKT by α Galcer decreased the number of infected cells.CD4+CD25+CCR4+ cells are a major HTLV-1 reservoir. Interferon expression by Foxp3lowCD4+CD25+CCR4+ cells was found to be increased and correlated with HAM/TSP disease severity . Removal of CD4+CD25+CCR4+ cells decreased the proliferation of CD4+ cells. Disease severity also correlated with the expression of CXCL10 and the soluble IL2 receptor. Fred Toulza  indicated that the frequency of FoxP3+ CD4+ cells was increased in ATL. He found a negative correlation between the frequency of FoxP3+ CD4+ and the rate of CTL lysis. CCL22, the ligand of CCR4, correlated with Tax expression and FoxP3 frequency. CCL22 expressed by infected cells exerted chemoattraction of CD4+FoxP3+ and increased their viability.Chemokine receptors may be potential targets for novel therapies. Indeed, inhibition of CXCR4 with AMD3100 suppressed the migration of ATL cells and murine lymphoblastoid cells from transgenic mice . AMD3100 decreased phosphorylation of ERK by SDF1α and inhibits cell migration. Another example was CCR4 against which a humanized antibody (KW0761) has been designed . The antibody exhibited high ADCC activity (antibody-dependent cellular toxicity) in cell cultures. In a phase I study, neutropenia and rash were the main side effects. Two partial and two complete responses were observed among 13 patients.In addition to HTLV-1, humans can be infected by 3 other members of the δ-retrovirus genus  -15. HTLVHTLV transmission has been a matter of intense debate. At first glance, it would appear that preventive measures prohibiting breast feeding are needed among HTLV-1 infected mothers. However, substitution with dry milk raises problems such as social habits, cost, availability of good quality water and protection against other pathogens. Recommendations by responsible pediatricians must individually take these parameters into account. As indicated by Soren Andersson , it is important to keep in mind that, when studying HTLV, subjects may also be co-infected by other pathogens such as pulmonary tuberculosis. The group of Achilea Lisboa Bittencourt  provided evidence that infectious dermatitis (ID), a severe recurrent infected form of eczema in children, may represent a prodromal stage of ATL. Indeed, a proportion of ID subjects had monoclonal proviral integration and characteristic flower cells. Cases of HAM/TSP with ATL were unusually frequent in the region of Bahia. Uveitis in the intermediate uvea was also frequently observed in HTLV-1 infected patients. A poster from Daniel Ceccaldi's group  provided evidence using in situ hybridization that muscle cells were infected in 4 out of 12 patients with myositis. Patients had myositis-associated auto-antibodies and muscle specific CD8+ T cells.Although HAM/TSP is usually a slow progressing disease, some patients exhibit a dramatic fast evolution. Eduardo Gotuzzo  described rapidly progressing HAM/TSP affecting 20% of Peruvian patients. Marco Lima  previously evaluated a treatment with AZT and prenidoslone without any significant improvement in these patients.Since the discovery of HTLV-1 three decades ago, apparently simple questions remain still unanswered: "Why do some subjects develop ATL and others HAM/TSP?"; "Why is there a predominance of females with HAM/TSP?" and "Why do some patients progress very rapidly?".In contrast to HTLV-1 and -2, HTLV-3 and -4 have not yet been associated with any pathology; this is likely due to their recent identification and to the low number of available isolates. Three HTLV subtypes have closely related simian viruses  while a STLV-5 strain is presently still devoid of a human counterpart. Contrasting with the homogenous HTLV-1/STLV-1 genotypes, STLV-2 and HTLV-2 are quite distant and form two distinct groups. Therefore, it is impossible to discriminate between STLV-1 and HTLV-1 without knowing the origin of the sample. Antoine Gessain  presented recent data from Central Africa, where HTLV-2 is endemic in Bakola pygmies. Intriguingly, there was no HTLV-1 in pygmies, who were infected by HTLV-2 subtype B. This genotype was also found in Amerindians tribes from the region of Amazonia. These data support evidence for an ancient origin of HTLV-2 in Central Africa. Some unanswered questions remain: "Why is the seroprevalence in hunter-gathered Bakola Pygmies higher than Bantu farmers living in the same region?" and "How were pygmies infected by HTLV-2?". HTLV-3 is also found in Central Africa and is most likely transmitted from a variety of monkey species to humans during hunting or, alternatively, through intrafamilial transmission.It thus appears that the PTLV family is composed of at least 5 genotypes. Although sequence divergence is more restricted, recent data show that this complexity also accounts for BLV where two new genotypes were described .Animal models are important to understand the mechanisms of pathogenesis and to test novel therapies . A stratAdditional strategies have been proposed at the meeting including minocycline , humanized mikβ1  and the immunosuppressant cisclosporin .In the absence of efficient treatment for HAM/TSP, all these approaches merit further evaluation in clinical trials.2O3) and IFNα which contributes to the degradation of Tax. Triple therapy arsenic trioxide+AZT+IFNα merits further consideration to achieve complete response thereby allowing interruption of the AZT+IFNα treatment.Olivier Hermine  summarized a survey of ATL chemotherapy and showed that the current optimal regimen is AZT+IFNα . In factConcomitant with improved chemicals, it is also essential to identify biomarkers predictive of treatment outcome . Masao Seto  presented different genomic profiles in acute ATL having 3p amplifications and lymphoma type showing gains of chromosome 7 and 13q loss. Other prognostic markers included high IL5, CCR4 expression, p53 mutation, p16 deletion and sIL2α .th HTLV-1 conference concluded successfully with a robust exchange of new data and information . As with a meeting report from the 13th conference [After four days of meeting, the 14ion Fig. . As withnference , and thiThe author declares that he has no competing interests.I collected the information and wrote the paper.


In the presence of carbonyl substituents (–COMe and –COPh), the largest Stokes shift  was observed in ethanol. Linear correlation was observed for the Stokes shifts in a Lippert–Mataga plot. Linear correlation of Stokes shift was also observed with ET(30) scale for protic and aprotic solvents but with different slopes. These results indicate that the fluorescence emission arises from excited state intramolecular charge transfer in these molecules where the triphenylene chromophore acts either as a donor or as an acceptor depending upon the nature of the substituent on the phenyl ring. HOMO–LUMO energy gaps have been estimated from the electrochemical and spectral data for these derivatives. The HOMO and LUMO surfaces were obtained from DFT calculations.Several 2-(phenylethynyl)triphenylene derivatives bearing electron donor and acceptor substituents on the phenyl rings have been synthesized. The absorption and fluorescence emission properties of these molecules have been studied in solvents of different polarity. For a given derivative, solvent polarity had minimal effect on the absorption maxima. However, for a given solvent the absorption maxima red shifted with increasing conjugation of the substituent. The fluorescence emission of these derivatives was very sensitive to solvent polarity. In the presence of strongly electron withdrawing (–CN) and strongly electron donating (–NMe Fluorescent molecular probes that emit in the visible region and whose fluorescence emission is sensitive to environment and solvent polarity are of significant interest due to their versatile applications in chemistry, biology and environmental science –4. ConsiPyrene is a prototypical example of a fluorophore and its monomer emission occurs around 380 nm. It has been shifted to as high as 600 nm by multiple substitution by groups that extend the conjugation and also by substituting donor-acceptor groups along the conjugation –25. In af) and solvent polarity (ET(30) scale). HOMO and LUMO surfaces of these derivatives, obtained from DFT calculations, help in identifying the triphenylene chromophore acting as an acceptor when substituted with electron donating groups and as a donor when substituted with electron withdrawing groups on the phenyl ring. HOMO–LUMO energy gaps have been estimated based on electrochemical studies and compared with those obtained from absorption spectroscopy.Herein we report the synthesis of a series of 2-(phenylethynyl)triphenylene derivatives with donor and acceptor substituents on the phenyl ring . The phe4) was synthesized by coupling 2-iodotriphenylene (2) with 1,1-dimethylpropargyl alcohol followed by deprotection with KOH in refluxing toluene (1a) and those bearing electron withdrawing substituent (1b–e) were synthesized by the Sonogashira coupling of 2-iodotriphenylene (2) with the corresponding phenylacetylene derivatives. Although 1f–g were also synthesized by this procedure, their purification proved difficult due to an inseparable minor product formed in these reactions. Therefore compounds 1f–g were synthesized via the Sonogashira coupling of 2-ethynyltriphenylene (4) and the corresponding iodoarenes  due to extended conjugation with the phenylethynyl group and loss of symmetry, respectively. Compared to 1a and 1b, the lowest energy absorption bands of 1c–g are further red shifted in both cyclohexane and acetonitrile. Although there is no significant solvent effect on the absorption bands of 1a–g in these two solvents, the lowest energy absorption bands are red shifted by 5–10 nm in cyclohexane compared to acetonitrile. Moreover, the vibrational fine structure is more clearly seen in cyclohexane than in acetonitrile where the bands are relatively broadened, especially for 1g. These observations led to the conclusion that irrespective of the substituents on the phenyl ring, the ground state of these molecules is relatively non-polar and devoid of significant solvent and substituent effects. By contrast, the fluorescence emission bands of 1a–g showed significant substituent and solvent effects.The UV–vis absorption spectra of 1a–g in cyclohexane, and DMSO are shown in 1a–f showed emission maxima around 380 nm which is characteristic of the local excited state emission of the phenylethynyltriphenylene chromophore. The emission maximum was independent of the substituent, which implied that there was no significant ICT in the excited state in cyclohexane. In the case of 1g, two emission bands were observed at 381 and 400 nm. The 381 nm band was assigned to emission from the local excited state of the phenylethynyltriphenylene chromophore and the band at 400 nm to ICT from the dimethylamino group to triphenylene moiety +, 293 [M − OH]+; HRMS calcd for C23H18ONa [M − Na]+ 333.1255; found, 333.1257.NMR) see , Pd(PPh3Synthesis of 2-ethynyltriphenylene (4). To a degassed solution of 3  in toluene (100 mL), KOH  was added and the reaction mixtureheated under reflux for 2.5 h. Upon completion of the reaction, the hot reaction mixture was filtered and the residue washed with toluene (10 mL). The combined filtrate and washings were washed with water (2 × 60 mL). The organic layer was separated and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure and the crude product purified by column chromatography on silica gel with hexane and dichloromethane  as eluant to give 4 as a colorless solid ; mp 149–151 °C; IR (neat) 3280, 2194 cm−1; 1H NMR  δH = 8.80 , 8.57–8.65 , 7.74 , 7.65–7.68 , 3.23  ppm; 13C NMR  δc 130.2, 130.1, 129.9, 129.1, 128.9, 127.7, 127.6, 127.5, 127.4, 127.3, 123.5, 123.4, 123.3, 120.6, 84.1, 77.9 ppm; MALDI-TOF MS m/z (%) 252 (72) [M+], 253 (100) [M+ + 1], 254 (22) [M+ + 2]; Anal. calcd. for C20H12 C, 95.23; H, 4.75. Found C, 95.04, H 4.60.General procedure for the synthesis of 1a–e. 1a–e were synthesized by coupling 2-iodotriphenylene (2 mmol) with the corresponding arylethyne (1.9 mmol) (see 3.mol) see  accordin2-(Phenylethynyl)triphenylene (1a). The crude product was purified by column chromatography with hexane as eluant to afford 1a as a colorless solid , mp 180–182 °C; IR (neat) 3069, 2217 cm−1; 1H NMR  δH = 8.80 , 8.55–8.64 , 7.75 , 7.61–7.66 , 7.36-7.40  ppm; 13C NMR  δc 131.7, 130.1, 130.0, 129.9, 129.7, 129.6, 129.3, 129.1, 128.5, 128.4, 127.59, 127.58, 127.4, 127.3, 126.8, 123.5, 123.43, 123.41, 123.36, 123.31, 121.9, 90.2, 89.9 ppm; The mass spectrum was recorded as the silver ion adduct of 1a by adding silver triflate to a solution of 1a in acetonitrile prior to measurement. ESI Q-TOF MS m/z 435 [M + Ag]+ along with the isotope peaks in the expected intensity ratios; HRMS calcd for C26H16Ag [M + Ag]+ 435.0303; found, 435.0298.2-(3-Trifluoromethylphenylethynyl)triphenylene (1b). The crude product was purified three times by column chromatography with hexane as eluant to yield 1b as a colorless solid , mp 137–139 °C; IR (neat) 3069, 2213 cm−1; 1H NMR  δH = 8.80 , 8.55–8.68 , 7.8 , 7.73–7.77 , 7.59–7.67 , 7.48-7.51  ppm; 13C NMR  δc 134.7, 131 , 130.1, 130.0, 129.9, 129.8, 129.7, 129.2, 129.0, 128.9, 128.5 , 127.7, 127.6, 127.41, 127.39, 127.0, 124.87 , 124.3, 123.5, 123.4, 123.3, 122.7, 121.1, 91.4, 88.6 ppm; MALDI-TOF MS C27H15F3m/z (%) 396 (100) [M+], 397 (84) [M+ + 1], 398 (22) [M+ + 2].4-(2-Triphenylenylethynyl)benzonitrile (1c). The crude product was purified by column chromatography with a mixture of hexane and dichloromethane  as eluant to yield 1c as a colorless solid , mp 192–194 °C; IR (neat) 3059, 2221 cm−1; 1H NMR  δH = 8.82 , 8.60–8.66 , 7.77 , 7.66–7.69  ppm;13C NMR  δc 132.1, 130.2, 130.0, 129.8, 129.1, 128.9, 128.2, 127.9, 127.8, 127.4, 127.2, 123.6, 123.5, 123.4, 123.37, 123.3, 120.8, 119.6, 111.5, 94.3, 88.8 ppm; ESI Q-TOF MS C27H15N m/z 376 [M +Na]+, 354 [M + H]+; HRMS calcd for C27H16N [M + H]+ 354.1283; found, 354.1287.4-(2-Triphenylenylethynyl)acetophenone (1d). The crude product was purified by column chromatography with a mixture of hexane and dichloromethane  as eluant to yield 1d as a colorless solid , mp 181–183 °C; IR (neat) 3067, 2218, 1669 cm−1; 1H NMR  δH = 8.86 , 8.62–8.68 , 7.97–8.0 , 7.80 , 7.66–7.71 , 2.64  ppm; 13C NMR  δc 191.3, 136.6, 131.8, 130.2, 130.0, 129.9, 129.8, 129.2, 129.0, 128.4, 128.2, 127.8, 127.7, 127.4, 127.1, 123.6, 123.4, 121.3, 93.2, 89.4, 26.6 ppm; ESI Q-TOF MS m/z 371 [M + H]+; HRMS calcd for C28H19O [M + H]+ 371.1436; found, 371.1433.4-(2-Triphenylenylethynyl)benzophenone (1e). The crude product was purified by column chromatography with a mixture of hexane and dichloromethane  as eluant to yield 1e as a colorless solid , mp 172–174 °C; IR (neat) 3080, 2211, 1658 cm−1; 1H NMR  δH = 8.85 , 8.61–8.66 , 7.79–7.86 , 7.59–7.73 , 7.49–7.53  ppm; 13C NMR  δc 196.0, 136.9, 132.6, 131.5, 130.20, 130.16, 130.06, 130.03, 129.9, 129.8, 128.4, 127.8, 127.7, 127.5, 127.1, 123.6, 123.5, 123.4, 121.4, 93.0, 89.5 ppm; ESI Q-TOF MS m/z 433 [M + H]+; HRMS calcd for C33H21O [M + H]+ 433.1592; found, 433.1593.General procedure for the synthesis of 1f–g. A Schlenk flask was charged with the corresponding aryl iodide (0.6 mmol), Pd(PPh3)2Cl2 , PPh3 , CuI , degassed THF (30 mL) and diisopropylamine (10 mL). The reaction mixture was stirred at room temperature for 15 min and a solution of 2-ethynyltriphenylene (4) (0.5 mmol) in THF (3mL) added dropwise. Stirring was continued for 2.5 h. Removal of solvent and other volatile materials under reduced pressure gave the crude product which was purified by column chromatography on silica gel.2-phenylethynyl)triphenylene (1f). The crude product was purified by column chromatography with a mixture of hexane and dichloromethane  as eluant to yield 1f as a colorless solid , mp 118–120 °C; IR (neat) 3084, 2959, 2918, 2850 cm−1; 1H NMR  δH = 8.82 , 8.58–8.65 , 7.78 , 7.64–7.68 , 7.20 , 7.15 , 6.87 , 4.05 , 1.83–1.88 , 1.47–1.52 , 1.27–1.29 , 0.88-0.92  ppm; 13C NMR  δc 149.9, 148.9, 130.0, 129.9, 129.8, 129.4, 129.3, 229.2, 127.5, 127.3, 126.6, 125.1, 123.5, 123.4, 123.35, 123.3, 122.3, 116, 115.4, 113.5, 113.5, 90.6, 88.2, 69.4, 69.2, 31.9, 29.7, 29.6, 29.4, 29.36, 29.30, 29.26, 26.1, 22.7, 14.1 ppm; MALDI-TOF MS: m/z (%) 640 (100) [M+], 641 (49) [M+ + 1], 642 (9) [M+ + 2].N,N-Dimethyl-4-(2-triphenylenylethynyl)aniline (1g). The crude product was purified by column chromatography with a mixture of hexane and dichloromethane  as eluant to yield 1g as an orange solid , mp 212–214 °C (decomposed during melting); IR (neat) 3064, 2798, 2190 cm−1; 1H NMR  δH = 8.80 , 8.61–8.66 , 8.59 , 7.77 , 7.65–7.69 , 7.51 , 6.71 , 3.02  ppm; 13C NMR  δc 150.2, 132.8, 130.0, 129.9, 129.8, 129.7, 129.3, 128.9, 127.5, 127.4, 127.3, 126.3, 123.5, 123.4, 123.3, 123.2, 122.9, 111.9, 110.0, 91.6, 87.9, 40.2 ppm; ESI Q-TOF MS m/z 372 [M + H]+; HRMS calcd for C28H22N [M + H]+ 372.1752; found, 372.1759.1a–g, 2–4), UV–vis data in various solvents, electrochemical data for 1a–g and calculated atomic coordinates for 1c and 1g.Supporting Information includes the NMR spectra of all compounds (File 11a–g and 2–4.Experimental data for compounds