Certain xenoestrogens (estrogenic compounds introduced from outside the body) are suspected of disrupting these activities. In a new study, xenoestrogenic alkylphenols and bisphenol A (BPA) interfered with normal estrogenic signaling in vitro, which suggests they could disrupt normal physiologic function at critical life stages .The body produces estrogens—including estrone . ERK-controlled pathways respond to many biochemical stimuli and integrate these signals to direct a cell toward division, differentiation, death, or malignant transformation. The structurally related alkylphenols and BPA interact weakly with nuclear estrogen receptors, but they can have pronounced effects on signaling pathways mediated by estrogen receptors in the cell membrane.1 and E2 prompted early, intermediate, and late surges in ERK activation at 5, 10–30, and >30 min, respectively; alkylphenols and E3 typically triggered early and late surges. Interestingly, a very low concentration of BPA (10−14 M) yielded a similar two-peak response, but a higher concentration (1 nM) induced a three-peak response like that of E1 and E2. Both BPA concentrations were typical of environmental exposures and, along with ineffective midrange doses, also illustrated the nonmonotonic dose–response relationship characteristic of many estrogenic compounds.In the current study, a rat pituitary cancer cell line was used to study the effect of alkylphenols and BPA on ERK1 and ERK2 activation (measured as phosphorylation), both alone and in combination with each physiologic estrogen. After treatment with each physiologic and environmental estrogen, the researchers measured time-dependent surges in ERK activation. In most cases, EWhen physiologic estrogens and xenoestrogens were combined, the response pattern generally shifted to a single major peak at an intermediate time. Xenoestrogens that caused a strong response when administered alone at a particular point in time or concentration tended to inhibit ERK activation in response to a physiologic estrogen. But at other times or concentrations, the same xenoestrogen might cause a weak response on its own, in which case it would tend to enhance ERK phosphorylation in response to physiologic estrogens.There were exceptions to these general patterns, however, which highlights the need to study effects of individual xenoestrogens at different points in time, at varying concentrations, and in different tissues. The effect of shifts in the patterns of ERK activation are only just beginning to be explored, although it is known that these patterns constitute an important component of information flow within a cell. The correct flow of information is likely to be especially critical during windows of vulnerability that are based in part on life stage.


Surgical instruments are often strongly contaminated with patients' blood and tissues, possibly containing pathogens. The reuse of contaminated instruments without adequate cleaning and sterilization can cause postoperative inflammation and the transmission of infectious diseases from one patient to another. Thus, based on the stringent sterility requirements, the development of highly efficient, validated cleaning processes is necessary. Here, we use for the first time synthetic single-stranded DNA (ssDNA_ODN), which does not appear in nature, as a test soiling to evaluate the cleaning efficiency of routine washing processes. Stainless steel test objects were coated with a certain amount of ssDNA_ODN. After cleaning, the amount of residual ssDNA_ODN on the test objects was determined using quantitative real-time PCR. The established method is highly specific and sensitive, with a detection limit of 20 fg, and enables the determination of the cleaning efficiency of medical cleaning processes under different conditions to obtain optimal settings for the effective cleaning and sterilization of instruments. The use of this highly sensitive method for the validation of cleaning processes can prevent, to a significant extent, the insufficient cleaning of surgical instruments and thus the transmission of pathogens to patients. The complexity of some surgical instruments and their inaccessibility during routine cleaning processes hamper the residue-free removal of contaminants from the surface of instruments. Thus, the use of these inadequately cleaned instruments can lead to postoperative sepsis and the transmission of infectious diseases, such as Creutzfeldt-Jakob disease (CJD) –3. ThereCleaning involves various methods, such as washing with water, with mechanical forces , enzymatically, or chemically . Test objects for cleaning validation can be contaminated with an artificial test soil by brushing, inserting, or pipetting a certain amount of contamination. Previous studies have used different test soils, such as citrated blood, mixtures of proteins, egg yolk, or semolina pudding, to evaluate the cleaning efficiency of medical cleaning and disinfection machines. The goal of the cleaning is to obtain an optically clean instrument in which the pathogen numbers are reduced. Residual contaminants on test objects can be determined qualitatively , semiquantitatively  method), or quantitatively  determination or the modified OPA  method).Every microorganism contains genetic information coded in DNA or RNA. DNA is a robust biological material that can survive unscathed for tens of thousands of years, as evidenced by the isolation of genetic material from archaeological finds of bones of long-extinct animals. Previous studies have generally validated the cleaning of medical instruments by the determination of residual proteins. However, residual viral DNA on medical instruments has not been a focus of study. Zhao and colleagues demonstrated the formation of complete viruses after the injection of naked human T-cell leukemia viral DNA into the bloodstream of rabbits . In otheAn ssDNA oligonucleotide library (ssDNA_ODN) containing a randomized sequence region of 49 nucleotides and two fixed primer hybridization regions, 5′-GCCTGTTGTGAGCCTCCTAAC-N49-CATGCTTATTCTTGTCTCCC-3′, was used for qPCR analyses. For the amplification of ssDNA_ODN, qPCR reaction mixtures contained the forward primer 5′-GCC TGT TGT GAG CCT CCT AAC-3′ and the reverse primer 5′-GGG AGA CAA GAA TAA GCA TG-3′. All oligonucleotides were ordered HPLC purified from Ella Biotech GmbH .μL ssDNA_ODN solution to 50 μL master mix, containing 30 μL iQ SYBR Green Supermix and 360 nM forward and reverse primer. The qPCR was performed using an iCycler PCR machine . PCR cycling conditions consisted of an initial hot start at 95°C for 3 minutes to activate the hot-start iTaq DNA polymerase and to denature the template DNA, followed by 30 cycles of denaturing at 95°C for 45 seconds, annealing at 58°C for 20 seconds, and elongation at 72°C for 20 seconds. An additional final elongation step was performed at 72°C for 5 minutes. At the end of the amplification process, a melt-curve analysis was performed consisting of 50 melt cycles, beginning at 70°C with increments of 0.5°C per cycle to control the specific length of the amplified ssDNA. After the amplification, the used ssDNA_ODN revealed a single melt curve with a maximum peak at 83°C.For the detection and quantification of ssDNA_ODN in solutions, qPCR was performed using iQ SYBR Green Supermix . All qPCR reactions were performed in 96-well plates. Each qPCR reaction mix was prepared by adding 10 μL sample volume. All qPCR data were collected and analyzed using iCycler iQ Multicolor Real-Time PCR Detection System Software Version 3.1 . Using the standard curve of the serial dilutions of ssDNA_ODN, the unknown quantities in the samples were calculated. For an exact determination of the amount of ssDNA_ODN in the solutions, samples were used in the correct dilution to measure within the linear range of the standard curve.Serial dilutions from 5 to 0.02 pg ssDNA_ODN and a negative control without template were included in each run. Each sample was tested in triplicate with, respectively, 15 To ensure that the used ssDNA_ODN specific primers cannot amplify parts of the genomic DNA (gDNA) material, which is present in the residual blood impurities on surgical instruments, qPCR experiments were performed with different gDNA isolated from peripheral blood of different species. For this purpose, 100 ng of isolated gDNA was added to the master mix of qPCR reactions containing ssDNA_ODN specific primers. Furthermore, spiked probes that contained additionally to the gDNA known amounts of ssDNA_ODN (5 or 2.5 pg) were used as a positive control to determine the influence of the gDNA on ssDNA_ODN detection.n = 3), pigs (n = 3), guinea pigs (n = 3), rats (n = 7), mice (n = 20), and chicks (n = 8). Because of the small amount of blood per rat, mouse, and chicken, blood was collected from each animal species and pooled for the isolation of cells. In contrast, the isolation of cells from human, porcine, and guinea pig blood was performed separately. Peripheral blood was collected using 1.6 mg EDTA/mL blood. To eliminate erythrocytes from the collected blood samples, whole blood was incubated in a fivefold volume of erythrocyte lysis buffer consisting of 0.15 M ammonium chloride, 10 mM potassium hydrogen carbonate, and 0.1 mM Na2-EDTA-2H2O for 15 minutes on ice and then centrifuged at 400 g for 10 min at 4°C. The supernatant was discarded, and the pellet was resuspended again in erythrocyte lysis buffer. After incubation on ice for another 5 minutes, the suspension was centrifuged at 400 g for 10 min at 4°C. This process was repeated 3-4 times. Subsequently, gDNA was isolated using the DNeasy Blood & Tissue Kit  according to the manufacturer's instructions. The concentration of the isolated gDNA was determined using a spectrophotometer .gDNA was isolated from the peripheral blood cells of humans .Stainless steel screw nuts , Larus Instrumente GmbH, Tuttlingen, Germany) for clinical surgery were used as test objects for the immobilization of ssDNA_ODN. These test objects were selected for experiments to mimic the adhesion of genomic material on stainless steel surgical instruments as used in the clinical praxis. Furthermore, ssDNA_ODN was applied to the surface of screw thread to imitate a location that would be difficult to access for cleaning, as in the case of many surgical instruments. The test objects were coated with 5 To determine the residual amount of ssDNA_ODN on test objects after the cleaning process, the ssDNA_ODN was eluted using nuclease-free water or 5 M urea solution from the surface of these objects. High ion concentrations or chemicals in the eluate can affect qPCR. Therefore, ssDNA_ODN in the eluates was concentrated and purified using Amicon Ultra 0.5 mL 30K centrifugal filters  according to the manufacturer's instructions to prevent an effect on qPCR. Urea concentrations less than 8 M are compatible with the filter. The used ssDNA_ODN had a length of 90 nucleotides and an approximate molecular weight of 29.7 kDa. Therefore, Amicon Ultra filters with a 30 kDa molecular weight cut-off were selected to ensure that the ssDNA_ODN remained in the collection tube. During centrifugation, molecules ≥ 30 kDa cannot flow through the filter, and they are collected, whereas all the smaller molecules are eluted.μL of water or 5 M urea solution containing 10 μg ssDNA_ODN for gel electrophoresis experiments was filtered using Amicon Ultra 0.5 mL 30K centrifugal filters. Thereby, a concentrate and flow-through were obtained. Both the concentrate and flow-through were filled up to 500 μL with nuclease-free water. For gel electrophoresis, 300 ng of the ssDNA_ODN as a positive control and 15 μL of concentrate and flow-through were run on 10% denaturing urea-polyacrylamide gel. The gel was stained with GelRed  and documented using the gel documentation system .To examine the efficiency of the purification method, 500 μL of nuclease-free water (n = 3) or 5 M urea solution (n = 3) at 93°C to determine the best elution method. Every 10 minutes, test objects were taken out of the elution solution and transferred to the next fresh elution solution. Positive controls were prepared by adding 5 μg ssDNA_ODN in 250 μL water (n = 3) or in 5 M urea solution (n = 3). Then, they were treated in accordance with the eluent for the test objects. After elution, the ssDNA_ODN in all elution solutions and positive controls was concentrated and purified using Amicon Ultra 0.5 mL 30K centrifugal filters. All concentrates with purified ssDNA_ODN were filled up to 500 μL with nuclease-free water. For gel electrophoresis, 15 μL of concentrates was run on 10% denaturing urea-polyacrylamide gel. The gel was stained with GelRed  and documented using the gel documentation system . The concentrates for qPCR experiments were diluted 1 : 10,000 in nuclease-free water, and the amount of ssDNA_ODN was quantified using qPCR. Measurements were repeated three times.To establish an efficient method for elution of the ssDNA_ODN from the surfaces of test objects, ssDNA_ODN coated test objects were incubated three times each for 10 minutes with 250 μL of 5 M urea solution (n = 3) without ssDNA_ODN was filtered using Amicon Ultra 30K centrifugal filters according to the manufacturer's instructions. The concentrated samples were filled up to 500 μL with nuclease-free water. The influence of concentrates on qPCR was tested by adding 10 μL of each sample to 40 μL of master mix and spiking this mixture with 10 μL of ssDNA_ODN solution containing defined amounts of ssDNA_ODN from 5 pg to 0.02 pg, as used for the standard curve. Detected Ct (cycle threshold) values of samples were then compared with the Ct values of the standard to determine possible changes.A possible influence of the used 5 M urea elution solution on qPCR was examined. Therefore, 500 n = 3) with 5 μg ssDNA_ODN coating and one negative control without ssDNA_ODN coating were washed at 60°C, 85°C, or 93°C for 10, 20, 25, and 30 minutes. The cleaning process was performed with and without detergent . After every cleaning process, elution of the residual ssDNA_ODN from the test objects was performed by heating them once at 93°C for 10 minutes in 250 μL of 5 M urea solution. Subsequently, the test objects were taken out of the hot solutions, and the eluates were purified and concentrated using Amicon Ultra 0.5 mL 30K centrifugal filters. Positive controls were prepared by adding 5 μg ssDNA_ODN in 250 μL 5 M urea solution and heating for 10 min at 93°C. They were also purified analogous to eluates from test objects using Amicon Ultra 30K filters. All concentrated samples were filled up to 500 μL with nuclease-free water and used in appropriate dilutions for qPCR.To verify the suitability of the used test method for the cleaning validation of medical cleaning machines, cleaning experiments were performed using a laboratory cleaning machine  with different cleaning temperatures and periods. Three test objects (http://dx.doi.org/10.1155/2014/632127) were detected sensitively and reproducibly by using the SYBR Green I-based qPCR method. The efficiency of the qPCR was 99.4%, which shows doubling of the amplicon at each cycle. The lower detection limit was 20 pg.In this study, an ssDNA library (ssDNA_ODN) that contained a randomized sequence region of 49 nucleotides and two fixed primer hybridization regions was used as a test soiling. The primer regions with known sequences enabled the specific amplification of ssDNA_ODN by qPCR. For the quantitative detection of ssDNA_ODN amounts in unknown samples, a standard curve presenting Ct values as a function of log amount of ssDNA_ODN was used. Per reaction mixture, ssDNA_ODN amounts from 5 pg to 0.02 pg .Cleaning of test objects was performed with or without detergent in a laboratory cleaning machine with different cleaning temperatures  and washing periods . In this study, ssDNA molecules with a length of 90 nucleotides were used as a test soiling on stainless steel test objects to evaluate the efficiency of medical cleaning processes. Using qPCR, residual amounts on test objects were quantified specifically and sensitively. The used ssDNA_ODN contained a randomized sequence region of 49 nucleotides and two fixed primer hybridization regions, which allowed the specific detection during qPCR. However, instead of randomized ssDNA molecules, a fixed sequence with a defined nucleotide sequence containing primer hybridization regions could also be used.2 for the ninhydrin assay, 30–50 μg for the BCA assay, or 0.003 μmol for the OPA assay [2, was developed [2, 100 pg/mm2, and 1 pg/mm2, respectively [The ISO 15883 specifies general requirements for the validation of washer disinfectors intended to be used for the cleaning and disinfection of reusable medical devices. Acceptable protein levels on processed instruments have to be below the detection limit of 2 mg/meveloped . More soectively . HoweverThe presence of gDNA in qPCR reaction mixtures had no influence on the accurate quantification of ssDNA_ODN amount. Thereby, the uniqueness of the primers for the detection of ssDNA_ODN on test objects was demonstrated. There was no interaction with the genomic material of humans, pigs, guinea pigs, rats, mice, or chicks. The primer regions of ssDNA_ODN, which were specifically designed by Mayer et al. for efficient and specific amplification, showed no interaction with the tested gDNAs . Thus, tTo elute immobilized ssDNA_ODN from test objects, 5 M urea solution or water was used. Urea is a good hydrogen bond donor and an excellent receptor. Thus, we suppose that the disruption of hydrogen bonds and/or disruption of base stacking led to the better elution of ssDNA_ODN from the surface of stainless steel test objects than when only water was used as an elution solution , 14. AddSeveral compounds of blood or tissue samples and of DNA extraction and purification methods, such as EDTA, hemoglobin, heparin, SDS, phenol, chloroform, ethanol, or salts, can influence PCR –17. TherThe detection of residual ssDNA_ODN on test objects washed for up to 20 minutes with detergent and the presence of ssDNA_ODN on all test objects washed for up to 30 minutes without detergent clearly showed the influence of detergents on the cleaning of medical instruments. Furthermore, the impact of temperature on the effectiveness of cleaning was clearly demonstrated at 93°C. Here, the detected amounts were the lowest after cleaning both with and without detergent. The detection of ssDNA_ODN on negative controls after washing at 93°C showed that during the cleaning process, the ssDNA_ODN on test objects can be thrown due to mechanical forces to other locations in the cleaning machine. This phenomenon can also happen in reality with protein or DNA contaminants on instruments. Thereby, instruments that are originally not contaminated can become contaminated during the mechanical cleaning process. This shows the importance of the validation of medical cleaning and disinfection processes. The necessary time, temperature, and rinsing agent have to be determined to ensure the use of only sterile instruments without infectious proteins and DNA for medical interventions.This study demonstrated for the first time the applicability of synthetic ssDNA oligonucleotides as a test soiling on stainless steel surfaces for the validation of medical cleaning processes. The established qPCR method enables the ultrasensitive and highly specific quantification of the residual synthetic ssDNA test soiling on test objects. The use of this highly sensitive method can greatly contribute to the prevention of pathogen transfer via insufficient cleaning of medical instruments.Supplementary Figure 1: Representation of the dynamic range of qPCR using 5, 2.5, 1.25, 0.625, 0.3125, 0.08, and 0.02 pg ssDNA_ODN.Supplementary Figure 2: Detection of the eluted ssDNA_ODN from test objects after three successive elutions in 5M urea solution or water using denaturing urea polyacrylamide gel electrophoresis.Supplementary Table 1: Detection of ssDNA_ODN amounts in the three successive elutions in 5 M urea solution or water using qPCR.Click here for additional data file.


The emergence of Human retroviruses, in Central Africa, may occur in contact with non human primates, during hunting and butchering. Recent studies described several cases of human infections with Human T-cell Lymphotropic Virus (HTLV) as the result of simian zoonotic transmissions, in south Cameroon corresponding to north Gabon.To search for new HTLV infections  and to investigate zoonotic infection, we conducted a study among individuals hunting and butchering of wild non-human primates, in rural area in north of Gabon, Central Africa.tax-rex region with specific primers. Phylogenetic analyses using Neighbour-Joining were done.This study was conducted during 2 weeks in May 2013, in rural villages in Gabon. We studied 222 people, with 15years and up, from the general adult population  experiencing hunting or butchering. Blood was collected in EDTA tube, and HTLV-1/2 immunoenzymatic test (ELISA) was performed as a screening test. Because of the unavailability of the Western Blot tests during the study, Positive samples were confirmed and discriminated by PCR in Among a total of 222 persons, seropositivity in HTLV was found in 18 (8.10%), 5 (2.25%) were women and 13 (5.85%) men. Men were hunters while women recognized butchering activities. PCR of 18 samples showed that 5 were HTLV-1 positive (2 men and 3 women); one HTLV-2, isolated from a hunter; 12 were not amplified (5 of them showed a high optic density by serology). Phylogenetic analysis confirmed PCR results obtained for HTLV-1 and HTLV-2.Our results show HTLV-1 and HTLV-2 Infections in a risk group in north of Gabon, corresponding to south of Cameroon, Central Africa. These results suggest a probable simian origin of HTLV-1 and HTLV-2 described subtypes. Further studies are needed to best understand the interspecies transmission of these complex retroviruses; and to explore the intra familial transmission.


Xiao-Xu-Ming decoction (XXMD) on neurovascular unit and to examine the role of PI3K (phosphatidylinositol 3-kinase)/Akt pathway in this protection. The cerebral ischemia was induced by 90 min of middle cerebral artery occlusion. Cerebral infarct area was measured by tetrazolium staining, and neurological function was observed at 24 h after reperfusion. DNA fragmentation assay, combined with immunofluorescence, was performed to evaluate apoptosis of neuron, astrocyte, and vascular endothelial cell which constitute neurovascular unit. The expression levels of proteins involved in PI3K/Akt pathway were detected by Western blot. The results showed that XXMD improved neurological function, decreased cerebral infarct area and neuronal damage, and attenuated cellular apoptosis in neurovascular unit, while these effects were abolished by inhibition of PI3K/Akt with LY294002. We also found that XXMD upregulated p-PDKl, p-Akt, and p-GSK3β expression levels, which were partly reversed by LY294002. In addition, the increases of p-PTEN and p-c-Raf expression levels on which LY294002 had no effect were also observed in response to XXMD treatment. The data indicated the protective effects of XXMD on neurovascular unit partly through the activation of PI3K/Akt pathway.In the present study, we used a focal cerebral ischemia and reperfusion rat model to investigate the protective effects of  Current biomedical research about stroke is focusing on treating neurovascular unit (NVU), and it is widely accepted that the key to effective therapy lies in restoring normal function of NVU.NVU is a functionally and structurally interdependent multicellular complex which consists of endothelial cells, basal lamina, pericytes, astrocytes, and neurons , and theβ (GSK3β) β (GSK3β) (GSK3β) . AlthougTo date, a large body of basic studies have indicated that numerous agents or options could reduce cerebral infarct area and improve neurological deficits with animal models of stroke –21 and tXiao-Xu-Ming decoction (XXMD), one classic TCM formula recorded by Sun Simiao of the Chinese ancient Tang Dynasty, has been widely used in the treatment of stroke. It has aroused extensively preclinical and clinical studies for its high efficacy [Traditional Chinese medicine (TCM) which is derived from natural plants and animals may act on multiple targets, and its flexible and comprehensive treatment strategies usually generate unexpected outcomes. These characteristics of TCM coincide with the view of NVU protection. Based on this fact, TCM has become the focus of medical research –27. Xiaoefficacy –32. Lineefficacy , 30. A refficacy . Most imefficacy , 32. Howβ , total GSK3β, GAPDH, and horseradish-peroxidase- (HRP-) linked anti-rabbit antibody purchased from Cell Signaling Technology  were used for Western blot analysis. Mouse monoclonal antibodies for NeuN and GFAP from Millipore , CD31 from Abcam , and Alexa Fluor 555 anti-mouse antibody from life technologies  were applied to immunofluorescence. LY294002 (PI3K inhibitor), the primary antibodies for phospho-PDK1 , total PDK1, phospho-PTEN , total PTEN, phospho-Akt , total Akt, phospho-c-Raf , total c-Raf, phospho-GSK3Herba Ephedrae, cassia twig, Radix Paeoniae Alba, Rhizome Chuanxiong, Radix Ginseng, Radix Stephaniae Tetrandrae, Radix Scutellariae, Semen Armeniacae Amarum, Radix Aconiti Praeparata, Radix Glycyrrhizae, Radix Ledebouriellae, and fresh Rhizoma Zingiberis Recens in a ratio of 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1.5 : 5. The crude drugs were purchased from Traditional Chinese Medicine Pharmacy of Zhongshan Hospital, Fudan University. XXMD was prepared as previously described with small modifications [The XXMD consists of twelve crude drugs including ications . After tOne hundred and six male Sprague-Dawley rats weighing 250–280 g  were housed in groups of four with free access to food and water and maintained in temperature (22 ± 2°C) and humidity-controlled (55 ± 5%) room with 12:12 h light-dark cycle. Prior to experimental manipulation, rats were handled daily for 3 days.drat = dhuman × 0.71/0.11 [We usually apply the water extract of XXMD in humans and the common human daily dosage of XXMD is 165 g/75 kg in body weight. According to the formula, .71/0.11 , the com.71/0.11 , we ther.71/0.11 . In the For drug treatment, the rats in XXMD-treated groups were orally administered with XXMD (60 g/kg/day) and others in the Sham, I/R groups were given the same volume of distilled water for 3 days before cerebral ischemic surgery. The drug administrations were performed twice a day at 8:00 and 18:00 and continued until animals were sacrificed. The above procedures were carried out by one investigator who was aware of the animal grouping but did not participate in the sequent investigations. All experimental protocols and animal handling procedures were approved by the Animal Care and Use Committee (ACUC) of Fudan University, and were consistent with the National Institutes of Health Guide for the Care and Use of laboratory Animals.Focal cerebral ischemia and reperfusion were performed as described previously . BrieflyIt should be noted that there are several technical factors in the MCAO rat model which may affect cerebral infarct area, such as physical differences in the employed monofilament suture, the depth of the insertion, and accidental premature reperfusion –38. In oμL of LY294002 solution or the small amount of vehicle (DMAO + ETOH) was injected intracerebroventricularly on the ischemic side at 30 min before operation. The experimental design for the present study was shown in  To investigate the function of PI3K after cerebral ischemia and reperfusion, several rats in XXMD-treated group were pretreated with LY294002, the PI3K special inhibitor, as described previously  with smaExamination of neurological deficits was performed at 24 h after reperfusion by one investigator who was unaware of the experiment design. The neurological deficits were scored on a four-point scale described by Hara et al.  with a mCerebral infarct area was measured by 2,3,5-triphenyl tetrazolium chloride  staining. Rats were killed at 24 h after reperfusion and perfused transcardially with normal saline. Brains were sectioned into six coronal slices from rostal to caudal and stained with 1% TTC staining at 37°C for 20 min away from light. Then the brain tissues were differentiated according to white-colored infarct area and red-purple noninfarct area. Cerebral infarct areas were calculated using microscope image-analysis software  according to the following formula: /contralateral hemisphere area) × 100%.μm on a rotary microtome. To assess brain injury, Nissl staining was performed with toluidine blue. Briefly, slices were dewaxed, dehydrated, and stained with 1% toluidine blue  at 50°C for 30 min. Then the slices were rinsed and cleared in graded ethanol and xylene and coverslipped under permount. The photomicrographs were captured with a light microscope. And the number of intact cells in penumbra of ischemic cortex was counted throughout five lesion regions randomly.At 24 h after reperfusion, the rats were deep anesthetized with 10% chloral hydrate  and perfused transcardially with 200 mL normal saline followed by 4% paraformaldehyde . The brains were removed and postfixed in the same fixative for 24 h at 4°C. After dehydration in graded ethanol and xylene, the brains were embedded in paraffin and sectioned into slices of 5 In order to detect the extent of DNA fragmentation of different cells in NVU, double staining of TUNEL and immunofluorescence labeling with different antibodies were performed and the positive cells were counted. In this study, we chose anti-NeuN, anti-GFAP, and anti-CD31 antibodies, the markers of neuron, astrocyte, and vascular endothelial cell, combined with TUNEL staining to evaluate apoptosis and its location, and to further clarify the roles of multiple cells in NVU after ischemia and reperfusion.μm. We performed immunofluorescence labeling with anti-NeuN, anti-GFAP, and anti-CD31 antibodies and TUNEL staining with an In Situ Cell Death Detecting Kit , respectively, according to the method described by Zhou et al. [At 24 h after reperfusion, rats were deep anesthetized and the brains were rapidly removed and cut into slices of 10 u et al.  and the  Western blots were used to evaluate expression levels of proteins involved in PI3K/Akt pathway at 24 h after reperfusion. Whole cell protein of ischemic penumbra was extracted from the fresh brain. Equal volumes and qualities of protein solutions were separated by electrophoresis of appropriate concentrations of polyacrylamide gels and transferred to the poly-vinylidnene fluoride membranes . Then the membranes were placed in 5% skim milk, prepared with Tris-buffered saline with 0.1% Tween-20 (TBST) to block nonspecific binding, and incubated with primary antibodies at 4°C overnight. In the following 2 h, the membranes were incubated with horse-radish peroxidase-conjugated secondary anti-rabbit antibody after washing with TBST for 10 min × 3. Eventually, the targeted antigens were detected by standard chemical luminescence methods  with Fluor Chem FC 2 gel imaging system . GAPDH protein was used as a loading control. Phosphorylation levels of the targeted proteins were analyzed by total levels of corresponding proteins. Western blots were duplicated with three independent sets. Band intensities were measured with Quantity One software .t-tests using SPSS 11.5 for Windows . All were considered statistically significant for P values < 0.05. When all the data have been collected, the animal grouping was released. Data are presented as the means ± standard error of the mean (SEM). Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test or unpaired Student's The occlusion for 90 min followed by reperfusion for 24 h led to an infarct area and marked neurological deficits in rats. The results indicated that XXMD treatment resulted in a significant reduction in infarct area compared to the I/R group . In addition, the rats subjected to cerebral ischemia and reperfusion showed noticeable neurological deficits which were markedly attenuated by XXMD treatment, while rats in the sham group performed normally after operation. To evaluate the contribution of PI3K/Akt pathway to cerebral infarct area and neurological deficits, we investigated the effects of inhibiting PI3K with the specific inhibitor LY294002 on cerebral infarct area and neurological function at 24 h after reperfusion. Larger infarct area was observed in LY294002-treated rats (39.01 ± 2.27%) compared with the rats in the XXMD60 group. Additionally, we found a smaller infarct area in the XXMD60 + vehicle group (24.25 ± 2.23%) compared with the I/R group Figures . LikewisNissl staining showed neuronal injury in penumbra of ischemic cortex at 24 h after reperfusion. The images showed that most cells with intercellular space enlarged were shrunk in the ischemic cortex and had deep color staining indicative of injury in the I/R group. However, these characteristic changes were not observed in the sham group and were improved by XXMD treatment. Interestingly, LY294002 blocked the effect of XXMD on neuronal injury. Furthermore, there were more intact cells in the ischemic cortex of the XXMD-treated rats in the absence of LY294002 compared to the I/R group .Double staining of TUNEL and immunofluorescence labeling with NeuN were performed to evaluate neuron apoptosis. As shown in To comprehensively determine apoptosis of different cells in NVU, we next performed double staining for TUNEL and immunofluorescence labeling with GFAP to evaluate astrocyte apoptosis. As represented in the ischemic cortex, the number of TUNEL and GFAP double stained cells was markedly increased in penumbra of ischemic cortex at 24 h after reperfusion. These changes were largely reversed in the XXMD60 group but worsened in the presence of LY294002. In all cases, double stained cells were hardly observed in the sham group. Percentages of apoptotic astrocyte in different groups were similar with the above findings .The data revealed that astrocyte, as the key component of NVU, was involved in apoptosis induced by stroke with reperfusion. However, astrocyte apoptosis was decreased by treatment with XXMD. In view of the important role of astrocyte, the results further indicated that there were more normal astrocytes which promoted neuronal survival by XXMD treatment.Intriguingly, the results of CD31 and TUNEL double staining in different groups were consistent with antiapoptotic effects of XXMD on neuron and astrocyte after ischemia and reperfusion. As reperfusion occurred, vascular endothelial cell, another considerable component of NVU, was subjected to severe damage unavoidably and showed the changes of apoptosis. However, XXMD treatment significantly reduced vascular endothelial cell apoptosis which was prevented by intracerebroventricular injection of LY294002 .β, whereas XXMD treatment maintained the phosphorylation levels of these proteins, which were reversed upon pretreatment with LY294002. Changes in total levels of PDK1, Akt, and GSK3β were not observed in different groups at 24 h after reperfusion. Additionally, our data indicated that p-PTEN levels were decreased after ischemia and reperfusion, and XXMD treatment attenuated the declines in p-PTEN after ischemia. However, LY294002 administration had no effect on the expression of p-PTEN in the XXMD-treated rats. Another surprising finding in this study was that XXMD treatment in the absence or presence of LY294002 effectively upregulated the expression of p-c-Raf, which was decreased after cerebral ischemia and reperfusion. Moreover, the data showed that total PTEN and c-Raf levels of different groups were not significantly different. Since the effects of the PI3K inhibitor indicated that XXMD may exert NVU protection via PI3K/Akt signaling pathway, we further examined whether XXMD regulated PI3K/Akt pathway following focal cerebral ischemia and reperfusion. The expression levels of related proteins in the penumbra of ischemic area were detected by Western blots. As shown in Figures Stroke, as one of the leading causes of human death and disability, has become a severe threat to human health. Although the researchers have conducted plenty of preclinical studies and obtained many striking outcomes, translating bench success to the bedside proof of efficacy and safety has been frustrating . There iIn recent years, several scientists have focused on TCM, a potential and promising therapeutic approach for cerebral ischemia and investigated its efficacy and mechanism. XXMD is beneficial for the patients with stroke. A spate of studies has indicated that XXMD is prepared from twelve medical herbs containing a number of active fractions –46. The In the present study, we further investigated the NVU protective effects of XXMD after ischemia and reperfusion. The results clearly demonstrated that PI3K/Akt pathway played the crucial roles in NVU protection of XXMD against focal cerebral ischemia and reperfusion injury in rats. Increased attention has recently been drawn to NVU protection which has quite significant implications for cerebral ischemia and reperfusion injury. Given to its important role, we focused on evaluating apoptosis of neuron, astrocyte, and vascular endothelial cell, respectively, which were important components of NVU. The results suggested that apoptosis was a common event after ischemia and reperfusion. Although apoptosis mainly located in neurons, other components of NVU were also involved in this event, such as astrocytes and vascular endothelial cells. These results further indicated that it was necessary to pay attention to NVU protection in treatment of stroke. XXMD had multiple targets to reduce apoptosis of different cells in NVU. Moreover, obvious improvements of neurological deficits and infarct area were also observed in XXMD-treated group at 24 h after reperfusion. β, and c-Raf were decreased in the penumbra of ischemic cortex at 24 h after reperfusion, and XXMD treatment improved phosphorylation levels of these proteins. The data also suggested that LY294002 reversed the effects of XXMD on phosphorylation levels of related proteins except for PTEN and c-Raf. These implicated that PI3K/Akt pathway contributed to neurovascular unit protection of XXMD after stroke with reperfusion  as the upstream of Akt may activate Akt by directly phosphorylating Thr308 and indirectly phosphorylating Ser473  and somePlenty of reports have shown that PTEN which prevents recruitment of Akt to the membrane for phosphorylation is a negative regulator of Akt , 66 and Akt, the serine-threonine kinase, is a key molecule in PI3K/Akt pathway and controls survival and apoptosis , 72 and β activity by phosphorylating it at Ser9 and further promotes neuronal survival [β phosphorylation of groups subjected to different administrations. The data showed a reduction in p-GSK3β at 24 h after reperfusion, but it was blocked by XXMD treatment. Additionally, PI3K inhibitor was involved in PI3K/Akt pathway and partly prohibited the expression of p-GSK3β in rats administrated with XXMD treatment. Activated Akt downregulates GSK3survival , 73, 74.c-Raf, a mitogen-activated protein kinase, is a crucial target of RAS/MARK pathway. Previous studies show that Akt mediates phosphorylation of c-Raf at Ser259 and inhibits Raf kinase activity , thus prAbove all, we highlighted NVU protection of XXMD and elucidated the role of PI3K/Akt pathway during this course in the current study. The findings indicated that XXMD reduced cerebral infarct area and improved neurological function after ischemia and reperfusion. More importantly, we found that XXMD could protect NVU from ischemia-induced apoptosis and may exert its effects, at least in part, via the PI3K/Akt signaling pathway. Whether other pathways or mechanisms were involved in the effects of XXMD on cerebral ischemia and reperfusion injury needs our further exploration.β, and c-Raf. Taken as a whole, these effects on PI3K/Akt pathway may help explain the NVU protection of XXMD. In light of these observations, our data revealed the underlying mechanisms of XXMD treatment for stroke and provided more support for clinical applications of XXMD.Focal cerebral ischemia and reperfusion injury were successfully induced by MCAO. Cellular apoptosis in NVU was increased in penumbra of ischemic cortex, and PI3K/Akt pathway dysfunction occurred after cerebral ischemia and reperfusion. The results indicated that treatment with XXMD reduced infarct area and cellular apoptosis and improved function outcomes after stroke. Moreover, we demonstrated that such protection may involve blunting the decreases in phosphorylation of PTEN, PDK1, Akt, GSK3


T-cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3) and programmed cell death-1 (PD-1) are T-cell exhaustion molecules. We investigated the expression of Tim-3 and PD-1 in HTLV-I infection. Tim-3 expression, but not PD-1 expression, was reduced on CD4+ and CD8+ T cells of HAM/TSP patients and HTLV-I carriers as compared to healthy controls. Tim-3 expression was also reduced in HTLV-I Tax-specific cytotoxic T lymphocytes (CTLs) as compared to cytomegalovirus-specific CTLs. Tim-3+, but not PD-1+, Tax-specific CTLs produced less interferon-γ and exhibited low cytolytic activity. However, we observed no difference in the expression of Tim-3 or cytolytic activity between Tax-specific CTLs of HAM/TSP patients or carriers. Moreover, HTLV-I-infected CD4+ T cells showed decreased Tim-3 expression. The decreased expression of Tim-3 in HTLV-I infection is a marked contrast to other chronic viral infections such as HIV and HCV infection, where Tim-3 expression is increased in T cells, including the virus-specific CTLs. In HTLV-I infection, CTL response may not be negatively regulated by Tim-3. Rather, immune cells such as HTLV-I-specific CTLs may be resistant to cell death through the Tim-3/galectin-9 pathway. In summary, our data suggest that Tim-3 expression is reduced in HTLV-I infection and that a high number of Tim-3- HTLV-I-specific CTLs preserves their cytolytic activity, thereby controlling viral replication.


HTLV indeterminate WB patterns are frequently observed in plasma/serum samples from persons living in intertropical areas.In the framework of ongoing projets on HTLV-1/2 and related viruses in central Africa, we systematically analysed by WB, plasma from villagers living in south Cameroun. The studied group included 2155 individuals , either Bantous (1258) or Pygmies (897). All plasma samples were tested by WB (HTLV 2-4 MPD) with interpretation done according to manufacturer instructions. Only clear bands were considered as positive/informative. DNA extracted from buffy-coat were subjected to PCR using several primer pairs known to detect HTLV-1/2/3/4. Positive PCR bands were sequenced.Among the 2155 plasma samples, 48 were HTLV-1, 20 HTLV-2, and 134 HTLV. Furthermore, 955 were indeterminate including 100 HGIP (HTLV-I Gag-indeterminate pattern) [Search for the origin of this frequent new WB is ongoing with special insights concerning cross-reactivities with parasitic antigens as suggested for the HGIP pattern .


Data suggests that antidepressants are useful in the management of depressive disorders, anxiety disorders, sexual dysfunction, eating disorders, impulse control disorders, enuresis, aggression and some personality disorders. Research focusing on the usefulness of antidepressants in India has more or less followed the trends seen in the West. Most of the studies conducted in India have evaluated various antidepressants in depression. In this article, we review studies conducted in India on various antidepressants. The data suggests that antidepressants have been evaluated mainly in the acute phase treatment and rare studies have evaluated the efficacy in continuation phase treatment. Antidepressants as a class of drugs are used primarily in the management of depressive disorders and anxiety disorders. However, this class of drugs is also used for the management of sexual dysfunction, eating disorders, impulse control disorders, enuresis, aggression and some personality disorders.Over the years many classes of antidepressants have become available in India, some of which have stood the test of time and are still in use and some, which are no more marketed or are no more a favorite of clinicians. The research focusing the usefulness of antidepressants in India has more or less followed the trends in the west; however, some of the antidepressants drugs which have been marketed have not been evaluated as thoroughly as others.Most of the studies done in India have evaluated various antidepressants in depression. There are very few studies which have evaluated antidepressants in conditions other than depressive disorders. In this article, we review studies done in India on various antidepressants. The review shall focus on the research published in Indian Journal of Psychiatry and studies reported in PubMed indexed journals on efficacy, effectiveness, usefulness and tolerability issues of antidepressants in human subjects.The trials done to evaluate the efficacy of antidepressants can be divided into studies evaluating an antidepressant (no comparator studies), efficacy of an antidepressant with placebo as a comparator, comparing efficacy of 2 active drugs and those evaluating the efficacy of antidepressants with other modalities of treatment like electro-convulsive therapy or psychological treatment.et al.[A total of 18 open trials without a comparator group have been conducted to evaluate the efficacy of various antidepressants .[1–18] Set al. in additSix placebo controlled trials have evaluated the efficacy of tricyclic antidepressants in depression .19–24] –24 19–24There have been 18 trials which have compared two antidepressants.–41 One tet al.[Of the 21 trials, 18 have assessed the outcome of depression at the end of trial on Hamilton depression rating scale. Findings from these trials suggest that imipramine is superior to Nialmide, phenelziet al. suggestset al.Active Comparator Group  Trials of Efficacy/Effectiveness.One study has compared the usefulness of antidepressants with respect to non pharmacological treatment and two 46Sudarshan kriya in the management of depression over the period of three weeks.[et al. reported quicker response with ECT compared to imipramine.One of these studies has shown that pharmacotherapy is more effective and more economical than non-pharmacological treatment. However,ee weeks. The studee weeks.47 howeveSeven trials have evaluated the different dosing schedules for treatment of depression.–56 These5254Chakrabarti and Kulhara58 evaluaOne double blind controlled trial has evaluated the efficacy of clomipramine in the treatment of OCD and showed that clomipramine was superior to placebo in the management of OCD. This stuOne study evaluated the efficacy of antidepressants in insomnia and showed that trimipramine was similar to nitrazepam for treatment of insomnia, especially in the presence of anxiety and depression; however, it had poor tolerability as compared to nitrazepam.One trial included subjects with generalized anxiety disorder, mixed anxiety depression and dysthymia and showed that imipramine was as effective as diazepam for anxiety symptoms and better than diazepam for the depressive symptoms.One trial used imipramine in combination of chlorpromazine and compared it with chlorpromazine alone in the treatment of depressive symptoms in schizophrenia and didn’t find any benefit of adding imipramine to chlorpromazine in the treatment of treatment of depressive symptoms in schizophrenia.et al.[Two studies have also studied the usefulness of antidepressants in common mental disorders. One study showed that treatment completion rates were higher with fluoxetine than imipramine. The triaet al. includedet al. (2003)[Various sexual side-effects of antidepressants have been utilized for the management of sexual dysfunction. In a recently published open trial Dhikav et al. comparedl. (2003) found clTwo studies have evaluated imipramine for management of enuresis in children.71 In oneBesides the above studies, case series and case reports have also shown usefulness of antidepressants in the management of trichotillomania with trichobezoar, AtypicalAs is evident from Tables 101109116One case report presented tricyclic withdrawal syndrome with amitriptyline 300 mg/day and anotet al.[In a case report, Gupta et al. describeMany studies have evaluated the efficacy of antidepressants in depression and have shown that most of the currently marketed antidepressants are useful. In addition, studies also suggest usefulness of antidepressants in generalized anxiety disorder, dysthymia and common mental disorders. Many of the recent studies have been of good design and have followed double blind randomized controlled design and had reasonable sample size. Further, several studies have been carried out at multiple sites throughout the country. The available data also suggest that antidepressants are more cost-effective than other modalities of treatment for depression.In addition, there is some evidence to suggest the usefulness of clomipramine in OCD and that of fluoxetine in management of OC symptoms in schizophrenia. However, some major limitations of the research have been that almost all the data available in relation to treatment of depression pertains to acute phase treatment and rarely studies have evaluated the continuation phase treatment. There is also lack of data with regard to the efficacy and effectiveness in the maintenance phase treatment. Surprisingly, no study has evaluated the efficacy/effectiveness of SSRIs in the management of OCD.There is a need to conduct studies to evaluate the usefulness of antidepressants in the management of panic disorder and depression in medically ill subjects. Studies are also required to evaluate the efficacy of SSRIs in the management of OCD, and to study the usefulness of polypharmacy in the management of depression and other disorders. Studies are few and sparse and there is a need for multi-centric studies in such a vast country.


N-acylethanolamines, oxidatively modified galactolipids, and others. In addition, advances in mass spectrometry-based analysis, which allow sensitive identification of lipids with structural information, have raised many new questions: How many lipids are there in plants? How do plant lipidomes change in response to growth, developmental, and stress cues? As a result, many lipid mediators remain to be identified, and this volume provides a current, baseline knowledge on lipid signaling molecules and their actions in plants.Membrane lipids provide both the structural basis for cell membranes and a rich source of cellular mediators that regulate many aspects of plant development and environmental interactions. Over recent years, lipids as hormones and signaling messengers have gained increasing attention in the plant biology community. This volume collected 20 articles, including both primary research articles and several timely reviews on lipid signaling pathways, from active researchers addressing various fundamental questions in lipid signaling in plants , abiotic stresses, plant-microbe interactions, and in plant growth and development. Some intriguing aspects of plant lipid mediators are also addressed, such as how lipids might play roles as long distance mobile signals in addition to their localized actions, contributing to processes such as flowering and defense against pathogens. One major challenge has been to elucidate mechanistically how lipid mediators carry out their functions. Recent advances in oxylipins, particularly metabolites in the jasmonate pathway, provide an excellent example of how some key players in the signaling cascade that have been identified and interact directly with target proteins to influence changes in gene transcription. At the same time, these articles on oxylipins emphasize the difficulty of assigning functions to lipid mediators when multiple metabolites within a pathway have biological actions . Identifying lipid-interacting proteins represents an exciting area for future research that will improve our understanding of how different signaling networks in plant cells are integrated. However, translating the milieu of lipid metabolite changes in cells into the mechanisms for regulation of physiological processes in plants will remain a formidable challenge in the coming years.myo-inositol phosphates, appear to play a role in energy homeostasis in plants. Furthermore, biophysical studies have provided information on how PA and its phosphorylated product, diacylglycerol pyrophosphate, interact with proteins and/or cell membranes, suggesting a means for different cellular effects of these two metabolically related classes of signaling lipids.Elsewhere in the volume, the contribution of lipid head-group differences and their potential for selective actions are suggested. The potential roles of phosphoinositides in nuclear function and in the dynamics of membrane trafficking and cell expansion are discussed. In addition, head-group modifications and their metabolites, like the The publication of this book would not have been possible without the efforts of many people. First and foremost are the authors who responded enthusiastically to the call to contribute to the special volume. And the essential critical comments from the many peer reviewers are gratefully acknowledged which provided valuable feedback to ensure the highest quality, and up-to-date information in the articles.


Chromatin remodeling is a rapidly emerging field with critical implications for the control of viral gene expression, especially for viruses with integrated genomes, such as HTLV-1. Recent observations indicate that there are as many as eight different BRG1 containing chromatin remodeling complexes highlight the advancement in the field, but also the necessity for future study especially when looking at viral infections. In the current study we focused on few of the Baf subunits that are common to most SWI/SNF complexes. We find that at least three Bafs, Baf53, Baf155 and Baf170, are highly regulated in HTLV-1 infected cells. Along these lines others have shown that depletion of Baf53 leads to the expansion of chromosome territories and decompaction of the chromatin. Here we show that there are clear varying differences between the Baf subunits after viral infection. These subunits also co-elute in different places from a sizing column and one particular form, Baf53 may be phosphorylated in HTLV-1 infected cells. Normally Baf53 is a suppressive complex and knock down experiments show increased level of virus gene expression from transfected or chronically infected cells.


Mesenchymal stem cells (MSCs) are multipotent cells mainly found in the bone marrow (BM) and are considered to support human hematopoiesis. During HTLV-1 infection, T cells that are infected may migrate to the BM, and establish a progressive viral invasion. The persistence of these cells in the BM may affect the function of the resident BM cells and the biological properties of this microenvironment. However, the exact mechanisms of this phenomenon and the relative contribution of HTLV-1 to BM cell dysfunction remain to be elucidated. In this study, we evaluated the ability of HTLV-1 to disturb MSCs normal functions.Human MSCs were cultured on the presence of MT-2 cell line. MSCs markers and adipogenic differentiation potential was evaluated.We observed that MSCs that had contact with HTLV-1 were able to differentiate into adipocytes. However, gene expression of PPARG, cell morphology and intracellular lipid formation analyses revealed that adipogenic differentiation of MSCs that were exposed to HTLV-1 was decreased compared to MSCs not exposed (p<0.03). In addition, analyses by flow cytometry showed a reduction of the expression of MSCs markers (CD271 and HLA-DR) in MSCs that had contact with HTLV-1.HTLV-1 caused significant changes in the characteristics and functions of MSCs. This finding suggests the importance of HTLV infection on MSCs, and this could contribute to the understanding the viral infection mechanisms and pathogenesis.


Other studies have reported high rates of depression and anxiety among human T-lymphotropic virus type 1 (HTLV-1) infected subjects, and have even suggested that HTLV-I causes psychiatric disease.We interviewed HTLV-I, HTLV-II and demographically similar HTLV seronegative blood donors with the Mini-International Neuropsychiatric Interview (MINI). Prevalences of major depression and generalized anxiety disorder in each group were calculated and compared to published U.S. population data. Adjusted odds ratios (aOR) and 95% confidence intervals (95% CI) controlling for educational achievement, alcohol intake and self-reported health status were calculated with multivariate logistic regression.Major depression was diagnosed in 5 (5.4%) of 93 HTLV-I positive subjects  and 17 (6.6%) of 256 HTLV-II positive subjects , compared to 12 (2.1%) of 585 HTLV seronegative blood donors. The prevalence of major depression among infected subjects was comparable to the 6.7% prevalence in the U.S. general population. Generalized anxiety disorder was diagnosed in 5 (5.4%) HTLV-I positive subjects  and 12 (4.7%) HTLV-II positive subjects (OR = 1.65 95% CI 0.68-4.01), compared to 15 (2.6%) seronegatives and 3.1% in the U.S. general population.We observed slightly higher prevalence of major depression and generalized anxiety disorder among HTLV-I and HTLV-II subjects that was not significantly elevated after controlling for health status and other confounding variables. Comparison to U.S. population data suggested that these findings are in part explained by a “healthy blood donor effect” among our controls.


ATL is a highly aggresive lymphoproliferative disorder caused by HTLV-1. While HTLV-1 proviral load increase with progression of the disease, changes following therapy in ATL have not been well characterized. We present a case of ATL in complete remission with persistent high HTLV-1 proviral load.6 PBMCs and log10 5.3 copies/106 PBMCs in September 2009 and December 2009, respectively. Due to persistent high proviral load, the therapy was changed to pegylated interferon, tenofovir and lamivudine (3TC). Proviral load levels were also high at 4 and 14 months on treatment .A 49-year-ol man was diagnosed as HTLV-1 carrier after a blood donation in 2004. He was born in Buenos Aires, a non-endemic area of Argentina. Four years later, he was diagnosed with T-cell lymphoma but rapidly blast cells were observed in peripheral blood and it was classified as T-cell leukemia . Chemotherapy was started in November 2008 and reached complete remission after 6 cycles. In May 2009, Interferon and zidovudine was started as maintainance therapy. HTLV-1 proviral load were log10 5.5 copies/10With the combination of interferon and reverse transcriptase inhibitors for more than one year, the patient is in complete remission, 27 month after the diagnosis, despite the fact that he never even attained reduction of HTLV-1 proviral load.


The level of HTLV-I expression is low in vivo. We found that recombinant interferon (IFN)-α and β suppressed viral expression in HTLV-I-infected cells. Non-lymphoid stromal cells also suppressed HTLV-I expression through type-I IFNs. The suppression was reversible after isolation of infected cells from the source of IFNs, mimicking the status of viral expression in freshly isolated ATL cells which is rapidly induced in culture. Nevertheless, HTLV-I-infected individuals maintain acquired immune responses against HTLV-I such as antibodies and cytotoxic T lymphocytes (CTLs), indicating the presence of HTLV-I proteins in vivo. Analysis on Tax-specific CTLs revealed that they were activated in HAM/TSP but unresponsive in ATL patients. We found that a subpopulation of HTLV-I carriers at asymptomatic stage exhibited impaired Tax-specific T-cell response and elevated HTLV-I proviral load. This combination is a feature of ATL and likely to be an underlying risk of ATL. Collectively, HTLV-I is doubly controlled by acquired and innate immunity; HTLV-I-specific CTLs eliminate infected cells, and IFNs suppress viral expression. Both would contribute the reduction of viral pathogenesis, while the efficiency of CTLs could be partial because of limited viral expression. An increase in viral expression would activate CTLs but also accelerate inflammation. When the viral expression is well-controlled, viral pathogenesis may not be apparent until infected cell clones with a malignant phenotype finally emerge, which may occur earlier without proper CTL responses. Diversity in innate and acquired immune responses among individuals might be important determinants of disease manifestation in HTLV-I infection.


It has been three decades since the discovery of human T-cell leukemia virus type 1 (HTLV-1), which is the first human retrovirus etiologically associated with an aggressive malignancy of CD4+ T cells known as adult T-cell leukemia (ATL) and a disabling chronic inflammatory disease of the central nervous system known as HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 infection is of particular interest to the field of immunology as well as virology, because HTLV-1 is never eliminated from the host in spite of a vigorous cellular and humoral immune responses against the virus, and it causes no disease in the majority (around 95%) of the infected subjects (asymptomatic carriers: ACs). Although accumulating evidence suggests the importance of complex virus-host interactions and the host immune response in determining the risk and timing of disease, the precise mechanism of disease pathophysiology is incompletely understood and the treatment is still unsatisfactory. The patients with ATL have a poor prognosis and approximately 40 percent of HAM/TSP patients become wheelchair-bound during their clinical course. In this special issue on “HTLV-1 Infection and Its Associated Diseases,” we have invited nine papers that address the recent developments in HTLV-1 research in order to elucidate the pathogenetic mechanisms and to identify effective means of treatment and prevention of HTLV-1 associated diseases. Three papers describe the important advances in understanding the molecular and virological aspects of HTLV-1. One paper explains the significant role of HTLV-1 basic leucine zipper factor (HBZ), a regulatory protein encoded in the minus strand of the HTLV-1 genome, in the viral pathogenesis. Another paper summarizes the comparative biology of HTLV-1 and HTLV-2. The paper by N. Aliya et al. discusses the possible interplay between HTLV-1 infection and miRNA pathways. Two papers describe the clinical trials and treatment for ATL from different groups, to give contrasting views on the current state of knowledge and current approaches to this subject. There is a paper that provides an overview of recent developments in HAM/TSP research, while another paper summarizes the current understanding of the host immune response against HTLV-1 infection, especially the potential importance of HTLV-1-specific CTLs in protection against ATL. Two clinical studies for ATL are also included. One of them describes the clinical and pathological characteristics of seven patients with HTLV-1-positive large B-Cell lymphoma, while another one discusses the hypercalcemia that is frequently observed in ATL patients. The papers in this special issue demonstrate important recent progress in HTLV-1 research and will bring the reader up to date with the current understanding of HTLV-1 infection and its associated diseases.Mineki SaitoMineki SaitoPooja JainPooja JainKunihiro TsukasakiKunihiro TsukasakiCharles R. M. BanghamCharles R. M. Bangham


Phosphorylation is the predominant language of cell signaling. And, as with any common language, an abundance of dialects has evolved to convey complex information. We discuss here how biosensors are being used to decode this language, affording an unprecedented glimpse into spatio-temporal patterns of protein phosphorylation events within the cell. Intracellular protein kinases relay information that originates from diverse chemical and ionic signals received at the cell surface, phosphorylating their substrates to elicit a variety of responses. A relatively small group of second messenger molecules, and the kinases they activate, function in a plethora of different signaling pathways, posing the question of how the specificity of the original signal is not lost in transmission. One answer to this appears to be the routing of signals with spatial fidelity and tight temporal control, through signaling complexes brought together on protein scaffolds. As we illustrate in this article, direct insights into local patterns of kinase activation have been made possible by the ingenious development of genetically encoded kinase activity reporters. These biosensors translate the language of cell signaling, as it occurs in real time, into observable bursts of light.2+ and lipid second messenger responses. Accordingly, this technology offers a relatively non-invasive way to monitor the action of diffusible chemical signals or protein phosphorylation events in real time.Nature has an abundance of fluorescent proteins that absorb and emit light . Since t2+ oscillations [The concept of using genetically encoded fluorescent reporters to visualize kinase signaling was pioneered by Tsien and others, who constructed kinase activity reporters for the epidermal growth factor receptor and for protein kinase A  using the modular structure shown in Figure llations .Genetically encoded FRET reporters have also been developed to measure dynamic changes in second messenger levels ,8. TheseBecause these signaling biosensors are genetically encoded, subcellular targeting motifs can be incorporated into their modular structure to direct these reporters toward organelles and intracellular membranes. Such targeted reporters have been useful to monitor the rates of signal transmission and kinase activity at the plasma membrane, Golgi, mitochondria, and recently in the nucleus ,10,11. FAnother means to monitor compartmentalized kinase activity is to fuse biosensors to scaffolding proteins. The best-characterized class of kinase scaffolds are the A-Kinase Anchoring proteins (AKAPs) that compartmentalize the cAMP-dependent protein kinase (PKA) to defined locations in the cell ,17. Thusphosphoinositide-dependent kinase-1 (PDK-1) activation reporter  activity is also under acute spatiotemporal control and this has been visualized by BKAR . AlthougTargeting of the PKC activity reporter CKAR to various intracellular membranes  has provided much insight into the rate, magnitude, and duration of agonist-evoked PKC signaling throughout the cell . In a stProtein scaffolds also serve as a nexus for signaling by PKD. Of the three PKD isoforms, PKD1 and PKD2 have distinct PDZ ligands that bind the PDZ domains of NHERF, a family of scaffolds that coordinates signaling by ion channels, G-protein coupled receptors, and tyrosine kinase receptors . Fusion A number of variants on FRET technology continue to expand our toolbox for visualizing signaling. One such variation is bioluminescent resonance energy transfer (BRET), which has been used to show dynamic interactions between the insulin receptor and the scaffold Grb14 . AnotherIn conclusion, the use of FRET-based reporters has allowed unprecedented insight into the cellular dialect of phosphorylation on protein scaffolds and membrane microdomains. By coordinating enzymes that propagate and terminate signaling, such macromolecular assemblies afford exquisite control of signaling in time and space. With the development of dual FRET pairs, the field is now poised to visualize the simultaneous activity of two kinases -33. Thes


Enhanced P-glycoprotein (P-gp) activity has been reported in epilepsy and this may confound interpretation of clinical scans if [11C]flumazenil is a P-gp substrate. The purpose of this study was to investigate whether [11C]flumazenil is a P-gp substrate.[11C]Flumazenil PET scans were performed in wild type (WT) (n = 9) and Mdr1a/1b, (the genes that encode for P-gp) double knockout (dKO) (n = 10) mice, and in naive rats (n = 10). In parallel to PET scanning, [11C]flumazenil plasma concentrations were measured in rats. For 6 of the WT and 6 of the dKO mice a second, [11C]flumazenil scan was acquired after administration of the P-gp inhibitor tariquidar. Cerebral [11C]flumazenil concentrations in WT and Mdr1a/1b dKO mice were compared (genetic disruption model). Furthermore, pre and post P-gp-blocking cerebral [11C]flumazenil concentrations were compared in all animals .[Mdr1a/1b dKO mice had approximately 70% higher [11C]flumazenil uptake in the brain than WT mice. After administration of tariquidar, cerebral [11C]flumazenil uptake in WT mice increased by about 80% in WT mice, while it remained the same in Mdr1a/1b dKO mice. In rats, cerebral [11C]flumazenil uptake increased by about 60% after tariquidar administration. Tariquidar had only a small effect on plasma clearance of flumazenil.11C]flumazenil is a P-gp substrate in rodents. Consequently, altered cerebral [11C]flumazenil uptake, as observed in epilepsy, may not reflect solely GABAA receptor density changes but also changes in P-gp activity.The present study showed that [ This may be caused by limited drug distribution across the blood-brain barrier (BBB) due to increased activity of multidrug efflux transporters, such as P-glycoprotein (P-gp) . Recent ubstrate . Current surgery ,4. If flumazenil is indeed a P-gp substrate in rodents, i.e., to assess whether changes in cerebral [11C]flumazenil uptake may reflect not only changes in GABAA receptor density, but also in P-gp activity. For that reason, [11C]flumazenil scans, before and after administration of the P-gp blocker tariquidar , were performed in both Mdr1a/1b dKO and wild type (WT) mice (genetic disruption model) and in rats.Potentially genetic disruption of the P-gp encoding genes may induce upregulation of other transporters 1), while chemical inhibition of P-gp function might also inhibit other transporters, such as Bcrp1, in the case of tariquidar . TherefoMdr1a/1b dKO  and WT  mice , and adult Sprague-Dawley rats  were used. Approval of the institutional animal ethics committee was obtained, and all experiments were carried out in accordance with the Dutch Law on Animal Experimentation and guidelines of the institutional committee on animal experimentation. All animals were kept at a constant temperature of 21°C and a 12-h light/dark cycle, in which the lights were switched on at 8 a.m. Animals had unrestricted access to food  and water. Studies were performed after approximately 1 week of habituation.-1 (S)-ketamine  and 1.0 mg·kg-1 medetomidineMaintenance anesthesia was administered via the intraperitoneal cannula (37.5 mg·kg-1·h-1 (S)-ketamine and 0.5 mg·kg-1·h-1 medetomidine). Rats first received an intraperitoneal injection of 60 mg·kg-1 ketamine  followed by a second intraperitoneal injection of 0.4 mg·kg-1 medetomidine. To maintain anesthesia, an intravenous infusion into the tail vein of 10 mg·kg-1·h-1 ketamine and 0.1 mg·kg-1·h-1 medetomidine was administered. This was started between 60 and 120 min after the first intraperitoneal injections and, in most animals, the infusion was then continued for the remainder of the experiment.In each mouse a cannula was inserted in the jugular vein and one in the intraperitoneal space 1-2 h prior to PET scanning. Each rat underwent cannulation of the femoral vein and artery 1-2 h before PET scanning. From the start of surgery until the end of scanning, animals were anesthetized with a combination of (S)-ketamine and medetomidine . Mice received an intraperitoneal injection of 75 mg·kg11C]Flumazenil was synthesized as previously described [2PO4 and 10% v/v ethanol. The specific activity was 178 ± 69 GBq·μmol-1 (mean ± standard deviation (SD)). The radiochemical purity was higher than 99% as assessed by analytical high performance liquid chromatography (HPLC) equipped with radio and ultraviolet detectors. The HPLC analysis did not detect any chemical impurities. The identity of [11C]flumazenil was confirmed by comparison of the retention times with the authentic isotopically unmodified (unlabelled) flumazenil.[escribed  and 3-10137Cs (662 keV) moving point source [11 C]flumazenil to each mouse or 24.4 ± 6.7 MBq [11 C]flumazenil to each rat. The injected activity for each of the different animal groups and scans is shown in Table Animals were positioned in pairs in a double lutetium oxyorthosilicate/Lu1.8Y0.2SiO5(Ce) or LSO-LYSO layer high resolution research tomograph  PET scanner . First, t source . Next, a11C]flumazenil PET scans separated by 100 min to allow for radioactive decay of carbon-11, with the exception of three Mdr1a/1b dKO mice and three WT mice who received only a baseline [11C]flumazenil scan. Tariquidar, 15 mg·kg-1 , a dose that completely blocks P-gp function, was administered as a 15-min infusion 35 min before the second scan to all mice and as a 10-min infusion 20 min before the second scan to all rats, providing optimal inhibition conditions of tariquidar [Mdr1a/1b dKO mice received tariquidar to ensure that tariquidar does not influence [11 C]flumazenil brain distribution in the absence of functional P-gp and exclude any P-gp-unrelated pharmacokinetic interaction.Each animal underwent two consecutive [riquidar . Mdr1a/111C]flumazenil was modified to study brain distribution of [11C]flumazenil at different GABAA receptor occupancies; this was done by adding four different doses, 5, 20, 100 or 400 μg, of isotopically unmodified flumazenil to the injection solutions of [11C]flumazenil . These doses were chosen based on previous studies of receptor occupancy by flumazenil in rats [11C]flumazenil and unmodified flumazenil were calculated based on the specific activity of the tracer solution and the flumazenil concentration in the solution containing the isotopically unmodified flumazenil and are shown in Table In rats the specific activity of flumazenil. Time activity curves were normalized for injected dose and body weight, generating standardized uptake value (SUV) curves as a function of time:PET image data were analyzed using the freely available software package Amide 0.9.2 . Time-acbrain and represents the value at pseudoequilibrium between brain and plasma concentrations.For comparison of results, the average SUV over the period from 20-30 min post injection was used. This average is denoted as SUVbrain was compared between Mdr1a/1b dKO and WT mice to assess whether [11C]flumazenil is a P-gp substrate (genetic disruption model). Furthermore, as a measure of the effect of tariquidar on brain uptake of [11C]flumazenil, SUVbrain was compared in all animals between pre and post tariquidar scans . The Mdr1a/1b dKO mice served as controls, as they are devoid of P-gp, which is the target of tariquidar. For rats, SUVbrain was compared for each flumazenil dose group separately to investigate whether P-gp inhibition is affected by receptor occupancy. In addition, in rats, plasma concentrations of flumazenil were compared before and after tariquidar treatment to determine whether tariquidar had an effect on plasma kinetics of flumazenil.In mice SUVbrain between Mdr1a/1b dKO and WT mice was carried out by means of independent samples t tests with Bonferroni correction to adjust for multiple comparisons. SUVbrain comparisons before and after P-gp inhibition for each animal group separately  were performed using paired samples t tests, again with Bonferroni correction to adjust for multiple comparisons.All statistical analyses were performed in SPSS . The comparison of SUVMdr1a/1b dKO mice and nine WT mice had successful baseline [11C]flumazenil scans. Of all animals, six Mdr1a/1b dKO mice, six WT mice and ten rats had two successful [11C]flumazenil scans. One WT mouse died during surgery, and oneMdr1a/1b dKO mouse and one rat died after tariquidar treatment. The baseline scan of the mouse that died during the second scan has been included in the comparison of Mdr1a/1b dKO and WT mice before P-gp blockade, as this animal appeared to be healthy before tariquidar treatment. Clearly, for the comparison before and after tariquidar treatment, both animals that died during the second scan had to be excluded. Table 11C]flumazenil injected.Ten Mdr1a/1b dKO mice showed higher brain uptake than WT mice throughout the entire PET scan . Figure brain images of one Mdr1a/1b dKO and one WT mouse, both before tariquidar administration. After P-gp blockade, SUVbrain increased by 79% in WT mice , while no significant changes were observed in Mdr1a/1b dKO mice . Furthermore, after tariquidar treatment, the SUVbrain was not significantly different between WT and Mdr1a/1b dKO mice .Before treatment with tariquidar, 11C]flumazenil . This increase in SUVbrain caused by tariquidar treatment was similar for all flumazenil doses used; 62%, 55%, 59% and 87% for the 4, 20, 100 and 400 μg dose, respectively. Flumazenil concentrations in rat plasma, before and after tariquidar treatment, are shown in Figure As expected for a receptor ligand, increasing the dose of unlabeled flumazenil resulted in reduced cerebral uptake of [l Figure . SUVbrai11C]flumazenil uptake as inhibition of P-gp  in WT mice and rats, showing an approximately 70% higher SUVbrain both in Mdr1a/1b dKO mice and in P-gp inhibited WT rodents compared with WT animals prior to P-gp inhibition. Furthermore, SUVbrain in Mdr1a/1b dKO mice did not change following pharmacological P-gp inhibition. Together, these results demonstrate that [11 C]flumazenil is a P-gp substrate in rodents. Furthermore, [11C]flumazenil does not appear to be a Bcrp1 substrate in rodents, as SUVbrain in Mdr1a/1b dKO mice did not change after tariquidar treatment compared to baseline.Taken together, absence of P-gp in mice (genetic disruption model) had a similar effect on cerebral [11C]flumazenil concentration was due to an increased plasma [11C]flumazenil concentration after tariquidar treatment. To rule out this possibility, plasma flumazenil concentration curves were compared in rats. There was a trend for slightly increased plasma concentrations of flumazenil after tariquidar treatment  did not play a significant role. Also, previous test-retest [11C]flumazenil studies show no influence of the first scan on the second scan flumazenil brain-to-plasma concentration ratios in mice treated with cyclosporine A than in control animals. Cyclosporine A, however, is a first generation P-gp inhibitor, which tends to be less potent and nonselective than tariquidar and may also influence metabolism and elimination of drugs/radioligands via interaction with cytochrome P450 enzymes [ex vivo rodent studies and in vitro studies using canine cells (Madin Darby canine kidney) transfected with human MDR1 gene could be due to species differences in BBB transport of [11C]flumazenil, which has been observed for other PET radioligands flumazenil can be affected significantly by P-gp activity.Previous man P-gp ,19. In vermeable . In linevo study , describ enzymes . Thus, toligands . Althougoligands . In the 11C]verapamil, show a much larger  increase in brain concentrations after the same dose of tariquidar [11C]flumazenil scans, particularly if there are regional differences in P-gp upregulation as indicated by some studies in people with epilepsy [11C]flumazenil uptake may not only be due to decreased GABA-ergic function, but may also be the result of increased P-gp activity. If both effects occur simultaneously, the cerebral [11C]flumazenil uptake could even be decreased further. Whether this will affect the interpretation of the data depends on the purpose of the study. If the aim is to localize the epileptic focus, decreased [11C]flumazenil will, in every case, still help to identify the affected brain region. However, one will not be able to interpret the underlying mechanism(s). Furthermore, if future therapies will be developed with the specific aim either to normalize increased P-gp activity or to compensate for decreased GABA-ergic inhibition, quantitative information is needed on P-gp activity or GABA-ergic function, which cannot be obtained with [11C]flumazenil PET scans alone.It should be noted that more avid P-gp substrates, such as flumazenil in epileptic rats are needed to clarify whether local changes in cerebral [11C]flumazenil uptake in epilepsy are due to decreased GABA-ergic function, P-gp upregulation, or both. Furthermore, a comparison between naive and epileptic rats is needed to assess to which degree P-gp upregulation affects cerebral uptake of [11C]flumazenil. Ultimately, [11C]flumazenil studies in both pharmacoresistant and pharmacosensitive patients with epilepsy, as well as in healthy volunteers are needed to elucidate this important issue.PET studies with [11C]flumazenil is a P-gp substrate (but most likely not a Bcrp1 substrate) in rodents. This finding could have implications for preoperative evaluation of people with epilepsy as altered cerebral [11C]flumazenil uptake may be due to changes in both GABAA receptor density and P-gp activity, potentially complicating interpretation of data.The present study showed that [BBB: Blood-brain barrier; Bcrp: Breast cancer resistant protein; dKO: Double knockout; Mdr: Multidrug resistance gene; PET: Positron emission tomography; P-gp: P-glycoprotein; SUV: Standardized uptake value; VOI: Volume of interest; WT: Wild type.The authors declare that they have no competing interests.Each author contributed significantly to the submitted work. FEF and SS were in charge of the study, acquiring, analyzing, interpreting the data and drafting the manuscript. NHH and CFMM contributed in conception and design of the mice study. MCH was responsible for PET image reconstruction and contributed to data analysis. YT analyzed the rat plasma data. CFMM contributed in to the acquiring of PET data. JCR, JJH, ECML and AAL, with expertise in epilepsy research and preclinical study design and PET, contributed to the design of the study, analysis and interpretation of data. JE was in charge of the radiochemistry. RAV contributed to the design of the study, analysis and interpretation of all data and in drafting the manuscript. All authors read and approved the final manuscript.


HTLV-1 and HTLV-2 trans-activating proteins  differ with respect to their ability to activate genes regulating viral replication, latency, and host cellular immune responses. Although HTLV-2 infections are usually asymptomatic, immunologic alterations are observed. We previously showed that HTLV-2 tax/rex viral load was upregulated in patients with HIV-1/HTLV-2 co-infection. This correlated with higher CD4+ T cell counts and improved health outcomes, possibly due to induction of CC-chemokines.In this study, recombinant Tax1 and Tax2 proteins were expressed in E. coli. PBMCs were incubated with different concentrations of Tax proteins (10-100 pM). Supernatant fluids and cells were harvested at multiple time points for quantitative determinations of MIP-1alpha/CCL3, MIP-1beta/CCL4, RANTES/CCL5, and CCR5 receptor expression.In preliminary experiments it was shown that PMBCs from HTLV-2 infected donors had significantly lower levels of CCR5 expression and higher levels of RANTES/CCL5 compared to HTLV-2 seronegative donors (p<0.05). Tax1 and Tax2-treated PBMCs showed increased viability over a seven day period compared to controls (p<0.01). Both Tax1 and Tax2 induced equally high levels of all three CC-chemokines compared to mock-treated controls (p<0.05). Tax2-treated PBMCs showed a significantly lower percentage of CCR5-PE positive cells compared to mock-treated PBMCs within the gated lymphocyte population (p<0.05).Recombinant Tax2 is a potent modulator of CC-chemokines and CCR5 in vitro. Further investigations are needed to determine the underlying mechanism(s), and whether Tax2 recapitulates the observed effects in vivo.


Interestingly, CysLT concentrations were increased in HAM/TSP patients. Also, the concentration of plasma LTB4 and LTC4 positively correlated with the HTLV-1 proviral load in HTLV-1-infected individuals. The gene expression levels of LT receptors were differentially modulated in CD4+ and CD8+ T cells of HTLV-1-infected patients. Analysis of the overall plasma signature of immune mediators demonstrated that LT and chemokine amounts were elevated during HTLV-1 infection. Importantly, in addition to CysLTs, IP-10 was also identified as a biomarker for HAM/TSP activity. These data suggest that LTs are likely to be associated with HTLV-1 infection and HAM/TSP development, suggesting their putative use for clinical monitoring.Leukotrienes (LTs) are lipid mediators involved in several inflammatory disorders. We investigated the LT pathway in human T-lymphotropic virus type 1 (HTLV-1) infection by evaluating LT levels in HTLV-1-infected patients classified according to the clinical status as asymptomatic carriers (HACs) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients. Bioactive LTB Human T-lymphotropic virus type 1 (HTLV-1), a complex retrovirus, is the causal agent of adult T cell leukemia (ATL), HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and other inflammatory disorders that develop after a variable period of latency ranging between months and decades HAM/TSP is a neuroinflammatory disease characterized by a chronic progressive myelopathy with infiltrating mononuclear cells in the areas of demyelination and axonal dystrophy 2 (PLA2) is oxidized by 5-lipoxygenase (5-LO) in combination with 5-LO-activating protein (FLAP) to generate the leukotriene A4 (LTA4). The downstream enzymes LTA4 hydrolase (LTA4H) and LTC4 synthase (LTC4S) give rise to leukotriene B4 (LTB4) and leukotriene C4 (LTC4). LTC4 is further converted to LTD4 and LTE4, which are collectively termed cysteinyl leukotrienes (CysLTs) respectively. LTB4 and CysLTs signal through distinct cell surface receptors named BLT1 and BLT2 and CysLT1 and CysLT2, respectively 4 is recognized as a potent leukocyte chemoattractant that also displays leukocyte activating functions, whereas the CysLT are better known for leading to airway constriction, increased vascular permeability, mucus secretion and cell trafficking Leukotrienes (LTs) are bioactive lipid mediators involved with inflammatory conditions Considering the importance of LTs as powerful mediators of inflammation, the present study was undertaken to test the hypothesis that HTLV-1 infection leads to an exacerbation of the 5-LO products formation and LT signaling in patients with HAM/TSP. We examined LT concentrations in plasma, the ability of PBMCs to produce LTs and LT receptor expression in lymphocytes from HTLV-1 patients. We also investigated the overall plasma LT, chemokine and cytokine signatures of HACs and HAM/TSP patients. Moreover, we investigated the correlations between LTs, chemokines and cytokines in HTLV-1-infected individuals and the capacity of LTs to modulate cytokine production. Our results demonstrate for the first time that LTs are upregulated during HTLV-1 infection, suggesting a role for LTs in HAM/TSP pathogenesis and presenting them as potential biomarkers for monitoring HAM/TSP development.4 was increased in the plasma of HACs and HAM/TSP patients when compared to that of NI donors; however, no difference was observed in LTB4 levels between HACs and HAM/TSP patients  (data not shown), and based on these values, each volunteer was classified as a low- (□) or high (▪)-mediator producer . On the other hand, LTs are involved in the control of host defense, including defense against HIV 4 generation observed in the PBMCs of HAM/TSP patients when compared to HACs may not represent a good method for controlling the high HTLV-1 set points seen in HAM/TSP patients. In addition, it was interesting to note the lack of correlation between 5-LO and LTC4S gene expression and LT generation by human PBMCs. Despite this dissociation between changes in 5-LO mRNA levels and protein expression, which has been reported previously LTs are produced primarily by neutrophils, eosinophils, mast cells and monocytes/macrophages + and CD8+ T cells 1 was upregulated in CD4+ T cells from HAM/TSP patients. Meanwhile, CysLT1 was upregulated in CD4+ T cells but downregulated in CD8+ T cells of HACs and HAM/TSP patients. In animal models, deletion of BLT11+ T cells may bias the host toward cellular infiltration of inflamed tissues, worsening the HAM/TSP disease. Moreover, as HTLV-1 preferentially affects CD4+ T cells, the migration of non-infected CD4+ T cells to inflamed sites containing HTLV-1-infected lymphocytes could facilitate cell-cell contact and consequently the spread of infection.The upregulation of LT receptors has been noted previously in experimental neuroinflammatory disease and is thought to be involved in the pathogenesis of this disease Studying the immunological response to HTLV-1 infection is important for the understanding of HAM/TSP pathogenesis. Others have shown that CXCL9 In this report, we demonstrate that HTLV-1 dysregulates the LT pathway. This finding has important implications for the understanding of HAM/TSP. First, CysLTs could be used as a biomarker for HAM/TSP development. Second, the fact that LTs are mediators of inflammation suggests that the LT pathway might be involved in HAM/TSP pathogenesis. Therefore, further experiments will be required to elucidate a potentially pathogenic function of LTs in HAM/TSP. Moreover, it will be interesting to determine whether drugs targeting the LT pathway ameliorate HAM/TSP symptoms.(tax and LTR regions). Subjects with HAM/TSP were selected from among a heterogeneous disease progression group. Individuals receiving therapies were excluded. All procedures were approved by the Ethical Committee of the University Hospital, School of Medicine of Ribeirão Preto, University of São Paulo (process number 1108/2008), and all subjects provided written informed consent.The study subjects were classified as asymptomatic HTLV-1 carriers or HAM/TSP patients in accordance with the criteria proposed by the World Health Organization. Non-infected (NI) healthy volunteers were included as a control . Biologi+ and CD8+ T cells by positive selection using the manufacturer’s protocol. Phenotypic analysis performed by flow cytometry (BD-FACSCanto) using anti-CD4-FITC, anti-CD8-FITC and anti-CD3-PE antibodies (BD Biosciences) demonstrated a minimum of 80% purity of CD4+ and CD8+ lymphocytes. A hemocytometer chamber was used to obtain absolute cell counts, and cell viability was determined by trypan blue exclusion.After separation of plasma from the heparinized venous blood, PBMCs were isolated using Ficoll-Paque  density gradient centrifugation. To isolate lymphocytes from PBMCs, magnetic beads conjugated with anti-CD4 or anti-CD8 antibodies  were used to separate CD4β-actin and HTLV-1 tax were performed in duplicate on the same plate. The HTLV-1 proviral load was calculated using the following equation: average of tax/average of β-actin×2×105. The values obtained were Log10 transformed for the correlation analysis.HTLV-1 proviral load was quantified as previously described 4 synthase: Hs_00168529_m1; BLT1: Hs_00175124_m1; CysLT1: Hs_00929113_m1) and analyzed using 7500 System SDS software. The relative mRNA expression was determined using the ΔΔCT method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH: 4310884-E) was used as an internal control for PBMCs. The geometric means of the values obtained for β-actin (ACTB: 4326315E), GAPDH, β2 microglobulin (B2M: 4333766-0710013) and ribosomal protein L13a (RPL13a: 185720330-7) were used as internal controls for CD4+ T cells, and ACTB was used as an internal control for CD8+ T cells.Total RNA was extracted from PBMCs and lymphocytes with TRIzol (Invitrogen) according to the manufacturer’s instructions, and reverse transcription was carried out with 2 µg of total cellular RNA using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). Thereafter, quantitative RT-PCR was performed using ABI 7500 Sequence Detection (Applied Biosystems). The reaction was performed in duplicate using TaqMan assay reagents (Applied Biosystems)  to remove cells and debris, and the virions were concentrated 10 times by ultracentrifugation for 2 hours at 100,000 g. The pellet containing virus particles was resuspended in RPMI and quantified before being subjected to HTLV-1 p19 ELISA (ZeptoMetrix).The human T cell line MT-2 was used as a source of HTLV-1 producing cells. For preparation of cell-free HTLV-I, cells were seeded at 5×106/well) in forty eight-well plates and maintained them overnight at 37°C and 5% CO2. The cells were provided with fresh RPMI 1640 containing 5% AB human serum (Sigma) and 100 U/mL penicillin and cultured for an additional 48 hours. For analysis of the effects of cell-free HTLV-1, cell cultures from healthy donors were challenged with 10 ng of virions particles  prior additional culture. For leukotriene detection, the supernatants were removed, and the cells were resuspended in HBSS containing Ca2+ and Mg2+ and stimulated for 30 minutes with 0.5 µM of the calcium ionophore A23187 (Sigma), and then, reactions were stopped using ice.We plated PBMCs  prior to performing the assay. Moreover, the cell-free supernatants were tested for IP-10 and TNF-α, and the plasma samples were tested for MCP-1, MIP1-α, IP-10, IL-8, IL-5, IL-4, IL-13, IL-1, IL-6, GM-CSF, TNF-α, IL-12, IFN-γ, and IL-10 using a Duoset ELISA Development kit (R&D Systems) and for IL-17 and IL-23 using an OptEIA ELISA kit (BD Bioscience) in accordance with the manufacturer’s instructions. The reactions were performed in 96-well ELISA plates (Corning), and the optical densities were determined at 450 nm using a microplate reader. The cytokine concentration in each sample was estimated by interpolation of sample optical densities with the cytokine standard using a four-parameter curve-fitting program.A specific enzyme immunoassay (Cayman) was used to quantify LTBA method for identifying low and high producers of mediators by analyzing cytokine profiles was previously reported by Luiza-Silva et al. t-tests or ANOVA with Bonferroni’s post-test as appropriate to identify significant differences between group means using GraphPad Prism version 5 (GraphPad Software).The data are presented as means ± SEM of values determined from the indicated number of samples. The data were analyzed by Student’s Spearman’s correlation test was performed to assess the association between the levels (pg/mL) of LTs, chemokines and cytokines while Person’s test was used to analyze the association of LTs and the HTLV-1 proviral load. In all cases, statistical significance was defined as p≤0.05. The cytokine signatures analyses were performed using the non-infected signature as the reference curve, and differences were considered significant when the values fell outside of the quartile of the reference signature. The use of the 50th percentile as the limit to identify relevant differences in the chemokine/cytokine/LT signatures between the groups has been adapted from a pioneering study by Luiza-Silva et al.