Human T-lymphotropic virus type 1 (HTLV-1) proviral load is related to the development of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and has also been shown to be elevated in the peripheral blood in HTLV-1-infected patients with uveitis or alveolitis. Increased proliferation of HTLV-1-infected cells in, or migration of such cells into, the central nervous system is also seen in HAM/TSP. In the present study, we evaluated the proviral load in a cohort of HTLV-1-infected patients with arthritic conditions.HTLV-1 proviral load in the peripheral blood from 12 patients with RA and 6 patients with connective tissue disease was significantly higher than that in matched asymptomatic HTLV-1 carriers, but similar to that in matched HAM/TSP controls. HAM/TSP was seen in one-third of the HTLV-1-infected patients with RA or connective tissue disease, but did not account for the higher proviral load compared to the asymptomatic carrier group. The proviral load was increased in the synovial fluid and tissue from an HTLV-1-infected patient with RA, the values suggesting that the majority of infiltrated cells were HTLV-1-infected. In the peripheral blood from HTLV-1-infected patients with RA or connective tissue disease, HTLV-1 proviral load correlated with the percentages of memory CD4+ T cells and activated T cells, and these percentages were shown to be markedly higher in the synovial fluid than in the peripheral blood in an HTLV-1-infected patient with RA.These biological findings are consistent with a role of the retrovirus in the development of arthritis in HTLV-1-infected patients. A high level of HTLV-1-infected lymphocytes in the peripheral blood and their accumulation in situ might play a central role in the pathogenesis of HTLV-1-associated inflammatory disorders. Alternatively, the autoimmune arthritis, its etiological factors or treatments might secondarily enhance HTLV-1 proviral load. Human T-lymphotropic virus type 1 (HTLV-1) is endemic in southern Japan, intertropical Africa, Melanesia, Latin America, and the Caribbean basin  and HTLVThe possibility that HTLV-1 may cause joint disease was initially raised by reports of arthralgia and polyarthritis in patients with adult T-cell leukemia ,11. Poly+ T cells, are the main target of HTLV-1 in vivo and carry the majority of the HTLV-1 proviral load [Several findings support the hypothesis of an etiopathogenic role for HTLV-1 in HAA: ATL-like T lymphocytes have been identified in the synovial fluid and synovial tissue ,18,23; hral load . The HTLral load  and the ral load .We postulated that HTLV-1 proviral load might also influence the initiation and course of HAA, and measured this marker in PBMCs from a previously described cohort  of HTLV-6 PBMCs ranged from 14,600 to 373,000 in the patients with RA , and the difference remained significant when the analysis focused on the RA subgroup . No differences were observed between the HTLV-1-infected group with RA or connective tissue disease and the matched HAM/TSP controls (P > 0.05 in both Wilcoxon's test and the paired t-test). As expected, the difference between the asymptomatic carriers and the HAM/TSP group was significant .The HTLV-1 proviral load was measured in the peripheral blood of HTLV-1-infected patients with RA or connective tissue disease and in matched asymptomatic and HAM/TSP controls , even when the analysis was restricted to the subgroup with RA alone . The patient with connective tissue disease and uveitis (patient 13) had 18,420 copies per 106 PBMCs. For one of the patients with RA and HAM/TSP (patient 10), 4 consecutive frozen dry pellets of PBMCs from 1996 to 2002 were available; in these, the proviral load was relatively stable at 92,100, 73,600, 143,300, and 106,500 copies per 106 cells, respectively.Of the 12 HTLV-1-infected patients with RA, 4  had HAM/TSP . HTLV-1 proviral load did not correlate with erythrocyte sedimentation rate or C-reactive protein level . No significant difference in HTLV-1 proviral load was seen in patients positive or negative for rheumatoid factor or antinuclear antibody  or receiving or not receiving either specific treatments for RA or corticotherapy .No correlation was found between HTLV-1 proviral load and the age of the patient, the duration of illness, or the Ritchie's index score . A positive correlation was found between HTLV-1 proviral load and the percentage of CD4+ T cells expressing HLA-DR (P = 0.008), while the correlation did not reach significance for CD8+ HLA-DR T cells.Lymphocytes subsets and activation status were examined in the peripheral blood of HTLV-1-infected patients with RA or connective tissue disease. When correlations were examined between HTLV-1 proviral load and the percentage of T lymphocytes expressing CD45RO, CD45RA, or HLA-DR in the HTLV-1 infected patients with RA or connective tissue disease Figure , HTLV-1 Lymphocyte distribution and activation were compared in the peripheral blood and synovial fluid of one HTLV-1-infected patient with RA (patient 5). The percentage of CD4+ T cells expressing CD45RO was dramatically increased in the synovial fluid (98%) compared to the peripheral blood (48%), as was the percentage of CD4+ T lymphocytes expressing HLA-DR (94% compared to 18%).The etiology of autoimmune diseases, such as RA or mixed connective tissue disease, has yet to be established, but appears to result from complex interactions between host genetic and environmental factors , and ret+ cells; (3) A high HTLV-1 proviral load was found in the synovial fluid and tissue cells in a patient with RA; (4) In this patient, the percentage of memory and activated CD4+ T cells was higher in the synovial compartment than in the peripheral blood.Nevertheless, the present study provides biological data suggesting a contribution of HTLV-1 to the development of some cases of RA or mixed connective tissue disease. We found that: (1) The circulating HTLV-1 proviral load was higher in HTLV-1-seropositive patients with RA or connective tissue disease than in asymptomatic HTLV-1 carriers and similar to that in patients with HAM/TSP; (2) In the peripheral blood of patients with arthritis, HTLV-1 proviral load correlated with the percentage of memory and activated T CD4The HTLV-1 proviral load is thought to be a major determinant of HTLV-1-associated diseases. The HTLV-1 proviral load is higher in the peripheral blood from patients with HAM/TSP than in blood from asymptomatic carriers . It is aIn HAM/TSP, the HTLV-1 proviral load reaches an equilibrium set-point that is correlated with progression of motor disability, and fluctuates by no more than 2- to 4-fold over a decade . In one + T lymphocytes and the proviral load is reported to correlate with the number of memory T cells [+ T cells expressing CD45RO and negatively with that of CD4+ T cells expressing CD45RA. The HTLV-1 proviral load also correlated positively with the percentage of HLA-DR-expressing T cells. Migration of HTLV-1-infected CD4+ T cells and HTLV-1-specific CD8+ cytotoxic T lymphocytes (CTL) into the central nervous system is a critical step in the pathogenesis of HAM/TSP [+ T cells in the joint fluid of an HTLV-1 carrier with RA supports the hypothesis of a pathogenic involvement of HTLV-1-infected T lymphocytes.The unusually high proviral loads in HTLV-1 infection results mainly from the Tax-driven activation and expansion of infected cells . The HTL T cells . In our  HAM/TSP ,32. Simi HAM/TSP ,30. ThusSeveral mechanisms are potentially involved in the occurrence of rheumatological disorders during HTLV-1 infection. Firstly, HTLV-1 infection upregulates the expression of adhesion molecules potentially involved in the migration of lymphocytes into the spinal and joint compartments . SecondlIn conclusion, our data in HTLV-1-infected patients with RA or connective tissue disease are consistent with a role of the proviral load in the development of these rheumatological disorders, although the direction of causality in this interaction remains open to question. HTLV-1 might cause a systemic immune-mediated inflammatory disease potentially involving tissues other than the central nervous system, HAM/TSP being only the major syndrome. The clinical expression of this disease might be determined by the amount of HTLV-1-infected T lymphocytes, their level of activation, and their capacity to accumulate in different body compartments. Further research is needed to increase our knowledge of the molecules involved in the homing of HTLV-1-infected CD4+ T lymphocytes and of anti-HTLV-1-specific CD8+ CTL to different target tissues.The study was performed in Martinique, an island in the lesser Antilles archipelago, with a population of 400,000. Between 1988 and 2001, 280 patients with RA, defined according to the American Rheumatology Association (ARA) criteria, and 335 patients with connective tissue disease were followed on an inpatient or outpatient basis at the Rheumatology Department of the Regional Teaching Hospital. Thirteen (4.6%) of the 280 patients with RA  were found to be HTLV-1 seropositive, confirmed by Western blotting  and peripheral blood was obtained from 12 of these. In addition, 6 patients with connective tissue disease , who were seropositive for HTLV-1, were included in the study. Samples were collected between September 2001 and June 2002. For one patient, sequential samples cryopreserved since 1996 were available.The mean age at the time of sampling was 63 years for the HTLV-1-seropositive RA patients compared to 60 years for the total RA cohort, while the mean age of the HTLV-1-seropositive patients with connective tissue disease was 58 years. The mean time interval between onset of the auto-immune disease and sampling for HTLV-1 proviral load determination were 8 years and 6 years in the patients with RA and connective tissue disease, respectively. Of the 12 HTLV-1-seropositive RA patients, 4 had HAM/TSP, defined according to the WHO guidelines . Two of Each HTLV-1-infected patient with RA or connective tissue disease was matched for age (± 5 years) and sex with 2 patients with HAM/TSP and 2 asymptomatic HTLV-1 carriers.PBMCs were isolated from EDTA blood by density gradient centrifugation. Synovial fluid samples were obtained by arthrocentesis and synovial tissue was obtained during arthroscopy. The synovial tissue was minced into small pieces, washed with phosphate-buffered saline, and passed through a wire mesh to collect synovial tissue cells. Cells were cryopreserved until use.6 cells using a phenol/chloroform procedure. The HTLV-1 proviral load was quantified using a real-time TaqMan PCR method [pol gene and the dual-labeled TaqMan probe (5' FAM and 3' TAMRA) was located at 4829–4858 bp of the HTLV-1 reference sequence (HTLVATK). Albumin DNA was quantified in parallel to determine the input cell number and was used as an endogenous reference to normalize variations due to differences in the PBMC count or DNA extraction. For both HTLV-1 and the albumin gene, amplifications were performed on 10 μl of DNA extract using the TaqMan PCR Core Reagent kit, data being acquired with the ABI Prism 7700 Sequence Detector System . Standard curves were generated using ten-fold serial dilutions of a double standard plasmid (pcHTLV-ALB) containing one copy of the target regions of both the HTLV-1 pol gene and the cellular albumin gene. The HTLV-1-infected human lymphocyte line MT2 (ECACC 93121518) was used as a control for quantification, the limit for an acceptable result being taken as 2.4–3.3 copies of the HTLV-1 pol gene per cell and the variation between series being normalized on the basis of three copies per MT2 cell. All standard dilutions and control and patient samples were run in duplicate for both HTLV-1 and albumin DNA quantification. Standard curves for HTLV-1 and albumin were accepted when the slope was between -3.322 and -3.743 (corresponding to amplification efficiencies of 100% to 85%) and the correlation coefficient, r2, was >0.992. If the variation between duplicate values of HTLV-1 or albumin DNA copy numbers was greater than 30%, the analysis was repeated. The normalized value for the HTLV-1 proviral load was reported as the HTLV-1 average copy number/albumin average copy number ratio × 106 and expressed as the number of HTLV-1 copies per 106 PBMCs.DNA was extracted from 10R method . SK110/S1), allophycocyanin-conjugated anti-CD4 , peridinin chlorophyll protein-conjugated anti-CD8 , phycoerythrin (PE)-conjugated anti-CD45RO , and PE-conjugated anti-HLA-DR  , and PE-conjugated anti-CD45RA . The analyses were performed on a FACSCalibur (BD Biosciences Immunocytometry Systems).Lymphocyte subsets in PBMCs were characterized using a panel of labeled anti-human monoclonal antibodies, consisting of fluorescein isothiocyanate-conjugated anti-CD3  declare that they have no competing interests.MY carried out most of the clinical and experimental work. AL was involved in the molecular biology work and contributed to the design of the study. FD and GL performed the flow cytometry analysis. SO and GJB participated in the neurological and rheumatologic evaluations, respectively. SA supervised the design and the course of the clinical study. RC conceived the study and drafted the manuscript. All authors read and approved the final manuscript.


A couple of years ago, a paper was published in a high-profile journal that challenged a long-established model of cell signaling. While researchers in the field mostly greeted the results with skepticism, some went into the lab to investigate the discrepancy. Many elements of this pathway, called the Wnt pathway, have been well characterized. The standard model of Wnt signaling holds that when the Wnt protein binds to its receptor, it initiates a labyrinthine signaling cascade that sends a protein called β-catenin into the cell's nucleus where, together with a protein complex, it initiates transcription. In the absence of this signal, β-catenin binds to an inactivating complex in the cytoplasm and is targeted for degradation. The paper that disputed this view suggested that β-catenin can effect gene expression without entering the nucleus and that it can activate the Wnt pathway while tethered to the cell membrane.Before that paper was published, Nicholas Tolwinski and Eric Wieschaus had shown that β-catenin, also known as Armadillo (Arm) in the fruitfly, is sent into the nucleus in response to Wnt signaling. Upon entering the nucleus, Arm interacts with a second protein complex to activate transcription. Now Tolwinski and Wieschaus have reexamined the function of Arm in the fruitfly and have demonstrated that the pathway “in fact does depend on the nuclear localization of β-catenin.” While their paper was in the final stages of acceptance, the dissenting paper was retracted, after it was learned that the results had been fabricated. Tolwinski and Wieschaus' findings confirm what had already been known about Arm's role in Wnt signaling and also fill in important details about how it works.Multicellular organisms rely on elaborate communication networks of signaling proteins and enzymes to exchange information between cells. The Wnt signaling pathway regulates the expression of a host of different genes during embryogenesis to control body patterning and cell differentiation in organisms from fruitflies to mammals. Miscommunications in this tightly regulated pathway contribute to a variety of developmental defects and cancers.In the developing fruitfly, Wnt signaling is normally restricted to the front of each larval segment, where it produces a smooth surface; the rear of the segments, where Wnt signaling is absent, is hairy. If Wnt signaling is overexpressed, it produces fruitfly larvae with only smooth segments; lack of Wnt signaling produces hairy segments. Using the smooth phenotype as a measure of Wnt signaling, Tolwinski and Wieschaus delved deeper into the role of Arm in this signaling process.These experiments are complicated because Arm functions not just in Wnt signaling, but also in cell adhesion. The trick is to make the endogenous Arm (the version encoded by the fly genome) defective for signaling, while leaving the cell adhesion functions fairly normal. Set against this “background,” an additional Arm protein is expressed that is tethered to the membrane; it still retains the protein domains required for signaling, but it's stuck on the inside of the membrane and can't move into the nucleus. In this background, any signaling in response to Wnt must mean that Arm can signal to the nucleus without actually having to get inside. Tolwinski and Wieschaus prove—again—that this is not the case. They do this by showing that the weak, medium, and strong endogenous Arm mutants—these classifications reflect the severity of the mutations' effects—have different effects on signaling in the presence of the membrane-tethered Arm.It's clear, they argue, that tethered Arm cannot signal on its own and must somehow be helping the weaker mutants signal. To further investigate how the tethered Arm activates the endogenous mutants, Tolwinski and Wieschaus developed two new and “cleaner” Arm mutants that impair Arm's signaling ability but have no effect on its cell adhesion function. The tethered Arm could not produce a completely smooth phenotype with these nonsignaling endogenous mutants. These experiments, the authors conclude, indicate that the tethered form of Arm produces its transcriptional effects by activating the endogenous Arm protein. Normal activation of the pathway liberates Arm proteins from the inactivating complex, which allows them to enter the nucleus and activate transcription. Tethered Arm appears to accomplish this by sequestering the inactivating complex at the cell membrane, preventing it from interfering with endogenous Arm.Even though Tolwinski and Wieschaus started these experiments based on what turned out to be fabricated results, their investigations produced valuable contributions. They not only reaffirm the standard model of Wnt signaling, but reveal important new insights into the workings of a major player in the pathway.


Human T-cell leukemia virus (HTLV) -1 and -2 are deltaretroviruses that infect a wide range of cells. Glut1, the major vertebrate glucose transporter, has been shown to be the HTLV Env receptor. While it is well established that the extracellular surface component (SU) of the HTLV envelope glycoprotein (Env) harbors all of the determinants of interaction with the receptor, identification of SU subdomains that are necessary and sufficient for interaction with the receptor, as well as critical amino acids therein, remain to be precisely defined. Although highly divergent in the rest of their genomes, HTLV and murine leukemia virus (MLV) Env appear to be related and based on homologous motifs between the HTLV and MLV SU, we derived chimeric HTLV/MLV Env and soluble HTLV-1 and -2 truncated amino terminal SU subdomains.bona fide interaction with the HTLV receptor as isolated SU subdomains specifically interfered with HTLV Env-mediated binding, cell fusion, and cell-free as well as cell-to-cell infection. Therefore, the HTLV receptor-binding domain (RBD) lies in the amino terminus of the SU, immediately upstream of a central immunodominant proline rich region (Env residues 180 to 205), that we show to be dispensible for receptor-binding and interference. Moreover, we identified a highly conserved tyrosine residue at position 114 of HTLV-1 Env, Tyr114, as critical for receptor-binding and subsequent interference to cell-to-cell fusion and infection. Finally, we observed that residues in the vicinity of Tyr114 have lesser impact on receptor binding and had various efficiency in interference to post-binding events.Using these SU constructs, we found that the 183 and 178 amino terminal residues of the HTLV-1 and -2 Env, respectively, were sufficient to efficiently bind target cells of different species. Binding resulted from The first 160 residues of the HTLV-1 and -2 mature cleaved SU fold as autonomous domains that contain all the determinants required for binding the HTLV receptor. Indeeiquitous . A cell iquitous .The corresponding domain in MLV Env SU – located upstream of a conserved K/R L L T/N L V Q motif in the SU of the HTLV-1 and F-MLV Env ,10 – is per se, as well as interference to receptor-binding, cell-to-cell fusion, syncytium formation, and infection involve several distinct cell surface-associated parameters [Several studies using synthetic peptides and neutralizing antibodies against the HTLV Env have shown that determinants within this proline rich region homologue (PRRH) are involved in interference to Env-mediated syncytium formation -21. The rameters -29. In tAs shown in Figure 183FEnv, comprises the 183 amino terminal residues of the HTLV-1 SU ending with the PSQL residues fused to the PIGP sequence of the F-MLV PRR  or rabbit immunoglobulin Fc (rFc) carboxy terminal tag Figure . The H12y Figure . The H11e Figure .215SU and H2211SU, containing the PRRH sequence, and H2178SU lacking this PRRH were all efficiently expressed in transfected cells . In contrast, no significant cleavage was observed with the other soluble domains released in the medium (not shown) . Immunoblots of cell extracts revealed two forms of intracellular H1215SU and H2211SU  Figure . As expeU Figure ; this waU Figure .397SU) and used as a heterologous control. A single form of this truncated SU was efficiently expressed in transfected cells  [178SU was similar to H2211SU, demonstrating that the first 158 residues of the mature HTLV-2 SU, without the 20 amino acids of the amino terminal signal peptide, are sufficient for cell surface binding, and therefore that the PRRH is not required for receptor binding  , HeLa (hoblasts) , none ofoblasts)  and datag Figure .bona fide binding to the HTLV receptor, we performed an Env-specific binding interference assay. In this assay, transfection of the above described chimeric Env and SU subdomains into 293T cells resulted in interference to cell surface binding by the soluble HA-tagged H2178SU subdomain   Figure . This efn Figure . Therefo183FEnv and the HTLV SU amino terminal subdomains to interfere with HTLV Env-mediated cell fusion. Interference to cell fusion was measured using a quantitative HTLV envelope cell fusion interference assay (CFIA), as previously described [Viral envelope interference occurs when cell surface receptors are occupied by receptor-interacting Env components -35. Sincescribed .215SU subdomain in target cells, 12% ± 2% of control fusion (P < 0.001), consistent with previous observations using the H1215FEnv chimera [183FEnv chimera, which lacked a PRRH, down to 26% ± 4% of control fusion (P < 0.001)   , although unnecessary for receptor binding, modulates the efficiency of interference to HTLV Env-induced cell-to-cell fusion (P < 0.03).HTLV-1 Env-induced cell fusion was significantly diminished upon expression of the H1 chimera . SignifiInterference, as described above, was based on the inhibition of cell-to-cell fusion induced by fusogenic Env expressed in the absence of other viral proteins. We further evaluated the abilities of the Env chimeras and soluble subdomains to specifically interfere with HTLV Env-mediated infection. HTLV Env-pseudotyped MLV virions, MLV(HTLV), were produced to infect 293T target cells. Because these recombinant cell-free virions are not competent for replication, this viral pseudotype infection assay tests a single round of infection, and does not measure replication and subsequent exponential viral dissemination. Therefore, relative infection values are expressed in linear rather than logarithmic scales.397SU  infection to 324 ± 98 iu/ml, 3.3% ± 1% of control infection, and to 307 ± 129 iu/ml, 3.1% ± 1.3% of control infection, respectively  infection to 191 ± 56 iu/ml and 215 ± 122 iu/ml, 1.9% ± 0.6% and 2.2% ± 1.3% of control infection, respectively , and this was taken as 100% control infection Figure . SimilarU Figure . ExpressBecause HTLV dissemination appears to occur mostly via cell-to-cell contact, we also tested envelope interference to infection by HTLV-1 SU amino terminal domains using a cell-to-cell transmission interference assay. In this assay, cells harboring interfering chimeric Env and soluble subdomains were cocultured with cells producing MLV(HTLV) virions. Transfection of either chimeric Env or soluble subdomains into HeLa target cells decreased MLV(HTLV) infection to levels similar to those observed in the cell fusion interference assay presented in figure 94) and serine 101 (Ser101) for HTLV-1 Env which correspond to Arg90and Ser97 in HTLV-2 Env, have been shown to alter cell-to-cell fusion and infection when mutated [215SU subdomains with either Arg94 or Ser101 mutated to Ala, yielding the mutant H1(R94A)SU and H1(S101A)SU subdomains, respectively. We also evaluated mutations of Asp106, mutant H1(D106A)SU, and Tyr114, mutant H1(Y114A)SU, both residues found to be highly conserved between all human and simian T cell leukemia viruses (unpublished observations). Surprisingly, cell surface binding profiles of H1(R94A)SU and H1(S101A)SU mutants were not significantly altered when compared to binding by the parental H1215SU, whereas the H1(D106A)SU mutant presented reduced binding to HTLV receptor-bearing cells and the H1(Y114A)SU mutant showed a nearly complete abrogation of cell surface binding . Altogether, these experiments demonstrated that Tyr114, and to a lesser extent Asp106, are key residues involved in HTLV Env receptor binding.Two key residues contained in the HTLV SU RBD and conserved between HTLV-1 and -2, arginine 94 . Thus, overexpression of mutant HTLV SU fragments with highly decreased receptor binding abilities can still exert, albeit to a significantly lesser extent, interference to HTLV Env-mediated cell fusion and infection.We next tested the abilities of these mutants to interfere with HTLV Env-mediated cell fusion and infection, using the assays described above. As mentioned above, all wild-type and mutant HTLV SU subdomains were produced and secreted with a similar efficiency Figure . Express215SU or the mutant H1(S101A)SU were expressed in target cells SU mutant inhibited cell fusion and infection, its effects were significantly lower than those of the wild-type H1215SU   Figure . Thus, a114 of the HTLV-1 Env, which corresponds to Tyr110 of the HTLV-2 Env, as a key residue in HTLV Env binding and for all the aforementioned HTLV Env-mediated functional assays. We could not detect binding of H1(Y114A)SU by flow cytometry, while this mutant exerted residual, albeit significantly decreased, interference to HTLV Env-mediated cell fusion and infection. Altered folding outside of the binding domain per se, rather than direct alteration of the receptor-binding site, could also account for the lack of binding of this mutant. However, we favor the latter hypothesis, since the H1(Y114A)SU mutant was properly folded and transported to the plasma membrane and secreted in the medium as efficiently as wild type RBD, thus arguing against gross misfolding of this mutant. Accordingly, Tyr114 appears to be conserved in all known human and simian T cell leukemia viruses strains, which share the same receptor.Here, we report the generation of MLV Env with chimeric HTLV/MLV SU and truncated HTLV-1 and -2 amino terminal SU subdomains that can be expressed in and secreted from eukaryotic cell lines in functional, soluble form. Using these constructs, we demonstrated that the amino terminal 163 and 158 residues  of the mature HTLV-1 and -2 Env SU, respectively, were sufficient to exert both HTLV receptor binding and efficient interference to diverse HTLV Env-mediated functions, including binding, cell-to-cell fusion and cell-free as well as cell-to-cell infection. Although the PRRH sequence comprising amino acid residues 180 to 215 of the HTLV-1 Env and 176 to 211 of the HTLV-2 Env was previously thought to be a receptor binding site, our data preclude a major role for this region in the binding properties described above. Indeed, whereas a synthetic peptide composed of amino acids 197 to 216 and located within the HTLV-1 PRRH, has been reported to interfere with HTLV Env-induced syncytia formation , this pe114 as a key HTLV-1 RBD residue points at this determinant as a very likely receptor-binding core. This, together with previous works relying on syncytia formation and cell-to-cell transmission [bona fide receptor binding determinants and determinants involved at a post-binding level.The receptor-binding site in MLV RBD is composed of a combination of several cysteine loops located upstream of the PRR ,39 whichsmission ,37, willAnother recently identified determinant, the Pro-His-Gln SU motif conserved among gammaretroviruses such as MLV and feline leukemia viruses (FeLV), has been determined to play a major role in viral entry during post-binding events . The mecThe recent identification of Glut1, the ubiquitous glucose transporter of vertebrates , as a reWe have generated truncated domains of the HTLV Env amino terminus, upstream of residues 183 and 178 of the HTLV-1 and -2 Env, respectively, that were sufficient to bind target cells of different species through interaction with the HTLV Env receptor. We also identified a tyrosine at position 114 and 110 in HTLV-1 and -2 Env, respectively, as a key determinant for this binding. In addition to their use for further exploration of the mechanisms involved in HTLV entry, the tagged HTLV-1 and -2 RBD subdomains described here are novel tools for the detection of Glut1 cell surface expression and intracellular trafficking. Indeed, we tracked intracellular expression of EGFP-tagged HTLV SU subdomains by time-lapse microscopy, and found that they are preferentially routed toward cell-cell contact areas (unpublished observations), where Glut1 is particularly abundant  and our MfeI restriction site in the HTLV-1 and F-MLV Env. Introduction of this site in F-MLV resulted in the substitution of a glutamine and leucine (QL) dipeptide for the parental arginine and valine (RV) residues of the GPRVPIGP motif, at the start of the MLV Env PRR. Introduction of the MfeI site in the PSQL motif of the HTLV-1 SU maintained the parental QL residues, at the start of the HTLV Env PRRH. By exchanging domains at the MfeI sites, we derived the H1183FEnv chimera containing the amino terminal 183 residues of the HTLV Env followed by the F-MLV PRR. In this chimera, the PSQL/PIGP hybrid sequence is generated at the exchange border, and the PRRH of HTLV is replaced by the F-MLV PRR  [To exchange the PRR and PRRH regions, we introduced an allelic R Figure . In cont HHproFc . The H11n vector . The HTL intron)  at the H215SU, H2211SU, H1179SU, and H2178SU subdomains, corresponding to the HTLV-1 and -2 SU amino terminus with and without their respective PRRH, were generated by PCR and subcloned into the pCSI expression vector as fusion proteins harboring a carboxy terminal rFc or HA tag  transfection protocol. Transfection medium was replaced with 3 ml of fresh culture medium twenty hours post-transfection. Forty-eight hours post-transfection cell culture medium (supernatant) was recovered and filtered through a 0.45 μm pore-size membrane to remove cell debris. Twenty μl were directly analyzed by SDS-PAGE (15% polyacrylamide gel), and the rest was aliquoted and stored at -20°C for later use in binding assays (see below). Cell extracts were collected 48 h post-transfection in 1 ml of cell lysis buffer  and clarified by two successive centrifugations at 13,000 rpm for 10 min at 4°C in a microcentrifuge. Approximately 20 μl of each extract, adjusted after normalization for protein concentration using the Bradford assay (Sigma), were subjected to electrophoresis on SDS-15% acrylamide gels, followed by transfer onto nitrocellulose . Membranes were blocked in phosphate-buffered saline (PBS) containing 5% powdered milk and 0.5% Tween 20, probed with a 1:1000 dilution of a goat anti-RLV gp70 polyclonal antibody (Viromed) followed by a horseradish peroxidase-conjugated anti-goat immunoglobulin (for detection of chimeric Env), or goat anti-rabbit-IgG-horseradish peroxidase-conjugated immunoglobulins (for detection of rFc-tagged SU subdomains). Immunoblots were subsequently washed three times with PBS-0.1% Tween 20 and revealed by chemiluminescence .Approximately 5 × 105 target cells were detached with a PBS-EDTA solution, collected by centrifugation, incubated for 30' at 37°C with 300 μl of rabbit Fc-tagged soluble HTLV-1, HTLV-2, or Ampho-MLV truncated SU, washed, labeled with an anti-rabbit-IgG FITC-conjugated antibody, and analyzed on a FACSCalibur (Becton Dickinson). Data analysis was performed using the CellQuest software (Becton Dickinson). For interference studies, 293T cells were transfected with 4 μg of Env or Env SU subdomain expression vectors  using the calcium-phosphate-HBS method. Under these conditions, transfection efficiencies ranged from approximately 80 to 90% of the target cells. Twenty-four and 48 hours post-transfection, cells were collected and transfected 293T cells expressing the different interfering HTLV or Ampho-MLV domains were incubated with a challenging HA-tagged soluble HTLV-2 SU amino terminal subdomain (H2178SU-HA). Cells were stained using a primary 12CA5 anti HA antibody followed by an anti-mouse-IgG FITC-conjugated antibody before detection by flow cytometry.Binding assays were performed as previously described . Briefly183FEnv, was used to interfere with challenging HTLV Env. The interfering non-fusogenic H1183FEnv and truncated HTLV SU subdomains were transiently transfected into HeLaCD4LTRLacZ, a cell line highly susceptible to HTLV Env-induced fusion that contains a stably integrated Tat-dependent LacZ expression vector [183FEnv Env and truncated HTLV SU subdomains plasmid DNA (2 to 3 μg) was transfected into HeLaCD4LTRLacZ cells, while challenging, fusogenic HTLV-1 Env plasmid (1 μg) was transfected into NIH3T3(TK-)Tat. The interfering Env or SU subdomain-presenting cells were detached 24 hours post-transfection and 1–2 × 105 cells were cocultured for 24 hours with 1–2 × 105 challenging HTLV-1 Env-presenting NIH3T3(TK-)Tat cells. Subsequently, the cocultured cells were fixed and stained for β-galactosidase expression as described previously [t test.Briefly, the HTLV/MLV Env chimera, H1n vector . These tn vector ,29. H118eviously . Transfe6 293T cells with 5 μg pCSI/Ampho or pCSIX/H2, respectively, 5 μg pCL/Gag-Pol [LacZ gene marker. pCSI/Ampho is an expression vector encoding the Ampho-MLV Env, and the HTLV-2 Env expression vector, pCSIX/H2, is described above.MLV(Ampho) and MLV(HTLV) pseudotyped virions were produced after transfection of 10/Gag-Pol , using aVirion-containing supernatants were used to infect target 293T cells expressing the chimeric Env or HTLV RBD subdomains. Transfection efficiencies of target 293T cells were >80% in all experiments. Infections were performed 36–48 hours post-transfection on cultures grown in 12 well plates (Costar) at 37°C, medium was changed 24 hours later, and confluent cell monolayers were fixed, stained for β-galactosidase activity before counting blue foci. Interference to infection was determined by infecting transfected target cells with approximately 100 and 1000 iu. Infection was evaluated as described above, and the number of LacZ-positive blue colonies counted was normalized by multiplying by the appropriate dilution factor. The resulting infection values were analyzed as iu/ml of virus containing supernatant. Subsequently the relative infection levels in cells expressing the HTLV SU domains were compared to those of mock transfected cells and were expressed as percentages of control infection (% control).HTLV Human T-cell leukemia virusSU envelope extracellular surface componentEnv envelope glycoproteinMLV murine leukemia virusF-MLV Friend-MLVRBD receptor-binding domainPRR proline-rich regionPRRH proline rich region homologueAmpho amphotropicHA influenza hemagglutininrFc rabbit immunoglobulin constant fragment397SU Ampho-MLV Env fused to a carboxy terminal rFc tagACFIA cell fusion interference assayiu/ml infectious units per ml94arginine 94Arg101serine 101Ser114tyrosine 114TyrFeLV feline leukemia virusesHBS Hepes buffered salinePBS phosphate-buffered salineSDS sodium dodecyl sulfateThe authors declare that they have no competing interests.FJK designed and realized or supervised most of the experiments and co-wrote the manuscript. NM participated to some molecular constructions, set up, realized and analyzed most binding assays and FACS analyses and participated to the redaction of the manuscript. ENG set up and performed the cell-to-cell transmission assay and performed the corresponding experiments, CV constructed some of the RBD point mutants and tested them, MS initiated the project, co-participated in the design of the study, co-coordinated its realization and co-wrote the manuscript, and JLB realized some of the molecular constructs, performed some of the experiments, co-participated in the design of the study, co-coordinated its realization and co-wrote the manuscript. All authors read and approved the final manuscript.


Adult T-cell leukemia (ATL) was first reported as a distinct clinical entity in 1977 in Japan. The predominant physical findings are skin lesions, lymphadenopathy and hepatosplenomegaly. The ATL cells are of mature T-helper phenotype and have a characteristic appearance with indented nuclei. There is striking frequent hypercalcemia with increased numbers of osteoclasts. Central to the identification of the disease is a striking geographic clustering in southwestern Japan and the isolation of human T-cell lymphotropic virus type-1 (HTLV-1) from the cell lines of patients. Worldwide epidemiological studies have been made through international collaborations. Several diseases were found to be related to HTLV-1 infection. Moreover, it was noted that an immunodeficiency state may be induced by HTLV-1 infection. In Japan, HTLV-1 carriers have been estimated to be 1.2 million, and more than 700 cases of ATL have been diagnosed each year. Adult T-cell leukemia (ATL) was described as a distinct clinical entity in 1977 in Kyoto, Japan . The illIt was around the 1973 that we came to recognize the existence of ATL, previously an unknown disease. I would like to give a retrospective view concerning the background of our studies on ATL.Many clinicians in Japan probably feel that the descriptions of diseases in the literature from abroad differ from the features of the diseases observed in their practices in Japan. We can mention many examples in the field of hematology. One of these is that the incidence of chronic lymphocytic leukemia is quite low, being only 2% of all hematological malignancies. What differs is not only the incidence but also the detailed symptoms and signs of diseases. This vague feeling that an autochthonous pathology exists in Japan may be considered as key factor in the background of our study of ATL.The second major factor is the recent progress made in basic immunology. Having an interest in immunoglobulin abnormalities, I studied multiple myeloma and related diseases in the days when even the word 'immunoglobulin' was not yet in use. The central theme of immunology at that time was to identify the structure of antibodies. Immunology has advanced rapidly, and myeloma was defined as a B-cell malignancy. Therefore, it was quite natural to enlarge the objectives of our clinical studies to all lymphoproliferative diseases.At this stage of our research in Kyoto, joint studies with young researchers were initiated. This was the third and most important element of the background of our ATL study. I became acquainted with Drs. Takashi Uchiyama and Junji Yodoi, who are currently both working in Kyoto, during their postgraduate training. Dr. Yodoi prepared an antiserum against human thymocytes and invited us to direct our attention to T-cells.In the course of examining patients with various lymphoproliferative disorders, we arrived at the conclusion that ATL was a disease which had not been described anywhere before. It was recognized that T-cell malignancy had a relatively high incidence among Japanese adults and that most of the patients with this disease were from Kyushu. This discovery was made from our bedside observations rather than from laboratory work. Thereafter, Dr. Uchiyama went to the National Cancer Institute, Bethesda to work with Dr. Thomas A. Waldmann and raised a monoclonal antibody, called 'Tac' antibody, which later played an important role in our ATL studies. Dr. Yodoi has developed a new field of research by identifying a novel cytokine, ATL-derived factor (ADF), which has proved to be important in redox regulation. At the end of 1981, I moved from Kyoto to Kumamoto, which is located in the middle of Kyushu. Our studies advanced remarkably there, due mainly to the efforts of excellent young co-workers including Drs. Kazunari Yamaguchi, Toshio Hattori, and Masao Matsuoka, who are now independently working in Tokyo, Sendai and Kyoto, respectively.th International Congress of Hematology in Kyoto, it was stated that 'attempt to elucidate leukemogenesis in this disease should be directed towards exploring the genetic background and a possible viral involvement'. HTLV (human T-cell leukemia virus), the pathogen of ATL, was first reported by Dr. Robert C. Gallo and his co-workers in Bethesda in 1980 and 1981. They isolated HTLV from cultured cells taken from a patient with an aggressive variant of mycosis fungoides and another with Sezary syndrome. Although both patients were said to have cutaneous T-cell lymphoma, they had some unusual features which, in retrospect, linked them to the clinical entity now called ATL.This discovery of ATL ushered in some dramatic developments in oncology, virology and, unexpectedly, neurology and other fields of medicine. When we reported a series of 13 patients with ATL in 1976 on the occasion of the 16In Japan, co-culturing of ATL cells with umbilical cord lymphocytes was first done successfully by Dr. Isao Miyoshi's group in Okayama, who obtained the cell line MT-1. Dr. Yorio Hinuma and his co-workers in Kyoto demonstrated that ATL patients have antibodies against presumed viral antigens on MT-1 cells by indirect immunofluorescence method. Subsequently, a retrovirus was isolated and called ATLV (adult T-cell leukemia virus). Since Dr. Mitsuaki Yoshida and his group in Tokyo showed that HTLV and ATLV are, in fact, identical, the term HTLV-1 (human T-cell lymphotropic virus type 1) has been commonly used.Furthermore, the following observations have been successively reported to support the etiologic association of HTLV-1.1. All patients with ATL have antibodies against HTLV-1.2. The areas of high incidence of ATL patients correspond closely with those of high incidence of HTLV-1 carriers.in vitro.3. HTLV-1 immortalizes human T cells 4. Monoclonal integration of HTLV-1 proviral DNA was demonstrated in ATL cells.Thus, HTLV-1 is the first retrovirus directly associated with human malignancy.The diagnostic criteria for HTLV-1 associated ATL have been defined as follows:1. Presence of morphologically proven lymphoid malignancy with T-cell surface antigens .2. Presence of antibodies to HTLV-1 in the sera.3. Demonstration of monoclonal integration of HTLV-1 provirus by Southern blot method.Virological study led us to classify the patients with ATL into four clinical subtypes according to the clinical features: acute, chronic, smoldering, and lymphoma. The acute type is the prototypic ATL in which patients exhibit increased number of ATL cells, frequent skin lesions, systemic lymphadenopathy, and hepatosplenomegaly. Most of these patients are resistant to chemotherapy and have a poor prognosis. In chronic ATL, white blood cell count is mildly increased, and skin lesions, lymphadenopathy, and hepatosplenomegaly are sometimes exhibited. In the past, chronic ATL was thought to be 'chronic T-lymphocytic leukemia'. Smoldering ATL is characterized by the presence of a few ATL cells in the peripheral blood over a period of years. Frequent symptoms are skin or pulmonary lesions. Chronic and smoldering ATL often progress to acute ATL after a long period (crisis). Lymphoma-type ATL is characterized by prominent systemic lymphadenopathy, with few abnormal cells in the peripheral blood. This type has been diagnosed as nonleukemic malignant lymphoma. Later, the Lymphoma Study Group in Japan proposed a practical diagnostic criteria for classifying ATL into these four subtypes.HTLV-1 is endemic in southwestern Japan, especially Kyushu and Okinawa, in the Caribbean Islands, and in Central Africa. It has been revealed that there are HTLV-1 carriers in South America, Papua New Guinea, the Solomon Islands, South China, and other isolated populations in the world, including Ainus in Hokkaido and Aborigines in Australia. These epidemiological studies have been promoted by international collaborations, to which Drs. William A. Blattner in Bethesda, Guy de The in Paris, Kazuo Tajima in Nagoya, Shunro Sonoda in Kagoshima, and many other epidemiologists have made important contributions. Dr. Daniel Catovsky and his colleagues in London delineated the clinical features of ATL patients originating in the Caribbean islands.On the other hand, several diseases have been found to be related to HTLV-1 infection. Drs. Antoinne Gessain and Guy de The first reported the association of tropical spastic paraparesis (TSP) and HTLV-1, and Dr. Mitsuhiro Osame and his co-workers in Kagoshima studied features of HTLV-1-associated myelopathy (HAM/TSP) in detail. HTLV-1 uveitis was subsequently described by a study group of ophthalmologists in Kyushu. HTLV-1 may also be associated with bronchopneumonitis, arthritis, polymyositis and other inflammatory conditions. Moreover, it was noted that an immunodeficiency state may be induced by HTLV-1 infection.In Japan, HTLV-1 carriers have been estimated to be 1.2 million, and more than 700 cases of ATL have been diagnosed each year. Majority of HTLV-1 transmission occurs via one of three routes, all of which require the passage of virus-infected cells. HTLV-1 carrier mothers transmit the virus to newborns mainly through breast milk. Dr. Shigeo Hino and his group in Nagasaki conducted intensive fieldwork regarding the mother-to-infant infection. Carrier mothers in Japan have been instructed to refrain from breastfeeding or modify the feeding procedures to prevent HTLV-1 infection. There is convincing evidence that HTLV-1 can be transmitted from individual to individual by sexual contact, especially males to females, and also through blood transfusions. All blood donated at the Red Cross Blood Centers in Japan has been subjected to HTLV-1 antibody testing beginning in November 1986.Treatment of ATL is the most difficult task. Dr. Masanori Shimoyama in Tokyo has organized a multicenter study group for the chemotherapy of ATL. Many other trials have been reported: deoxycoformycin, α-interferon combined with azidothymidine, and so on. The use of humanized monoclonal antibodies against the interleukin-2 receptor has been championed by Dr. Waldmann's group. More recently, successful allogeneic bone marrow transplantation for patients with ATL has been reported from many institutions.th International Conference of Human Retrovirology held in Kumamoto on May 11–13, 1992.Before my retirement from Kumamoto University in 1996, I edited a book titled "Adult T-cell Leukaemia" . Many ofA companion article by Robert C. Gallo recollects the events surrounding the discovery of the first human retrovirus, HTLV-I .


HTLV-1 infection is currently restricted to endemic areas. To define the prevalence of HTLV-1 infection in patients living in Italy, we first carried out a retrospective serological analysis in a group of people originating from African countries referred to our hospital from January 2003 to February 2005. We subsequently applied a real time PCR on peripheral blood mononuclear cells from subjects with positive or indeterminate serological results.All the sera were first analysed by serological methods (ELISA and/or Western Blotting) and then the peripheral blood mononuclear cells from subjects with positive or inconclusive serological results were analyzed for the presence of proviral DNA by a sensitive SYBR Green real time PCR. In addition, twenty HTLV-I ELISA negative samples were assayed by real time PCR approach as negative controls.Serological results disclosed serum reactivity by ELISA  in 9 out of 3408 individuals attending the Sexually Transmitted Diseases Clinic and/or Oncology Department, and 2 out 534 blood donors enrolled as a control population. Irrespective of positive or inconclusive serological results, all these subjects were analyzed for the presence of proviral DNA in peripheral blood mononuclear cells by SYBR real time PCR. A clear-cut positive result for the presence of HTLV-1 DNA was obtained in two subjects from endemic areas.SYBR real time PCR cut short inconclusive serological results. This rapid and inexpensive assay showed an excellent linear dynamic range, specificity and reproducibility readily revealing and quantifying the presence of virus in PBMCs. Our results highlight the need to monitor the presence of HTLV-1 in countries which have seen a large influx of immigrants in recent years. Epidemiological surveillance and correct diagnosis are recommended to verify the prevalence and incidence of a new undesirable phenomenon. HTLV-1 (Human T-cell lymphotropic virus type 1) is etiologically linked with adult T-cell leukemia (ATL) . HTLV-I To date, blood screening for HTLV-I has not been mandatory in Italy, but a more careful screening of the population might be justified by several literature reports -24. ScreConsidering the need to update information on HTLV-1 incidence in Italy, we investigated the presence of HTLV-1 infection in a selected group of patients originating from endemic areas using serological methods and a SYBR Green real time PCR technique able to verify and quantify the HTLV-1 proviral load.From January 2003 to February 2005 we enrolled in the study a group of HIV-1/2 negative 3408 recent immigrants from African countries referred to the Sexually Transmitted Diseases Clinic and/or Oncology Department (group 1) and a group of 534 blood donors (group 2) undergoing laboratory analysis for serological diagnosis of other infectious diseases. Recent immigrants were defined as people stating they had lived in Italy for less than five years at the time of HTLV-I serological analysis. All patients, after informed consent, were screened for HTLV-1 antibodies by ELISA assay  as described by the manufacturer. In addition, all ELISA borderline or positive samples and some randomly selected ELISA negative samples were analyzed by Western-blot technique  following the manufacturers' procedure.MT2 lymphoblastoid T cell line the most commonly used cell line for HTLV-I production, previously characterized as having 2.1 HTLV-I copies of virus/cell ,30, Jurk6, were prepared and stored at -80°C until use. Plasmid pHP1 is represented by the pCR2.1-TOPO vector  where it was cloned the HTLV-I 117 bp amplicon as indicated by the manufacturer. The plasmid pHP1 was purified by Midi plasmid extraction kit (Qiagen) following the manufacturer's procedure. Genomic and plasmid DNA concentration and purity were determined by spectrophotometric analysis at 260/280 nm.Peripheral blood mononuclear cells (PBMCs) from twenty HTLV-1 negative patients and eleven patients with indeterminate or positive HTLV-I serological assay were isolated from whole blood by Ficoll-HistoPaque gradient separation (Amersham Pharmacia). As previously described , DNA was5 to 1 copies of HTLV-I genome) and clinical samples.SYBR green real time PCR assay was performed in 20 μl PCR mixture volume consisting in 2× Quantitect SYBR green PCR Master Mix (Quiagen) containing HotStarTaq DNA polymerase, 200 nM of each oligonucleotide primer and 5 μl of DNA extracted from scalar dilution of MT-2 cell line (from 10pol gene amplification was carried out as follows: one cycle of 15 min at 95°C (hot-start PCR) and 45 cycles in four step each . At the end of amplification cycles, melting temperature was analyzed by a slow increase in temperature (0.1°C/s) up 95°C. The property elicited an accurate analysis of the melting temperature curve of the amplified fragments generated by real time PCR to determine the detection and quantification of specific products [HTLV-1 products . Real tipol primer sequences were: 5' GTG GTG GAT TTG CCA TCG GGT TTT 3' and: 5' GTA CTT TAC TGA CAA ACC CGA CCT AC 3'. The amplification with this pair of oligonucleotides yielded a 117 bp pol fragment. Three replicates were done for each scalar dilution for intra-assay validation whereas three experiments were performed in triplicate for inter-assay analysis. All MT-2 DNA scalar dilutions were equalized at 600 ng of total DNA by Jurkat cell DNA addition in all experiments. In addition, 600 ng of total DNA from patients' PBMC or HTLV-I negative cell lines were amplified. All patients' samples were also analysed by SYBR Green real-time PCR for globin gene in a parallel run to check the equal amount in all samples determined by spectrophotometric data as described [6 PBMC. Exact values were used for calculations, ruling out decimal values.HTLV-I escribed . All staElectrophoresis agarose gel, and Southern blot of PCR amplicons were carried out as previously described . The hybWe analyzed the sera from 3408 immigrant African individuals HIV-1/2 negative, attending the Sexually Transmitted Diseases Clinic and/or Oncology Department (Group 1), and 534 blood donors by ELISA assay. Among the patients selected, serum reactivity  was disclosed in 9 out of 3408 patients (0.26%) and 2 out of 534 blood donors (Group 2). Hence, we assayed the reactive serum of these 11 individuals by HTLV-I specific Western-blot. As shown in Table Western-blot assay results Figure  demonstrIn particular the two positive samples with a clear antibody pattern reacting to all HTLV-1 proteins belonged to patients originating from Nigeria (samples n°3) and Ghana (sample n°7) respectively. Interestingly, none of blood donors tested were Western-blot reactive or indeterminate for HTLV-I.5 to 10 HTLV-I genome equivalent copies) of MT2 cell line genome containing 2.1 copies of proviral HTLV-I genomes/cell  by using an oligonucleotide specific pair able to amplify a 117 bp conserved region of HTLV-I pol gene. The assay encompasses at least five orders of magnitude with a high linear relationship (r2 > 0.99) between the Ct values and the cell line input copies. The specificity of amplified products was assessed by melting curve analysis: all specific PCR amplicons showed the same dissociation temperature (79.35C°).We evaluated indeterminate and reactive Western-blot patients by in house SYBR green based Real time PCR technique to determine the presence of HTLV-I proviral genome in the peripheral blood mononuclear cells. This technique was validated on scalar dilutions  of our assay disclosed 10 HTLV-I equivalent genome copies in 100% of replicates . The specificity of SYBR Green real time PCR was determined by Jurkat or 8E5LAV cellular DNA analysis. These HTLV-I negative cells did not show any positive signal. Of note, this method shows a good intra- and inter- assay reproducibility determined by scalar dilutions of HTLV-I-positive MT-2 cell DNA equivalent genome analysis. In particular, intra-assay reproducibility was evaluated by three replicates of each point of scalar dilutions between 105 and 10 HTLV-I genome equivalent copies. The coefficient of variation (CV) of Ct was <3.8 % for all scalar dilutions tested. The CV of copy number was <35% for 10 copies and <25% for all more-concentrated dilutions (from 105 to 102 copy/sample)  and 5.9 × 102 (n°7 patient) per 106 PBMC genomes. Electrophoresis agarose gel and Southern blot assay analysis of amplicons indicated the presence of a specific band at 117 bp , a high number of lymphocytes , severe hypercalcemia  and elevated LDH plasma values . Hematological findings showing a monoclonal T-cell lymphocytosis  confirmed the diagnosis of ATL.Oncologic clinical investigation of patient n°3 revealed that the positive sample belonged to a 27-year-old Nigeria-born female prostitute living in Italy since 2001. History-taking disclosed ATL disease with diffuse exfoliative dermatitis followed by a rapid deterioration of the patient's general condition, high white blood cell (WBC) count  revealed clear-cut positive results in two samples but Western blot did not rule out inconclusive results in three serum samples from high risk individuals. However, the finding of inconclusive results both by ELISA and immunoblotting analysis suggests the need to confirm virus presence by molecular methods. The identification of specific viral sequences in infected cells is essential to confirm the serological diagnosis in subjects with positive or indeterminate results ,21,30. IThe clinical interest in molecular biology assays for HTLV diagnosis is increasing since proviral DNA levels represent a measure of integrated genome  and a su5 to 101 copies with a detection limit established at ten copies: a sensitivity comparable to other PCR formats for HTLV-I. SYBR Green was chosen instead of different real time approaches such as TaqMan or beacons to generate fluorescence signals, for several reasons. In particular, SYBR Green is less expensive than labelled probes that could also determine PCR artefacts beyond the 30th cycle during the amplification. In addition, probe selected sequences may be prone to specific mutations [Our study also shows the application of a SYBR Green based real time PCR committed to HTLV-I provirus detection and quantitation. We optimised the conditions of SYBR Green real time PCR for HTLV-I DNA proviral detection with a high level of specificity . Moreover, the assay has excellent dynamic range from 10utations .The lack of any signal both in seronegative blood donors, used as negative controls and in samples with inconclusive serological results suggests a high specificity level. Some literature data suggest that an indeterminate serological profile  could reflect true HTLV-1 infection . InterprSince our SYBR green based real time PCR technique is a specific and simple assay for quantitative detection of HTLV-I proviral DNA, its application to monitor disease progression and verify the effectiveness of therapy offers an interesting option not only for first level diagnosis, but also for ongoing epidemiological surveillance.HTLV-1 proviral DNA quantification opens interesting prospects. Cell-free viremia in plasma is not a prominent aspect of HTLV-associated diseases, unlike HIV infection, where the quantitative determination of RNA copies, the main prognostic parameter for disease evolution, directly mirrors viral replication . MoreoveOur method may also be useful to analyze PBMCs from blood donors with serological indeterminate results. Blood donor screening for HTLV was introduced in Japan in the mid 1980s, in the United States and Canada in 1988 and finally in France in 1991 , and eveEven though our data demonstrated that HTLV-I infection is mainly confined to African immigrants, the feasibility of a simple and effective real time approach like our SYBR Green real time PCR suggests a possible application to cut short doubtful diagnosis of HTLV-1 infection and to monitor proviral and viral load during the course of infection and the efficacy antiviral therapy.The author(s) declare that they have no competing interests.MCR and DG conceived and designed the study. FV, PS and IB developed the HIV-1 DNA real time assay and performed all the experimental work. AD and LG provided blood samples and clinical information on the patients enrolled in this study. MCR drafted the manuscript and DG reviewed it. All authors contributed to the final version of manuscript, read and approved it.The pre-publication history for this paper can be accessed here:


The virus, that we tentatively named HTLV-3, originated from a 62 years old Bakola Pygmy living in a remote settlement in the rain forest of Southern Cameroon. The plasma was reactive on MT2 cells but was negative on C19 cells. The HTLV 2.4 western-blot exhibited a strong reactivity to p19 and a faint one to MTA-1. On the INNO-LIA strip, it reacted faintly with the generic p19 (I/II), but strongly to the generic gp46 (I/II) and to the specific HTLV-2 gp46. The molecular relationships between Pyl43 and STLV-3 are thus not paralleled by the serological results, as most of the STLV-3 infected monkeys have an "HTLV-2 like" WB pattern. In the context of the multiple interspecies transmissions which occurred in the past, and led to the present-day distribution of the PTLV-1, it is thus very tempting to speculate that this newly discovered human retrovirus HTLV-3 might be widespread, at least in the African continent.Human T-cell Leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) are pathogenic retroviruses that infect humans and cause severe hematological and neurological diseases. Both viruses have simian counterparts (STLV-1 and STLV-2). STLV-3 belongs to a third group of lymphotropic viruses which infect numerous African monkeys species. Among 240 Cameroonian plasma tested for the presence of HTLV-1 and/or HTLV-2 antibodies, 48 scored positive by immunofluorescence. Among those, 27 had indeterminate western-blot pattern. PCR amplification of  Three types of Primate T-cell lymphotropic viruses (PTLVs) have been discovered so far in primates . While tCameroon has a remarkable diversity of retroviruses. All the subtypes of HIV-1 group M (A to H) are present, subtype-recombinant strains co-circulate, and HIV-1 groups O and N have been reported. Besides, HTLV-1 subtypes B and D as well as HTLV-2 type A and B are also present in Cameroonian individuals, while STLV-1 and STLV-3 strains have been isolated from several non-human primates (NHPs) species living in this region ,8. We thtax and pol primers designed on highly conserved regions that are common to all PTLVs. The tax primers are the following: SCTaxoutse: 5'-CTHTAYGGRTACCCHGTCTACGT-3' and SCTaxoutas: 5'-AGGGGAGBCGAGGGATAAGG-3' corresponding to nucleotides 7279 to 7301 and 7455 to 7474 respectively of the prototype STLV-3PHA969 sequence (GenBank accession number Y07616). The pol primers are SCPOL1outse: 5'-TTAAACCDGARCGCCTCCAGGC-3' (nt 2485 to 2506) SCPOL1outas: 5'-GGDGTDCCYTTRGAGACCCA-3' (nt 3201 to 3220) and SCPOL1inse: 5'-TAYHHAGGRCCAGGMAATAACCC-3' (nt 2556 to 2578).The 240 plasma were tested at a 1/40 dilution for the presence of HTLV-1/2 antibodies with a highly sensitive immunofluorescence test (IF), that uses MT2 and C19 as HTLV-1 and HTLV-2 viral antigen producing cells respectively. This test also allows the detection of STLV-3 positive samples . The 48 tax sequences were obtained for 4 and 11 samples which exhibited complete HTLV-1 and HTLV-2 WB profiles respectively, but none of the WB indeterminate sample gave a PCR signal. Consistent results were obtained for these HTLV-1 and HTLV-2 strains with the pol semi-nested PCR. However, a faint band (665 bp) was also obtained for one sample (Pyl43), which was previously found to be tax PCR negative. Sequencing of this fragment indicated the presence of an HTLV pol sequence that is highly related to STLV-3 strains (86.6% to 99.2% nucleotide identity). Based on an alignment of different STLV-3 sequences, a tax semi-nested PCR was then designed using SCTaxoutse (see above) and Mac4 followed by Mac2 and Mac4 as inner primers  respectively.The The author(s) declare that they have no competing interests.SC and SAC performed the laboratory work. SB did the serological assay and RD the phylogenetic analyses. AF and AG organized and performed the field studies, AG and RM designed, implemented and coordinated the study, wrote the manuscript. All authors have read and approved the manuscript.Wolfe et al. recently reported in an abstract the presence of two novel HTLV viruses . Whether


International Conference on Human Retrovirology: HTLV and Related Viruses sponsored by the Association supports clinicians and researchers in the exchange of research findings and stimulation of new research directions. This years conference will be held from June 22 to 25, in Montego Bay, Jamaica . Since its inception in 1988, these conferences have provided a highly interactive forum for the global community of HTLV scientists. This is of particular importance as HTLV research enters its third decade and a new generation of scientists takes over this important work. Many of the scientists attending the meeting will be from developing countries where HTLV is endemic, consistent with the history of international collaborations that have characterized HTLV research. The International Conference on Human Retrovirology provides a unique opportunity for researchers of all disciplines interested in HTLV infections to meet their peers and to address the questions facing clinicians and scientists who study retroviruses, like HTLV.Human T-lymphotropic virus type 1 (HTLV-1) and HTLV-2 were among the first human retroviruses discovered in the early 1980's. The International Retrovirology Association is an organized effort that fostered the efforts of scientists and clinicians to form interdisciplinary groups to study this group of retroviruses and their related diseases. The Association promotes excellent science, patient education, and fosters the training of young scientists to promote "bench-to-bedside" research. The  This organized effort from its beginning fostered the efforts of scientists and clinicians to form interdisciplinary groups to study HTLV and its related diseases in a cooperative and innovative manner. With the growth of the conference over the next decade  scientists, representing such diverse disciplines as epidemiology, virology, immunology, and clinical medicine, came together at the 6The Association has evolved since these humble beginnings to now promote research and education in the field of human retrovirology at the international level, including scientific conferences, interdisciplinary research collaborations, and educational exchanges related to the study of HTLV and related viruses. The Association has chosen to focus on HTLV and other related human and nonhuman primate retroviruses, in part, because numerous other organizations and conferences already exist to study human immunodeficiency virus (HIV). It strives to promote excellent science in the field of HTLV and related viruses and to facilitate the communication of scientific results. The Association fosters the education and training of young scientists who will contribute to and expand the field. It promotes "bench-to-bedside" research that translates findings from the laboratory into clinical trials that benefit HTLV-infected patients. Finally, it promotes awareness of and education about HTLV and related viruses to non-specialist physicians and the broader public.Human T-lymphotropic virus type 1 (HTLV-1) and the closely related HTLV-2 were among the first human retroviruses discovered in the early 1980's. HTLV-1 The International Retrovirology Association accomplishes its goals through a variety of activities. Principally among these is the sponsorship of its biennial general scientific conferences held at rotating international venues, generally in areas with endemic HTLV infection. This unique meeting brings together basic scientists, epidemiologists and clinical researchers in a free form exchange of data to discuss approaches to prevent HTLV infection or develop new therapies against HTLV-mediated diseases. The Association also sponsors smaller symposia and regional meetings directed at specific topics such as disease pathogenesis, treatment of HTLV diseases and regional epidemiology of this group of retroviruses. The group also honors the contributions of leading scientists through endowed awards to leaders in the HTLV research field and promotes partnerships with professional journals to promote the publication of HTLV research and conference proceedings. By funding travel scholarships for the biennial conference to young investigators the association encourages the next generation of retrovirologists and physician-scientists.HTLV and Related Viruses is a biennial conference that fulfills the continuing scientific need for the exchange of research findings and stimulation of new research directions. Basic scientists who study the molecular biology of HTLVs continue to grapple with the problem of how these viruses cause cancer. They have discovered important clues in this process by studying the viral gene product called Tax, and other factors that support virus replication. The conference also brings together those scientists that seek to understand the pathogenesis of HAM/TSP, which occurs presumably via aberrant immunologic response to the viral infection. The immune-mediated nature of HAM/TSP in infected subjects with particular HLA genotypes suggest important host factors in understanding this disease and provides a comparative model for other neuro-immunologic diseases such as multiple sclerosis. Clinicians and other scientists employing traditional and molecular epidemiologic tools have been reasonably successful at defining important public health issues related to HTLV infections. These discoveries have led to improved preventative measures to block mother to child transmission, better confirmatory test strategies for blood donor screening, and the prevention of HTLV-2 infection among injection drug users[rug users. Despitend to 25th, 2005, the 12th International Conference on Human Retroviruses: HTLV and Related Viruses meeting will be held in Montego Bay, Jamaica (Fig. From June 22th anniversary of the discovery of the first identified human retrovirus, HTLV-1. HTLV-1 and HTLV-2 infect a wide range of cells in cell culture. Recent reports that indicate that Glut1, the major vertebrate glucose transporter, acts as a HTLV receptor provides new and exciting directions in research of the pathogenesis and anti-viral therapy against HTLV-1[I, p13II, and p30II, which continue to provide important clues into virus replication and T-lymphocyte activation [This year it is appropriate that the conference is held in context to the 25st HTLV-1,11. Despst HTLV-1. Althougtivation . The conth International Conference on Human Retrovirology. Partnership between clinicians and scientists is key not only to the development of clinical trials but also to pathogenesis studies, which will inform the development of novel interventions. The biannual conference provides a unique opportunity for researchers of all disciplines interested in HTLV infections to meet their peers and truly look beyond their fields to elevate their "level of thinking" to address the questions facing clinicians and scientists who study retroviruses, like HTLV.The International Retrovirology Association through its many varied approaches, including its international conference, aims to encourage research in HTLV infections and disease, foster collaborations between research groups, provide a platform for critical analysis of new data and contribute to the dissemination of knowledge about these infections. While the biannual scientific meeting has been the cornerstone of the Association's activities there is increasing recognition of the need for more rapid progress in improving the management of HTLV-associated malignant and inflammatory diseases. The Association is ideally positioned to facilitate this process through its membership and the inclusion of a workshop on clinical trials in ATLL and HAM/TSP in the program of the 12HTLV, human T-lymphotropic virusesHTLV-1, human T-lymphotropic virus type 1HTLV-2, human T-lymphotropic virus type 2HAM/TSP, HTLV-1-associated myelopathy/tropical spastic paraparesisATLL, adult T-cell lymphoma/leukemiaThe authors have no competing financial or other interests involved in the data, methods, or writing of this manuscript.Edward Murphy, Steven Jacobson, Genoveffa Franchini, Graham P. Taylor, Barrie Hanchard, Owen Morgan and Michael D. Lairmore have all met the definition of author as outlined by the Retrovirology journal. Each has made substantive intellectual contributions to the commentary. Each author has given final approval of the version to be published. Each author have participated sufficiently in the work to take public responsibility for appropriate portions of the content.


When the first issue of Cell Communication and Signaling was launched in 2003, I had the opportunity to discuss the potential implications of CCN proteins in the control of cell-cell communication that is required for balanced tissue organisation, organ development, and species communication with their surrounding medium .Since then, the CCN family of proteins has emerged as a central hub for extracellular matrix signaling and CCN proteins were found in different subcellular compartments where they interact with a variety of signaling ligands and receptors  as an essential media for the exchange and diffusion of scientific information regarding inter cellular signaling.The publication of the "Journal of cell Communication & Signaling [JCCS published by Springer]" as the official organ journal of ICCNS is major step along the establishment of the field.For ethical reasons, the editorial board of CCS and myself had to present our resignation to BMC, effective september 2007.We wish to inform that according to BMC policy manuscripts published in CCS will remain in the BMC database, and will remain freely accessible to readers.The editorial board and myself wish to thank all authors for their contribution and members of the publishing team at BMC for their expertise and support during all these years.Pr. Bernard PerbalEditor in Chief


Sera from a small sample of adult blood donors, healthy school children and patients with lymphoma, leukaemia, non-haematologic cancer, congenital and inflammatory disorders from Ibadan, Nigeria were screened for HTLV-I antibody by an enzyme-linked immunoabsorbent assay and confirmed by investigational Western blot. Seventy-nine of 236 positively screened samples could not be tested for confirmation. Seropositive reactivity was observed in nine of 123 blood donors, and 3 of 46 healthy school children but banding patterns on Western blot were often sparse. Among non-Burkitt's non Hodgkin's lymphoma patients six of 30 were HTLV-I positive including four of four with clinical features of adult T-cell leukaemia (ATL). Other clinical conditions had a frequency of positivity indistinguishable from healthy donors. Western blot patterns ranged from strong with multiple bands, which were uncommon, to those with only p24 and p21 envelope positive which were frequent. Given the relative paucity of clinical ATL and the unusual Western blot patterns the true rate of HTLV-I infection may be lower than estimated. It is possible that a cross-reactive HTLV-I-like virus accounts for this pattern.


Recent studies have reported that T cells can integrate signals between interrupted encounters with Antigen Presenting Cells (APCs) in such a way that the process of signal integration exhibits a form of memory. Here, we carry out a computational study using a simple mathematical model of T cell activation to investigate the ramifications of interrupted T cell-APC contacts on signal integration. We consider several mechanisms of how signal integration at these time scales may be achieved and conclude that feedback control of immediate early gene products (IEGs) appears to be a highly plausible mechanism that allows for effective signal integration and cytokine production from multiple exposures to APCs. Analysis of these computer simulations provides an experimental roadmap involving several testable predictions. The orchestration of the adaptive immune response is predicated on the integration of signals derived from peptide fragments that bear the molecular signature of an invading pathogen. T cells become activated by integrating signals derived from such peptides that are presented with proteins from the Major Histocompatibility complex (MHC) that are present on the surface of antigen presenting cells (APCs). Upon engagement with an APC, signal transduction is initiated by the interaction of the T cell Receptor (TCR) with MHC-peptide complexes. Successful signal integration results in numerous phenotypic outcomes and allows for the T cell to coordinate an appropriate immune response. In contrast, the failure of such priming processes leads to deleterious consequences such as autoimmunity.The advent of two-photon imaging technologies has allowed for the study of real-time, in vivo T cell activation in lymph nodes in the presence of an antigenic challengein vitro experiments were conducted to specifically address the question of whether T-cells can integrate multiple interrupted signals and utilize the accumulation of these signals for a biological responseIn a parallel investigation into the nature of T cell signaling, a Control T cell Activation dithFseries of provocative In addition, the authors report several other unexpected findings. They first demonstrate that thirty minutes of stimulation is initially insufficient for cytokine  production. They then show that subsequent rounds of TCR signaling, after interruption of the signal, produce significant amounts of cytokine within thirty minutes. These results suggest that T-cells have the ability to integrate interrupted signals from multiple encounters with antigen and also suggests that T-cells can exhibit a “short-term” (the phrase “short-term” is used to distinguish from the ‘long-term’ development of the T cell memory phenotype) memory of past exposures to antigen in that the first exposure seems to prepare the T cell for subsequent exposures to antigen. The acquisition of ‘short-term’ memory is an emergent property that requires the coordination of cooperative, dynamic events involving many interacting cellular components. Thus, it is often difficult to intuitively assess how different molecules act in concert to exhibit collective, system-wide cellular behaviors such as signaling memory. In this regard, theoretical and computational studiesIn the scope of this work, we present results from computer simulations of mathematical models that explore the physiological consequences of different mechanisms that can account for the biochemical memory observed in several experiments. We first briefly discuss some key observations from existing data in the literature and in particular, the experimentOur goal was not to simulate every detail of the T-cell signaling network but rather to construct the simplest possible models that can investigate how the basic structure of several different molecular mechanisms could lead to the collective property of biochemical memory in T cell signaling. Other computational models have considered many elements of T cell activation such as how sensitivity to antigen is establishedInstead, our modeling efforts specifically aim to demonstrate how the first round of signaling can seemingly prepare the T cell for signal integration so that later exposures to antigen do not require a long lag time for cytokine production. Towards this end, we focus on events at later times that occur downstream of Erk activation. We derive from these models, testable predictions that can ascertain many aspects of such memory phenomena in T cell signal integration. The predictions that emerge from our calculations suggest several experiments that could further elucidate the possible mechanism for this ‘short-term’ biochemical memory. Finally, it is our hope that such calculations serve as a template for further quantification and modeling of memory phenomena and signal integration that are observed in T cell signaling.While initial signaling events, such as the mobilization of intracellular calcium and activation MAPK pathways, occur within minutes of the initial T cell/APC contact, at least thirty minutes of signaling is required for cytokine production. One hypothesis for the existence of this waiting period is that there is a time required for the accumulation of immediate early gene products (IEGs) such as Jun and Fos proteins which comprise the AP1 transcription factor complex. IEGs are synthesized de novo upon TCR signaling and their presence is a necessary condition for cytokine productionStudies on the duration of Erk signaling leading to the accumulation of IEGs suggest that a hyper-phosphorylated state of the IEG product Fos can remain active for long timesOne general way to generate sustained activity of signaling intermediates is to exploit positive feedback in signaling networksde novo upon TCR signaling and are necessary for cytokine productionThe first model that we studied is derived primarily from experimental results relating signal duration with the stability of IEG productsHowever, since all cytokine production appears to cease once TCR signaling is aborted, active IEGs alone cannot be a sufficient condition for IFN gamma production. For example, calcium mobilization another necessary condition for cytokine productionAs it stands, this mechanism for signaling memory is not without difficulties. Degradation mechanisms that are mediated by various ubiquitin pathways have been shown to occur with members of the AP-1 complex such as JunThe three scenarios examined in detail, are depicted in The calculations aim to mimic the experiments by periodically interrupting signaling by “inhibiting Lck” in the simulation for a period and then removing the “inhibitor”. This is accomplished by disallowing any contribution of triggered T cell receptors to the activation of downstream pathways for a specified time interval. The “strength” of the signal is determined by the duration of initial signaling, the number of agonist pMHC molecules, or the affinity of agonist molecules. Two general cases .Finally, we observe the case where IEG products are embedded in an autocatalytic feedback loop . For strQualitative differences among the three models are further illustrated by monitoring the time evolution of probability distributions of pertinent signaling species. Such distributions are the analog to monitoring the statistics of the cell population. In In the presence of a feedback loop and sufficiently strong stimulation, we obseFor the case of weak stimulation, signal In the absence of a feedback loop , the disThese results appear to suggest that experiments that probe the dose-dependent response of the signaling system would give qualitatively different results for each of the three models. The presence of a feedback loop would allow for an “all or nothing” memory effect and cytokine response, as would the presence of a cooperative “switch-like” enzymatic stabilization mechanism. Alternatively, in the case of a model with linear enzymatic kinetics, a graded response would be predicted. A graded dose response is expected because there is no mechanism to give rise to a threshold in the signaling network. Such behaviors are displayed in What might be the biological consequence of hysteretic effects present in the production of IEG products? We first consider the dependence of hysteresis on the strength of such a feedback loop. Such an effect in Our computational analysis suggests specific experiments that could provide insights into the mechanisms that underlie the ability of T cells to integrate signals and retain a “memory” in the signaling process. The most significant experiments will be ones that monitor the stability of transcription factors in and out of the nucleus and determine whether individual activated molecules are stable or rather, constantly turning over when signal memory is exhibited. Signaling “memory” then can be assessed by the persistence of nuclear transcription factors after inhibition of the signaling pathway. Experiments with the Lck inhibitor PP2, in conjunction with immunofluorescence assays that make use of fluorescent secondary linked antibodies, can monitor the nuclear translocation of the relevant transcription factors such as Fos, Jun, NF-κB, and NFAT upon disruption of TCR mediated signaling. These experiments will be essential to understanding the relevant transcription factors that enable short-term biochemical signaling memory.Feedback loops are ubiquitous in T cell signalingFurthermore, one prediction obtained from this model suggests that one could distinguish between the two possible mechanisms for sustained activity by carrying out photobleaching experiments using GFP constructs of the relevant transcription factors. If signaling memory is due to a long half-life of the signaling intermediate, photobleaching will largely eradicate the ability to observe nuclear localization of the transcription. If, on the other hand, signaling memory is due to existence of positive feedback in the signaling circuit, then one would expect that nuclear fluorescence will rapidly recover after photobleaching.in silico. Should it be found that a particular model where individual activated molecules are rendered stable for long times is appropriate, the importance of cooperative enzymatic modifications can be determined by measurements of dose-response curves for the amount of activated cFos as a function of signal strength, which could be modulated by the amount or quality of the agonist pMHC as well as the duration of the initial signal.These experiments will first help to first determine whether AP1 is the transcription factor that is the source of biochemical memory, and should allow one to discriminate between the models of biochemical memory we have studied Directly observable predictions about how the kinetics and strength of TCR signaling affects signaling memory could be tested. Since a model involving either a cooperative mechanism or a feedback loop predicts a threshold for a memory effect in signal transduction, the strength of signal will determine whether or not T cells can integrate signals during multiple exposures to antigen. These models propose that there exists some crossover between weak and strong agonists, short and long durations of TCR signaling, and concentrations of low and high numbers of agonist; this crossover will determine whether or not a lag time is required for cytokine production during subsequent rounds TCR signaling after the signal has been disrupted.Signaling memory implies the persistence of sustained activity of some biophysical or molecular signaling intermediate even after signaling is interrupted by removing the stimulus. Such an intermediate could be the persistence of spatially clustered signaling components (e.g. as the formation of microclusters)in silico, several molecular models which can explain the mechanism of biochemical memory in T cell signaling and activation. Each model involves the sustained activation of a certain transcription factor in the presence of disrupted signaling. Furthermore, our computer simulations make several predictions that we have briefly outlined.In summary, we have explored, in vivo along with genetic and biochemical experiments to investigate the underlying mechanisms in T-cell activation across multiple length and time scales, from the molecular features governing the dynamics of signaling pathways to the clearance of infection occurring at higher levels of biological organization.It is our hope that this work will serve to motivate as well as guide future experimentation into mechanisms underlying biochemical memory in T-cell signaling and activation. Once these mechanisms are better understood, further elaboration on the details of our computer models will be necessary to provide a better quantitative analysis of the mechanism governing the memory phenomenon. Also, it will be important to address in the near future how signaling memory at the cellular level functions in the context of T-cell activation in vivo where T-cell migratory patterns in lymph nodes are important in controlling the overall outcome of the physiological response. Integration of a more detailed computational model of the signaling pathways that maintain short-term memory with a computational model for T-cell trafficking in lymph nodes will be essential for understanding this problem. A model of this sort can then be used in conjunction with two-photon imaging experiments The signaling models that we chose to simulate consist of the following half reactions and the basic set of molecular processes common to each of the three models is as follows (cFOS is taken to be the example of the Immediate Early Gene product):a.) Linear modelWe simulated these models by solving a master equationThe set of kinetic parameters used in the simulations is shown in sigτ, pτ1, pτ2, mem τcytτ}, that emerge in the modular network architecture that we constructed.The mathematical models of cell signaling that we analyzed are comprised of several modular components. Therefore, the sensitivity of the qualitative results of our models to the choices of kinetic parameters may best be understood by considering the key competing time scales, {sigτ is the time scale for signals derived from TCR-MHC to propagate to downstream messenger pathways. sigτ emerges from kinetic constants and initial concentrations in reactions (1) and (2). sigτ then, is a measure of the overall signal strength, which can be varied by adjusting the agonist concentration. For example, high strength (1000 pMHC molecules) and low signal strength (10 pMHC molecules) as well as long and short durations of signal map onto a value of sigτ. pτ1 and pτ2 are the characteristic time scales involved in activating the two parallel messenger pathways in our model. pτ1 is the time scale to activate the fast pathway (e.g. Ca2+ Mobilization and active NFAT). pτ2 is the time scale required to activate the other pathway that leads to the synthesis of unstable IEG products. memτ is the time needed to establish a biochemical memory in the signaling circuit. A model assumption is that (pτ1∼pτ2)<<memτ. If this were not the case (i.e. (pτ1∼pτ2)>memτ) then subsequent rounds of signaling would not quickly produce cytokine. Thus, pτ1 and pτ2 as well as the the time scale for cytokine production cytτ then limits the speed at which productive signaling can recover from interrupted stimulation. A mechanism involving the stabilization of IEGs as a source of memory requires that memτ be large–at least on the order of minutes.memτ were varied and results are either presented in the main text or are discussed below. For the linear model, memτ changes in response to the kinetic parameters in reactions 10a, 11a, 12a and subsequently determines the amount of stable IEG but does not affect qualitative findings. For the cooperative model, parameters in reaction 10b then control the positioning and sharpness of the threshold. α controls the amount of stable IEG that can be obtained. The behavior of the parameters involved in the feedback loop is discussed in the results section. Finally, cytτ is a time required for cytokine production once signaling intermediates (available IEGs and Transcription factors come that from the other pathway) are available. Changing cytτ then results in changing the amount of cytokine produced in the simulation in a monotonic fashion.Parameters from each model contributing to For the most part, we found that many of the qualitative results obtained from our models are robust to large (greater than 10-fold) variations in key individual parameters. However, two key parameters in our model could potentially change the qualitative results of our computer simulations; these parameters are the rate constant for the de novo synthesis of IEGs (reaction 7) and the rate constant for IEG decay (reaction 8). This is because, in our model, there exists a competition between the synthesis of cFOS and its decay. Upon varying these rates, we find that our key result can be sensitive to the rate of cFOS production. If cFOS synthesis is too slow, then no stabilized cFOS will be present once the stimulus is removed at t = 30 minutes in our simulations.