HTLV-1 causes adult T-cell leukemia (ATL). Although there have been many studies on the oncogenesis of the viral protein Tax, the precise oncogenic mechanism remains to be elucidated. Recently, a new viral factor, HTLV-1 basic Zip factor (HBZ), encoded from the minus strand mRNA was discovered and the current models of Tax-centered ATL cell pathogenesis are in conflict with this discovery. HBZs consisting of non-spliced and spliced isoforms (HBZ-SI) are thought to be implicated in viral replication and T-cell proliferation but there is little evidence on the HBZ expression profile on a large scale.To investigate the role of HBZ-SI in HTLV-1 provirus-positive cells, the HBZ-SI and Tax mRNA loads in samples with a mixture of infected and non-infected cells were measured and then adjusted by dividing by the HTLV-I proviral load. We show here that the HBZ-SI mRNA level is 4-fold higher than non-spliced HBZ and is expressed by almost all cells harboring HTLV-1 provirus with variable intensity. The proviral-adjusted HBZ-SI and Tax quantification revealed a characteristic imbalanced expression feature of high HBZ and low Tax expression levels in primary ATL cells or high HBZ and very high Tax levels in HTLV-1-related cell lines (cell lines) compared with a standard expression profile of low HBZ and low Tax in infected cells. Interestingly, according to the mutual Tax and HBZ expression status, HTLV-1-related cell lines were subcategorized into two groups, an ATL cell type with high HBZ and low Tax levels and another type with high Tax and either high or low HBZ, which was closely related to its cell origin.This is the first comprehensive study to evaluate the mutual expression profile of HBZ and Tax in provirus-positive cells, revealing that there are quantitative and relative characteristic features among infected cells, primary ATL cells, and cell lines. Adult T-cell leukemia (ATL) is a unique T-cell malignancy derived from T-cells infected with a retrovirus of human T-cell leukemia virus type-1 (HTLV-1) . ATL is I, p13II, and p30II [Although it is a fact that HTLV-1 reaches an oncogenic event and causes ATL, the oncogenic mechanism of HTLV-1 is not fully understood. The HTLV-1 genome, in addition to the structural and enzymatic proteins gag, pol, and env, encodes the regulatory and accessory proteins tax, rex, p12nd p30II ,5. Amongnd p30II ,7. Indeend p30II . On the A novel viral protein, HTLV-1 basic zipper factor (HBZ), which is encoded by the minus strand RNA of the HTLV-1 genome, has been identified recently ,10. We aIn this study, to investigate the role of HBZ in the multi-step development of ATL, the quantitative expression levels of HBZ and Tax transcripts were measured by real-time reverse-transcription PCR using HTLV-1-infected cells well characterized by HTLV-1 proviral integration status. Consequently, HBZ transcripts were observed ubiquitously in almost all cells harboring HTLV-1 provirus, and primary ATL cells were characteristic with the very high HBZ transcript levels relative to Tax.Blood specimens with cells carrying HTLV-1 provirus from ATL patients and healthy persons were collected in our hospital under the approval of the Research Ethics Committee of our Institute.+T-cell line derived from MT1 during many passages [According to the status of cytomorphological and clinico-oncological findings, HTLV-1 proviral load and the HTLV-1 proviral integration status were determined by Southern blot analysis and classified into 31 asymptomatic carriers (AC), and 35 patients with ATL. ATL was subtyped according to the JLSG criteria ; acute apassages . All of passages ,18.HTLV-1 infection was demonstrated by a commercial anti-HTLV-1 assay kit . In this study, infected cells were defined as non-malignant T-cells randomly integrated with HTLV-1 provirus, while ATL cells were defined as malignant T-cells monoclonally integrated with the provirus.High molecular weight DNA was extracted from mononuclear blood cells and cell-lines using a QIAmp DNA Blood Mini kit . Total RNA was extracted using ISOGEN . After removing contaminating-genomic DNA using a Message Clean kit, two types of anti-sense cDNA and sense cDNA were synthesized. Sense cDNA was synthesized using Oligo(dT)12–18 Primer and SuperScriptTM RT (Invitrogen). The first anti-sense strand cDNAs used to amplify both HBZ and HBZ-SI mRNAs were reverse-transcribed using a minus-strand-specific primer, 5'-cccatgtctcaatactacaagaaag-3', in order to avoid contamination of cDNA from the HTLV-1 sense strand genome.Real-time RT-PCR was performed using a LightCycler Technology System (Roche Diagnostics) as described previously . BrieflyThe HTLV-1 Tax mRNA load was measured from a template of sense cDNA using the same LightCycler PCR System as described previously . BrieflyUsing restriction enzymes of EcoRI and PstI and a digoxigenin-labeled whole HTLV-1 probe, SBH analysis was performed as described previously ,21. Visi-3 (one infected cell relative to 1000 non-infected cells). Normalization was done by using β-globin quantification as an internal control. Assuming one provirus per infected cell , proviral load was considered to be equivalent to the number of infected cells, namely infected-cell number per 10000 cells = (copy number of Tax)/(copy number of β-globin/2) × 10000.Next, HTLV-1 proviral load was quantified using a real-time DNA PCR LightCycler Technology System according to our previously described method ,23. The Using the Stat View software, the Mann-Whitney U test or Student's t-test were used to compare data between two groups, and Spearman's rank correlation was used to examine the two groups.4 cells was 316 in ACs, 2739 in ATL patients, and 7600 in cell lines.The HTLV-1 proviral load represents the population of infected cells in blood mononuclear cells when one cell harbors one provirus. Accordingly, we first examined the band status of SBH analysis. Using EcoRI digested DNA samples, SBH analysis revealed one sharp band in 28 ATL samples, two bands in 2 ATL samples, and smeared-bands in 36 AC samples including 5 ATL patients in remission. The samples with two bands were adjusted when the infected cell number was estimated based on the HTLV-1 proviral load. The cell lines used here were also demonstrated to contain multiple proviruses in their genomes, e.g. 8 bands in HUT102 and 2 bands in ST1. Subsequently, the mean value of the HTLV-1 proviral load per 103 and 0.2 × 103 in ACs, 1.3 × 103 and 6.0 × 103 in ATL samples, respectively. Of all samples tested, the expression load of HBZ-SI was about 4-fold higher than that of HBZ . Accordingly, HBZ-SI was analyzed in this study.HBZs are known to consist of non-spliced and spliced isoforms, HBZ and HBZ-SI, as shown in Figure 5. As shown in Figure 4. Consequently, the HBZ-SI mRNA expression level after adjustment, as shown in Figure HBZ-SI mRNA load was detected in all but 5 of the 72 samples ,2),3), and 4)) distributed in the area of high HBZ-SI and low Tax (the ATL cell area) all originated from an ATL cell clone, while three other lines ,7), and 8)) out of 4 distributed in the area of high-Tax and either high or low-HBZ-SI were derived from infected cells.Then, to investigate the mutual expression status in the three provirus-positive cell types of non-malignant infected cells, ATL cells, and HTLV-1-related cell lines, the ratio of HBZ-SI/Tax was calculated. The mean ratio of HBZ-SI/Tax was 3.7 in infected cells, 330 in ATL cells, and 0.02 in the cell lines, representing an imbalanced expression between HBZ and Tax in ATL cells and cell lines compared to the base line of the 3.7 in infected cells. This feature is depicted as a twin dot plot of HBZ-SI and Tax loads in Figure Since loss of HBZ-SI or Tax transcripts in MT1s and underestimation of proviral copy number in KK1 was observed in this study, we examined the genomic structure of the provirus by DNA PCR amplifying between nucleotides (nt) 6461 to 8853 including the region of the HBZ gene. Interestingly, as shown in panel B of Figure Many studies have indicated that Tax is likely to be a central player in the induction of ATL. However, nobody has answered the paradoxical question why T-cell transformation and clonal proliferation of ATL cells is associated with a Tax-low or -negative phenotype. HBZ, a novel viral factor encoded from the minus-strand RNA of HTLV-1, is expected to play an important role in HTLV-1 biology by counteracting the action of Tax. Indeed, HBZ has been shown to interact with the cellular transcription factor CREB to inhibit HTLV-1 transcription . HoweverThis study demonstrated a ubiquitous expression of HBZ isoforms, mainly the spliced isoform of HBZ-SI, in almost all provirus-positive cells. Furthermore, in contrast to Tax, up-regulation of HBZ was characteristic of primary ATL cells, although the increase in level was subtle. These results were supported by previous studies describing that HBZ mRNA is expressed in all fresh ATL cells and HTLV-1 cell lines ,12,25, bInterestingly, HBZ-SI mRNA was detectable in samples from almost ACs and ATL patients. Furthermore, the HBZ-SI mRNA load was significantly correlated with the HTLV-proviral load, but not the Tax mRNA load. On the other hand, although Tax mRNA was also subtly detectable in blood samples from ACs and patients with ATL, it was not correlated with the HTLV-1 proviral load. These results indicate that the expression profile of HBZ is different from Tax, namely HBZ is near-equally expressed by all provirus-positive cells, while Tax levels are variable and can be actively up-regulated when necessary. In other words, the absolute amount of HBZ-SI mRNA load is dependent on the total of infected cells estimated by the proviral load within samples consisting of a mixture of infected and non-infected cells. Accordingly, in order to compare the expression intensity per provirus-positive cells only, it is reasonable to adjust by dividing the HBZ-SI mRNA load by the proviral copy number estimated by the HTLV-1 proviral load . ActuallWhat does the difference in the mutual expression patterns, such as low-HBZ and low-Tax in infected cells, high-HBZ and no or subtle-Tax in primary ATL cells, and variable high or low HBZ and Tax in cell lines mean? Currently, only the role of Tax is stressed for oncogenic pathogenesis of HTLV-1, so the co-operative occurrence of up-regulated HBZ and down-regulated Tax may be closely associated with the oncogenic process in the early stage and with the persistent maintenance of malignancy in the late stage. Interestingly, recent reports on HBZs encoded from the minus strand of HTLV-1 seem to be providing a new insight in the current models of Tax-centered HTLV-1 pathogenesis, such as ATL oncogenesis and viral replication. In particular, the bimodal function of HBZs is of interest, in which the HBZ protein suppresses Tax transactivation of E2F1 and the HBZ mRNA promotes T-cell proliferation ,25. Furt4 fold less than that in MT2 cells, which is consistent with previous results as reported by Furukawa et al. [Another important point of this study is a mutual correlation of Tax and HBZ mRNA expression level in HTLV-1-associated cell lines. Although Tax mRNA is generally said to be low or negative in all ATL cells and extremely high in cell lines, our Tax mRNA quantification clarified that Tax mRNA was detectable in almost ATL cells at the intensity of approximately 10a et al. . In contKK1 and MT1s were found to harbor defective proviruses involving the pX/HBZ gene region, probably resulting in the loss of tax and HBZ mRNA expression in the MT1s. Despite the absence of HBZ, the cells have immortalized and survived for many generations, suggesting the possibility that HBZ may not be required in ATL cells, at least in cell lines.In conclusion, our study provides better understanding of multi-step leukomogenesis in ATL through the characteristic expression of HBZ isoforms. Among the isoforms of HBZ, HBZ-SI is dominant over non-spliced HBZ. HBZ-SI is constantly and ubiquitously expressed in all cells harboring HTLV-1 provirus and is more highly expressed in ATL cells than in infected cells. To address ATL cell pathology induced by viral factors, it is of importance to evaluate simultaneously Tax and HBZ mRNA levels and proviral load.HBZ: HTLV-1 basic zipper factor; SBH: Southern blot hybridization; PCR: polymerase chain reaction; AC: asymptomatic carriers; HTLV-1: human T-cell leukemia virus type-1; ATL: adult T-cell leukemia.The authors declare that they have no competing interests.TU designed the study, and performed the analysis. KY, KT, KM and HH recruited and monitored the subjects. YY provided the cell lines. SK made substantial contributions to the conception and design of the study, wrote and drafted the manuscript, and contributed to data interpretation.


Binding of the HIV-1 envelope to its chemokine coreceptors mediates two major biological events: membrane fusion and signaling transduction. The fusion process has been well studied, yet the role of chemokine coreceptor signaling in viral infection has remained elusive through the past decade. With the recent demonstration of the signaling requirement for HIV latent infection of resting CD4 T cells, the issue of coreceptor signaling needs to be thoroughly revisited. It is likely that virus-mediated signaling events may facilitate infection in various immunologic settings in vivo where cellular conditions need to be primed; in other words, HIV may exploit the chemokine signaling network shared among immune cells to gain access to downstream cellular components, which can then serve as effective tools to break cellular barriers. This virus-hijacked aberrant signaling process may in turn facilitate pathogenesis. In this review, we summarize past and present studies on HIV coreceptor signaling. We also discuss possible roles of coreceptor signaling in facilitating viral infection and pathogenesis. Infection by the human immunodeficiency virus (HIV) causes severe depletion of the CD4 T cell population, which eventually leads to acquired immunodeficiency syndrome (AIDS). The selective nature of the infection immediately prompted speculation and the subsequent identification of the CD4 molecule as the main surface receptor for HIV entry The discovery of the HIV chemokine coreceptors opened up a new avenue for AIDS research. The fact that both CXCR4 and CCR5 are chemokine receptors raised interesting questions regarding the role of chemokine receptor signaling in viral infection and pathogenesis s, Gαi, Gαq, Gα12/13), and CXCR4 seems to be specifically coupled to Gαi and Gαq. Gαq proteins activate phosphatidylinositol-specific phospholipases such as phospholipase C-γ (PLC-γ), which hydrolyzes phosphatidylinositol-4,5-biphosphate (PIP2) to generate inositol triphosphate (IP3) and diacylglyerol (DAG). These events lead to calcium flux and the activation of several PKC isoforms that have been shown to be important for SDF-1-induced chemotaxis i protein activation, which inhibits adenylyl cyclases that in turn lead to a reduction in cAMP levels as well as the activation of phospholipases and phosphodiesterases. These events result in the activation of the lipid kinase PI3K via Gαi-coupled Src-family kinases as well as PI3Kγ through direct binding of Gβγ to the regulatory subunits of PI3Kγ Chemokine receptor signaling is known to be diverse and is coupled to distinct signaling pathways that mediate cell migration, transcriptional activation, and cell growth and differentiation . For exaSDF-1 binding to CXCR4 also results in GTP-bound Gβγ, which can stimulate guanine nucleotide exchange factors (GEFs) such as TIAM1 and PREX1 specific for Rho family GTPases such as Rac, Rho Consistent with the signaling diversity of chemokine–coreceptor interaction, binding of HIV-1 gp120 to CCR5 or CXCR4 has also been shown to trigger the activation of Pyk2 Given this array of targets that can be activated by gp120 in vitro, one question is whether these signaling events are physiological at the low gp120 dosages present in vivo. The presumption based on plasma viral load is that the physiological levels of gp120 are likely to be significantly lower than most of those used in in vitro experiments In our recent studies of gp120 signaling in peripheral resting CD4 T cells, we did notice dosage- and conformation-dependent differences in gp120 signaling The direct involvement of chemokine coreceptor signaling in HIV infection has been speculated right from the identification of the chemokine coreceptors Despite growing evidence supporting a possible role for coreceptor signaling, contradictory findings remained. Amara et al. compared R5 HIV infection of CCR5 wild-type and signaling-deficient T cells derived from CCR5Δ32 individuals Recently, we examined the requirement for CXCR4 signaling in HIV-1 latent infection of human resting CD4 T cells using an in vitro system to mimic in vivo latent infection of resting T cells With the identification of the molecular mechanism of CXCR4 signaling, the remaining issues are to confirm the requirement for CCR5 signaling, and to determine the molecular basis for this requirement. There appears to be a clear distinction in the requirement for signaling between R5 and X4 viruses. For example, the B-oligomer of PTX inhibits R5 virus at entry and reverse transcription, but inhibits the X4 virus at multiple steps post reverse transcription CCR5 signaling has also been known to trigger distinctive signaling cascades that activate kinases and transcription factors associated with cell activation It has long been suggested that HIV-1 envelope plays a central role in HIV-mediated CD4 T cell depletion and pathogenesis. Prior to the identification of the chemokine coreceptors, gp120 was proposed to directly trigger activation-dependent T cell apoptosis through its binding to surface CD4 6 resting CD4 T cells HIV envelope–chemokine receptor signaling has been shown to mediate chemotaxis in both CD4 and CD8 T cells In the human immune system, cofilin is involved in two hallmark activities of T cells, namely chemotaxis and T cell activation The utilization of different chemokine coreceptors, either CCR5 or CXCR4, for entry largely differentiates HIV into two distinctive phenotypes with either M- or T-tropism. The high pathogenic potential of the late emerging T-tropic viruses in causing rapid CD4 depletion is clearly a demonstration of the pathogenic significance of the CXCR4-engaging viruses Understanding HIV envelope–coreceptor interaction holds the key to unlocking the mystery of HIV-mediated CD4 depletion and pathogenesis. The signaling capacity of the viral envelope, along with its pathogenic potential, has been a subject of great interest and intense investigation. Yet, the critical role of the coreceptor signaling in HIV disease had not been clearly defined. This lack of appreciation has been largely attributed to the complexity of the chemokine signaling network itself, as well as the complication of many in vitro systems used, in which cellular states are derailed from the genuine signaling circuitries of natural viral targets in the body. Given these complexities, a critical function of the coreceptor signaling in promoting viral infection has only recently been demonstrated in the latent infection of human resting CD4 T cells. The deciphering of the molecular mechanisms involved starts to offer exciting fresh perspectives and opens a new avenue for examining HIV pathogenesis. However, a great deal is yet to be tested regarding the in vivo importance of coreceptor signaling in mediating CD4 T cell dysfunction and pathogenesis.Human CXCR4 – Entrez Gene ID # 7852; Human CCR5 – Entrez Gene ID # 1234; Human non-muscle Cofilin 1 – Entrez Gene ID # 1072.


The central ritual of Holi involves throwing of colors on one another. Playing with toxic industrial dyes often results in various dermatological complaints in a significant number of people immediately following the celebration.To describe patterns of various skin manifestations directly or indirectly related to the use of different colors in the celebration of Holi.Observational clinical study on consecutive patients presenting to a teaching hospital in Kolkata, India.Forty-two patients with a mean age of 24.2 years were studied. Itching was the commonest symptom , followed by burning sensation, pain, oozing, and scaling. Eleven patients’ symptoms were attributed to activities related to preparation of colors and the removal of colors from the skin surface. Eczematous lesions were the most common pattern  followed by erosions, xerosis and scaling, erythema, urticaria, acute nail-fold inflammation, and abrasions. Thirteen (30.9%) patients reported aggravation of preexisting dermatoses . Secondary pyoderma occurred in 3 (7.1%). Face was the commonest site affected , followed by dorsum of the hands, scalp, forearm, palms, arms, and trunk. Ocular complaints in the form of redness, watering, and grittiness occurred in 7 (16.7%) patients.Various forms of cutaneous manifestations, often associated with ocular complaints, occur commonly due to Holi colors. Public awareness and regulatory actions are needed to avoid these preventable conditions. Holi, an annual festival celebrated all over India since ancient times. Holi begins on the day after the full moon in the Hindu month of Phalguna (February-March). Originally, an agricultural festival of fertility and harvest to welcome the spring, Holi also commemorates some Hindu mythology and legends. Holi is one of the most popular annual events in India, observed with great festivity and hilarity. In the eastern Indian state of West Bengal and in the surroundings of the city of Kolkata, it is popularly known as Doljatra or Basonto- Utsav . On the day of Holi, people gather together in a common place where they traditionally celebrate by applying colors in different forms on friends, family, and even complete strangers. Originally, the bright flowers that blossomed during spring acted as raw materials from which the different shades of Holi colors were made. Most of these trees were supposed to have medicinal properties beneficial to the skin. Over the years, with the spread of industrialization and urbanization, natural colors came to be replaced by inexpensive industrial dyes manufactured through chemical processes. A large number of patients consult dermatologists over the few days immediately following Holi for skin problems arising out of celebration with these colors. Although widely recognized and discussed as an annually recurring problem involving many people, lack of any formal study on it has prompted us to undertake the present work. We intend to describe the patterns of cutaneous manifestations in patients resulting from the colors and associated complications.Cultural and religious practices may have significant impact on the health of people. Some practices may often introduce health hazards, including skin diseases. One such cultural and religious practice is Holi colors to the Dermatology Clinic of a teaching hospital at Kolkata, India, were included in this observational clinical study. This year (2008), the Holi was observed on 21st March. The study duration included the next two weeks, because this was the most probable time of presentation for Holi-related dermatoses. Detailed history regarding age, gender, symptoms, type of color used, activities related to preparation and removal of colors from the skin, and history of aggravation of preexisting dermatoses were recorded. Every patient then underwent a thorough clinical examination with special reference to the morphology of the lesions and sites of involvement.Consecutive patients presenting with dermatological problems directly or indirectly attributed to the A total of 42 patients were evaluated. All the patients were residents of the state of West Bengal comprised by both urban population of the city of Kolkata and its surrounding rural districts. Their ages ranged from 4 to 46 years with a mean of 24.2. One third of the patients (14) belonged to the 20–30 years age group. There was a slight female preponderance (F: M = 1:0.8). Pruritus was the commonest symptom found in 25 (59.5%) patients followed by burning sensation , pain , oozing  and scaling . Thirty-four (80.9%) patients were exposed to more than one color. Red color was the commonest  color used, followed by green , blue , black , pink , silver , violet , and golden yellow . Cutaneous involvements were limited to the sites of application of colors. Among different types of cutaneous affections , eczematHoli is now a symbolic commemoration of a legend from Hindu mythology which provides some of the ingredients for the celebrations.[Holi as a symbolic mark of this event from mythology. This vibrant festival is also associated with the legends of immortal love between Lord Krishna and Radha.Originally, a festival to celebrate good harvests and fertility of the land and to welcome spring with its new life and colors, brations. The legeHoli involves throwing of colors in various forms on one another. The colors used during the Holi come in different forms including pastes, colored powders, and watercolors. Colors are often thrown at people with the help of long syringes called pichkaris [gulals’ or ‘abeer’, have two components – a colorant and a base, both of which may cause cutaneous problems. Mica dust is often added as a sparkling agent to the dry powders that can lead to multiple microtraumas of skin and predisposition to infections.[Despite its religious roots, little religious activities are involved in the celebration and the central ritual of ichkaris  or balloichkaris  The dry fections. Use of cHoli colors,[Holi-related cutaneous affections have hitherto been unreported in literature. Although definite cause-effect relationship was not established in our cross-sectional observational study, it has emerged that skin problems occur quite frequently following the celebration with colors. Most of the patients complained of pruritus and burning sensation and eczematous reaction pattern was found to be the commonest clinical findings in our patients (54.1%). There was no definite correlation between the hue of the color reported to be used and the cutaneous reaction patterns. The face was found to be the most commonly affected site presumably due to the fact that it was the usual site to which colors were applied. Hand involvement in some patients was attributed to immersion of hands in colored solutions during their preparation. In addition, different measures including vigorous scrubbing to remove the stains after the celebration also caused abrasions of skin. Thus, the dermatoses related to the celebration of Holi were presumably due to irritant or allergic contact dermatitis as well as mechanical factors. A limitation of our study has been our inability to perform appropriate patch tests to detect the precise etiology because the colorants were not available with the patients at the time of presentation and the reactions could have been due to unknown industrial dyes. A significant number of patients reported exacerbation of pre-existing dermatoses like acne vulgaris, eczema, and paronychia. Associated mild ocular involvements were also observed in some patients in the form of redness, watering, and grittiness.Although various ocular problems including conjunctivitis, corneal abrasions, and periorbital necrotizing fasciitis have been described to occur due to contact with i colors,–5 patterHoli colors are produced in India without any quality checks and are sold freely in the market. Even when these are available in a packaged form, there is little information for the consumer about the source of the colors, their contents, and possible toxic effects. In recent years, several nongovernmental organizations have started campaigning for safe practices related to the use of colors. Some are producing and marketing ranges of safer colors derived from natural sources such as vegetables and flowers.[ flowers. We belie


Pesticide ingestion is a common method of self-harm in the rural developing world. In an attempt to reduce the high case fatality seen with the herbicide paraquat, a novel formulation (INTEON) has been developed containing an increased emetic concentration, a purgative, and an alginate that forms a gel under the acid conditions of the stomach, potentially slowing the absorption of paraquat and giving the emetic more time to be effective. We compared the outcome of paraquat self-poisoning with the standard formulation against the new INTEON formulation following its introduction into Sri Lanka.p = 0.002, log rank test). Cox proportional hazards regression analyses showed an approximately 2-fold reduction in toxicity for INTEON compared to standard formulation. A higher proportion of patients ingesting INTEON vomited within 15 min . Median survival time increased from 2.3 d (95% CI 1.2–3.4 d) with the standard formulation to 6.9 d (95% CI 3.3–10.7 d) with INTEON ingestion ; however, in patients who did not survive there was a comparatively smaller increase in median time to death from 0.9 d (interquartile range [IQR] 0.5–3.4) to 1.5 d (IQR 0.5–5.5); p = 0.02.Clinical data were prospectively collected on 586 patients with paraquat ingestion presenting to nine large hospitals across Sri Lanka with survival to 3 mo as the primary outcome. The identity of the formulation ingested after October 2004 was confirmed by assay of blood or urine samples for a marker compound present in INTEON. The proportion of known survivors increased from 76/297 with the standard formulation to 103/289 with INTEON ingestion, and estimated 3-mo survival improved from 27.1% to 36.7%  to 37% (new formulation), an effect that was unlikely to be due to chance. More patients vomited within 15 minutes of taking the new formulation of paraquat. Patients who died generally survived longer if they had taken the new rather than the standard formulation. The researchers estimated that the new formulation is just over half as toxic as the standard formulation, meaning that a patient was likely to suffer the same level of ill effects after taking twice as much of the new formulation compared to the standard formulation.This study was designed, funded, and led by Syngenta, the manufacturer of the standard and new formulations of paraquat but the study team included a number of independent Sri Lankan and international scientists. As the researchers observed the effects of the introduction of the new formulation across the entire country at the same time, they could not completely rule out other possible reasons for the differences in outcomes for those who had taken the two formulations, such as differences in treatment.Despite this inherent drawback, the researchers estimate that during the study the new formulation saved about 30 lives. They conclude that the the new formulation does reduce the amount of paraquat absorbed by the body, although the study does not answer the question whether this was due to the gelling agent, the increased emetic in the new formulation or a combination of factors. The researchers suggest that the new formulation, by keeping patients alive longer, may allow doctors more time to treat patients. As no effective treatment exists at present, this benefit relies on a treatment being developed in the future.The researchers note that the most important factor in predicting the outcome when paraquat has been taken deliberately is the dose. As a result, they suggest that the new formulation can only be one part of a wider strategy to reduce deaths by deliberate self-poisoning using paraquat. They suggest that such an integrated approach might include generic measures to reduce incidents of self-harm, reduced access to paraquat, reduced formulation strength, and improvements in treatment.http://dx.doi.org/10.1371/journal.pmed.0050049.Please access these Web sites via the online version of this summary at US Environmental Protection Agency has published its Reregistration Eligibility Decision for paraquatThe fact sheet on how to handle paraquat and suspected cases of exposureThe Department of Health and Human Services of the US Centers for Disease Control and Prevention provides a Poisons Information Monograph for paraquatThe World Health Organisation has recently finished consulting on a draft review of paraquat in its Environmental Health Criteria SeriesThe International Programme on Chemical Safety (IPCS) has published a MedlinePlus provides links to information on health effects of paraquat Self-poisoning with pesticides is a major public health problem in many developing countries, accounting for up to one-third of all suicides worldwide according to recent estimates . While oSurvival after acute paraquat poisoning is related to the ingested amount, the circumstances of poisoning, and the formulation ingested . While iGRAMOXONE INTEON is a novel paraquat formulation specifically developed to decrease toxicity through a reduction in the amount of paraquat absorbed from the gastrointestinal tract following ingestion . A naturWe carried out an observational study to compare the 3-mo survival of patients admitted to hospital following paraquat ingestion before and after the introduction of the new INTEON formulation in Sri Lanka.The study was conducted in nine large hospitals , covering the main agricultural areas in Sri Lanka, with the exception of the northern and eastern regions. The protocol  was apprData on the exposure, treatment, and outcome of patients ingesting paraquat were collected prospectively from December 2003 to January 2006. Following review and approval of the registration package by the Office of the Registrar of Pesticides, the new INTEON formulation was introduced in October 2004 and stocks of the existing formulation were actively withdrawn from distributors and retailers. The pesticide, bottle, and label were similar to the standard formulation, the only differences being that the INTEON formulation was slightly more viscous, and the batch numbers differed. INTEON also included a tracer compound (500 ppm diquat) that could be detected in blood and urine following oral ingestions.Data were collected by trained research assistants using a standardised questionnaire. Upon admission, demographic data  were recorded together with information relating to previous treatments and transfer from a primary hospital. Details relating to the ingestion were taken: time of exposure; circumstances ; time to emesis; and number and force of vomiting episodes. The patient was asked to state the ingested volume from a range of quantities (<5 ml to >150 ml) with a variety of measuring schemes .A plasma and/or urine sample was taken soon after admission, where possible. Samples were stored frozen and sent to Syngenta CTL  for determination of paraquat ion concentration and detection of the tracer compound diquat ion to classify the case as either standard formulation or INTEON. Analysis was conducted using HPLC, LC-MS-MS, and LC fluorescence .Details of treatments and clinical observations throughout the patients' stay in hospital and clinical outcome were recorded; if the patient was discharged from hospital, study doctors visited the patient at home at least 3 mo after the initial exposure to ascertain survival.Cases were initially recorded on paper and then transferred to a Microsoft Access database. For quality control, a separate database was created from data collected from the medical notes by an auditor . The two databases were compared to assess completeness of case ascertainment and to highlight differences in recording of details.To find out whether the pattern of patient admissions to, and referrals from, hospitals not participating in the survey had changed over time, the study team contacted 147 hospitals and care units towards the end of the survey in the provinces where the study hospitals were located. Using a structured questionnaire, information was obtained from physicians who were in charge of admitting patients, or, in the case of central dispensaries, from the pharmacists.Both standard and INTEON formulation cases were classified as ‘confirmed' on the basis of blood or urine analysis and as ‘probable' when bottle or label were presented . The recThe power calculation was based on the Mantel-Haenszel risk ratio estimate stratified for three ingestion groups and indicated that a total of 210 cases would give > 85% power to detect a 2-fold reduction in potency for a two-sided test with significance level of 5%. It was decided to use the number of confirmed INTEON cases to close the survey in order to achieve adequate power for the sensitivity analyses. The number of confirmed cases fell below 210 after some patients were identified with admission records at more than one hospital after transferring between hospitals and other patients had to be excluded because they did not meet the study entrance criteria. However, the total number of INTEON cases  included in the analyses exceeded 210.t test for continuous variables and the χ2 test for categorical variables. The primary analysis compared survival among standard formulation cases before 1 October 2004 with survival among confirmed, probable, and possible INTEON formulation cases after the washout period. In sensitivity analyses, survival among all confirmed and probable standard formulation cases was compared with survival among all confirmed and probable INTEON formulation cases.Means and proportions for baseline variables were compared between the two ingestion groups using Student's Time to death analyses were performed using both nonparametric analysis methods  and semiparametric methods . Standard errors for 3-mo survival estimates were obtained using Greenwood's method . All staCox PH regression models were used to estimate unadjusted and adjusted hazard ratios for the INTEON formulation. Adjusted analyses always included terms for the following covariates: (a) sex, age, and weight of participant; (b) treatments received; (c) use of adsorbent; and (d) time from ingestion to presentation at a medical centre.Estimated ingestion amount was an important factor influencing survival, but information was not available for a number of cases. Consequently, unadjusted and adjusted hazard ratios were also derived for the subset of patients who had ingestion information. Adjustment was performed with and without estimated ingestion amount in the regression model. Ingestion amount was included as a categorical variable (eight levels) but also as a continuous variable using the logarithms of the midpoint of ingestion categories. Models were also fitted to examine whether the relationship with ingestion amount differed between the two groups. Estimates of relative potency were derived using the slope of the relationship with the logarithm of ingestion amount and term for formulation group in the Cox PH model.Variation in survival characteristics between the nine study hospitals was investigated using a gamma frailty model . In addition, evidence of nonproportional hazard functions was assessed by visual methods and by testing the significance of the interaction with the logarithm of survival time. Stratification was used to account for nonproportionality of the hazard functions.Information was collected by the nine study hospitals on 774 patients over the study period. The numbers of participants eligible for the primary analysis and sensitivity analyses broken down by formulation are given in The two primary study populations were similar for demographic and ingestion variables at baseline . Most paThe clinical characteristics of the two groups were generally similar , but a sp = 0.002). Median survival time increased from 2.3 d (95% CI 1.2–3.4 d) with the standard formulation to 6.9 d (95% CI 3.3–10.7 d) with INTEON ingestion .Follow-up of patients was generally good , but it The overall improvement in survival among patients who had ingested the INTEON formulation was seen in every ingestion group except the <5 ml group, in which survival was already high. p = 0.002) in an unadjusted analysis  and there was no evidence of a different relationship with ingestion amount for the standard and INTEON formulations. The HR for a doubling of ingestion amount was 1.57 (95% CI 1.46–1.69). The strong relationship with the logarithm of ingestion amount enables an estimate to be made of the potency (toxicity) of the INTEON formulation relative to the standard formulation. Based on the subset of patients with ingestion information, the potency of INTEON was estimated to be 0.54 of the standard formulation.Replacing the eight-level categorical variable for ingestion amount with the logarithm of the midpoint of ingestion in each category made little difference to the fit of the model  and an HR of 0.64 (95% CI 0.50–0.82) with a potency estimate for INTEON of 0.47 of the standard formulation.p = 0.02. This effect was more pronounced in the sensitivity analysis, restricted to confirmed and probable cases, where the median time to death was 1.1 d (IQR 0.5–3.9) for the standard formulation but 2.5 d for INTEON (IQR 0.8–9.0); p = 0.001.Among patients who died there was an increase in median time to death from 0.9 d (interquartile range [IQR] 0.5–3.4) for the standard formulation to 1.5 d for INTEON (IQR 0.5–5.5); Monthly admissions of patients with paraquat poisoning to study hospitals showed some seasonal variability, related to the use pattern of paraquat in Sri Lanka . HoweverIn Sri Lanka, pesticides are the most common means of self poisoning, with case fatality ratios more than 10-fold higher than those from self-poisoning in industrialised countries . AlthougDespite much research into the mechanism of toxicity and the potential for treatment of paraquat poisoning, no specific therapy has so far been shown to affect outcome in controlled clinical studies ,6,17. CoParaquat causes mucosal damage and increases passive flux across the mucosal barrier at high concentrations , and peaAlthough steps were taken to actively withdraw the old product from the market when the new formulation was introduced, we recognised that there would be a period in which the old product would still be with farmers. It was therefore important to unequivocally identify as many cases as possible through analysis of the marker that had been added to the INTEON product in a plasma or urine sample. However, this identification was possible only in two-thirds of the INTEON cases due to a combination of samples not being taken  and samples with plasma paraquat concentrations so low that the diquat marker could not be detected. To reduce the number of standard formulation cases incorrectly included in the INTEON group we introduced washout periods for the centres. During the washout periods only 6/44 (14%) of patients with sample confirmation were INTEON ingestions. In contrast, 195/242 (81%) of patients with samples after the washout period had ingested INTEON. Hence, it is likely that the majority of the 89 possible INTEON cases after the washout period were correctly classified as INTEON cases. Only 18 cases with no sample information or equivocal results occurred during the washout period and had to be excluded from the survival analyses. Importantly, the sensitivity analyses excluding those patients without sample or bottle confirmation gave very similar results to the primary analysis, providing further evidence that our overall classification of cases was largely correct. The possible inclusion of a small number of standard formulation cases in the INTEON group may have had a small impact on the survival rate. However, the effect of not including possible INTEON formulation cases would have been far greater because of (a) missing cases with large ingestion volumes because of the difficulty of collecting samples from very sick patients, and (b) missing ingestions too small for the marker to be detectable in samples.Ingestion information was not available for 26% of standard formulation cases and 14% of INTEON ingestions. The higher proportion of standard formulation cases with missing ingestion information resulted because information was not routinely collected at the start of the survey at one hospital. Many of the other patients without an ingestion amount were too ill to supply this information. Standard formulation patients with ingestion information tended to have ingested more than INTEON patients, and 29% had ingested more than 100 ml compared with 17% of INTEON patients. However, this difference in ingestion amounts would only explain a small part of the observed improvement in survival since standardising the survival rate of the standard formulation cases with ingestion information to the ingestion amount distribution of the INTEON patients only increased the estimated survival probability of standard formulation cases from 27.1% to 27.7%. Furthermore, standard formulation cases without ingestion information appeared to have ingested less than INTEON patients without ingestion information based on their higher survival rate, and the ingestion distributions of the full groups were probably closer than those of the subgroups with ingestion information.Since the INTEON formulation was introduced in the whole country at the same time we had to rely on a before-and-after design for the survey. It is therefore possible that changes in treatment, hospital admissions, or referrals may have occurred over the period of the survey. There were some differences between the two groups in terms of treatment, with fewer INTEON patients receiving gastric lavage and prednisolone, but none of the differences were major confounders of the observed beneficial effect of INTEON on survival. The monthly admissions over the study period suggest an overall decrease of the number of cases over time. In the survey of peripheral hospitals and care units there was no indication of a change in their referral practices over time. Changes in case ascertainment and management are therefore unlikely to have substantially contributed to the improved survival noted with INTEON. However, many hospitals indicated that the number of paraquat cases had decreased. This change may relate to shifts in the general pattern of self-harm incidents, but it is also possible that fewer patients ingesting the INTEON formulation were seeking health care.For those patients who did not survive, there was an increase in time to death for INTEON compared to the standard formulation. This difference may become important when trying to achieve improvements in the treatment of paraquat poisoning, as it may allow more time for new or existing therapies to become effective. Our data show that in Sri Lanka self-harm patients reach hospital reasonably quickly (nearly 60% are treated within 4 h), so improved treatment of poisoning cases in addition to the INTEON formulation could have a further positive effect on survival.While our finding of improved survival of patients in the INTEON group is encouraging the data also show that the beneficial effect of the formulation is limited by the amount of product ingested, since this was the single most important predictor of survival in both groups. It is therefore apparent that formulation changes in themselves will not be sufficient to comprehensively address the problem of mortality from self-harm with paraquat. An integrated approach has recently been proposed including generic measures to reduce self-harm incidents, as well as focusing on reducing access, reducing formulation toxicity , and improving the treatment of poisoning . HoweverIn conclusion, this survey shows that the introduction of a new paraquat formulation with INTEON technology has led to a significant improvement in survival of patients with paraquat poisoning. Our statistical analyses indicate that this effect is due to a real difference between the two formulations. Patients who ingested a lethal amount of the formulation survived longer with INTEON, raising the prospect of more opportunities for treatment. These encouraging results were achieved despite suboptimal homogeneity of the formulation, and future improvements in formulation technology may reduce overall toxicity even further.Text S1(152 KB DOC)Click here for additional data file.Text S2(284 KB PDF)Click here for additional data file.Text S3(322 KB PDF)Click here for additional data file.Text S4(284 KB PDF)Click here for additional data file.Text S5(627 KB PDF)Click here for additional data file.


Cell proliferation in multicellular organisms must be coordinated with pattern formation. The major signaling pathways directing pattern formation in the vertebrate limb are well characterized, and we have therefore chosen this organ to examine the interaction between proliferation and patterning. Two important signals for limb development are members of the Hedgehog (Hh) and Fibroblast Growth Factor (Fgf) families of secreted signaling proteins. Sonic hedgehog (Shh) directs pattern formation along the anterior/posterior axis of the limb, whereas several Fgfs in combination direct pattern formation along the proximal/distal axis of the limb.shh gene, proliferation in the pectoral fin buds is initially normal, but later is strongly reduced. Correlating with this reduction, Fgf signaling is normal at early stages, but is later lost in shh mutants. Furthermore, pharmacological inhibition of Hh signaling for short periods has little effect on either Fgf signaling, or on expression of G1- and S-phase cell-cycle genes, whereas long periods of inhibition lead to the downregulation of both. In contrast, even short periods of pharmacological inhibition of Fgf signaling lead to strong disruption of proliferation in the fin buds, without affecting Shh signaling. To directly test the ability of Fgf signaling to regulate proliferation in the absence of Shh signaling, we implanted beads soaked with Fgf protein into shh mutant fin buds. We find that Fgf-soaked beads rescue proliferation in the pectoral find buds of shh mutants, indicating that Fgf signaling is sufficient to direct proliferation in zebrafish fin buds in the absence of Shh.We used the genetic and pharmacological amenability of the zebrafish model system to dissect the relative importance of Shh and Fgf signaling in regulating proliferation during development of the pectoral fin buds. In zebrafish mutants disrupting the Previous studies have shown that both Shh and Fgf signaling are crucial for outgrowth of the vertebrate limb. The results presented here show that the role of Shh in this process is indirect, and is mediated by its effect on Fgf signaling. By contrast, the activity of the Fgf pathway affects proliferation directly and independently of its effect on Shh. These results show that Fgf signaling is of primary importance in directing outgrowth of the limb bud, and clarify the role of the Shh-Fgf feedback loop in regulating proliferation. During the development of multicellular organisms, pattern formation must be precisely coordinated with proliferation and differentiation. Given that only a relatively small number of signaling pathways are used to direct both pattern formation and cell proliferation during development, it is clear that cell fate specification and cell division are highly context-dependent read-outs of signaling in a given tissue or organ. Activation of a particular signaling pathway, such as the Hedgehog pathway, can stimulate proliferation in one cell type, while activation of the same pathway in another cell type has no effect on proliferation. Moreover, the observation that identical signaling pathways can regulate both pattern formation and cell proliferation provides a mechanism for coordination of these distinct behaviours.The vertebrate limb is an excellent model system in which to study the interplay between pattern formation and cell proliferation. Limb development is highly amenable to experimental and genetic manipulation in several model organisms, and the main signaling pathways that direct limb development are well characterized (reviewed in -3). Threfgf-4, which is expressed in the posterior AER, can be induced in the anterior AER of the chicken limb bud by ectopic Shh protein [fgf4 and fgf8 expression in the AER [shh expression in the ZPA [The apical ectodermal ridge (AER) is another major signaling center of the limb bud which runs along its distal margin, and which is the site of expression of several Fgf genes (reviewed in ). The AE protein ,14. Furt the ZPA , indicatin vivo during embryonic development. Thus FGF-4 is necessary for proliferation of the inner cell mass during early post-implantation development in the mouse [Members of both the Hh and Fgf family of signaling proteins have been shown to function as mitogens in several contexts. Indeed, Fgf1 and Fgf2 were initially identified as mitogenic factors in fibroblast tissue culture, and subsequently, other members of the FGF protein family were found to have a similar activity . Furtherhe mouse , and FGFhe mouse . Additiohe mouse , of lenshe mouse , and durhe mouse .Drosophila eye [Like the Fgf family, members of the Hh family function as mitogens in a number of contexts. The Hh signaling pathway has been linked to several cancers, including basal cell carcinoma, pancreatic tumors, and digestive tract tumors, and may be upregulated in as many as 25% of tumors -25. In ahila eye , in mammhila eye , and in hila eye . In sevehila eye ,30-32. Thila eye ,32,33.shh-expressing virus [cyclin D1 expression in the mesoderm of chicken wing buds after AER removal. This observation raises a third possibility: that Shh and Fgf signaling both contribute to the regulation of limb bud proliferation.Since there is clear evidence that both Shh and Fgf signaling are important for outgrowth of the vertebrate limb bud, and since both signaling pathways are known to have a mitogenic effect during development, this raises the question of the relative contribution of Shh and Fgf signaling to regulation of proliferation in the limb bud. This issue is complicated by the feedback loop operating between the two signals, as inhibition of either signaling pathway leads to loss of the other signaling pathway. Laufer and colleagues have previously addressed this issue by removing the AER from chicken wing buds and adding back either FGF4-soaked beads, or ng virus ,14. Theing virus  shows thshh mutants. Our data confirm that Fgf signaling is of direct and crucial importance for growth and cell-cycle progression in the limb bud, whereas the effect of Shh on proliferation is indirect, and is mediated via its effect on Fgf expression in the AER.To distinguish between these possibilities, we have made use of the genetic, embryological, and pharmacological tools of the zebrafish model system to uncouple the activity of Shh and Fgf signaling in the pectoral fin buds, and to investigate their individual effects on proliferation. In order to categorically uncouple the effect of Fgf signaling from Shh, we implanted FGF4-soaked beads into the limb buds of shh mutants. We focused on cyclinD1, pcna and mcm5, which are generally used as markers of proliferating cells in zebrafish [cyclinD1 is necessary for G1-progression and S-phase entry, while pcna and mcm5 are necessary for DNA replication during S-phase [replication protein A1 gene (ra1), which is expressed constitutively in all cells of the fin bud. We find that at 32 hpf, cyclinD1, pcna, mcm5 and ra1 are expressed at indistinguishable levels in wild-type and in shh mutant fin buds , a member of the Tgf-β family of secreted signaling proteins [Drosophila retina, on the other hand, Hh signaling has both positive and negative effects on cell-cycle progression [Since Hh signaling has also been shown to have a direct mitogenic effect on some cell types, it is perhaps surprising that Shh directs proliferation indirectly in the vertebrate limb bud. However, there is at least one previous example of such an indirect effect of Hh signaling on proliferation. During proteins -46. Furtproteins . Interesproteins . In the gression . Cell-cygression ,50.Our results also show a context-dependent effect of Fgf signaling on cell-cycle progression. Thus Fgf signaling is clearly essential for cell-cycle progression in the pectoral fin buds and in the branchial arches, since expression of G1- and S-phase cell-cycle genes in these tissues is lost after only 3 hours of inhibition of the Fgf pathway. Inhibition of Fgf signaling fails to affect cell-cycle progression in other organs, however, such as the retina and the optic tectum, or at earlier stages of development. The Fgf signaling pathway is therefore not a global mitogenic signal in the zebrafish embryo, but instead directs proliferation in a highly tissue-specific manner. Altogether, the evidence thus indicates that both the Hh and Fgf pathway affect cell-cycle progression in some cell types but not in others, and that this effect can be either direct or indirect. The control of cell-proliferation in multicellular organisms can therefore only be understood in a context-dependent manner, and our results help to shed light on this question in the context of the vertebrate limb bud. The molecular mechanisms by which different cell types respond distinctly to the same signal are still poorly understood, but will undoubtedly be unravelled by future research.Our data show that Shh and Fgf signaling have distinct effects on proliferation during vertebrate limb development. While both genes are necessary for outgrowth of the limb bud, the role of Shh in this process is indirect, and is mediated via its effect on expression of Fgf genes in the AER. In contrast, the effect of Fgf signaling on cell-cycle progression in the limb bud appears to be direct, and Fgf signaling is both necessary and sufficient to direct proliferation, even in the absence of Shh. Together with the work of others, our results indicate that the role of both signaling pathways in regulating proliferation is highly context dependent, and our results shed light on their function in directing proliferation in the context of limb development.Tupfel Long Fin (TLF) and sonic-yout4 heterozygous fish were used. Fish were maintained according to standard protocols. Embryos were grown in E3 embryo medium at 28°C with or without the addition of 0.003% 1-phenyl-2-thiourea  to inhibit pigmentation. Staging was performed according to hours post-fertilization (hpf) [Wild-type on (hpf) .FGF signaling inhibitor SU5402  was dissolved in DMSO at 8 mM. Treatment was performed with a 10 μM solution of SU5402 or a corresponding control DMSO solution in E3 embryo medium. Cyclopamine  was dissolved in ethanol at 20 mM. Treatment was performed with 100 μM solution of cyclopamine or control ethanol solution in E3 embryo medium.ptc1, pea3 [cyclin D1 [pcna (cDNA was ampplified using pcna_for: CCTACTCCAAACTAAGAAAGCAGCA and pcna_rev: ATCGGGAATCCATTGAACTGG), mcm5 (cDNA was amplified using mcm5_for: TGGTGGAGGAGAAAGCGTCG and mcm5_rev: GGCCTCATGGATTGCGACTC and cloned into pGEM-T-easy (Promega)), ra1 (NM199811) cDNA was ampplified using ra1_for: CCATTGGAGGAAACAGGAGA and T7_ra1_rev: TAATACGACTCACTATAGGGcagcgtgtacctggtaagca and transcribed from the PCR product using T7 polymerase).RNA in situ hybridisations were performed according to Jowett . Probes c1, pea3 , cyclin yclin D1 , pcna (cAntibody stainings were performed on 14 μm cryosections. Cryosections were rehydrated in PBS with 0.1% Tween-20 (PBST), treated for 20 min with 4N HCl, washed several times in PBST and blocked for 1 hour with blocking solution . Mouse anti-BrdU antibody (1:100)  and secondary anti-mouse Alexa Fluor 488 antibody (1:400)  were diluted in blocking solution and incubated with the sections for 2 hours each. Images were taken using Leica SP2 confocal microscope and processed using Adobe Creative Suite CS2.Bead implantation was performed as described before . RecombiCJN suggested the idea of checking the relative importance of Shh and FGF signaling pathways in driving proliferation in zebrafish fin buds, guided the project and helped drafting the manuscript. SVP designed and performed experiments and drafted the manuscript. All authors read and approved the final manuscript.


However, there is no systematic method to identify the attributes of a protective CD8+ T cell response. Here, we combine theory and experiment to identify and quantify the contribution of all HLA class I alleles to host protection against infection with a given pathogen. In 432 HTLV-1-infected individuals we show that individuals with HLA class I alleles that strongly bind the HTLV-1 protein HBZ had a lower proviral load and were more likely to be asymptomatic. We also show that in general, across all HTLV-1 proteins, CD8+ T cell effectiveness is strongly determined by protein specificity and produce a ranked list of the proteins targeted by the most effective CD8+ T cell response through to the least effective CD8+ T cell response. We conclude that CD8+ T cells play an important role in the control of HTLV-1 and that CD8+ cells specific to HBZ, not the immunodominant protein Tax, are the most effective. We suggest that HBZ plays a central role in HTLV-1 persistence. This approach is applicable to all pathogens, even where data are sparse, to identify simultaneously the HLA Class I alleles and the epitopes responsible for a protective CD8+ T cell response.CD8 + T cells may contribute towards the inflammatory disease associated with HTLV-1 infection. This observation has led to the hypothesis that it is the ‘quality’ of the immune response towards HTLV-1 that is important, and not simply a response in itself. Using a combination of computational and experimental methods we have investigated T cell ‘quality’. We have found that specificity is an important determinant of CD8+ T cell quality with recognition of the viral protein HBZ enabling the host to make a more effective immune response. This approach can be used for other pathogens to identify what HLA class 1 alleles and the parts of the pathogen they bind to are responsible for a protective CD8+ T cell immune response. This work informs basic immunology: “what constitutes a protective CD8+ T cell response?”; vaccine design: “which antigens elicit the most effective response” and virology: “which viral proteins are key players in the strategy of persistence?”.A large immune response to the retrovirus HTLV-1 does not always prevent HTLV-1-associated diseases. Indeed, it has been shown that CD8 Human T cell lymphotropic virus-type 1 (HTLV-1) is an oncogenic retrovirus that infects between 10 and 20 million people worldwide. Of these infected individuals, 1–6% develop adult T cell leukaemia/lymphoma (ATL/ATLL) and a further 2 to 3% develop a variety of chronic inflammatory syndromes including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP); the rest remain lifelong asymptomatic carriers (ACs) of the virus.+ T cell response to HTLV-1 and it is unclear why this fails to eradicate the virus. Furthermore, there is evidence for both protective + T cells. The attributes of a protective anti-HTLV-1 response in vivo are unknown, although specificity for the viral protein Tax is a strong candidate. There are good reasons to believe that a Tax-specific CD8+ response HLA-A*02, which is associated with protection in southern Japan in vitro, that CD8+ T cells specific to early viral proteins are particularly effective in viral control Most HTLV-1-infected individuals mount a large, chronically activated CD8+ T cell response is difficult to ascertain in any infection. Measurements of CD8+ T cell frequency, phenotype, function and specificity are informative but, because antigen load influences each of these factors, it can be difficult to ascertain if a particular immune profile is the cause or effect of good pathogen control + T or NK cells. However, why one particular allele should be protective remains unclear and so a class I association provides no information about how to manipulate the immune response to enhance protection.What constitutes an effective CD8The aim of this study was to develop a method to test the hypothesis that the effectiveness of an individual's HTLV-1-specific response and thus their proviral load and HAM/TSP risk was determined by the epitope binding properties of their HLA class I alleles. This method resulted in the identification of the viral protein HTLV-1 basic leucine zipper factor (HBZ) as a significant immunogenic target for both proviral load reduction and reduced disease risk. The HBZ gene was identified recently P <0.00001, Spearman's rank correlation; P <0.001, Spearman's rank correlation; Fig. S1 in Approximately 50 HLA class I-epitope pairs have been identified for HTLV-1 A*0201 and Cw*0801, with those of the known detrimental allele, B*5401 . These P values needs to be treated with caution because the rank of the binding affinity of one HBZ peptide for A*0201 may not be independent of the rank of the binding affinity of a second peptide to A*0201 and similarly for Cw*0801 and B*5401 . This statistic is based only on the top binding peptide so it does not assume different peptides have independent binding affinity ranks. Henceforth, we only considered the top binding peptide to avoid the potential problem of dependence  and HAM/TSP patients (n = 230) from the Kagoshima cohort. We predicted the HTLV-1 peptides bound most strongly by each individual, given their HLA class I types and then tested for differences between the two subject groups , so this result does not follow trivially from our previous observation than asymptomatic carriers bind HBZ significantly more strongly than HAM/TSP patients. An alternative metric, the binding strength of the top HBZ-binding peptide to each allele instead of the number of strongly binding alleles, yielded an identical conclusion i.e. there was a significant negative correlation between the proviral load and the strength of binding to HBZ peptides .Next we investigated why strong binding of HBZ peptides was associated with remaining asymptomatic. One of the strongest correlates of HAM/TSP is a high HTLV-1 proviral load A*02 was a significant predictor of load in ACs but not in patients with HAM/TSP. We therefore directly compared the ability of the novel peptide binding method and the traditional genotype method to predict proviral load in ACs and HAM/TSP patients , but confirmed the finding that in a traditional analysis HLA-A*02 (presence/absence) was a significant predictor in ACs only (P = 0.01) and HLA-B*54 for HAM/TSP patients only (P = 0.019). The proportion of variance in proviral load explained was also marginally higher for the peptide binding analysis than the traditional analysis. The observation that HBZ binding strength correlated with proviral load in both ACs and HAM/TSP patients suggests that peptide binding is the more fundamental predictor than HLA genotype.Next we compared our peptide-binding analysis of HLA class I genotype with a traditional frequency-based “presence or absence of an allele” analysis. Previously a “traditional” analysis yielded inconsistent results S = 0.86, P = 0.0005, Spearman's rank correlation; S = 0.66, P = 0.02, Spearman's rank correlation; Fig. S4 in + efficiency  is determined by protein specificity and we have established a list of protein targets of the most efficient response to the least efficient response. Secondly, the fact that across all alleles and across all proteins, peptide binding associated with immune control  is strongly correlated with prevention of HAM/TSP is the strongest evidence yet that the CD8+ T cell response can have a beneficial role in HTLV-1 infection.Our findings demonstrate that the HTLV-1 protein that is associated with the most significant reduction in HAM/TSP risk when bound by HLA class I molecules (i.e. HBZ) is also, independently, associated with a significant reduction in proviral load when bound. We wished to investigate whether this relationship held across all proteins. We therefore produced two ranked lists of proteins. In the first list we ranked the HTLV-1 proteins according to whether they were bound more strongly by asymptomatic carriers or HAM/TSP patients (p)≈48% (12.3% SD) of potential cases of HAM/TSP in the study population. However, although we found that a high proportion of potential HAM/TSP cases are prevented by strong HBZ binding, it should be noted that the strength of HBZ binding is not the only determinant of disease status: in a logistic regression model, the strength of HBZ binding alone could only correctly classify 55% of cases of HAM/TSP.We calculated the prevented fraction of disease attributable to the possession of one or more strong binding alleles to HBZ + T cells play a protective role in HTLV-1 infection. HBZ immunogenicity has been studied in ATL patients + T cell response is generated or even whether HBZ protein is expressed in asymptomatic carriers and HAM/TSP patients. We therefore sought to identify HBZ-specific CD8+ T cells in fresh PBMCs from HTLV-1 infected individuals. We assayed IFN-γ production by ELISpot following stimulation in vitro with a pool of overlapping peptides that spanned the entire HBZ protein. Of 45 subjects tested, 31% had detectable HBZ-specific CD8+ T cells , confirmed that HBZ-specific CD8+ T cells are present in PBMC from infected individuals. We conclude that HBZ protein is expressed in vivo and is immunogenic.These results strongly imply that HBZ-specific CD8 T cells . An inde+ T cell clone that recognised HBZ26–34 (GLLSLEEEL) in the context of HLA-A*0201 + T cell clone was able to kill naturally-infected cells from non-leukemic HTLV-I-infected individuals. First we confirmed the finding of Sumeori et al that autologous B-LCL loaded with HBZ26–34 peptide could be lysed by the CD8+ T cell clone (data not shown). Then we demonstrated, by a classical chromium release assay, that naturally-infected CD4+CD25+ cells from the PBMCs of 3 out of 4 +HLA-A*0201 non-leukemic patients were lysed by the CD8+ T cell clone but that cells from 3 out of 3 HLA-mismatched donors were not lysed . Peptides from Tax were predicted to bind significantly more strongly than peptides from HBZ (HLA-A*02 and HLA-Cw*08), but is generally associated with HLA class I alleles that bind strongly to HBZ. Given that a protein-specific HLA-restricted association is more likely to be mediated by CD8+ T cells than NK cells which show limited protein specificity we interpret this work in the context of the CD8+ T cell response.We show that strong predicted binding of peptides from the HTLV-1 protein HBZ is associated with a reduced risk of HAM/TSP and a reduced proviral load in a population with endemic HTLV-1 infection in southern Japan. We demonstrated that protection is not limited to a small subset of HLA class I alleles previously associated with disease status and proviral load  that HBZ-specific CTL are present in PBMC from HAM/TSP patients and ACs. We further show that naturally infected cells, isolated directly from HAM/TSP patients and ACs, are susceptible to lysis by an HBZ-specific CTL clone. Suemori A*02 is associated with these outcomes is a simultaneously more fundamental and more applicable level of understanding. Secondly, identification of protective epitopes immediately suggests a practical approach to measure and enhance, via therapeutic vaccination, the efficiency of an individual's anti-viral response. Thirdly, because the same effect (e.g. HBZ binding) can be identified for many alleles it is less likely to be a spurious result of linkage disequilibrium or genetic stratification. Finally, effects due to multiple low-frequency alleles can be captured because analysis is made at the level of peptide binding rather than allelic frequency.This approach to studying the association between HLA class I genotype and the outcome of infection has a number of strengths compared with a traditional frequency-based analysis. Firstly, it is more mechanistic: knowing that binding HBZ is associated with a reduced proviral load and disease risk compared with knowing that + T cell response in HTLV-1 infection.In summary, using a novel and generalizable approach, we have identified one of the constituents of an effective CD8Two hundred and thirty individuals with HAM/TSP were compared with two hundred and two randomly selected HTLV-1 seropositive asymptomatic carriers (ACs) from the Kagoshima Red Cross Blood Transfusion Service. All cases and controls were of Japanese ethnic origin and resided in Kagoshima Prefecture, Japan. Full details of the cohort can be found in Ethics statement: All subjects attended the National Centre for Human Retrovirology at St. Mary's Hospital and donated blood after giving written informed consent as approved by the St. Mary's Hospital Research Ethics Committee.IFNγ ELISpot, CD107a mobilisation and the chromium release assays were all carried out using cells from HTLV-1-infected individuals attending St Mary's Hospital. We used two different algorithms to predict HLA class I epitopes: Metaserver and Epipred. Figures based on Metaserver predictions are in the main text, the corresponding figures for Epipred are in Metaserver is a combination of two web-based prediction methods that use artificial neural nets, NetCTL v1.2 In order to validate our results, we used a second, independent method of epitope prediction Other than our initial comparison , analysis was limited to A and B loci for two reasons: Metaserver does not have algorithms for the C loci and C loci predictors tend to be less accurate because of the lack of peptide-HLA-C experimental binding affinities to train the software. Metaserver provided coverage of 84% of the total count of A/B alleles in the Kagoshima cohort.The missing alleles were: A*0207, A*0210, A*2603, A*3201, B*1301, B*1501, B*1508, B*1511, B*1518, B*2704, B*3701, B*3802, B*4005, B*4006, B*4601, B*4801, B*5201, B*5501, B*5504, B*5601, B*5603, B*5605, B*5705, B*5901, and B*6701.We were able to obtain predictions for , A*2603 and  to a resolution of 2 digits by combining the predictions of other A02*, A26* and B40* predictors according to their frequency in Kagoshima.We estimate that approximately 2,200 peptides could be bound by the alleles present in the Kagoshima cohort. This figure is 1% + T cell responses against these epitopes experimentally. Using this technique has yielded true-positive (correctly predicted) estimates of 62–80% The accuracy of epitope prediction algorithms has increased to such an extent that the correlation between predicted binding affinities and measured binding affinity is as strong as the correlations of measurements between different laboratories Both prediction methods that we use produce a score for each peptide-HLA that represents the binding strength of that complex. In theory this score would allow us to compare predicted binding affinities between alleles. However, between allele comparisons can be problematic. Firstly, within-allele comparisons  are thought to be more comparable than predictions between alleles We were concerned that the binding of the top 8 peptides from a protein to an allele may not be independent of one another. Since, the strength of the strongest binder provides information (i.e. an upper bound) about the strength of the second highest binder. For this reason, apart from The REVEAL HLA-peptide binding assay  was used to quantify peptide-HLA binding. For each allele-peptide combination that was tested, assembly of peptide-HLA complexes was quantified by ELISA with a conformation-dependent anti-HLA antibody. Samples of assembling peptide-HLA complexes were taken at a defined time point and snap-frozen in liquid nitrogen prior to analysis. The assembly for each peptide-HLA complex was then compared against a positive control peptide for that allele as the percentage of assembled peptide relative to that control. We selected four HLA class I alleles and 50 HTLV-1 peptides for each allele. The allele choice was based on allele frequency in the Kagoshima database and included 2 A alleles and 2 B alleles as well as alleles for which we knew that the epitope prediction tended to be poor. The 50 HTLV-1 nonamer peptides for each allele were selected to represent a range of predicted binding affinities, from weak to strong binding peptides. They originated from 4 HTLV-1 reference strain proteins: Tax, HBZ, Gag and Polymerase.Due to allele coverage (see above), it was necessary to use Metaserver for A*0201 and B*5401 and Epipred for Cw*0801. As the rank values were derived for each allele separately, it was acceptable to use different prediction methods for each allele in this case. Epipred predicts binding to allele families rather than individual alleles and so we calculated binding to Cw*08. The ranks of the strongest binding 8 peptides from each protein to the alleles A*0201 and Cw*08  were compared against the ranks of the strongest binding 8 peptides to the allele B*5401 . A Wilcoxon-Mann-Whitney test was performed for each protein to test for differences between the two sets of rank values. The analysis was repeated using top 5 and top 10 as well as top 8 binding peptides, conclusions were robust to the choice of number of peptides . These ranks were then split into two groups – those from HAM/TSP patients and those from asymptomatic carriers (AC). The two sets of ranks (HAM/TSP vs. AC) were then compared for each protein using a Wilcoxon-Mann-Whitney test .P values using Fisher's combined test (rather than simply looking at the relationship in the whole cohort). Therefore we could be confident that any relationship between protein binding and proviral load that we found did not follow trivially from a relationship between protein binding and disease status and the fact that asymptomatic carriers have a significantly lower load than HAM/TSP patients.We considered each HTLV-1 protein in turn. Firstly, we split the cohort by disease status (AC or HAM/TSP). Then, for each individual, we counted the number of alleles they possessed that were strong binders to the protein of interest and then tested for a correlation between the number of strong binders to the protein and proviral load using the Spearman rank correlation. A strong binding allele to a particular protein was defined as one that was in the top 40% of alleles. That is, the rank of the top binding peptide from the HTLV-1 protein to each of the individual's A and B HLA class I alleles was found (see The Rank Measure). This set of rank values (pooled HAM/TSP and AC) was then ordered from highest to lowest rank and the alleles that were represented in the top 40% of these ranks were defined as strong binding alleles to that protein. Importantly, for each protein, we looked at the relationship between strength of binding and proviral load separately in HAM/TSP patients and ACs and then combined the Our alternative metric for this method used the Rank Measure to quantify the strength of binding of peptides from each HTLV-1 protein to each individual's A and B alleles. We then tested for any correlation between these values and the individuals' proviral load for HAM/TSP patients and asymptomatic carriers.All analysis was performed with two independent epitope prediction algorithms (Metaserver and Epipred) and with two different methods ; additionally an alternative approach to comparing protective v detrimental alleles  and to comparing proviral load with strength of binding were investigated. Conclusions were highly robust  of disease p) of potential cases of HAM/TSP in the population that is prevented by the protective genotype is given by Fp = (1−R)×[1−(d×r1/b×r2)], where R = prevalence rate of HAM/TSP in the population (estimated as 1% of the HTLV-1-infected population), r1 = a+b and r2 = c+d. Fp is approximately normally distributed: the standard deviation is given by SD (Fp) = (1−R−Fp)×√[(c/d×r2)+(a/b×r1)].To calculate the prevented fraction  were isolated from whole blood from HTLV-1 infected individuals by density gradient centrifugation.+ T cells using MACS beads (Miltenyi Biotec). The resulting cells were cultured in duplicate at a density of 100,000 cells per well in the presence of a range of concentrations of pooled overlapping 20mer peptides (offset by 6 amino acids) spanning HBZ, Tax, or with medium alone. After 6 hours, IFN-γ producing cells were detected by ELISpot (Mabtech). The threshold for a positive response to peptide was defined as greater than the mean plus two standard deviations of the number of spots in the medium only control.PBMC were depleted of CD46 PBMC were cultured in a 400 µl volume in the presence of 5 µl anti-CD107a-PE  1.4 µg/ml monensin (eBioscience), 20 µg/ml DNase  with 2 µM HBZ peptide pool , or the equivalent volume of peptide solvent, acetonitrile. After 5h, the cells were harvested and stained for 30 min with Live/Dead red , a fixable viability stain. Cells were fixed and permeablised using ebioscience FoxP3 staining buffer set according to the manufacturer's instructions, then stained with anti-CD3-APC-eFluor780, anti-CD4-eFluor450, anti-CD8-PECy5  anti-IFN-γ-FITC, anti-CD14-ECD, anti-CD19-ECD . Samples analysed by flow cytometry using a Cyan ADP (Beckman Coulter), and summit software (DAKO). Doublets, dead cells, monocytes and B cells were excluded from the analysis on the basis of forward and side scatter, pulse width, viability staining, and CD14 or CD19 expression. Antigen-specific CD8+ T cells were identified as CD3+CD8+ cells capable of producing IFN-γ and/or mobilising CD107 to the cell surface.1×10+HLA A*0201 and −HLA A*0201 HTLV-1 infected individuals were depleted of CD8+ cells, then enriched for CD25+ cells using MACS beads , according to manufacturer's instructions. CD25+ cells were cultured for 16h to allow for viral antigen expression and presentation, then labelled with 51Cr by incubating for 1h in the presence of 50–100 µCi Na2CrO4 . Labelled cells were washed extensively and placed in culture in triplicate  in the presence of defined ratios of HBZ-1, a CTL clone which recognises HBZ 26–34 in the context of HLA A*0201 51Cr labelled B-LCL (autologous to the CTL clone) were cultured at the same ratios, with and without 1 µM HBZ 26–34 peptide. After 4h, culture supernatants were harvested, placed on a scintillation plate, and 51Cr release was assayed using a beta counter. Total specific lysis was calculated using the following formula: [chromium release (test well)−chromium release (no CTL control)]/ [chromium release (Triton−100% lysis)−chromium release (no CTL control)]*100, expressed as a percentage specific lysis of total cells. As not all CD4+CD25+T cells are infected, and thus do not represent targets for the CTL line, an estimate of specific lysis of infected cells was also calculated, making the conservative assumption that all the viral load is present in CD25+ cells, +CD25+ cells that are infected i.e. provirus positive].PBMC from The reference strain is from Supporting Information S1Supporting information(0.78 MB DOC)Click here for additional data file.


Cellular molecules possess various mechanisms in responding to oxidant stress. In terms of protein responses, protein S-glutathionylation is a unique post-translational modification of protein reactive cysteines forming disulfides with glutathione molecules. This modification has been proposed to play roles in antioxidant, regulatory and signaling in cells under oxidant stress. Recently, the increased level of protein S-glutathionylation has been linked with the development of diseases. In this report, specific S-glutathionylated proteins were demonstrated in human embryonic kidney 293 cells treated with two different oxidative reagents: diamide and hydrogen peroxide. Diamide is a chemical oxidizing agent whereas hydrogen peroxide is a physiological oxidant. Under the experimental conditions, these two oxidants decreased glutathione concentration without toxicity. S-glutathionylated proteins were detected by immunoblotting and glutathione concentrations were determined by high performance liquid chromatography. We further show the effect of alteration of the cellular thiol pool on the amount of protein S-glutathionylation in oxidant-treated cells. Cellular thiol concentrations were altered either by a specific way using buthionine sulfoximine, a specific inhibitor of glutathione biosynthesis or by a non-specific way, incubating cells in cystine-methionine deficient media. Cells only treated with either buthionine sulfoximine or cystine-methionine deficient media did not induce protein S-glutathionylation, even though both conditions decreased 65% of cellular glutathione. Moreover, the amount of protein S-glutathionylation under both conditions in the presence of oxidants was not altered when compared to the amount observed in regular media with oxidants present. Protein S-glutathionylation is a dynamic reaction which depends on the rate of adding and removing glutathione. Phenylarsine oxide, which specifically forms a covalent adduct with vicinal thiols, was used to determine the possible role of vicinal thiols in the amount of glutathionylation. Our data shows phenylarsine oxide did not change glutathione concentrations, but it did enhance the amount of glutathionylation in oxidant-treated cells. The generation of reactive oxygen species (ROS) is part of physiologically metabolic processes in cells. For example, this process can occur either in mitochondria during the electron transport chain or in NADPH oxidase of neutrophils. The redox state of cells is determined by the balance of generation of ROS and the capacity of antioxidant systems. Oxidant stress has been defined as an imbalanced redox state and favors ROS generation In general, there are two cellular pools of thiol molecules that possess antioxidant functions. One thiol pool is composed of low molecular weight (non-protein) molecules, ascorbic acid, tocopherol and glutathione. Glutathione is the representative molecule of the non-protein antioxidant molecules because of its abundance in cells Oxidative effect on proteins has received considerable attention especially cysteine residues as they are sensitive to oxidative modifications It is known that protein post-translational modifications play a significant role in many biochemical functions. The best example of various modifications is protein phosphorylation and dephosphorylation. Although protein S-glutathionylation is a new addition to the list of modifications, a large body of data has shown the importance of glutathionylation. Initially, many in vitro studies have shown glutathionylation is a switch to turn on/off enzymes' activities In this report, our data shows specific S-glutathionylated proteins in HEK 293 cells using different oxidants. Since S-glutathionylation is the linkage of two cellular thiol pools, we also show the amount of protein S-glutathionylation in response to the alteration of cellular thiol pools by two different means. Futhermore, our results suggest the role of dethiolase in contributing to the amount of glutathionylation. Finally, the possible rational of the specific protein S-glutathionylation is discussed.Although protein S-glutathionylation has been studied by various means The overall pattern of S-glutathionylation suggests different degrees of sensitivities of proteins in response to oxidative stress. The predominately S-glutathionylated proteins may be due to the number of reactive cysteine residues  associated with protein, or simply, the reflection of the abundance of a protein level. Based on the presented data, our results suggest that different oxidative mechanisms stimulate different sets of proteins responding to stress. In our studies, there were significant amounts of S-glutathionylation, even in the presence of more reduced GSH than GSSG. Our data supports a previously proposed mechanism t-Butyl hydroperoxide also induced 43 kDa S-glutathionylation in HEK 293 cells (data not shown). To our knowledge, this is the first evidence that shows specific protein S-glutathionylation by different oxidative mechanisms in cells. It is necessary to determine the identity of this 43 kDa protein to understand its function and propose a mechanism for the specific S-glutathionylation by hydrogen peroxide. We speculate that this 43 kDa protein could be actin since actin has been shown as a target protein for S-glutathionylation In theory, diamide should induce all possible proteins that undergo S-glutathionylation. However, a significantly modified protein (43 kDa) is only induced by hydrogen peroxide. These data suggest the 43 kDa protein is only sensitive to a free- radical related mechanism because another oxidant, Protein S-glutathionylation serves as the linkage between the pools of protein and non-protein thiols. We took two approaches to determine whether the amount of S-glutathionylation changes if the pools of cellular thiols are altered. Since glutathione is the most abundant non-protein antioxidant molecule in cells, we studied the effect of cellular glutathione concentration on protein S-glutathionylation. Buthionine sulfoximine (BSO) irreversibly inhibits gamma-glutamylcysteine synthetase, a required enzyme for GSH biosynthesis, thereby depleting cells of glutathione. Cells were incubated with BSO (100 µM) overnight before treating with oxidants. In Another non-specific approach was used to examine the effect of cellular thiol concentrations on protein S-glutathionylation. Cells were incubated in cystine-methionine deficient media overnight before treating with oxidants. This is a non-specific method to deplete thiol concentration versus the specific method using BSO. The data in Our data also shows two different mechanisms to deplete cellular glutathione: oxidative (diamide and hydrogen peroxide) and non-oxidative (BSO and cystine-methionine deficient medium). Although the non-oxidative mechanism depletes approximately 60% of cellular glutathione, only protein S-glutathionylation can be detected in an oxidative mechanism. A possible explanation for the amount of protein S-glutathionylation in BSO and cystine-methionine deficient media-treated cells is that there are still enough glutathione for glutathionylation under these conditions. Either BSO or a cystine-methionine deficient medium depletes approximately 60% cellular glutathione. Upon adding oxidants, the remaining 40% of glutathione was able to form the same amount of S-glutathionylation found in non-depleted cells to prevent proteins from irreversible damage. Furthermore, the 40% glutathione observed may also be enough to maintain a physiological redox environment in cells, i.e. most proteins are still in reduced state. This could be the reason why protein S-glutathionylation was not enhanced in BSO and cystine-methionine deficient medium alone cells.Protein S-glutathionylation is known to be an equilibrium process that depends on the rate of adding glutathione and the rate of removing glutathione, i.e. dethiolation. Since the mechanism for the addition of glutathione is not well understood, experiments were performed to study the possible role of removing glutathione on the amount of S-glutathionylation. Dethiolation has been shown to be an enzyme-dependent reaction; the enzyme contains vicinal dithiols. Phenylarsine oxide (PAO) is a specific vicinal dithiol-binding agent and hardly binds to monothiols. Cells were pretreated with a sub-toxic concentration of PAO before oxidant treatment. The data in Although our data show the specific effect of PAO under the experimental condition, the depletion of glutathione by oxidants was more dramatic in PAO-pretreated cells than non-pretreated cells. This observation suggests some vicinal-dithiol containing molecules play an antioxidant role in HEK 293 cells. The potential target for the PAO effect in our condition could be glutaredoxin since it is a known dethiolase. Glutaredoxin contains vicinal dithiols in the catalytic site and its function is usually characterized in vitro using purified S-glutathionylated proteins. To our knowledge, this is the first data to show increased S-glutathionylation by possibly inhibiting the activity of dethiolase (glutaredoxin) in vivo. Our in vivo data also support the perception that protein S-glutathionylation is a dynamic reaction. In future experiments, we will attempt to assay the activity of glutaredoxin in PAO-treated cells to reveal the role of glutaredoxin in protein S-glutathionylation in vivo.N-ethylmaleimide (NEM), DL-buthionine sulfoximine (BSO), Dulbecco's modified eagle's medium (DMEM), cystine-methionine deficient DMEM, glutathione disulfide (GSSG), reduced glutathione (GSH), iodoacetic acid (IAA), 1-flouro-2,4-dinitrobenzene (FDNB), diamide, fetal bovine serum (FBS), and phenylarsine oxide (PAO) were all purchased from Sigma-Aldrich . Goat anti-mouse antibody conjugated with alkaline phosphatase and its substrate were purchased from BioRad . Anti-glutathione monoclonal antibody was purchased from Virogen . Hydrogen peroxide was purchased from Calbiochem, a brand of EDM Bioscience, Inc . Waters Spherisorb NH2 (5 µm 250 mm×4.6 mm) high performance liquid chromatography (HPLC) ion exchange column was purchased from Supelco Inc. . Human embryonic kidney cells (HEK 293) were kindly provided by Dr. Anthony J. Koleske .Dithiothreitol (DTT), HEK 293 cells were seeded into 60 mm culture plates and grown until 70% confluent in Dulbecco's modified eagle's medium (DMEM) containing 8% fetal bovine serum (FBS) at 37°C supplemented with 5% carbon dioxide. The medium was changed to 0.5% FBS before the treatment of oxidants so the oxidant's effect would not be quenched by FBS.N-ethylmaleimide (NEM). NEM irreversibly alkylates thiol groups to prevent further S-glutathionylation during sample processing. Soluble fractions of cell lysate were separated by centrifugation at 4°C and protein concentration was determined via Bradford Assay S-glutathionylation was induced using two oxidants: diamide and hydrogen peroxide. After the oxidant treatment, the cells were harvested in a lysis buffer  containing freshly prepared 50 mM Cells were pretreated with BSO (100 µM) for 16 hours to alter cellular thiol pools prior to medium change. BSO specifically inhibits glutathione synthesis by inhibiting gamma-glutamylcysteine synthetase After oxidant treatment, the cells were harvested in 5% perchloric acid for 20 min on ice. Acid soluble and insoluble fractions were separated by centrifugation. Acid soluble fractions were used to determine GSH and GSSG concentration and acid insoluble fractions were used to determine the protein concentration. Glutathione samples were prepared and determined by a previously described method