Adult T-cell leukemia-lymphoma (ATL) is a distinct mature T-cell malignancy caused by chronic infection with human T-lymphotropic virus type 1 with diverse clinical features and prognosis. ATL remains a challenging disease as a result of its diverse clinical features, multidrug resistance of malignant cells, frequent large tumor burden, hypercalcemia, and/or frequent opportunistic infection. In 2009, we published a consensus report to define prognostic factors, clinical subclassifications, treatment strategies, and response criteria. The 2009 consensus report has become the standard reference for clinical trials in ATL and a guide for clinical management. Since the last consensus there has been progress in the understanding of the molecular pathophysiology of ATL and risk-adapted treatment approaches.Reflecting these advances, ATL researchers and clinicians joined together at the 18th International Conference on Human Retrovirology—Human T-Lymphotropic Virus and Related Retroviruses—in Tokyo, Japan, March, 2017, to review evidence for current clinical practice and to update the consensus with a new focus on the subtype classification of cutaneous ATL, CNS lesions in aggressive ATL, management of elderly or transplantation-ineligible patients, and treatment strategies that incorporate up-front allogeneic hematopoietic stem-cell transplantation and novel agents.As a result of lower-quality clinical evidence, a best practice approach was adopted and consensus statements agreed on by coauthors (> 90% agreement).This expert consensus highlights the need for additional clinical trials to develop novel standard therapies for the treatment of ATL Incidence of ATL is rising in nonendemic regions of the world.3 In 2009, ATL researchers joined together and published an ATL consensus report that has been a standard reference for clinical trials of new agents for ATL and that focused on definition, prognostic factors, clinical subtype classification, treatment, and response criteria.4Adult T-cell leukemia-lymphoma (ATL) is an intractable mature T-cell malignancy with diverse clinical features, etiologically associated with a retrovirus designated human T-cell leukemia virus type I or human T-lymphotropic virus type 1 (HTLV-1), which is endemic in several regions, including the southwest region of Japan, Central and South America, central Africa, the Middle East, Far East, central Australia, and Romania.5 The ATL clinical workshop held during the 18th International Conference on Human Retrovirology—HTLV and Related Viruses—held in Tokyo, Japan, March, 2017 focused on discussion and revision of the 2009 consensus report. Consensus methodology and its limitations are detailed in the Data Supplement.Since publication, additional progress has been made in the molecular pathophysiology of ATL and risk-adapted treatment approaches.4 An aim of this report is to recommend good practice where there is a limited evidence base but for which a degree of consensus or uniformity is likely to benefit patient care and may be used as a tool to assist policymakers.Some therapeutic agents used in the treatment of ATL are not universally available and treatment strategies will therefore differ among countries, which is reflected in these recommendations . For exa6 In the 2009 report, primary cutaneous tumoral (PCT) ATL without leukemic, lymph node, and other lesions was frequently included within smoldering ATL and was considered a poor prognostic factor by univariable analyses.7 PCT-ATL is distinct, with cutaneous lesions appearing as tumors that grow rapidly and whose histology shows large, atypical cells with a high proliferative index7  with prominent perivascular infiltration and scant epidermotropism.8 Such cases could be considered for immediate treatment per aggressive ATL protocols with intensive chemotherapy with or without skin-directed therapies, including phototherapy or radiation followed by allogeneic hematopoietic stem-cell transplantation  or interferon with zidovudine (IFN/AZT) with or without arsenic trioxide with or without skin-directed therapies. It should be noted, however, that precisely defining cutaneous tumoral type is difficult—size, height, number, with or without ulceration or subcutaneous extension, and so on—although the nodulotumoral type was defined as nodules or tumors with diameters > 1 cm and the multipapular type as multiple papules with a diameter < 1 cm.6 Furthermore, other types of cutaneous lesions can be aggressive.6 Papules, nodules, and tumors are considered as solid, palpable, and raised lesions with a diameter of < 1 cm, < 3 cm, and ≥ 3 , respectively. Papules usually occur as multiple lesions, whereas tumor(s) may be seen even as a solitary lesion. Recently, a prognostic index (PI) for chronic- and smoldering-type ATL identified soluble interleukin-2 receptor (sIL-2R) levels as an independent prognostic factor. sIL-2R may therefore be useful but requires additional prospective validation.9 Additional clinicopathologic and molecular studies are warranted in cutaneous ATL, including the application of recent new agents for MF and PTCL.8Cutaneous lesions of ATL are variable and may resemble those of mycosis fungoides (MF), with mostly an indolent course, but some are associated with a poor prognosis. Therefore, ATL should be distinguished from cutaneous T-cell lymphomas, including MF, and peripheral T-cell lymphoma (PTCL), especially in endemic areas, by HTLV-1 serology and genomic analysis as necessary. In a large Japanese retrospective study of ATL with cutaneous lesions, 5-year survival rate was 0% in nodulotumoral and erythrodermic types compared with more than 40% in multipapular, plaque, and patch types.e index7 . In thisHTLV-1 serology should be undertaken in cases of cutaneous T-cell lymphoma and PTCL, particularly in HTLV-1 endemic regions.Active monitoring is not appropriate for PCT-ATL, and intensive treatment should be considered.29 Thus, even in patients without a CNS lesion, it is important to incorporate CNS prophylaxis. Intrathecal prophylaxis was incorporated into sequential Japan Clinical Oncology Group (JCOG) studies for aggressive ATL  following both the poor results of earlier study JCOG9109, which did not contain intrathecal prophylaxis,13 and a previous retrospective analysis that revealed that more than one half of relapses that occurred at a new site after chemotherapy occurred in the CNS.30 CNS involvement occurred in 1.6% of patients in JCOG9109 without intrathecal prophylaxis, in 6.3% of patients in JCOG9303 with intrathecal methotrexate (MTX)/prednisone, and in 3.5% of patients in the VCAP-AMP-VECP arm and 8.2% in the CHOP-14 arm  of JCOG9801, both with intrathecal ara-C/MTX/prednisone(JCOG Prognostic Index and Characterization of Long-Term Survivors of Aggressive Adult T-Cell Leukaemia-Lymphoma [JCOG0902A]).13 The reason for the higher incidence in the JCOG9303 trial compared with the JCOG9109 trial might be associated with simultaneous CSF examination in all patients even without symptoms associated with CNS disease after one cycle of chemotherapy.11 In asymptomatic patients with aggressive ATL, diagnostic lumbar puncture/intrathecal chemotherapy should be performed at the end of the first chemotherapy or equivalent antiviral therapy (AZT/IFN) cycle upon successful disease control.Ten percent to twenty percent of patients with aggressive ATL will experience CNS progression.32 However, the scantiness of reports for these options for ATL precludes a more specific recommendation.Options for treating active CNS disease at initial presentation include incorporating high-dose (HD) MTX into combination chemotherapy regimens  for intracerebral tumors or adding intrathecal chemotherapy to the standard induction chemotherapy, as reported for other aggressive lymphomas.33 Numbers of such cases are small and the indication of allo-HSCT for ATL with CNS involvement remains controversial. No specific transplantation conditioning regimen can be recommended, but those that incorporate drugs that can cross the blood-brain barrier  should be considered.Retrospective analysis revealed that allo-HSCT with local treatment of ATL with CNS involvement achieved long progression-free survival (PFS) in four of 10 patients (> 2.5 years) despite high transplantation-related mortality (TRM).Prophylactic CNS therapy should be considered for all patients with aggressive ATL.38 Nontransplantation treatment regimens alone are suboptimal for the majority of patients, although a proportion of patients with aggressive ATL who achieve complete remission after intensive chemotherapy or AZT/IFN therapy may achieve a long (> 5 years) PFS.14 After several promising case series were published,19 several large retrospective analyses of allo-HSCT in Japan reported favorable outcomes, with long-term survival achieved in approximately one third of allo-HSCT recipients after chemotherapy,39 but with significant TRM. The European Society for Blood and Bone Marrow Transplantation registry also demonstrated similar outcomes.40 Thus, treatment strategies that include allo-HSCT as consolidation are recommended in transplantation-eligible patients with aggressive ATL, although there have yet to be any prospective randomized trials to support this approach. Three PIs for aggressive ATL after chemotherapy have been reported as useful with validation cohorts: JCOG-PI, on the basis of corrected calcium and performance status (PS)13; ATL-PI, on the basis of age, serum albumin, sIL-2R,41 Ann Arbor stage, and PS; and modified ATL-PI, on the basis of corrected calcium, clinical subtype, PS, C-reactive protein, and sIL-2R level.39 However, long-term overall survival (OS) in the low-risk group was still poor and a group for whom up-front allo-HSCT might not be recommended could not be clearly identified. A proportion of patients with ATL with localized lymphoma had a long PFS after chemotherapy as well as chronic and a subgroup of acute cases treated with IFN/AZT.41 Several reports demonstrated a graft-versus-ATL effect which contributed to long-term relapse-free survival.43 ATL with abnormalities in tumor suppressor genes, such as p53, was reportedly resistant to IFN/AZT therapy and chemotherapy.45The prognosis of aggressive ATL remains dismal with nontransplantation treatments.46 Thus, early referral to a transplantation center at diagnosis is strongly recommended, particularly in patients with high-risk features as described.41 With progressive disease, clinical outcome after allo-HSCT is poor48 and it is crucial to conduct an up-front allo-HSCT while ATL is controlled to maximize the cure rate. It is recommended that allo-HSCT for progressive disease be performed within the setting of a prospective clinical trial to investigate novel HSCT conditioning regimens or post-HSCT treatment strategies to improve current limited treatment results. When considering allo-HSCT, the standard approach involves searching for an HLA-matched related donor (MRD) or an HLA-matched unrelated donor (MUD) at diagnosis. However, only a proportion of patients will have an MRD. HTLV-1 seronegative donors are also preferred to avoid the risk of donor-derived ATL.49 When only HTLV-1 seropositive related donors are available, it is recommended to exclude the presence of abnormally abundant HTLV-1–infected clones using Southern blotting or polymerase chain reaction on the basis of clonality methods.51 Furthermore, outside of Japan, HTLV-1 infection often arises in minority immigrant populations, which makes it difficult to obtain a suitable MUD from registry panels.53 In these cases, other possible approaches include cord blood transplantation (CBT) or haploidentical HSCT (haplo-HSCT). Early experience with haplo-HSCT or CBT was unsatisfactory; however, protocols for both CBT and haplo-HSCT have been modified in recent years, although the efficacy of these protocols in ATL is not known. Preliminary anecdotal experience suggests that the TRM remains high, although several reports have suggested that CBT provides results that are similar to other transplantation procedures for selected patients with ATL.56 Although experimental in the ATL setting, the addition of post-transplantation cyclophosphamide to haplo-HSCT protocols has improved transplantation outcomes for other hematologic malignancies. HSCT from alternative donor sources earlier in the disease course with good disease control may increase the potential to achieve outcomes that are similar to MUD transplantation outcomes. Without additional evidence from the results of ongoing trials (Data Supplement), it remains difficult to prioritize one donor source over another when an MRD or MUD is unavailable.As responses to intensive chemotherapy in general are not durable and long-term continuation of intensive chemotherapy is not feasible because of complications and cumulative toxicities, early allo-HSCT is recommended after response to first-line therapy.59 Intensity of conditioning should be determined by attendant comorbidities and patient fitness at transplantation.Both myeloablative and reduced-intensity conditioning (RIC) have been used in patients with ATL; however, as the median age in Japan is > 60 years, RIC regimens are increasingly used and sequential prospective trials have revealed the relative safety and promising efficacy of allo-HSCT with RIC.60 The roles of AZT/IFN in this setting or in prophylaxis to prevent relapse are yet to be determined.Although allo-HSCT has the potential to cure ATL, relapse/progression of ATL after allo-HSCT remains a major obstacle and conveys a poor prognosis. In patients with focal relapse  radiotherapy alone with or without the reduction of immune suppression or donor lymphocyte infusions can achieve durable disease control.61 In general, if up-front allo-HSCT is planned after induction chemotherapy, intensive chemotherapy without mogamulizumab should be considered as a result of the described risk of GVHD. For relapsed/refractory patients treated with mogamulizumab, the interval between the last salvage mogamulizumab and allo-HSCT should be long (at least 50 days) to decrease drug levels in vivo61 and additional intensification of GVHD prophylaxis should be considered. Data on allo-HSCT after up-front IFN/AZT are limited, but allo-HSCT is generally feasible and recommended for patients with acute ATL.In Japan, where mogamulizumab and lenalidomide are available for the treatment of relapsed/progressed ATL, reports after allo-HSCT remain limited. In patients with aggressive ATL who received mogamulizumab before allo-HSCT, there seems to be a significantly increased risk of severe and steroid-refractory graft-versus-host disease (GVHD).Pre-emptive treatment after the detection of minimal residual disease post-transplantation should be considered, although methods of monitoring for minimal residual disease of ATL have not been well established after allo-HSCT. In patients in whom HTLV-1 proviral load (PVL) and/or sIL-2R levels begin to increase (no clear threshold exists to detect patients who are at high risk of relapse) chimerism analysis should be considered as well as early taper of immunosuppression and donor lymphocyte infusions.59 Strategies have been undertaken in the United Kingdom and France to minimize neoinfection of donor stem cells after allo-HSCT, including the addition of integrase inhibitors, such as raltegravir at engraftment, or close PVL monitoring and the early addition of zidovudine, although there is no published evidence to support this approach.Kinetic changes in PVL post–allo-HSCT are variable, but three patterns have been observed in patients with full donor chimerism. The first pattern, observed in recipients of allo-HSCT from infected or uninfected donors, was that changes became undetectable after allo-HSCT and remain so. The second pattern, from uninfected donors, was that PVL became undetectable but returned to detectable levels thereafter, usually 6 to 12 months post–allo-HSCT. The third pattern was in those who had detectable PVL throughout.Up-front allo-HSCT should be considered for all suitable patients with aggressive ATL.Early referral to a transplantation center is recommended.HTLV-1 seronegative donors are preferred to reduce the risk of donor-derived ATL.Mogamulizumab should not be used within 50 days of allo-HSCT.23 The regimen was modified with dose reductions that were typically between 50% and 80% in 31 (91%) of 34 patients , and overall response rate [complete response (CR) or partial response (PR)] was 75% after two or three cycles of VCAP-AMP-VECP treatment; however, the completion rate of planned chemotherapy was only 19%. Eleven (32%) of 34 patients who achieved objective responses to initial treatment were switched to an oral maintenance chemotherapy regimen that contained oral etoposide or sobuzoxane and/or prednisolone therapy. With the exception of platelet count, there were no significant differences in the background between patients who were treated with the maintenance treatment and those not. Median survival time and 2-year OS rate of those who received maintenance oral chemotherapy were 16.7 months and 33%, respectively, which suggests that such a treatment strategy is a viable option for elderly and/or non–transplantation-suitable patients, warranting prospective trials.23A retrospective study of elderly patients with aggressive ATL (age > 70 years) observed that elderly patients who were treated with the VCAP-AMP-VECP regimen as initial treatment demonstrated OS that was similar to that in the trial reports of patients age 56 to 70 years.12Alternatively, dose-reduced CHOP-14–like regimens could be considered in elderly and/or non–transplantation-suitable patients, as the subgroup analysis of the JCOG9801 trial demonstrated that the OS rate was similar between VCAP-AMP-VECP and CHOP-14 regimens in patients age 56 to 70 years in contrast to the superiority of the VCAP-AMP-VECP regimen in patients younger than 56 years of age with a 45% OS rate at 1 year.21 Despite the lack of published reports on AZT/IFN outcomes for elderly patients, as with younger patients, this combination should be considered as a first-line option followed by maintenance where available.22Combination mogamulizumab and chemotherapy  could be considered as initial therapy for such patients, although the combination was only evaluated in relatively nonelderly patients.Given the lack of a preferred or compelling treatment strategy for elderly patients with aggressive ATL, alternative options, such as the selection of less intensive regimens that can be used in a continuous fashion, maintenance strategies using oral agents , or AZT/IFN, may be appropriate. There may be future roles for monoclonal antibodies  as a maintenance strategy, but these require additional evaluation before recommendations can be made.Less intensive induction therapy with or without maintenance therapy with either oral chemotherapy or AZT/IFN may be appropriate for elderly and/or non–transplantation-suitable patients with aggressive ATL.63 However, whether these agents work in vivo via an antiviral effect has remained controversial. Several studies have shown little, if any, viral structural, enzymatic, or regulatory gene expression in ATL, with the exception of the antisense gene HBZ,65 and it remains controversial whether the continued expression of non-HBZ viral genes is required for tumor maintenance. However, recent data demonstrate that the extinction of Tax expression leads to total growth inhibition of ATL-derived cells even if Tax protein is undetectable at baseline.67 A possible explanation for these contradictory results is the recent demonstration on the basis of single-cell analysis of transient bursts of Tax oncoprotein, and presumably other viral proteins, which has led to the suggestion that transient Tax expression is essential for ATL survival.67 Thus, immunotherapy against Tax might be theoretically promising. A recent phase I study that used vaccination with Tax peptide-pulsed dendritic cells in three patients with aggressive ATL in PR or stable disease after chemotherapy demonstrated an induction of immune response against Tax with promisingly long-term remission.68 This warrants larger studies to demonstrate clinical effectiveness.Several antiretrovirals used for HIV have demonstrated proven efficacy against HTLV-1 in tissue culture, including the reverse transcription inhibitors zidovudine and tenofovir, as well as the integrase inhibitor raltegravir.69 Of note, doses of AZT used for the treatment of ATL—600 to 900 mg/d and up to 3,000 mg/d in certain reports—are generally higher than those used for HIV treatment (600 mg/d) although the inhibitory concentrations of AZT for HTLV and HIV replication are similar.72 It is also unclear whether reactivation of HTLV-1 expression in malignant or nonmalignant cells after induction therapy leads to chemotherapy resistance73; however, it is notable that a recent chemotherapy trial that included raltegravir demonstrated results that were similar to a trial that lacked antiviral agents during the same induction therapy.37 Conversely, a recent report demonstrated that the combination AZT/IFN induced a significant inhibition of HTLV-I reverse transcription activity in responding ATL patients but not in resistant patients.74 These results are in line with a direct antiviral effect, likely in the HTLV-1–infected nonmalignant cells, which may play a major role in the survival of ATL cells.Although the combination of AZT/IFN has shown activity, it is uncertain whether these drugs function via antiviral activity in cells in the tumor or microenvironment or through mechanisms other than antiviral activity.5 These include genes that are involved in T-cell receptor pathway activation, including phospholipase γ, protein kinase Cβ, caspase recruitment domain-containing protein 11, and interferon regulatory factor 4, affecting the nuclear factor κB (NF-κB) pathway, which is activated in ATL and contributes to cell proliferation and resistance to apoptosis. Inhibitors of these pathway mediators, including protein kinase C or NF-κB inhibitors, could be useful in ATL therapy; however, a trial that included bortezomib, as an NF-κB inhibitor with induction chemotherapy, did not provide significant benefit.37 IRF4 expression could have oncogenic potential in ATL and may be associated with interferon resistance,75 which warrants therapeutic targeting of its function. Of note, lenalidomide, an active agent in ATL, may function via the inactivation of IRF4 by enhancing the degradation of the IRF4 gene activators Ikaros and Aiolos.76New insights into the molecular biology of ATL have provided ideas for future clinical trials. Although there was no detectable viral Tax expression in ATL samples, many of the cellular proteins affected by Tax (Tax interactome) were found to be mutated in ATL.5 In addition, genes that are involved in antigen presentation and immune surveillance are often mutated,5 including aberrant programmed death ligand-1 expression.77 These findings suggest that immune checkpoint therapy could play a role in ATL treatment. Currently, a trial of a programmed death-1 inhibitor, nivolumab, is underway in Japan (Data Supplement), although a study in the United States was recently halted as a result of concerns over accelerated disease progression.38Genomic studies of ATL have revealed high rates of genetic damage compared with other hematopoietic malignancies.28 Mogamulizumab has been also approved for the treatment of newly diagnosed aggressive ATL in combination with intensive chemotherapy.21Recently, two new agents, mogamulizumab, an anti-CCR4 monoclonal antibody, and lenalidomide, an immunomodulatory drug, have been approved for the treatment of ATL in Japan after pivotal trials for relapsed aggressive ATL78 In phase I and II studies conducted in Japan, a response rate of approximately 50% was observed with manageable toxicities, including moderate-to-severe skin reactions and other immunopathology, even in nontransplantation patients, possibly by depleting nontumor regulatory T cells.28 CR rates varied among target lesions—rates were high in peripheral blood, intermediate in skin, and low in lymph nodes. Median PFS and OS were 5.2 months and 13.7 months, respectively. The findings of a subsequent randomized phase II study of intensive chemotherapy (modified LSG15) with or without mogamulizumab for the treatment of untreated aggressive ATL has recently been reported.21 The combination was well tolerated and produced a higher CR rate compared with chemotherapy alone . However, PFS and OS were identical in both arms, although the sample size and duration of follow-up were relatively short and median OS was not reached in either arm. Recently, a phase II trial in Europe, the United States, and South America of single-agent mogamulizumab  versus investigator’s choice of salvage chemotherapy for relapsed aggressive ATL revealed a 15% confirmed overall response rate in the mogamulizumab arm versus 0% for investigator’s choice.25CCR4 is expressed in neoplastic cells in approximately 90% of ATL cases and mutated in 26%. This expression has been associated with cutaneous manifestations and poor prognosis.26 A subsequent phase II study evaluated the efficacy and safety of lenalidomide monotherapy at an oral dose of 25 mg/d continuously until progressive disease or unacceptable toxicity in patients with relapsed or recurrent aggressive ATL.78 Objective responses were noted in 11 of 26 patients , including four patients with CR and one with unconfirmed CR. Rate of disease control, including overall response and stable disease, was 73%. Median time to response and duration of response were 1.9 months and not estimable, respectively. Median PFS and OS were 3.8 months and 20.3 months, respectively. The most frequent grade 3 or greater adverse events were neutropenia (65%), leukopenia (38%), lymphopenia (38%), and thrombocytopenia (23%), which were all manageable and reversible.26 In conclusion, lenalidomide demonstrated clinically meaningful antitumor activity and an acceptable toxicity profile in patients with relapsed or recurrent aggressive ATL. Lenalidomide also demonstrated activity with CNS involvement by diffuse large B-cell lymphoma,79 which could have implications for its use in ATL despite the lack of data in the previous trial as CNS involvement was an exclusion criterion.A phase I study of lenalidomide demonstrated preliminary antitumor activity in patients with relapsed ATL or PTCL at a higher dose than that used for the treatment of multiple myeloma.+ cases (NCCN grade 2A). Pralatrexate was included as an investigator choice drug in the recently completed randomized phase II trial of single-agent mogamulizumab versus salvage chemotherapy for the treatment of relapsed aggressive ATL, but no confirmed responses were reported.25 Histone deacetylase inhibitors are known to activate HTLV-1 expression; therefore, these drugs might be used with caution in ATL, with consideration given to the addition of an antiretroviral agent  and under clinical trials desirably.80 Epstein-Barr virus reactivation in patients with extranodal natural killer/T-cell lymphoma was reported as a previously unrecognized serious adverse event in a pilot study with romidepsin.81Other US Food and Drug Administration (FDA)–approved drugs are now available for the treatment of relapsed/refractory PTCL and have been used for ATL in the United States, including the antifolate agent pralatrexate and the histone deacetylase inhibitors belinostat and romidepsin . Mogamulizumab has recently received FDA approval in MF or Sézary syndrome after at least one prior therapy (August 2018). Although not FDA approved, current NCCN guidelines support the use of brentuximab vedotin in CD30All patients should be considered for entry into clinical trials where available.+ ATL, remains incompletely understood. Clinical trials are required to understand the roles of these agents in the front-line setting, including indolent subtypes, in maintenance therapy for transplantation-ineligible patients, in maintenance therapy after allo-HSCT, and in the prevention or treatment of CNS disease. As ATL is characterized by early relapses, clinical trials of therapies that might eradicate minimal residual disease are warranted. Preliminary data from France have shown that combining arsenic trioxide with low-dose interferon is feasible and an effective consolidation therapy that is capable of selectively eliminating the malignant clone and restoring oligoclonal architecture. This has raised hopes that the extinction of viral replication (AZT) and Tax degradation (arsenic/IFN) may eradicate the disease.83 Addition of arsenic trioxide to IFN/AZT was promising in a phase II study for the treatment of chronic ATL.16Whereas the backbone of ATL treatment has remained largely unchanged since the 2009 consensus report, there have been several advances in the clinical management of these patients, particularly for patients treated in Japan. These include the increased role of allo-HSCT after first-line treatment and the use of mogamulizumab and lenalidomide as novel single agents, both licensed in Japan. The precise roles of these agents and others, such as brentuximab vedotin for CD30Since 2009, there has been more widespread use of PET/CT in the diagnosis and follow-up of patients with non-Hodgkin lymphoma. ATL is frequently associated with extranodal disease, and it might be recommended that PET/CT be used at diagnosis, where available, and that PET/CT assessment should be incorporated in future studies. The significance of negative interim PET/CT should be evaluated as it is not yet clear how this might affect treatment strategy.Reported prognostic factors at diagnosis  require validation in the context of treatment with AZT/IFN, chemotherapy, or active monitoring to assist in tailored treatment decisions. New methodologies to detect clonal HTLV minimal residual disease have been established using next-generation high-throughput sequencing methods, and the challenge now remains to validate these observations within clinical trials and bring them into the clinical domain in a rapid, cost-effective manner.ATL continues to have a dismal prognosis with current therapies, and clinical trials that incorporate molecular and prognostic factors will remain paramount to advances in ATL treatment. We have proposed a strategy for ATL treatment stratified by subtype classification, including an updated opinion on the management of patients with tumorous skin lesions. Future clinical trials should remain a priority to ensure that the consensus is continually updated to establish evidence-based practice guidelines.


This study aims to describe factors associated to treatment continuity and psychiatric relapses in patients treated with Long Acting Injectable antipsychotics (LAIs) in Bologna Community Mental Health Centers (CMHCs).New LAI treatments administered between July 1, 2010 and June 30, 2015 in CMHCs were selected. The cohort was followed-up for 6 months; predictors of continuity and psychiatric admissions were investigated by using logistic regression- and Cox- analysis respectively.Among the cohort of 1 070 patients, only 222 (21%) continued LAI treatment during the follow-up. LAI continuity was higher with first generation agents  and in case of previous psychiatric hospitalizations . Incidence of psychiatric hospital admissions showed a sharp reduction in the follow-up compared with 6-month period before initiation (from 458 to 212), and was associated with previous psychiatric hospitalizations , immigration  and LAI discontinuation .Psychiatric hospital admission before LAI initiation was the main predictor both of LAI continuity and hospitalization during the follow-up. The long-acting injectable antipsychotics (LAIs) have aroused new interest, especially with the introduction of second generation agents (SGA) in LAI formulations. Some aspects are still controversial, including delayed recovery from adverse reactions and possible perception of stigma and coercion, which could compromise the quality of the therapeutic relationship .NICE guidelines recommend that treatment with LAIs should be considered, after oral antipsychotic (AP) medication, for patients with psychosis or schizophrenia who would prefer such treatment after an acute episode or when avoiding covert non-adherence to antipsychotic medication is a clinical priority . SGA-LAIThe present study aims to provide real-world evidence on LAI treatments in Community Mental Health Centers (CMHCs) of a Northern Italy area from 2010 to 2015. It is first intended to outline the five-year trend in LAI initiation and to point out if the recent expected LAIs increase in psychiatrists’ prescriptions was observed. Moreover, this study will describe social and clinical characteristics of LAI new users, and will investigate over a 6-month follow-up LAI continuity along with its principal predictors, as well as the relationship of LAI discontinuation and other risk factors with hospitalization.The study was carried out in the Community Mental Health Centers (CMHCs) of the Bologna Local Health Authority (LHA) in Emilia-Romagna Region. Bologna LHA covers both urban  and rural areas, and serves approximately 860,000 inhabitants, that represent a fifth of the regional population. CMHCs are run within the Department of Mental Health by multi-professional teams and are responsible for community and residential treatments of adults, as well as for coordinating with hospital psychiatric units and for continuity of psychiatric care.Data on patients and treatments are recorded in the local mental health information system, established in 2007 for administrative and clinical-epidemiological purposes. Data selected for the present analysis include socio-demographic characteristics, diagnoses, length of CMHC care, hospital admissions and AP medication delivered by CMHC staff. Since in Italy public and private licensed hospitals record admissions and discharges in the Hospital Discharge Register (HDR), the local information system has been cross-checked with the regional HDR, so as to include all admissions for psychiatric reasons, both in the public and the private sector. Oral APs are usually supplied by CMHC, whereas LAI injections are directly administered by CMHC nurses.Diagnoses are codified according to the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) and drugs according to the Anatomical Therapeutic Chemical classification (ATC).FGA-LAIs marketed in Italy from 2010 to 2015 were fluphenazine decanoate (ATC: N05AB02), haloperidol decanoate (N05AD01), perphenazine enantate  and zuclopenthixol decanoate (N05AF05). SGA LAIs included risperidone long-acting (N05AX08), paliperidone palmitate (N05AX13), olanzapine pamoate (N05AH03), aripiprazole long-acting (N05AX12). It should be mentioned that only drugs in the Regional Formulary Drugs (RFD) are usually provided by CMHCs, while other marketed drugs can be exceptionally delivered if specific clinical reasons are detailed. Paliperidone entered in the RFD in 2014, while olanzapine and aripiprazole were not yet included in the RFD when the study ended.The study protocol was approved by the Ethics Committee of Bologna-Imola Local Health Authorities in September 2015. Data extracted from the local mental health information system have been anonymized by the Department of Mental Health office before the analysis; ethics committee waived the requirement for informed consent.The study cohort was represented by patients aged 18 years or more, residents in the area of Bologna LHA, receiving a new treatment with LAIs between July 1, 2010 and June 30, 2015 and in contact with CMHC for at least 6 months before starting LAIs. A “new-start LAI treatment” was defined when no LAI had been administered by a CMHC within the previous 6 months. In case of subsequent cycles of LAIs for the same patient over the study period, only the first one was considered. The first LAI administration date was used as a reference to collect socio-demographic and clinical data.Information on sex, age, nationality, living area , years of education, employment, marital status and living arrangement  were extracted from the local mental health information system. From the same source, we also collected psychiatric diagnoses, grouped into the following categories: schizophrenic-like disorders , bipolar disorders , depressive disorders , anxiety disorders , personality disorders , other diagnoses (other codes not previously listed).Data on hospital admissions for psychiatric reasons and oral AP utilization  were collected over the six-month period before the entry date and during the 6-month follow-up together with possible discharge from CMHCs.Rate of LAI utilization in CMHCs was estimated and each treatment was assessed for continuity along the six-month follow-up: it was considered “discontinued” when either intervals > 45 days between two LAI administrations or switches to other LAIs occurred; all other treatments were considered “continuous”. Discontinued treatments were further distinguished into “definitively discontinued”, if no more LAI administrations were found before the end of the six-month follow-up, and”intermittently discontinued”.Psychiatric hospital admissions were assumed as a proxy of severe clinical relapses. Data on drug treatments during hospital stay were not available.Rates of LAI utilization within Bologna CMHCs from 2011–2015 were evaluated.A descriptive analysis of socio-demographic and clinical characteristics of the study cohort was performed, including psychiatric admissions and oral APs prescribed in the six month-period before initiation. Continuity of LAI medication and hospital admissions during the six-month follow-up were then analyzed.p< 0.05) at 95% confidence intervals (95%CI) was considered a predictive factor of treatment continuity.In order to identify predictors of LAI continuity, a logistic regression model was used including the following characteristics as categorical variables: sex, age, nationality, living status, diagnosis, LAIs generation and previous hospital admission. Any independent variable with an adjusted odds ratio (ORadj) statistically significant  version 21.0 for Windows.The annual frequency of new treatments with LAIs did not increase from 2011 to 2015, involving on average 2.1% of CMHC patients and 5.4% of patients on AP treatment .A moderate prescribing shift to SGA-LAIs was found over the five-year period, though FGA-LAIs remained the most frequent option for initiating new treatments, from 71.7% (n = 281 out of 392) in 2011 to 64.4% (n = 237 out of 368) in 2015. In particular, in 2011 the most prescribed formulations were fluphenazine (36%) and haloperidol (27%), whereas in 2015 fluphenazine decreased to 27%, haloperidol increased to 32% and paliperidone attained 21%.st July 2010 to 30th June 2015 , AP users were first selected  and LAI users were then identified . The study cohort included only those subjects who initiated a LAI treatment over the period and had received CMHC care for at least 6 months before then . For patients initiating more than one LAI treatment during the study period , only the first one was considered  steadily continued LAI treatment, 517 (48.3%) discontinued it definitively, whereas 331 (30.9%) discontinued “intermittently”. Moreover, 646 subjects (60.4%) were prescribed also oral APs during LAI treatment, and 455 (42.5%) received oral AP after the last LAI injection . It should also be noted that 97.4% of patients remained in care at CMHC all along the six-month follow-up .adj 1.71; 95%CI 1.18–2.49) and previous hospitalization  were associated with LAI continuity All other variables included in the logistic regression model  did not result in any statistically significant impact on LAI continuity.As reported in During the follow-up, 20% of patients experienced hospitalization for psychiatric reasons compared with 43% in the 6-month period before LAI initiation . The risk of hospital admission, hence of severe clinical relapse, increased mainly for those patients admitted before LAI initiation , for immigrants  and to a less extent in case of free-of-treatment days during the follow-up . Also age was found to influence this outcome (p< 0.05), with higher risks for older patients .The annual incidence of treatments with LAIs in Bologna remained substantially unchanged from 2011 to 2015. Only a moderate shift toward initiation with SGA-LAIs was observed, despite other drug-utilization studies documenting the increasing use of these drugs in recent years , 11. It Patients with schizophrenic-like disorders were 54% of the study cohort. The remaining proportion was partly represented by bipolar and personality disorders, for which evidence of effectiveness of LAIs is also available , 13. As In the six-month period before the entry date, less than two-thirds of patients had used some oral APs, at least in terms of out-of-hospital treatments. This rate seems to be low if we consider that guidelines and practices, as well as recent appraisals , usuallyForty percent of patients of our cohort had been hospitalized with psychiatric diagnosis for at least a day in the six-month period preceding the entry date. This is remarkable, because only 7% of patients treated by Bologna CHMCs have psychiatric admissions in 1-year period. Since hospitalization can be considered a proxy of acute clinical impairment, these data confirm that Bologna CMHCs are more likely to use LAIs for patients with the most severe psychiatric conditions [Although it is widely accepted that LAI administration improves adherence –18, in oThe large proportion of cases (60%) with concomitant oral and long-acting AP medication at follow-up highlighted the well-known tendency of patients with psychiatric diseases to receive AP polypharmacy.Only two factors were identified through a multivariate analysis as predictors of the six-month continuity of LAI utilization: initiation with FGA-LAI and, most importantly, psychiatric admissions in the previous six-months. As regards the former, this generation is likely to be used in case of higher severity of illness, since some of these agents are deemed to have greater sedative or incisive effect, no matter the scientific evidence and the considerable heterogeneity across single APs. Our findings cast doubts on the alleged lower tolerability of FGA medications, as already highlighted in CATIE and CUtLASS studies , 24. As A recent hospital admission, being a proxy of clinical seriousness, is also the principal determinant of re-admission at follow-up, although other variables play a part: early discontinuity of LAIs, as admissions after LAI initiation more likely occurred during free-of-treatment days. Nevertheless, our findings show that starting a LAI treatment strongly decreased hospital admissions, while discontinuation had a lower association with next hospitalisation. An expeditious pattern of LAI prescriptions, more crisis-oriented and briefer than expected, may also be evinced by the fact that 555 out of 1,625 cases initiating a new LAI treatment (34.1%) were excluded from the final cohort because they had been in contact with CMHC less than 6 months before initiation.A few remarks are needed for immigrant subjects: despite their limited number in this study, there was a significant risk of hospitalization at follow-up for this population. Their higher risk of hospital admissions at follow-up could be partially explained by a tendency to provide immigrants with briefer admissions to hospital or residential settings, as showed by a recent 12-month investigation in Bologna, on CMHC patients with severe mental disorders . CombiniThe higher risk of hospital admission for psychiatric disorders found for older age could be explained by a general frailty of this population, which may be susceptible to hospitalization also for less severe conditions, in comparison to younger patients.Both limitations and strengths of this study stem largely from the same source, insofar as data were drawn from the ordinary database of CMHCs and no supplementary clinical information was collected. The short follow-up, the lack of standardized measures of clinical severity, the absence of information on comorbidities, the uncertainty on the actual intake of oral APs supplied by the CMHCs are examples of consequent shortcomings. In addition, an “immeasurable time bias” , due to On the whole, it may be envisaged that the information retrieved from current databases allows a first, essential level of quantitative and qualitative evidence. Further research with longer follow-up and more detailed information should assess if LAIs can play a transient but effective role in more complex and recovery-oriented strategies of care, favouring and triggering other therapeutic factors, such as a closer involvement of patients and families in person-centred treatments, integrated with talking therapies, psychosocial activities and rehabilitation programs.In conclusion, the study provides new evidence on LAI utilization in a vast Italian Department of Mental Health. LAIs were used not only for schizophrenic like disorders and not necessarily after previous stabilization with AP therapy. The continuity of treatment seems associated with the severity of clinical symptoms  and with the choice of FGA-LAIs over SGA-LAIs. The incidence of LAI treatments in CMHCs did not increase along five years, with a surprisingly low proportion of cases with continuous treatment for at least 6 months. Psychiatric hospital admission before LAI initiation was also the main predictor of hospitalization during the follow-up. Studies replicating this analysis in other Departments of Mental Health would allow to compare evidence across different areas and strengthen our findings.S1 TableLAI administration at hospital admission was assumed.(PDF)Click here for additional data file.


This work tests bioenergetic and cell-biological implications of the synthetic fatty acid Minerval (2-hydroxyoleic acid), previously demonstrated to act by activation of sphingomyelin synthase in the plasma membrane (PM) and lowering of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) and their carcinogenic signaling. We show here that Minerval also acts, selectively in cancer cell lines, as an ATP depleting uncoupler of mitochondrial oxidative phosphorylation (OxPhos). As a function of its exposure time, Minerval compromised the capacity of glioblastoma U87-MG cells to compensate for aberrant respiration by up-modulation of glycolysis. This effect was not exposure time-dependent in the lung carcinoma A549 cell line, which was more sensitive to Minerval. Compared with OxPhos inhibitors FCCP (uncoupler), rotenone (electron transfer inhibitor), and oligomycin (F1F0-ATPase inhibitor), Minerval action was similar only to that of FCCP. This similarity was manifested by mitochondrial membrane potential (MMP) depolarization, facilitation of oxygen consumption rate (OCR), restriction of mitochondrial and cellular reactive oxygen species (ROS) generation and mitochondrial fragmentation. Additionally, compared with other OxPhos inhibitors, Minerval uniquely induced ER stress in cancer cell lines. These new modes of action for Minerval, capitalizing on the high fatty acid requirements of cancer cells, can potentially enhance its cancer-selective toxicity and improve its therapeutic capacity. MinervaAnti-cancer drugs such as Minerval, which interact with membrane lipids and which modify the composition and structure of cell membranes, belong to a conceptually new approach for treating cancer called membrane lipid therapy (MLT) . MinervaIn addition to modulation of the PM, Minerval also specifically modulates the activity of sphingomyelin synthase (SMS1), leading to an increased concentration of sphingomyelin (SM) in cancer cells but not in non-cancer cells, in which SM levels are already high . These cDue to the promising anti-cancer effect of Minerval in lung and leukemic cancer cells and its lipid nature, it was decided to explore it as a treatment against glioma in a phase IIb clinical trial currently in progress . This decision is based on the demonstration that Minerval has greater efficacy than the first-line standard of care temozolomide. It is expected that concurrent treatment with both will increase the patients’ progression-free and overall survivals. Moreover, in contrast with temozolomide, there is no apparent relapse in the tumor formation after treatment with Minerval ,14.As Minerval is known to increase membrane fluidity and to be selectively toxic to cancer cells, we set out here to explore the possibility that Minerval, like other hydroxylated fatty acids ,16, can Glucose, paraformaldehyde (PFA) and sodium pyruvate were procured from Sigma. DMEM (with and without phenol red (for microscopy)), RPMI and EMEM media, as well as L-Glutamine, cell culture antibiotics (penicillin–streptomycin–neomycin), fetal calf sera, trypsin, trypan blue (for live cell counting in seeding), and phosphate buffered saline (Ca and Mg free) were all procured from Biological Industries . CellROX Green, MitoTracker Red (CMXRos), MitoSox red, ER-Tracker red, LysoTracker Deep Red, DAPI, Calcein-AM Green, and TMRE  were all purchased from Thermo Fisher Scientific.We used three human cell lines: primary glioblastoma (U87-MG), non-small cell lung adenocarcinoma (A549), and non-cancerous fibroblast-like MRC5 cells derived from fetal lung. U87-MG cells were grown in DMEM medium supplemented with 4.5 g/l glucose, 10% fetal bovine serum and 1 mM L-glutamine, sodium pyruvate, and penicillin–streptomycin–neomycin. A549 and MRC5 cells were respectively grown in RPMI and EMEM media with the same supplements.Bioenergetic assays  were performed using the XF Cell Mito Stress Test Kit  following manufacturer’s instructions. These measurements are based on extracellular monitoring of oxygen and proton fluxes by fluorescence-based probes. Specifically, for our experiments, cells were seeded in XF96 cell culture plates at 40,000 cells/well. Minerval  using a 60/1.35 NA PlanApochromat oil-immersion lens. The ImageJ software was used for analysis and quantification of the fluorescence intensity of the confocal images. To permit comparison of images, the same laser power and detector gain and offset were used for all compared images. The following fluorophores were used: The CellROX® Green Reagent  to measure overall cellular reactive oxygen species (ROS) production; TMRE . In FCCP treatments, TMRE was added to the FCCP containing medium after 30 min without washing). TMRE readily accumulates in active mitochondria due to their relative negative charge. However, TMRE is poorly retained in fixed cells and was therefore only used by us to detect MMP dynamically in live cells. For fixed cells, we used the CMXRos (MitoTracker red) stain . MitoSox , which is a mitochondrion-targeted dihydroethidium, was used to detect superoxide production  2 incubator. This mix included ER-Tracker red  stain), LysoTracker Deep Red , DAPI  stain), and Calcein-AM Green . Cells were then fixed with 4% paraformaldehyde (PFA), washed with PBS and plates were transferred to an InCell2200  machine for image acquisition at 40× magnification. The output produced was based on comparative fluorescence intensity. Object segmentation was carried out using Multi-target analysis in the GE analysis workstation to identify the nuclei (DAPI) and cell boundary . We further identified ER (ER-Tracker) and lysosomes (LysoTracker) as intracellular ‘organelle’ objects. All the assay parameters  were kept constant for all assay repetitions.U87-MG, MRC5, and A549 cells were cultured in specialized microscopy-grade 96-well plates . Minerval (200 μM) dissolved in DMSO was added only for 72 h, in order to maximize its effects. Total DMSO concentrations were always kept below 0.1%. After a process of optimization, taking into account the growth of cells during the 72 h period of Minerval (200 µM) exposure, U87-MG and A549 cells were seeded at a concentration of 800 per well, and MRC5 at a concentration of 15,000 per well. In experiments where the effects of Minerval were compared with those of OxPhos inhibitors, these inhibitors were added to cells, pre-treated for 72 h with DMSO as a vehicle control, for 30 min in the same concentrations specified in The inhibitors oligomycin, FCCP, and rotenone plus Antimycin A were sequentially injected by the Seahorse XF machine to measure OCR driving ATP production, maximal respiration, and non-mitochondrial respiration, respectively. In parallel, the extracellular acidification rate (ECAR) was also measured in response to these inhibitors. These Seahorse bioenergetic profiling experiments were applied to the U87-MG (glioblastoma), A549 (lung adenocarcinoma) and, for comparison, MRC5 (non-cancerous) cell lines. Minerval at 200 µM was added for 24, 48, and 72 h to all cell lines. The choice of these times and concentration is based on previous work in these cell lines showing time-escalation of various cancer growth-diminishing parameters ,12,18. FIn the glioblastoma U87-MG cells, basal OCR A and maxIn contrast with U87-MG cells, Minerval did not significantly affect basal A and maxMinerval-triggered increases in proton leak E and cony-axis) and glycolytic (x-axis) contributions to overall cell energetic status, both at basal (empty squares) and maximal (filled squares) respiration. According to these diagrams, Minerval treatment rendered U87-MG cells more energetic, or closer to the upper right-hand corner of the energy diagram , which restricts proton flow through the complex down its electrochemical gradient.Having established that Minerval acts as a partial uncoupler, we decided to confirm these results microscopically by comparing the effect of Minerval to that of established mitochondrial inhibitors. +-ATPase blocker OM can prevent proton re-entry through the inner mitochondrial membrane and consequent MMP dissipation and thus also hinders electron flow and generates ROS. These differences among the inhibitors and the observation that, like the uncoupler FCCP, Minerval too mitigates mitochondrial ROS production are shown in MMP depolarization by the uncoupler FCCP occurs by a mechanism different than that of the respiratory chain blocker rotenone: while FCCP is a protonophore that mitigates MMP by mobilizing protons down their electrochemical gradient, rotenone blocks respiratory complex I thus inhibiting electron flow that generates this gradient. This fundamental mechanistic difference is expressed by the difference in ROS production: while FCCP facilitates electron flow through the respiratory chain thus diminishing premature reduction of dioxygen into superoxide and ROS production, rotenone slows down this electron flow thus enhancing mitochondrial ROS production. Similarly, the HTo obtain a more comprehensive picture of the influence of Minerval at the cell level, we have analyzed and quantified in U87-MG cells the effect of the lipid on cellular features other than the mitochondria using the unique high content, multi-parametric image analyzer InCell 2200 . Interestingly, Minerval has increased the area, vesicle number and staining intensity of the endoplasmic reticulum (ER) in U87-MG cells . This efThe main finding of this work is that Minerval serves as a partial uncoupler of oxidative phosphorylation (OxPhos). We have shown that, similar to the well-established uncoupler FCCP, Minerval increases proton leak and reduces coupling efficiency in the cancer cell lines U87-MG and A549, while not affecting leak and coupling efficiency in the non-cancerous cell line MRC5. In addition, in U87-MG cells we have demonstrated that the modular kinetics of Minerval correspond only to those of an uncoupling agent, and not to other OxPhos modulators A. ExpectAlso at the cell biological level Minerval was demonstrated to act as an uncoupler and not as nutrient excess, as the mitochondrial fragmentation it triggered was accompanied by a size decrease, and not increase, of the solitary mitochondria formed by the fragmentation B. Apart http://www.uniprot.org/uniprot/Q86VZ5#subcellular_location) and SMS2 (http://www.uniprot.org/uniprot/Q8NHU3#subcellular_location), are not expressed in mitochondrial membranes. Moreover, the modified lipid composition of mitochondria derived from tumors, mainly consisting of reduced levels of mature cardiolipins, is considered irreversible [+-ATPase) [As an MMP depolarizing agent, Minerval is also expected to facilitate electron flux through the respiratory chain and thus reduce mitochondrial ROS production, mainly caused by stalled or back flowing electrons that prematurely reduce dioxygen. This MMP depolarization may be associated with the extent of cancer selective Minerval toxicity, as the largest MMP depolarization was observed in the cancer cell line most susceptible to Minerval, A549 . We specversible . Possibl-ATPase) . This mo-ATPase)  associat-ATPase) , and, es-ATPase)  are depeCancer cells have important energetic needs and fatty acids are one of the sources they use to fulfill their growth requirements. Previous studies have shown that intake of the fatty acid analog Minerval is markedly faster in cancer cells with respect to non-tumor cells . Possibl2 reduced/min/nucleus for MRC5 cells). The higher basal OCR in U87-MG cells suggest they have higher ATP demand and are therefore more susceptible to perturbation of energy production by an uncoupler such as Minerval. Hence, U87-MG cell viability or total ATP is more heavily affected by Minerval as compared with MRC5 cells  than in U87-MG (ca. 400 μM) cells . As the glycolytic compensatory response as a whole in A549 cells was significantly less pronounced than in U87-MG cells, some of the relative resilience of U87-MG cells to Minerval (as compared with A549 cells) may be related to their higher capacity to compensate for the respiratory holdback by glycolytic up-regulation. However, at longer exposures to Minerval, this capacity is eventually dwindled. Our observations that A549 cells are more sensitive to Minerval than U87-MG cells suggest that in the clinical settings, where Minerval has only been tested in glioblastoma (and not on any other type of cancer), there is more room for improvement in glioblastoma than in lung cancer. We will discuss this possibility in the next section.Within cancer cell lines, there appears to be a difference between the glioblastoma cell line U87-MG and the lung carcinoma cell line A549. Minerval seems more cytotoxic to A549 than to U87-MG. This difference is most apparent if the energy phenotypes of the two cell lines are compared . Minerva. increase in selective toxicity) might be improved with glycolytic inhibitors . Further inhibition of glycolysis, especially if Minerval dosage used for treating glioblastoma patients is equivalent to cell exposure times of below 72 h, may impede the recovery of cancer cells from Minerval and thus make the drug more efficient. In addition to compromised glycolytic compensation, another possible Achilles heel of cells treated with Minerval is ER stress  and A549 (lung carcinoma)—suggest that the selective cancer toxicity of Minerval has some weak spots that can be improved. As described above, in contrast with A549 cells showing constant and less pronounced up-regulation of glycolysis following Minerval, in U87-MG cells at long enough exposure to Minerval (72 h), the ability to compensate reduced coupling efficiency with up-modulated glycolysis is compromised. This phenomenon in U87-MG cells is manifested both in


The Coxsackie and adenovirus receptor (CAR) is a transmembrane cell-adhesion protein that serves as an entry receptor for enteroviruses and may be essential for their ability to infect cells. Since enteroviral infection of beta cells has been implicated as a factor that could contribute to the development of type 1 diabetes, it is often assumed that CAR is displayed on the surface of human beta cells. However, CAR exists as multiple isoforms and it is not known whether all isoforms subserve similar physiological functions. In the present study, we have determined the profile of CAR isoforms present in human beta cells and monitored the subcellular localisation of the principal isoform within the cells.Formalin-fixed, paraffin-embedded pancreatic sections from non-diabetic individuals and those with type 1 diabetes were studied. Immunohistochemistry, confocal immunofluorescence, electron microscopy and western blotting with isoform-specific antisera were employed to examine the expression and cellular localisation of the five known CAR isoforms. Isoform-specific qRT-PCR and RNA sequencing (RNAseq) were performed on RNA extracted from isolated human islets.An isoform of CAR with a terminal SIV motif and a unique PDZ-binding domain was expressed at high levels in human beta cells at the protein level. A second isoform, CAR-TVV, was also present. Both forms were readily detected by qRT-PCR and RNAseq analysis in isolated human islets. Immunocytochemical studies indicated that CAR-SIV was the principal isoform in islets and was localised mainly within the cytoplasm of beta cells, rather than at the plasma membrane. Within the cells it displayed a punctate pattern of immunolabelling, consistent with its retention within a specific membrane-bound compartment. Co-immunofluorescence analysis revealed significant co-localisation of CAR-SIV with zinc transporter protein 8 (ZnT8), prohormone convertase 1/3 (PC1/3) and insulin, but not proinsulin. This suggests that CAR-SIV may be resident mainly in the membranes of insulin secretory granules. Immunogold labelling and electron microscopic analysis confirmed that CAR-SIV was localised to dense-core (insulin) secretory granules in human islets, whereas no immunolabelling of the protein was detected on the secretory granules of adjacent exocrine cells. Importantly, CAR-SIV was also found to co-localise with protein interacting with C-kinase 1 (PICK1), a protein recently demonstrated to play a role in insulin granule maturation and trafficking.The SIV isoform of CAR is abundant in human beta cells and is localised mainly to insulin secretory granules, implying that it may be involved in granule trafficking and maturation. We propose that this subcellular localisation of CAR-SIV contributes to the unique sensitivity of human beta cells to enteroviral infection.The online version of this article (10.1007/s00125-018-4704-1) contains peer-reviewed but unedited supplementary material, which is available to authorised users. Epidemiological and pathological studies have linked enteroviral infections with the development of type 1 diabetes mellitus –4. AdditIn order for enteroviruses to infect cells, they must bind to specific cell surface proteins that serve as vehicles to mediate their entry. Such binding is thought to alter the conformation of the viral capsid to facilitate the entry of viral RNA into the target cell. An endogenous cellular protein known as the Coxsackie and adenovirus receptor (CAR) is one such viral receptor. CAR is a transmembrane protein involved in homotypic cell adhesion and tight junctional integrity  but alsoCXADR gene, which comprises eight exons and yields a protein with an extracellular domain (ECD) linked by a single transmembrane region to a cytoplasmic tail. Differential splicing yields at least five different isoforms  is produced from a cryptic splice site within the seventh exon linked to exon 8, and is expressed apically [Interestingly, the two isoforms bearing a transmembrane domain vary only in the sequence of the final 26 (CAR-SIV) or 13 (CAR-TVV) amino acids, but are differentially localised within cells and may fulfil different functions [apically . In suppapically , 16 and apically , but onlapically , 18. ThuTo date, few studies have examined the expression and distribution of CAR isoforms in human beta cells , 19. Ylihttp://foulis.vub.ac.be/) and from the Network for Pancreatic Organ donors with Diabetes  were studied. Samples were from 21 non-diabetic individuals (age range 4–47 years) and ten individuals with type 1 diabetes  pancreatic sections from the Exeter Archival Diabetes Biobank  of CAR-SIV to examine the specificity of the CAR-CT antiserum ESM .The production of CAR in human islets and EndoC-βH1 cells and the specificity of the CAR-CT and CAR-RmcB antisera were assessed using western blotting ESM .The specificity of the CAR-CT and CAR-RmcB antisera was assessed using flow cytometry ESM .This was performed using a standard immunoperoxidase approach . Bright-https://imagej.nih.gov/ij/plugins/track/jacop2.html).To examine multiple antigens within the same section, FFPE samples were probed in a sequential manner with up to three different antibodies  islets were determined using quantitative (q)RT-PCR ESM .RNA sequencing (RNAseq) was performed using islets obtained from normoglycaemic human islet donors or EndoC-βH1 cells as previously described . Genes aWe used cryoimmune electron microscopy to assess and quantify the localisation of CAR-SIV, zinc transporter protein 8 (ZnT8), insulin and proinsulin in human pancreas samples as described previously  and in tThis was performed in EndoC-βH1 cells and human islets as described in ESM p value <0.05 was considered statistically significant.Immunofluorescence images of islets were selected randomly from stained pancreas sections. The Pearson correlation coefficient was used to estimate the co-localisation between proteins using the JACoP plugin from Image J. Values for the Pearson correlation coefficient ranged from 0 for no correlation, to 1 for a positive correlation. GraphPad Prism 5.04  was employed for all statistical analysis, and data are expressed as means ±SEM. Statistical significance was calculated using one-way ANOVA, and the Bonferroni multiple comparison test was used for multiple comparisons. A The availability of antisera directed against different regions of the CAR protein Fig. b enabledTwo further CAR antibodies, CAR-ECD  and RmcB , both of which recognise the ECD of human CAR, were also used. By deploying a panel of approaches , it was found that the CAR-ECD antiserum worked effectively in both western blotting and immunohistochemistry, whereas the RmcB clone was most suitable for immunofluorescence staining of fixed cells or flow cytometry. As noted above, both the CAR-ECD and RmcB antibodies recognise multiple isoforms of CAR including CAR-SIV, CAR-TVV and CAR4/7 ESM Fig. b,c.Use of these differentially specific antisera revealed that various isoforms of CAR are expressed among the exocrine and endocrine compartments of the human pancreas Fig. c,d. ImpoCXADR isoform expression , this did not hold in reverse  because little proinsulin escaped into mature secretory granules. Together, these findings suggest that CAR-SIV is present in both immature and mature insulin secretory granules in human beta cells.To confirm more directly that the CAR-SIV isoform localises to insulin secretory granules, immunogold labelling was performed on thin frozen sections using the post-embedding Tokuyasu method  with antQuantification of 841 CAR-SIV immunogold particles from 21 different micrographs, across 1291 different membrane intersections, revealed that CAR-SIV was most abundant on mature insulin secretory granules (79%). CAR-SIV was also observed in immature insulin granules (14.4%), but was rarely observed in beta cell nuclei 2.5%), mitochondria (1.0%) or endoplasmic reticulum (1.1%) or on the plasma membrane , CAR-SIV (10 nm gold particles) and insulin (5 nm gold particles) was performed ESM Fig. . This rePICK1 may play an important role in insulin granule trafficking and maturation , and theTo examine the co-localisation of PICK1, CAR-SIV and insulin in more detail, confocal microscopy coupled with Hyvolution software was employed to provide improved resolution. This confirmed the intimate association between PICK1 and the SIV isoform of CAR within insulin secretory granules Fig. c. PICK1 The present study reveals that the SIV isoform of CAR is expressed selectively by beta cells in the human pancreas. This finding was confirmed at both the RNA and protein levels. We also discovered that the subcellular localisation of CAR-SIV was atypical: the protein was found mainly within the cytoplasmic domain of beta cells rather than at the cell surface.We used both immunological and molecular biological approaches to investigate CAR expression in human islet cells, and the results were concordant. Thus, analysis of RNA extracted either from isolated human islets or laser capture microdissected islets revealed that two major isoforms of CAR, CAR-SIV and CAR-TVV, were present. Both of these contain a single transmembrane domain, implying that they could each be localised within defined, membrane-limited, compartments in the islet cells, as in other cell types . The resThe primary subcellular localisation of CAR in beta cells has not been extensively addressed in previous work , 20 but,High-resolution confocal microscopic analysis was used to examine in more detail the unexpected subcellular localisation of CAR-SIV in beta cells. This revealed a punctate, cytoplasmic immunolabelling pattern for CAR-SIV, consistent with its distribution in a distinct intracellular organelle compartment. Additional studies demonstrated that this immunolabelling profile correlated with that of insulin, as well as with two additional secretory granule proteins, ZnT8 and PC1/3, thereby implying a localisation within beta cell insulin secretory granules. Direct confirmation of this was provided by cryoimmune electron microscopy studies in which immunogold methods allowed the visualisation of CAR-SIV, principally in association with the dense-core granules characteristically found in beta cells. By contrast, no labelling was seen in the equivalent secretory granules found in adjacent exocrine cells, implying that CAR-SIV is not absolutely required for secretory granule biogenesis in all cell types.trans-Golgi network.Importantly, and consistent with results obtained in other cells , we alsoIf this model is correct, it has additional important consequences. In particular, the model predicts that the C-terminus of CAR-SIV must face the extragranular environment, since its PDZ-binding domain is located in this region and would only be available to interact with cytoplasmic PICK1 (or other cytoplasmic PDZ-binding proteins) in this orientation. As such, the putative ECD of CAR-SIV would then face the lumen of the granule, with the single transmembrane region serving to anchor the protein in this orientation in the limiting membrane surrounding the granule. Thus, the region of SIV required for the binding of enteroviruses would face the interior of the secretory granule during maturation. It follows from this that the ECD of CAR-SIV would become displayed on the external face of the plasma membrane following the fusion of the secretory granule and plasma membranes during exocytosis.These considerations are summarised in Fig. In support of our conclusions, Ylipaasto et al  demonstrIn summary, we show that human beta cells express the SIV isoform of CAR in the insulin granules. We propose that the biochemical properties of CAR-SIV that confer the physiological importance of this protein within beta cells may also represent an ‘Achilles heel’ by which the entry and replication of enteroviruses is facilitated.ESM(PDF 962 kb)


Studies aimed at improving the provision of evidence-based care (EBC) for the management of acute whiplash injuries have been largely successful. However, whether EBC is broadly provided and whether delivery of EBC varies based on risk of non-recovery, is uncertain. Receiving EBC should improve recovery, though this relationship has yet to be established. Further, mitigating the effect of EBC is the relationship with the practitioner, a phenomenon poorly understood in WAD. This study aimed to determine the proportion of individuals with whiplash, at differing baseline risk levels, receiving EBC. This study also aimed to determine whether receiving EBC and the therapeutic relationship were associated with recovery at 3 months post injury.Participants with acute whiplash were recruited from public hospital emergency departments, private physiotherapy practices, and State Insurance Regulatory Authority (SIRA) databases. Participants completed questionnaires at baseline  and 3-months  post injury. Primary health care providers (HCPs) treating these participants also completed questionnaires at 3-months. Recovery was defined as neck disability index ≤4/50 and global perceived effect of ≥4/5.Two-hundred and twenty-eight people with acute whiplash, and 53 primary care practitioners were recruited. The majority of the cohort reported receiving EBC, with correct application of the Canadian C-spine rule (74%), and provision of active treatments (e.g. 89% receiving advice) high. Non-recommended (passive) treatments were also received by a large proportion of the cohort (e.g. 50% receiving massage). The therapeutic relationship was associated with higher odds of recovery, which was potentially clinically significant . EBC was not significantly associated with recovery.Guideline-based knowledge and practice has largely been retained from previous implementation strategies. However, recommendations for routine risk identification and tailored management, and reduction in the provision of passive treatment have not. The therapeutic relationship was identified as one of several important predictors of recovery, suggesting that clinicians must develop rapport and understanding with their patients to improve the likelihood of recovery. Recovery following whiplash injury, if it is to occur, will occur for the most part within the first 3 months post injury . CurrentThe first key recommendation identified from the Australian whiplash guidelines was the appropriate application of the Canadian C-Spine rule. The Canadian C-Spine rule is a widely validated rule used to assist clinicians in determining whether an x-ray is required to confirm the diagnosis of a clinically important cervical injury . The appThe provision of active treatments, such as advice and neck exercises, and the reduction of passive treatments with little or no evidence supporting their use, was also a key recommendation. The relative efficacy of active versus passive treatments is well established, –16 and wThe final key recommendation from the Australian whiplash guidelines was a differential process of management based on risk of non-recovery. Individuals at risk of non-recovery should be identified early, using a recommended prognostic tool, to facilitate timely and appropriate multidisciplinary management. Recommended prognostic tools include measures of pain intensity using the visual analogue scale (VAS), or the numeric rating scale (NRS), and measures of neck pain-related disability using the neck disability index (NDI). Scores of > 5/10 on the NRS or VAS, and > 15/50 on the NDI are associated with poorer recovery . AdditioFollowing early identification of risk, the guidelines recommend differential management. For example, it is recommended that individuals at low-risk of non-recovery have minimal care, and individuals at high risk be referred to WAD specialists, including rehabilitation physicians, specialist physiotherapists, psychologists and occupational physicians . The proFinally the relevance of the provision of EBC may be important only if it relates to recovery. Given recovery following whiplash injury is poor, it is important to establish the relationship between provision of EBC and recovery. Mitigating the effect of EBC is how it is delivered, namely the relationship with the practitioner. The therapeutic relationship refers to the sense of trust, warmth and support between patient and clinician,  and is pThis study was a prospective, multi-centre inception cohort study of patients with acute WAD.participants with whiplash], and their primary HCPs. Whiplash is defined, according to the Quebeck Task Force, as an acceleration-deceleration mechanism of energy transfer to the cervical spine, commonly resulting in a variety of clinical manifestations (whiplash-associated disorders) [Participants included individuals with whiplash [sorders) . Particisorders)  were at sorders) .Primary HCPs were included if they were a primary care practitioner treating a participant with whiplash, were contactable, and provided informed consent.Participants with whiplash were recruited from public hospital emergency departments, private physiotherapy practices and State Insurance Regulatory Authority (SIRA) databases in NSW, Australia. The SIRA databases include the Personal Injury Register (PIR), where it is mandatory for insurance claims to be reported in NSW, and the Claims Advisory Service (CAS) database, which provides support for the assessment and resolution of motor vehicle accident compensation claims.Participants with whiplash were contacted by telephone by the research team. Eligibility was established and informed consent obtained. Participants with whiplash completed baseline and 3-month follow-up questionnaires. At 3 months, participants were asked to identify their primary HCP, defined as their main primary health care provider following injury. This study was approved by the Sydney Local Health District Ethics Committee; reference number HREC/13/CRGH/67.Baseline questionnaires assessed demographic information, pain and disability, risk status, psychological factors and general health. Demographic data captured included age, gender, weight, height, martial status, education level and country of birth.Pain was assessed at baseline using the NRS. The NRS is frequently used in healthcare settings, , 27 and Baseline risk status was assessed using a validated CPR . This CPPsychological factors assessed at baseline included post-traumatic stress symptoms, negative expectations of recovery, and depression, anxiety and stress. These constructs were chosen due to their association with non-recovery after whiplash . Post-trGeneral health questionnaires assessed at baseline included the Short Form-12  Health Survey and the European Quality of Life – 5 Dimensions – 3 levels (EQ5D3L) questionnaire.Participants with whiplash completed three questionnaires at follow-up, assessing treatment received, recovery, and the therapeutic relationship  to + 5/5 (completely recovered) [Recovery at 3 months was assessed using the NDI, and the Global Perceived Effect (GPE) scale. The GPE quantifies a patient’s covered) . Therapecovered) . Scores Primary HCPs completed similar questionnaires regarding treatment provided and therapeutic relationship. Practitioners were additionally asked to self-nominate the perceived risk status of their patient, as either low-, medium- or high-risk of non-recovery.The Whiplash Evidence-based Care tool (WAD-Evidence tool) was derived for use in the study, and based on previous questionnaires used to measure adherence to Australian whiplash guidelines [content and process for management. Content-based recommendations included the correct application of the Canadian C-Spine Rule and the provision of recommended activating treatments. These treatments include advice and exercises. Process-based recommendations referred to risk identification and referral decisions. Scores were assigned for compliance with recommendations, with higher scores assigned if the practice was compliant with Grade A/B recommendations [idelines , 9. It widelines . Recommendations , 40 and The primary outcome at 3-month follow-up was recovery measured by NDI and GPE. Recovery was defined as a score of 4 or more on the 11-point GPE, and a score of ≤4/50 on the NDI, based on consensus decision and previous definitions , 37, 41.Additional secondary outcomes included the proportion of participants with whiplash receiving EBC via the WAD-Evidence tool, and the therapeutic relationship, according to primary HCP report. Correct identification of risk level of participants with whiplash and the appropriate decision-making processed around referral on the part of the primary HCP were also measured.α = 0.05.A post hoc sample size calculation showed that the present design would provide 83% power to detect recovery at Statistical analyses were performed using SPSS v22 . BaselinA separate logistic regression model was derived for each outcome through a forward-stepwise process to assess the individual contribution of each predictor variable to the model.Both known  and potential  predictors of recovery were assessed.p <  0.05) associated with recovery, alone, were ranked in order of significance. Odds ratios with 95% confidence intervals, Nagelkerke R2 and p-values were reported. These predictors were then combined sequentially in a forward stepwise manner, retaining only those that continued to contribute significantly to the model. A final ‘reduced’ model, containing only those variables with significant contributions, was created for each outcome. The potential predictors of therapeutic relationship and WAD-Evidence tool score were then added to each model to assess their individual contribution to the model.Predictors significantly (y = therapeutic relationship + WAD-Evidence tool score + therapeutic relationship*WAD-Evidence tool score. The significance of the interaction term (therapeutic relationship*WAD-Evidence tool score) in this model was then assessed. Mixed effects models were not suitable for use in the case of these data.In order to determine whether the two predictor variables, in combination, had a different contribution to the model than they would in isolation, their interaction was assessed. A model was run in the form of A total of 228 participants with whiplash, and 53 primary HCPs, were recruited. Three-month data collection was completed for 160 participants, with 68 participants with whiplash lost to follow-up. Contact details were received for 123 primary HCPs, with 93 contactable and 53 consenting to participate  and presented to hospital more frequently post-injury (p = 0.040). A significantly greater number were single (p <  0.001), and not currently in paid employment (p <  0.001). They also had a higher mean body mass index (BMI)  were most frequently consulted (84%), followed by physiotherapists (60%), chiropractors (13%), massage therapists (6%), psychologists (3%) and osteopaths (3%).p = 0.018; Table According to participant self-report, the Canadian C-Spine Rule was applied correctly more frequently for individuals at high-risk than for individuals at low- and medium-risk  received recommended active treatments. A large proportion of individuals received treatments with no evidence for or against their use . Only 49% of individuals at high-risk received the appropriate number of treatments  low-risk: 8(6), medium-risk: 9(9), high-risk: 10(7); p = 0.931; Table WAD-Evidence tool scores ranged from − 10 to 135 and did not vary between risk groups (mean (SD) low risk: 59(17), medium-risk: 60(28), high-risk: 63(32); p <  0.001; Table p <  0.001; Table Self-reported disability due to neck pain significantly increased with risk status (mean NDI/50 (SD) low-risk: 5(7), medium-risk: 10(9), high-risk: 22(10), p = 0.169).The therapeutic relationship was high in all groups (mean (SD) low-risk: 38 (7), medium-risk: 42 (2), high-risk 39 (7); Table Details were received for 123 primary HCPs, of which 93 were contactable. Of those contactable, 53 completed the 3-month questionnaire. Reasons for difficulties in contacting and obtaining questionnaires from primary HCPs are shown in Fig. Primary HCPs reported that the Canadian C-spine rule was correctly applied to a large proportion of their patients, across all risk groups  Table . The EQ5p = 0.881).The first multivariate logistic regression model aimed to determine predictors of GPE. This model contained the CPR, SF-12 mental component summary (MCS) and SF-12 physical component summary (PhCS). The CPR was a good predictor of recovery within this cohort, with individuals at medium-risk of non-recovery 35% less likely to recover than those at low-risk. Individuals at high-risk of non-recovery were 80% less likely to recover than those at low-risk. The model correctly classified 75% of participants, and when therapeutic relationship was added, the model correctly classified 78% of participants. Therapeutic relationship was associated with higher odds of recovery of between 2 and 34% , representing a potentially clinically significant improvement. The WAD-Evidence tool score did not contribute significantly to the model  and NDI (p = 0.905) outcomes.The second model measuring recovery via NDI scores contained the CPR, EQ5D3L post injury, and OMPQ expectations of recovery. Again, the CPR was an important predictor of recovery, with individuals at medium-risk 53% less likely to recover than individuals at low-risk. Further, individuals at high-risk of non-recovery were 69% less likely to recover than individuals at low-risk. The model correctly classified 81% of participants. When therapeutic relationship was added to the model, it correctly classified 83% of participants. It was again associated with higher odds of recovery, between 11 and 62%, and potentially clinically significant  compared to other cohorts, which are typically 60–62%, , 43–46 tThe Canadian C-Spine rule was applied correctly for the majority (74%) of this cohort, with a very low rate (4%) of unnecessary imaging identified. This is consistent with findings from a multi-centre cluster randomised controlled trial,  where thProvision of the guideline-recommended active treatments by primary HCPs was excellent. This suggests that the implementation of messages around the provision of these treatments have been effective. Previous implementation strategies undertaken in Australia to ensure these messages were implemented included the provision of online  and faceA high proportion of this cohort received passive treatments that are not routinely recommended or have no evidence supporting their use, including massage (received by 50%) and manual therapy and manipulation (received by 33%). This recommendation was made on the basis of several trials highlighting a lack of evidence supporting the use of passive treatments including acupuncture,  manual tAlternate strategies to reduce the provision of passive treatments are warranted. In other populations, guideline adherence has improved with concurrent patient and clinician education. Targeted patient education strategies, including practice-based and mailed household educational material, has reduced unnecessary antibiotic prescription for bronchitis by 40%, when run concurrently with clinician education . AdditioOrganisation-targeted strategies may be more effective. In a single-site before-and-after study,  the CanaOrganization changes may also have an important role in improving practices related to process of care. Since their inception in 1999, the Australian whiplash guidelines have recommended care be provided according to risk of non-recovery. Specifically, individuals at lower risk should receive less intervention, and individuals at medium to high risk, more. We found no difference in the number of treatments received by individuals in different risk groups. The implementation of the validated CPR may be the first step in improving the process of care in WAD. Given the CPR was an accurate predictor of recovery in this cohort, its use in facilitating prognosis-matched management strategies by identifying risk of non-recovery should improve recovery, and is proposed in our recent trial protocol . MandatiIndividuals at high-risk of non-recovery should also receive referral to a specialist clinician with expertise in the management of WAD. Our data indicated that very few individuals are referred , and largely to traditional medical specialists such as rheumatologists and neurologists (49% of those referred). The lack of recommended referral practices observed in the present study could be explained by the poor ability of primary HCPs to identify individuals at high-risk of non-recovery, and poor awareness of those with expertise in whiplash . PreviouFinally, the therapeutic relationship was identified as an important predictor of recovery following WAD, yet higher scores on the WAD-evidence tool were not. The relationship between therapeutic relationship and improved outcomes identified in this cohort is consistent with previous studies in populations including low back pain, , 64 postThe majority of the literature suggests that patients possessing a shared sense of goals and trust with their therapist can utilise these emotions in their recovery , 68. SinOur WAD-Evidence tool was not associated with recovery, suggesting either that receiving evidence-based care is not associated with outcome, or that our tool was not sensitive.content of care. The issues identified with the provision of care related predominantly to the process of care, which may be more difficult to capture within an assessment tool.The known predictors of outcome in WAD are pain, disability and psychological factors , 17, as Messages related to the provision of guideline-recommended active treatments for acute WAD appear to have been successfully implemented. Therefore focus must shift to reducing the provision of passive treatments, and improving processes around the identification and appropriate management of at-risk individuals. Future work should investigate whether changes to CTP insurance funding for rehabilitation, interactive clinician education sessions, and the use of structured referral sheets disseminated within the Australian whiplash guidelines, may improve compliance.Additional file 1. Questionnaire tool used to assess provision of evidence-based care.


The pathophysiology of malaria-related anaemia is not fully understood although increased destruction of parasitized and nonparasitized erythrocytes, as well as inadequate erythropoiesis, has been proposed. Circulating antierythropoietin (anti-EPO) antibodies have also been implicated in malaria and malaria-related anaemia in mice. However, studies on this association have not been investigated in humans. This study therefore determined the prevalence of anti-EPO antibody production and assessed its association with malaria and malaria-related anaemia in humans. A total of 86 children aged 1-10 years , all residents of Duayaw Nkwanta, Ghana, were recruited for this case-control study. Venous blood was collected for thick and thin films for malaria microscopy, full blood count by automated haematology analyzer, and antierythropoietin antibody and erythropoietin estimation by sandwich ELISA method. Out of the 86 participants recruited, only 3 (3.5%) were positive for anti-EPO antibody; 2.3% of the case group; and 1.2% of the control group. There was no association between the cases and the controls in the production of anti-EPO antibodies. Erythropoietin concentration was significantly higher in malaria-related anaemic subjects (p=0.032). Antierythropoietin antibodies are not associated with malaria infection and malaria-related anaemia in humans. Erythropoietin concentration is associated with malaria-related anaemia. Plasmodium berghei, where they established that malaria infection was associated with anti-EPO antibody production in some strains of mice [Recent studies have also shown an association between antierythropoietin antibodies and malaria-related anaemia in murine models. A study by Tsubata et al. found that treating infected mice with exogenous antierythropoietin (anti-EPO) antibodies provides protection against malaria infection . Another of mice . These sThis study observed the expected high temperature among participants with malaria compared to the controls , consistVarious mechanisms of malarial anaemia have been investigated including increased RBC destruction through rupture of parasitized red blood cells (PRBCs) , phagocy Plasmodia sp. do not affect only red blood cells but also platelets as these cells originate from a common stem cell [α is elevated in malaria due to inflammation [α has also been reported to inhibit EPO production and EPO-induced erythroid progenitor cell proliferation [α in this study, we could not conclude about the impact of TNF- α on EPO concentration; thus this association requires further studies.Furthermore, as an obligate intracellular parasite,tem cell . A signitem cell , Patel etem cell , and Shatem cell . Additioammation , 41. On feration . This co Plasmodium berghi Anka was the malaria parasite examined in the study by Helegbe et al. in mice; however, the predominant Plasmodium species responsible for malaria infection in humans in Ghana is Plasmodium falciparum.The prevalence of anti-EPO antibodies production among the entire study population was 3.5%; 2.3% in the case group; and 1.2% in the control group . Chi-squAdditionally, during falciparum malaria infection, there is loss of both parasitized and nonparasitized erythrocytes through parasite maturation and antibody sensitization, respectively, leading to anaemia. Erythropoietin (EPO), on the other hand, is crucial to sustain the proliferation and differentiation of erythroid cells. EPO is bound by circulating erythrocytes and studies have shown that reduction in circulating RBCs results in a relatively high concentration of unbound EPO which in turn stimulates erythrocyte production in the bone marrow , 44. In This study is however limited by the small sample size used which may have encumbered the full exploration of the association between anti-EPO and malaria. Future studies may focus on a larger sample size.Antierythropoietin antibodies are not associated with malaria infection and malaria-related anaemia in humans. Erythropoietin concentration is associated with malaria-related anaemia.