Uric acid (UA) is synthesized mainly in the liver, intestines, and vascular endothelium as the end product of an exogenous purine from food and endogenously from damaged, dying, and dead cells. The kidney plays a dominant role in UA excretion, and the kidney excretes approximately 70% of daily produced UA; the remaining 30% of UA is excreted from the intestine. When UA production exceeds UA excretion, hyperuricemia occurs. Hyperuricemia is significantly associated with the development and severity of the metabolic syndrome. The increased urate transporter 1 (URAT1) and glucose transporter 9 (GLUT9) expression, and glycolytic disturbances due to insulin resistance may be associated with the development of hyperuricemia in metabolic syndrome. Hyperuricemia was previously thought to be simply the cause of gout and gouty arthritis. Further, the hyperuricemia observed in patients with renal diseases was considered to be caused by UA underexcretion due to renal failure, and was not considered as an aggressive treatment target. The evidences obtained by basic science suggests a pathogenic role of hyperuricemia in the development of chronic kidney disease (CKD) and cardiovascular diseases (CVD), by inducing inflammation, endothelial dysfunction, proliferation of vascular smooth muscle cells, and activation of the renin-angiotensin system. Further, clinical evidences suggest that hyperuricemia is associated with the development of CVD and CKD. Further, accumulated data suggested that the UA-lowering treatments slower the progression of such diseases. Uric acid (UA) is synthesized mainly in the liver, intestines, and vascular endothelium as the end product of an exogenous purine from food 100–200 mg/day), and endogenously (500–600 mg/day) from damaged, dying, and dead cells, whereby nucleic acids, adenine and guanine, are degraded into UA 00 mg/day. UsuallyHyperuricemia is induced by UA over-production due to acquired factors such as high purine diet, fructose ingestion, alcohol intake, myeloproliferative disorders, and also rare genetic causes such as hypoxanthine-guanine phosphor-ribosyl-transferase (HPRT) deficiency and phosphor-ribosyl-pyrophosphate (PPRP) synthetase (PRS) hyperactivity . Renal eHyperuricemia has been previously classified into the “UA overproduction type”, “UA underexcretion type” and “combined type”, and approximately 10%, 60%, and 30% of hyperuricemic patients have been classified into each type, respectively ,17. AbcgHyperuricemia was previously thought to be simply the cause of gout and gouty arthritis. Further, the hyperuricemia observed in patients with renal diseases was considered to be caused by UA underexcretion due to renal failure since the kidney excretes approximately 70% of daily produced UA . TherefoHyperuricemia is significantly associated with the development and severity of metabolic syndrome. Meta-analysis showed that higher serum UA levels led to an increased risk of metabolic syndrome regardless of the study characteristics, and were consistent with a linear dose-response relationship . Choi HKp < 0.001), and insulin resistance was also inversely related to urinary UA clearance , and urinary UA clearance was inversely related to serum UA concentration   [p < 0.001]  . The met< 0.001] . In this< 0.001) . No signn = 2221 patients) did not demonstrate a significant difference in non-Anti-Platelet Trialists’ Collaboration CV events  or all-cause mortality  [p = 0.01) in patients allocated to allopurinol [p = 0.05) and cardiovascular mortality  were significantly lower in patients receiving allopurinol during CABG compared to patients in the control group [p = 0.50) and , respectively. Similarly, the use of allopurinol did not reduce the odds of recurrent acute cardiovascular syndrome events at two years .Zhang, T, et al. performed the meta-analysis to determine if ULT in gout can reduce CV outcomes. Comparing any ULT to placebo  . There wopurinol . In seveopurinol . In the ol group . By contp = 0.96) compared to the control. The RR of CV-related death for febuxostat was 1.29 . The meta-analysis was done to determine the association of 2 ULT commonly used in clinical practice  on MACE [p < 0.0001) and stroke  [p = 0.91), cardiovascular related mortality  and all-cause mortality . No significant differences in cardiovascular related mortality and all-cause mortality were observed across any subgroup. The cardiovascular safety of febuxostat compared to allopurinol for the treatment of gout remains equivocal.Febuxostat is approved for the management of hyperuricemia in patients with gout. In November 2017 the FDA released a warning alert on a possible link between febuxostat and CVD reported in a single clinical trial . A meta- on MACE . No sign on MACE . The rec= 0.009) . However2; 95%CI, 3.50 to 10.15) and high methodological quality (blind design)  [The meta-analyses which studied whether ULT reduces the progression of CKD showed insufficient data on incidence of end-stage renal disease (ESRD) for analysis and the heterogeneity across included studies, suggesting that adequately powered RCTs are needed to establish whether ULT has beneficial renal effects ,88. Anotto 4.99) . No defito 4.99) .Such meta-analyses cannot prove renal protective effect of ULT due to the main limitation by the heterogeneity across included studies, and did not include trials using non-purine XO inhibitors (febuxostat and topiroxostat) and uricosuric agents. We show the renal outcome by ULT in The UA lowering mechanism of non-UA-lowering drugs were shown in Increased levels of serum UA in postmenopausal women are thought to be caused by a change in renal urate elimination associated with the loss of female hormones. In the study to evaluate the regulation of renal urate transporter expression by female hormones using ovariectomized mice with or without hormone replacement, estradiol suppressed the protein levels of URAT1 and GLUT9 . The rolLosartan, an angiotensin II receptor antagonist, inhibits URAT1 and GLUT9 . In the p < 0.001) [The effects of fenofibrate, an anti-lipidemic drug, on UA metabolism and URAT1 was investigated in healthy male subjects . Fenofib< 0.001) . Another< 0.001) .Our previous study showed that SGLT2i, oral anti-diabetic drug, significantly reduced serum UA levels . In the SGLT2i improve insulin resistance by reducing body weight . The incHyperuricemia is significantly associated with the development and severity of metabolic syndrome. Increased URAT1 and GLUT9 expression and glycolytic disturbances due to insulin resistance may be associated with the development of hyperuricemia in the metabolic syndrome. Emerging evidence suggests a pathogenic role of hyperuricemia in the development of hypertension, CKD, and CVD, by inducing inflammation, endothelial dysfunction, proliferation of vascular smooth muscle cells, and activation of the RAS. Highly evidenced studies show a significant association of hyperuricemia to the development of CKD and CVD. Beneficial effects of XO inhibitors, especially, allopurinol, for CVD and CKD have been reported. Further studies should be performed to elucidate the effects of non-purine XO inhibitors (febuxostat and topiroxostat) and uricosuric agents on the development and progression of CVD and CKD. Estrogen, losartan, fenofibrate and SGLT2i reduce serum UA by modulating urate transporters and XO.


This article addresses the Human T-lymphotropic virus (HTLV). This subject comprises the Clinical Protocol and Therapeutic Guidelines for Comprehensive Care for People with Sexually Transmitted Infections, published by the Brazilian Ministry of Health. HTLV-1/2 infection is a public health problem globally, and Brazil has the largest number of individuals living with the virus. HTLV-1 causes several clinical manifestations of neoplasm (adult T-cell leukemia/lymphoma) and inflammatory nature, such as HTLV-1-associated myelopathy and other manifestations such as uveitis, arthritis, and infective dermatitis. These pathologies have high morbidity and mortality and negatively impact the quality of life of infected individuals. This review includes relevant information for health authorities professionals regarding viral transmission, diagnosis, treatment, and monitoring of individuals living with HTLV-1 and 2 in Brazil. HTLV-1/2 transmission can occur through blood transfusion and derivatives, injectable drug use, organ transplantation, unprotected sexual intercourse, and vertical transmission. This article addresses Human T-lymphotropic virus (HTLV) infection. This subject comprises the Clinical Protocol and Therapeutic Guidelines (PCDT) for Comprehensive Care for People with Sexually Transmitted Infections (STI), published by the Health Surveillance Department of the Brazilian Ministry of Health. To elaborate the PCDT, selection and analysis of the evidence available in the literature were performed, and a panel of specialists discussed it. The document was approved by the National Committee for the Incorporation of Technologies in the Brazilian National Health System (Conitec)-Retroviridae family, genus Deltaretrovirus---HTLV-1 was described in patients with adult T-cell leukemia/lymphoma and, like HTLV-2-,HTLV-1/2 infection results from the transmission of infected lymphocytes, present in body fluids , by transfusion of blood and derivatives, intravenous drug use, organ transplantation, unprotected sexual intercourse, and vertical transmission. Vertical transmission can occur by the placental route, during birth, and mainly by breastfeeding,,-,,-Sexual contact is an important route of HTLV-1 and HTLV-2 dissemination in urban, rural, and indigenous areas,,,,,,HTLV-1 and HTLV-2 are distributed worldwide,The seroepidemiological studies for HTLV-1/2 are based on the detection of specific antibodies. It is important to emphasize that few population studies were conducted adequately. Therefore, a significant part of the epidemiological information about HTLV-1/2 derived from old studies, which often do not sufficiently define incidence and prevalence rates, shows conflicting results and does not allow the definition of precise prevention and control measures,-,HTLV-2, considered an ancestral infection, is apparently well adapted to humans, with rare clinical manifestations,-Retroviruses integrate with the nucleic acid in the infected cell and establish a viral persistence, leading to the virus maintenance and the different outcomes of the infection. HTLV-1 is associated with an aggressive malignant disease, adult T-cell leukemia/lymphoma (ATL)-,,--,,,,,,,,HTLV-1 infection shows a great variety of interactions with the human host and important clinical manifestations have been recognized in the eye,HAM occurs in about 4% of HTLV carriers, although clinical manifestations may affect more than 10% of them,--Proviral load levels correlate with the progression of the disease, especially with muscle weakness. Although the magnitude of the proviral load in peripheral blood is associated with HAM, it is not the sole diagnostic or prognostic factor of the pathology,,The neoplasm of peripheral T-cells caused by HTLV-1 presents itself with leukocytosis, characterized by the presence of abnormal lymphocytes (flower cells) and, clinically, by lymphadenopathies, skin lesions, dysfunction of multiple organs resulting from the invasion of the neoplastic cells, in addition to the presence of opportunistic infections. Elevated levels of the enzyme lactate dehydrogenase and hypercalcemia are characteristic. In Japan, there are over one million carriers and the incidence of ATL varies from 0.6 to 0.7 per 1000 persons/year,-Four clinical forms of ATL are recognizedIn addition to the clinical manifestations classically associated with HTLV-1 in the skin, such as infective dermatitis and the cutaneous manifestations of ATL, other dermatological affections attributed to the infection have been described as serious forms of scabies Streptococcus beta-hemoliticus and Staphylococcus aureus. According to a case series study, almost half of the individuals who had long-term follow-up were also diagnosed with HAMAt first, infective dermatitis was described in Jamaican children infected by HTLV-1Dermatological alterations in ATL vary in presentation  and depend on the disease stage; nodulations are more frequent in severe forms, especially in the acute, lymphomatous, or cutaneous primary tumoral formIn Japan, uveitis was first reported in 1989HTLV-infected individuals may present some coinfections, more frequently than the general population, either by sharing infection routes or as a consequence of the immunological alterations induced by the infection itself. Moreover, HTLV can alter the natural course of some coinfections.+ cells count caused by HTLV-1. Coinfected individuals treated with antiretroviral therapy and with HIV-1 viral suppression present similar survival time to those monoinfected under the same conditions; however, in those with a detectable viral load, the survival of coinfected individuals is significantly lowerIn HIV coinfection, for example, the evidence suggests a neutral or even protective role for those coinfected by HTLV-2,Regarding coinfection with hepatitis C virus (HCV), existing data are conflicting: while some studies show an increase in HCV viremia and a lower probability of spontaneous clearance of the infectionStrongyloides stercoralis coinfection suffer a negative impact in the course of both infections, becoming more susceptible to more severe forms of strongyloidiasis, therapeutic resistance, in addition to presenting a higher HTLV-1 proviral load and a higher risk of HTLV-1 vertical transmission-Individuals with HTLV-1 and Mycobacterium tuberculosis-Individuals with HTLV-1 present a higher risk of infection by ,-In Brazil, routine testing for HTLV-1/2 in blood and organ donors has been performed since 1993 and 2009, respectively,-The screening tests are used for detecting antibodies against HTLV-1/2 in plasma or serum. The laboratory techniques for performing these tests include (i) immunoenzymatic reaction, (ii) chemiluminescence, and (iii) particle agglutinationThe confirmatory tests identify antibodies against different HTLV-1 and HTLV-2 antigens or amplify and identify proviral genetic material, usually in peripheral blood lymphocytes. Confirmatory and viral typing tests are (i) Wb, (ii) line immunoassay (LIA), and (iii) PCR-,,,,,,-,Usually, Wb and LIA are sufficient for diagnosis; however, in some cases, undetermined or undefined results may occur regarding the type of HTLV,-,-At the time of this publication, a molecular test for HTLV-1/2 is not commercially available. The tests used are in-house, requiring prior validationThere is evidence that the duration of the immunological window period in HTLV-1/2 infection for antibody detection varies from 16 to 39 days after organ transplantation, and for the proviral genetic material, from 16 to 23 days after infection,,,The therapy for HTLV-1 infection consists of interventions directed to the complications resulting from the diseaseInfected people must be accompanied in the specialized service to receive psychological support, with particular attention to the early diagnosis of clinical manifestations associated with the infection.Despite being described some decades ago, HTLV infection remains relatively unknown to the general population and health professionals. In the services that assist the infected individuals, the approach should focus not only on the aspects of the risk of becoming sick,After a positive diagnosis for HTLV-1/2 infection, the sexual partners should be invited to undergo serological screening, and those with positive tests must be referred for counseling and appropriate follow-up. Such counseling should include information about the chronicity of the infection and the relevance of long-term clinical follow-upIn HIV and other STI specialized clinical centres, it is important to include HTLV screening in the routine of care. HTLV-infected individual must be oriented about the risk of sexual transmission, serodiscordant sexual partners, and condom use - which may be interrupted during the fertile period when there is a firm decision to become pregnant and following medical counselling and recommendationIn Brazil, given the scarcity of material available for health professionals and the general population, several initiatives have been developed by academic groups and non-governmental organizations to disseminate information about HTLV-1/2. Among the organizations and initiatives with this purpose, the following should be highlighted: the Research Support Center on Retroviruses (NAP-Retroviruses) of the University of São Paulo; the Hemominas Foundation Journals on HTLV infection; the HTLVida Association; and the Vitamóre Group - Association of HTLV Carriers.-The lack of a national register system impairs the identification of the actual scenario of the infection in the country and, therefore, the implementation of specific public health policies. It is essential to highlight that case notification is one of the pillars of confrontation and research about HTLV-1 in countries like Japan, England, Spain, and Martinique island,-,,,,In Brazil, HTLV-1/2 prevalence in pregnant women can reach 1% in certain regions of the country 159,,-Since breastfeeding is the main mother to child transmission route of HTLV-1/2,,-,-The vertical and sexual transmission routes are essential for HTLV maintenance in epidemiologically closed or semi-closed communities, as it occurs with HTLV-2c, which is prevalent among indigenous people residing in the Brazilian Amazon and urban areasAlthough HTLV infection is neglected, Brazil has produced several initiatives directed towards the prevention of HTLV-1 infection and disease. The complications with relevant clinical consequences, such as HTLV-1 associated myelopathy and T-cell leukemia/lymphoma, can be minimized with access to services offered by the SUS. The low complexity cases can be assisted at the health centers and, when necessary, forwarded to the specialized centers for treatment, rehabilitation, and social support. Despite the severe consequences that the infection can have on people's lives, its control still represents a public health challenge. National epidemiological studies, development and validation of diagnostic tests, and elaboration of clinical protocols with new therapeutic options can define public policies and specific actions towards the approach, prevention, control, and adequate treatment of HTLV-1/2 infection in Brazil.


Strongyloides stercoralis. Strongyloides stercoralis infection has been reported to be impacted by co-infection with HTLV-1. Disseminated strongyloidiasis and hyperinfection syndrome, which are commonly fatal, are observed in HTLV-1 co-infected patients. Reduced efficacy of anti-strongyloidiasis treatment in HTLV-1 carriers has been reported. The aim of this meta-analysis and systematic review is to better understand the association between HTLV-1 and S. stercoralis infection.The distribution of human T cell lymphotropic virus type 1 (HTLV-1) overlaps with that of S. stercoralis, those evaluating the frequency of mild or severe strongyloidiasis, and treatment response in people living with and without HTLV-1 infection were included. Data were extracted and odds ratios were calculated. Random-effect meta-analysis was used to assess the pooled OR and 95% confidence intervals.PubMed, Embase, MEDLINE, Global Health, Healthcare Management Information Consortium databases were searched. Studies regarding the prevalence of S. stercoralis and HTLV-1. The odds of S. stercoralis infection were higher in HTLV-1 carriers when compared with HTLV-1 seronegative subjects (OR 3.2 95%CI 1.7–6.2). A strong association was found between severe strongyloidiasis and HTLV-1 infection . Co-infection with HTLV-1 was associated with a higher rate of strongyloidiasis treatment failure .Fourteen studies were included after full-text reviewing of which seven described the prevalence of Strongyloides stercoralis infection is more prevalent in people living with HTLV-1. Co-infected patients are more likely to develop severe presentation and to fail treatment. Screening for HTLV-1 and Strongyloides sp. should be routine when either is diagnosed. Deltaretrovirus genus in the Retroviridae family (Human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus that belongs to the e family . HTLV-1 Asymptomatic infection can progress to severe, or even fatal diseases. Adult T-cell leukemia/lymphoma (ATL/ATLL) with a median survival <1 year, develops in 5% HTLV-1 infected patients , 4. HTLVStrongyloides stercoralis has two life cycles, a free-living cycle in soil and a parasitic cycle in the host (mainly human). In the free-living cycle, eggs are hatched as rhabditiform larvae, transformed into infectious filariform larvae, which penetrate the skin and migrate to the intestine where they mature into adult females and produce eggs parthenogenetically. The eggs hatch as non-infective rhabditiform larvae and are excreted in feces. In the soil or feces rhabditiform larvae are fertilized to filariform larvae. However, autoinfection also occurs in which the rhabditiform larvae fertilize into filariform larvae in the large bowel, migrate to the lungs and return to the bowel through swallowed sputum, allowing S. stercoralis to persist  and disseminated strongyloidiasis (DS) are severe clinical presentations with 85–100% mortality –17. HS iHere, we conducted a systematic review and meta-analysis of the association between HTLV-1 and strongyloidiasis infections, the impact of HTLV-1 co-infection on strongyloidiasis severity, and on anti-helminth treatment efficacy.Strongyloides stercoralis,” “S. stercoralis,” “Strongyloides,” “Strongyloidiasis,” “Human T-lymphotropic virus,” “Human T-cell Leukemia Virus,” “Human T-cell Leukemia Virus,” “HTLV-1,” and “HTLV”. Studies were included if they focused on at least one of these areas: (1) the prevalence of Strongyloides stercoralis and HTLV-1 in the population; (2) the severity of strongyloidiasis in people living with HTLV-1 and HTLV-1 uninfected individuals; (3) the efficacy of anti-helminthic treatment in S. stercoralis-infected patients co-infected with HTLV-1 or without HTLV-1. Duplicate studies, review articles and articles: that were not written in English, Portuguese, Spanish or Chinese; where full text was not available; or had not been peer reviewed, were excluded. Studies without a control group and those without confirmatory test for HTLV-1 infection , those focusing only on changes in biomarkers, for example, eosinophils count and IgE levels, and HTLV-1 proviral load, were also excluded. Severe strongyloidiasis was defined as hyperinfection, dissemination and the identification of S. stercoralis-larvae in specimen other than stool sample as well as the definition used by the authors.This systematic review and meta-analysis were performed following the guideline of PRISMA . PubMed and OVID  were used as the electronic databases . Keywords used in the search were “S. stercoralis and HTLV-1 diagnosis. To analyze the clinical outcome, the number of patients with severe strongyloidiasis and those without in case and control groups was extracted. For studies relating to efficacy of treatment, the number of patients with evidence of treatment failure, the dosages and the types of drugs used were collected.For each paper that met the selection criteria, the following data were extracted when available: year of publication, locations, regions and country of the study, total number of participants, number of positive subjects (infected by HTLV-1 and/or Strongyloides), methods for Strongyloides stercoralis infection among the HTLV-1 positive and HTLV-1 negative populations and 95% confidence intervals (CI). The OR of S. stercoralis infection among people living with HTLV-1 (PLHTLV) when different diagnostic methods were used was calculated in the subgroup analysis . A forest plot for this subgroup analysis was plotted. Odds ratio (OR) and 95% CI were calculated for the effect of HTLV-1 on S. stercoralis infection, i.e., severity and treatment results. Heterogeneity in all meta-analyses was calculated. The statistical significance level considered was p < 0.05.Meta-analysis was performed using software Revman5. A random-effect model was used to calculate the odds ratio of One thousand hundred and seventy studies had been retrieved according to the keywords. Three hundred and twenty-four studies were full-text available articles and written in English, Portuguese, Spanish or Chinese. Eighty-five papers were screened using full-text and 14 studies that met the inclusion criteria were used in the meta-analysis .Strongyloides stercoralis co-infection: two from Australia  vs. 0/29 (0%), p = 0.036]  vs. HTLV-1 seronegative: 18/154 (11.7%),  = 0.79] . In a re= 0.063] . In Guad= 0.063] .Strongyloides and HTLV-1 infection  were co-infected with HTLV-1. In contrast, 4 of 45 (8.9%) individuals with S. stercoralis-seropositive but larva negative were HTLV-1 positive and only 9 out of 145 (6.2%) participants not infected by S. stercoralis  were HTLV-1 positive. This study gave evidence that being a S. stercoralis carrier was highly associated with the presence of HTLV-1  compared with 16.6% in HTLV-1 uninfected individuals , with a pooled odds ratio 3.22 [95% Confidence interval (95%CI) = 1.66–6.25]. However, large heterogeneity was observed between different studies . The association was stronger when analyzing patients diagnosed by stool methods [OR (95%CI) = 8.43 (2.4–29.2)] than those diagnosed with serological examination [OR (95%CI) = 2.12 (1.2–3.8)]. A high heterogeneity was observed within subgroups and among the studies that used serology  . Strongyserology .Three studies reporting the association between the severity of strongyloidiasis, and HTLV-1 co-infection met the inclusion criteria  25–27).–27.25–27p < 0.001) and to age-matched healthy individuals with negative stool samples for Ss  . On the other hand, only 4 of 42 (9.5%) patients with asymptomatic or mild strongyloidiasis had concurrent HTLV-1 . This gives the odds of severe strongyloidiasis to be 214 times 10.7–4,256) higher among PLHTLV compared to those without HTLV-1 infection, although the 95% CI was broad . In anot1, 4.7%) . The gro1, 4.7%) . In bothpecimen” , 27. Sevpecimen”  (25). In6 higher Furtado et al.  reportedIn total, 33/68 patients with HTLV-1 and Strongyloids had severe strongyloidiasis, while only 3/157 individuals without HTLV-1 co-infection had severe presentation of Ss infection, resulting in a OR = 59.9 (18.1–198).S. stercoralis treatment response, three studies from Japan and one study from Peru found association between treatment failure and HTLV-1 infection (p < 0.05) (p < 0.02). Even when treated with Ivermectin , which was considered the most effective drug among the three analyzed in this study, patients co-infected with HTLV-1 failed treatment more frequently than those seronegative for HTLV-1   anthelminthic effect of 110 μg/kg Ivermectin in HTLV-1 co-infected patients was 50% (28/56) and significantly increased to 90% , when the dosage increased to 200 μg/kg (When considering the impact of HTLV-1 on anti-nfection  28–31).–31.S. stnfection . The poonfection . Most stnfection . The hig < 0.05) . Respons< 0.002] . Zaha etI2 = 60.6%, p = 0.05)  and for those treated with Ivermectin 110 μg/kg  was 13.07 (95% CI 5.36–31.87). In total the OR of treatment failure was 5.05 (95% CI = 2.5–10.1). The heterogeneity between subgroups was large and reaching statistical significance ( = 0.05) .Strongyloides and HTLV-1 infection  amongst HTLV-1 infected persons. This extends the earlier observations of Schär et al.  is higher than that determined by stool examinations (10%). However, the odds for being HTLV-1 seropositive are higher when Ss is detected in stool (OR 8.43), compared to those positive for S. stercoralis by serology (OR 2.12). This suggests that persistent parasite excretion contributes to these associated prevalence rates, rather than incidence of infection. However, it remains possible that patients infected with HTLV-1 are at higher risk of becoming infected with S. stercoralis, although this may be due to co-factors such as socio-economic and environmental characteristics. The heterogeneity across the studies that used serological tests was large and significant , while studies using stool examinations were more homogenous . Although the findings are robust, the number of studies remain limited and further investigation from a wider range of countries, using robust diagnostics test and an adequate number of participants are required to better understand the contribution of each (risk of infection or parasite persistence) to the overall association.In total, 14 studies from both r et al.  . The number of manuscripts that matched the inclusion criteria is small and were restricted to two South American countries (Peru and Brazil). However, there are enough biological data to support this finding. Experimental data suggest that HTLV-1, unlike HIV, activates, rather than kills, CD4+ T cells which upregulates Interleukin (IL)-2/IL-2 receptor and subsequently induce Th1 responses  = 0.23 vs. HTLV-1 negative (n = 827) = 0.11, p < 0.0001]; Adjusted negative binominal regression coefficient = 0.563, p = 0.005). This epidemiological data also supports the findings of our study.In addition to the evidences showing plausible immunological basis underlying the association between HTLV-1 infection and increased disease severity in Ss co-infected patients, Einsiedel et al.  observedS. stercoralis (Another concern that needs attention is that the impact of this co-infection is not restricted to unfavorable outcome of Ss, but it may also negatively impact the outcome of HTLV-1 infection. A number of studies reported higher HTLV-1 proviral load among co-infected individuals, a known risk factor for the development of HTLV-1 associated disease , 38, 39.rcoralis , 40. Higrcoralis .S. stercoralis infection in the outcome of HTLV-1 infection and the interplay between Ss and HTLV-1 in the pathogenesis of both infections was recently reviewed. Nevertheless, the authors highlighted that in the absence of suitable animal models, the mechanisms in disrupting host's immune system between HTLV-1 and S. stercoralis is poorly studied (Altogether, these findings point to a negative impact of  studied .S. stercoralis drugs in patients with or without HTLV-1 infection was also assessed. Efficacy varied by treatment with some being very poor and perhaps now obsolete. Nevertheless, regardless of treatment type, HTLV-1 co-infected patients had higher odds of having failed antiparasitic therapy than those without HTLV-1. The effective of treatments were determined by stool examinations with a post-treatment follow-up duration ranging from 3 to 12 months, with a PCR test used in a proportion of patients in one study. It is important to undertaken proper follow-up of patients under/post-treatment and to perform multiple samplings to avoid false-negative stool examination results caused by the intermittent excretion of larvae (S. stercoralis autoinfection cycle, which takes about 3 or 4 weeks for eggs fertilizing to larvae and these to settle into the small bowel (S. stercoralis, especially in immunocompromised patients (Strongyloides treatment is essential. Also, prompt treatment of strongyloidiasis in patients with HTLV-1 is important, as it resulted in increased TNFα levels and decreased the risk of recurrence (S. stercoralis. Whatsoever, as discussed above, there is not such a “golden standard” of diagnosis of S. stercoralis, which poses difficulties for routine diagnosis. As both S. stercoralis and HTLV-1 may cause chronic and asymptomatic infection, active surveillance on those patients who are diagnosed with these pathogens is important. In addition, co-infection cases may also occur in non-prevalent areas due to influx of immigrants or prior travel to endemic countries.Treatment efficacy of different anti-f larvae , 44. Allpatients . Hoces ecurrence . This shStrongyloides and have higher risk of developing severe strongyloidiasis and anthelmintic treatment failure. Patients who have been infected with S. stercoralis, especially those who had traveled to or live in an endemic area, should be advised to be tested for HTLV-1. HTLV-1 patients who live in a S. stercoralis endemic area should be tested for S. stercoralis, especially before starting treatment for HTLV-1 associated diseases, in order to prevent the development of hyperinfection and disseminated strongyloidiasis. All patients who had developed a HS or DS should be screened for HTLV-1 as well as those who failed treatment to strongyloidiasis. Increasing awareness about the impact of this coinfection among general population and healthcare workers is needed, as well as further studies about the impact of HTLV-1 in other co-infections.People living with HTLV-1 have a higher risk of being infected by The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.LY conducted the literature search, undertook the analyses, and wrote the first draft. CR and GPT designed and supervised the work and edited the final version. All authors contributed to the article and approved the submitted version.GPT was supported by Imperial NIHR Biomedical Research Center.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The reconstruction of critical size bone defects is still clinically challenging. Even though the transplantation of autologous bone is used as gold standard, this therapy is accompanied by donor site morbidities as well as tissue limitations. The alternatively used allografts, which are devitalized due to thermal, chemical or physical processing, often lose their matrix integrity and have diminished biomechanical properties. High Hydrostatic Pressure (HHP) may represent a gentle alternative to already existing methods since HHP treated human osteoblasts undergo cell death and HHP treated bone cylinders maintain their mechanical properties. The aim of this study was to determine the biological effects caused by HHP treatment regarding protein/matrix integrity and type of cell death in trabecular bone cylinders. Therefore, different pressure protocols  and end point analysis such as quantification of DNA-fragmentation, gene expression, SDS-PAGE, FESEM analysis and histological staining were performed. While both protein and matrix integrity was preserved, molecular biological methods showed an apoptotic differentiation of cell death for lower pressures and shorter applications (250 MPa for 10 and 20 min) and necrotic differentiation for higher pressures and longer applications (300 MPa for 30 min). This study serves as a basis for further investigation as it shows that HHP successfully devitalizes trabecular bone cylinders. Bone is one of the few human tissues which can regenerate and repair itself . HoweverA gentle alternative to already existing processing methods could be the treatment of bone specimens with high hydrostatic pressure (HHP). Several studies have shown that HHP is able to devitalize cells and various tissues . An inhiThe purpose of this experimental study was to characterize the biological effects of HHP on human trabecular bone in terms of cell death and protein integrity. On basis of preliminary work, pressure applications of 250 and 300 MPa were chosen. In addition to a treatment period of 10 min, pressures were also applied for 20 and 30 min as a longer application could be necessary for complete devitalization due to the three-dimensional tissue structure of bone blocks. DNA fragmentation caused by HHP was analysed by gel electrophoresis. Furthermore, the occurrence of apoptosis-specific genes was determined at gene expression level. In addition, an SDS-PAGE was used to examine the protein integrity. Histological analyses were performed to assess whether HHP causes a homogeneous distribution of apoptosis in the bone blocks. Finally, field emission scanning electron microscopy (FESEM) was used to estimate possible superficial damage to the bone matrix after HHP-treatment.An overview of the methods used to answer the present research question is shown in 3 using a diamond coated bone saw . For High Hydrostatic Pressure (HHP) -treatment, bone blocks were then transferred into 2 ml CryoTubes  filled with Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% fetal calf serum , 1% amphotericin B, 1% penicillin/streptomycin and 1% HEPES buffer . The samples were treated with different pressure protocols  at a constant temperature of 30°C. The control groups were prepared in the same way as the HHP treated groups but received no HHP treatment. During HHP application, untreated controls were incubated at 30°C. Afterwards, samples were incubated for 0, 1, 2 and 3 h at standard culture conditions . These incubation periods after HHP application were chosen to track time courses of apoptotic signaling cascades, since previous studies showed that DNA fragmentation could only be detected 2 h after apoptosis inducing treatments   or with fixation buffer (FESEM) .Trabecular bone specimens were harvested from femoral heads of patients undergoing a total hip joint replacement. Before surgery, patient consent and ethical approval from the ethics committee of the University Rostock, Germany were obtained . Specimens were stored at −20°C until further preparation. Prior to sample manufacturing, femoral heads were rinsed with sterile phosphate-buffered saline (PBS) , supplemented with 1% penicillin/streptomycin . Afterwards, the cancellous parts of the tissues were sawed into bone blocks of a size of 125 mmeatments . After t® 200 Pro microplate reader and NanoQuant™ Plate  with TE-buffer as blank.Frozen bone specimens were thawed at 37°C using a water bath. Afterwards the blocks were digested overnight, using 1 ml collagenase A  per bone block. Non-digestible components were removed and the suspension was centrifuged at 1,000x g for 5 min. After removal of the supernatant, the pellet was resuspended with 500 µL isolation buffer . After centrifugation at 12,000x g for 5 min and 4°C the supernatant was transferred into a 1.5 ml reaction tube following the addition of 750 µL ice cold 100% ethanol. The samples were centrifuged at 12,000x g for 5 min at 4°C, the supernatant was discarded and the precipitated DNA was dissolved in 50 µL TE-buffer (pH 7.5). DNA concentration was measured using a Tecan Reader InfiniteDNA-fragmentation was analysed by performing a gel electrophoresis using a 1.7% agarose gel with 0.01% SYBR™Safe DNA gel stain . Samples were mixed with TriTrack loading Dye (ThermoFisher Scientific) in a 1:5 dilution. A Gene Ruler 100 bp DNA ladder (ThermoFisher Scientific) was used as a marker and was prepared according to the user manual’s loading protocol. Per sample 500 ng DNA was loaded on the gel and a voltage of 80 V was applied till the sample left the gel wells. Afterwards a voltage of 120 V was applied for at least 20–30 min. The imaging was performed using the ChemiDoc™ Molecular Imager  with the Image Lab Software (BioRad Laboratories) using the application “Nucleic Acid Gels”.For RNA isolation from trabecular bone blocks, samples were digested overnight and afterwards centrifuged as described above. The received pellet was resuspended with 1 ml TRIzol Reagent™ (ThermoFisher Scientific) and incubated for 5 min at room temperature. 200 µL chloroform were added and the samples were again incubated for 2–3 min at room temperature following a centrifugation step at 12,000 x g for 15 min at 4°C. The aqueous phase, which contained the RNA, was removed and 1 ml of ice-cold 100% ethanol was added. After an incubation period of up to 3 h at -20°C, samples were centrifuged at 12,000 x g for 10 min at 4°C. The supernatant was discarded and the pellet was washed with 500 µL ice-cold 70% ethanol following a centrifugation at 13,500 rpm for 10 min at 4°C. The RNA was precipitated again using 500 µL ice-cold 100% ethanol and centrifuged under the same conditions as described before. The supernatant was discarded and the pellet was dried at room temperature. Afterwards, the RNA was resuspended in 100 µL TE-buffer and RNA concentration was measured analogous to DNA concentration measurement.18 primer, reaction buffer, RiboLock RNase Inhibitor, dNTP Mix and RevertAid M-MulV RT, were mixed and the PCR protocol was carried out as follows: 1 h at 37°C, 5 min at 70°C. Afterwards, samples were stored at −20°C for further use.For reverse transcription polymerase chain reaction (RT-PCR), 100 ng RNA was used. The RT-PCR was performed according to manufacturer’s recommendations of the Revert Aid First Strand cDNA Synthesis Kit (ThermoFisher). The template RNA and the master mix containing Oligo (dT)fas) and Caspase-8 (casp-8) . The primers are listed in The regulation of apoptosis-specific genes was evaluated at the gene expression level by the detection of the Fas Cell Surface Death Receptor (2O and 10 µL DreamTaq Green PCR Master Mix (ThermoFisher). Next, 19 µL Master mix was added to 1 µL cDNA and PCR was performed  applying the following protocol: 1 cycle of 95°C (5 min), 40 cycles of 95°C (30s), 58°C (30s), 72°C (1 min) and 1 cycle of 72°C (5 min).A master mix was prepared for each gene and sample, containing 1 µL forward primer, 1 µL reverse primer, 7 µL HSamples were then loaded on a 1% agarose gel with 0.01% SYBR™Safe DNA gel stain. Gel electrophoresis and subsequent imaging was carried out as described above.All steps during protein isolation were performed on ice. After a gentle defrosting of the specimens, 600 µL of ice-cold Pierce™ RIPA buffer (ThermoFisher Scientific) was added, containing 0.02% animal component free protease inhibitor cocktail . After an incubation period of 10 min, specimens were homogenized with an ultrasonic homogenizer UP100H  with an amplitude of 80% and a cycle of 0.5 for 90s. Samples were cooled again on ice for 5 min. Larger tissue debris was then removed and the lysate was centrifuged at 12,500x g for 15 min. The supernatants were collected for measurement of protein concentration and SDS-Page.®Quant solution . Thus, 499 µl solution and 1 µl protein solution were incubated for 10–15 min and the absorption at 595 nm was measured. Using a BSA standard curve, the protein concentration per sample was calculated.Determination of protein concentration was performed via Bradford assay using RotiIn preparation for SDS-PAGE, protein samples were mixed with an equal volume of Lämmli Buffer (Sigma-Aldrich) and incubated at 95°C for 5 min. Afterwards, samples were put on ice immediately. The SDS-PAGE was performed using a Mini-PROTEAN TGX Stain-Free Any kD Precast Gel (BioRad Laboratories). Per lane 10 µg total protein was loaded on the gel. In order to determine the band size, the Precision Plus Protein™ Dual Color Standard (BioRad Laboratories) was carried along with every SDS-PAGE. First, a voltage of 60 V was applied until the proteins had left the gel pockets, then the voltage was increased to 120 V for at least 30 min. The imaging was performed as already described above, now using the application “Protein Gels”.The bone blocks stored in fixation buffer were prepared for FESEM analysis as described in . Images 2PO4  was combined with 0.08 M Na2HPO4  and both were diluted in 860 ml Aqua dest. Finally, 140 ml formalin  was added to the solution. The samples were treated with the fixation medium for 24 h at room temperature. After fixation, the tissue was washed for 2 h with tap water. EDTA-decalcification solution was produced by combining 14 g EDTA  with 9 ml ammonia solution  and 76 ml Aqua dest. During incubation, the solution was kept in constant motion via a stirring magnet to reduce saturation effects. At high turbidity medium was renewed. Decalcification time differed from 1 to 3 weeks, depending on the size of the bone plate. To verify sufficient decalcification, a needle test was performed. For sectioning, tissue samples were placed in embedding medium consisting of a 1:1 dilution of TissueTek  with 1xPBS and shock frozen in liquid nitrogen in square Peel-A-Way containers . Then, 10 µm thin cryosections were created with a CM 3050 S Cryotome . Sections were fixed with 4% paraformaldehyde for 10 min at room temperature, stained with an in situ cell death detection kit  and counterstained with DAPI . Stained sections were stored at 4°C for at least 24 h before microscopy. All images were captured on the same day at a constant exposure time  and magnification (20x). Afterwards images were merged to create whole sample images with a high resolution. From single images, apoptotic cells were identified by positive TUNEL staining and quantified using QuPath . First of all, each picture was inverted and lanes and bands were detected using the automatic lane and band finder. If any problems arose here , they were corrected manually. Using the “Volume Tools”, the size of the bands could be determined and thus the number of pixels of each band was calculated. To quantify DNA fragmentation, all bands per lane were detected and categorized according to the following: < 200 bp, 200–500 bp, > 500 bp. For each group, the numbers of pixels were summarized and the groups were then set in relation to each other to determine the distribution over all groups.p-values ≤ 0.05 were deemed as significant.To evaluate gene expression, lanes and bands of the gels were detected as described before. The number of pixels for each band was calculated with the “Volume Tools” of the software. Then, the relative gene expression related to the control group was calculated using the following equation:To investigate the effect of HHP on trabecular bone, the first step was to analyse the DNA degradation by gel electrophoresis. For further quantification, the detected bands were categorized into three different groups  and a densitometric analysis was performed. The results are shown in In case of category 2  led to a fluctuation in gene expression; while a higher expression was observed at incubation times 0 and 1 h after HHP, it dropped to the control level at incubation time 2 h and was clearly above the control level again at incubation of 3 h after HHP.For all depicted groups, an overexpression of reatment . Regardlfas gene expression, the analysis of caspase-8 gene expression showed a progression in dependence of the incubation periods following HHP  was used as a method for assessing protein integrity after HHP-treatment.The relative protein content of alpha-1 chains compared to the control group was located at a similar level over the incubation period. Only after an incubation period of 3 hours after HHP a significant increase in the protein content and thus in the band strength could be detected in some treated groups compared to the control . In caseField Emission Scanning Electron Microscopy was carried out following HHP-treatment to determine the surface matrix integrity of trabecular bone blocks.To support our findings regarding the influence of HHD treatment on trabecular bone tissue apoptosis, we created a stochastic model that displays apoptotic cell density correlated to its location in the tissue when applied onto our image data. The performed analysis visualizes qualitative information about the spatial distribution of formerly observed increased apoptosis rates. We were able to show a higher apoptosis rate in HHD treated tissue  comparedBone grafting procedures are the second most common tissue transplantations right after blood transfusions . AutologHigh hydrostatic pressure (HHP) represents an alternative to these methods as it has already been shown that at least biomechanical features of bone specimens were not influenced negatively by HHP . Two posIn addition to the preserved protein integrity, it was also shown that resident tissue cells react apoptotic or necrotic to HHP. Necrosis leads to a rupture of cells, releasing intracellular components. This may cause immunogenic reactions in the recipient of the transplanted graft. Apoptosis results in the formation of apoptotic bodies in which intracellular debris is enclosed. Released debris can easily be found and phagocytosed by macrophages which makes an apoptotic decellularization more desirable . Via DNAHistological analysis of the HHP treated tissue answers the question whether the HHP application is uniformly effective all across the tissue. The highest density of apoptotic cells was noticed in the center of the samples. However, this should not lead to the conclusion that the cells at the outer edge were unaffected by HHP. During apoptosis cell form apoptotic bodies which therefore lose their original structure and attachment to the tissue matrix . This lefas) of cell A to a membranous death receptor of cell B. This leads to the activation of an intracellular signaling cascade in cell B. With the activation of caspase-8 two signaling ways are possible. On the one hand a direct activation of caspase-3 is feasible. On the other hand, caspase-8 can activate the BH3 interacting-domain death agonist (BID) which is part of the intrinsic pathway. Further signaling then leads to an activation of caspase-9 and results again in an activation of caspase-3 (fas and caspase-8 were overexpressed after HHP-treatment it can be suggested that HHP initially leads to the activation of the extrinsic pathway. Regarding gel electrophoresis and histological staining, it can be assumed that the signaling cascade reaches the DNA cleavage step. However, the question of whether caspase-3 is activated directly or via an intermediate route via the intrinsic pathway cannot be answered with the available data.Gene expression also confirmed the hints regarding the apoptotic behavior of bone-associated cells. To interpret gene expression analysis, the apoptotic signaling pathway should be examined more closely. Apart from a few modifications, apoptosis is basically classified into an extrinsic and an intrinsic pathway, both resulting in the cleavage of DNA . If an easpase-3 . Since tfas and caspase-8. Globally, both fas and caspase-8 were overexpressed at 0 h after HHP. Short HHP-treatments of 10 min led to a steady decrease of this overexpression over time, longer HHP-treatments of 20 and 30 min led to a fluctuation of gene expression. A possible explanation for this is that the signal of apoptosis is passed on from cell to cell. It should also be noted that apoptosis is a reversible process if, e.g., the stimulus is not sufficient or is removed too early (It is also striking that the period of the selected incubation time after HHP is related to the degree of gene expression of oo early . It is tcaspase-8 at a pressure of 250 MPa for 30 min at the incubation time 0 h with the expression at a pressure of 250 MPa for 10 min at the incubation time 1 h, gene expressions are nearly at the same level. This also applies to the same pressures and fas expression. Therefore, a longer HHP application without subsequent incubation has a comparable effect to a shorter HHP application with subsequent longer incubation. This leads to the assumption that apoptosis signaling pathway in HHP-treated tissue progresses further with a prolonged HHP application.Comparing the gene expression data of longer HHP applications with the data of a short HHP-treatment, it can be suggested that the cells in the tissue already activate the signaling cascade during HHP-treatment. This can be demonstrated by the following example. Comparing the expression of Like all studies, the one at hand also has its limitations. Although there are various indications that pressures applied here lead to apoptosis, the complete signaling cascade could not be reconstructed using the available methods. Therefore, possible approaches could be further gene expression analysis of BID or caspase-9. A conceivable alternative would also be a specialized live-cell imaging analogous to the work of in vitro studies in which PBMCs are incubated with HHP treated and untreated bone blocks would be conceivable. With this, e.g., the released pro-an anti-inflammatory cytokines could be detected, which could provide information regarding the type of immune response. If the results of these studies are promising, more comprehensive in vivo studies could follow to give an overview of the reaction to the transplant, locally in the peri-implant tissue, as well as systemically, in the whole organism.An essential aspect, that was not taken into account in the present study is the remaining immunological potential of the bone grafts. Besides resident proteins, which could possibly lead to graft rejection, increased immune reaction of the recipients to the devitalized cells cannot be ruled out. Although the collected data provide information and indications about the type and distribution of cell death, it is not possible to estimate the exact immune responses from this data alone. Future research should focus on this remaining immunological potential. In order to answer this question Staphylococcus aureus but this required a pressure of 600 MPa (Another point of interest which has not been considered here is residual microbiota. Like different cell types, various bacteria and virus strains react differently to HHP. Investigations showed a complete inactivation e.g., of  600 MPa . At thisin vitro and in vivo studies regarding immunological reactions must be carried out. Based on this, revitalization of HHP treated materials with e.g., stem cells could be performed to determine the differentiation capacity and therefore the osteogenic and osteoinductive potential of bone grafts.In conclusion, HHP-treatment has a devitalizing effect which is a gentle alternative compared to other applications. For further application, pressure levels and durations should be chosen carefully as a too short treatment may not lead to complete devitalization and too high pressure may lead to a necrotic reaction which among other aspects, could lead to a strong immune reaction. Based on this study, a reasonable application would be 250 MPa for 20 min. On the one hand, apoptosis is presumably induced to such an extent that the signaling cascade is not interrupted. On the other hand, the probability of necrosis is lower than at 300 MPa. In order to be able to discuss HHP treatment as an alternative to already existing methods of preparing bone substitute materials, further


Human T-cell lymphotropic virus type 1 (HTLV-1) infection affects millions of individuals worldwide and can lead to severe leukemia, myelopathy/tropical spastic paraparesis, and numerous other disorders. Pursuing a safe and effective immunotherapeutic approach, we compared the viral polyprotein and the human proteome with a sliding window approach in order to identify oligopeptide sequences unique to the virus. The immunological relevance of the viral unique oligopeptides was assessed by searching them in the immune epitope database (IEDB). We found that HTLV-1 has 15 peptide stretches each consisting of uniquely viral non-human pentapeptides which are ideal candidate for a safe and effective anti-HTLV-1 vaccine. Indeed, experimentally validated HTLV-1 epitopes, as retrieved from the IEDB, contain peptide sequences also present in a vast number of human proteins, thus potentially instituting the basis for cross-reactions. We found a potential for cross-reactivity between the virus and the human proteome and described an epitope platform to be used in order to avoid it, thus obtaining effective, specific, and safe immunization. Potential advantages for mRNA and peptide-based vaccine formulations are discussed. Human T-cell leukemia virus-1 (HTLV-1) infection is associated with a broad spectrum of clinical manifestations such as bronchiectasis, bronchitis and bronchiolitis, dermatophytosis, community acquired pneumonia, strongyloides hyperinfection syndrome, liver, lymphoma and cervical cancer and several autoimmune/inflammatory disorders such as Sjögren’s Syndrome (SS), arthropathies, and uveitis . HAM/TSPWhile in certain endemic regions the prevalence and incidence of clinical symptoms among persons infected with HTLV-1 is significantly elevated, HTLV-1 infection leads to changes in the systemic immune response even in asymptomatic patients . The HTLTo evoke proper immune responses against HTLV-1, especially cellular immunity without undesired side effects, development of an effective and safe vaccine is essential. Synthetic peptide-based vaccines have been developed to evoke immune responses with various advantages in comparison to conventional vaccine formulations. Peptide vaccines are intended to induce cellular and humoral immunity and are chemically stable without oncogenic potential .Several peptide vaccines against HTLV-1 have been proposed  and seemWith the aim of investigating possible immunotherapeutic approaches against HTLV-1, we analyzed the HTLV-1 proteome in an attempt to extract peptide sequences belonging uniquely to the virus and absent in the human proteome. The research rationale was that immunotherapeutic approaches based on peptide sequences unique to the virus might be highly immunogenic by being unknown to the immune system, thus representing the “non-self” . In addihttps://www.uniprot.org/proteomes/?query=11926&sort=score, accessed on 1 September 2020 [https://research.bioinformatics.udel.edu/peptidematch/, accessed on 1 September 2020) [http://www.iedb.org/, accessed on 1 September 2020) was searched for peptide sequences unique to the virus in order to assess their immunological relevance [The analyzed HTLV-1 polyprotein sequence (accession NCBITaxId11926) is described at er 2020) . The Immelevance .As a second step in our investigation, we searched among pentapeptides absent in the human sequences and eventually selected unique consecutive viral pentapeptides overlapping each other by four residues. The results are reported in Finally, the peptides described in Data from B cell and T cell assays as retrieved from the IEDB are reported in The data reported above not only open new scenarios for effective and specific anti-HTLV-1 vaccine formulations, but can also provide a potential mechanistic link between HTLV-1 infection and related disorders including lymphomas and leukemias, respiratory disorders, fibromyalgia, rheumatoid arthritis, arthritis, tuberculosis, kidney and bladder infections, dermatophytosis, community acquired pneumonia, strongyloides hyperinfection syndrome, liver cancer, lymphoma, cervical cancer, and neurological disorders .Indeed, analyses of the experimentally validated epitopes described in Generation of autoantibodies following peptide sharing-induced cross-reaction would also explain the multitude of disorders that, to a greater or lesser extent, characterize HTLV-1 infection. Indeed, analyses of the peptide overlap between the viral epitopes and the human proteome show that, on the whole, 64 epitope-derived hexapeptides repeatedly occur among 143 human proteins , thus prA-kinase anchor protein 1, mitochondrial precursor (PSLALP) has a pivotal role in mitochondrial physiology, and its degradation leads to an increase in reactive oxygen species production, mitochondrial dysfunction, and ultimately cell death .A-kinase anchor protein 7 isoform gamma (HLTLPF) modulates L-type Ca2+ channels . AlteratAtaxin-2-like protein (QAIVSS) is involved in spinocerebellar ataxia type 2 , cutaneoB-cell CLL/lymphoma 6 member B protein and B-cell CLL/lymphoma 9-like protein, both of which share the peptide PPTAPP with HTLV-1. When altered, these two proteins relate to tumorigenesis .Cadherin-2 (APQVLP) may be associated with reduction in bone mineral density or vertebral fracture prevalence in survivors of childhood acute lymphoblastic leukemia .Constitutive coactivator of PPAR-gamma-like protein 2 (QLPPTA) deletion is associated with autism .Dedicator of cytokinesis protein 9  contributes to both risk and increased illness severity in bipolar disorder .DNA methyltransferase 1-associated protein 1 (APPLLP) is an essential regulator of activity and function of Ataxia Telangiectasia Mutated (ATM) kinase . AlteratDNA polymerase nu (PPPGPC) plays a role in translesion DNA synthesis during interstrand cross-link repair in human cells .E3 ubiquitin-protein ligase HECTD1 (APGYDP) is an important factor in promoting base excision repair (BER), which is the major cellular DNA repair pathway that recognizes and excises damaged DNA bases to help maintain genome stability .Fertilization-influencing membrane protein (PSQLPP) plays a role in sperm–oocyte fusion during fertilization .FLYWCH-type zinc finger-containing protein 1 (PSLALP) has a possible tumor suppressive role in preventing colorectal cancer metastasis .Heterogeneous nuclear ribonucleoprotein C-like 1 , alterations of which are found in sporadic and suspected Lynch syndrome endometrial cancer .Histone acetyltransferases KAT2A and KAT2B (DGRVIG), when altered, are associated with cardiovascular pathology .Islet cell autoantigen 1 (MKDLQA) Islet autoantibodies are typically associated with type 1 diabetes, but have been found in patients diagnosed with type 2 diabetes in whom they are associated with lower adiposity .La-related protein 1B (AIKQEV) is dysregulated in hepatocellular carcinoma .Mitochondrial dynamics protein MID49 (QPRPPP) is related to myopathies .Mucin-5AC precursor  is a major macromolecular component that determines the rheological properties of mucus; otherwise abnormal not-flowing mucus results in defective lung protection and leads to infection and inflammation .Neurofascin precursor (TGAVSS), which is a cell adhesion, ankyrin-binding protein, may be involved in neurite extension, axonal guidance, synaptogenesis, myelination, and neuron–glial cell interactions. Anti-neurofascin antibodies relate to optic, trigeminal, and facial neuropathy .Palmitoyltransferase ZDHHC1 (LALPAP) is involved in innate viral immune response .Protein piccolo (PPNHRP) is required for the development and function of neuronal networks formed between the brainstem and cerebellum. Alterations of protein piccolo may result in impaired motor coordination, cerebellar network dysfunction and pontocerebellar hypoplasia .Serine/threonine-protein kinase WNK1 (APQVLP) can cause hereditary sensory and autonomic neuropathy .Suppressor of tumorigenicity 7 protein (SLILPP) deficiency promotes laryngeal squamous cell carcinoma .Finally, to conclude this survey of pathologies that may be associated with HTLV-1 infection following cross-reactivity and autoantibody generation, we highlight the HTLV-1 epitope-derived VNFTQE peptide that is present in seventeen zinc finger proteins (ZFP)  and the related pathologies that could arise in case of cross-reactivity. Examples among the many are:Vaccines are a crucial tool for fighting viral infections and their profile of efficacy and safety is of paramount importance. Minimizing the risk of collateral autoimmune cross-reactivity might be advantageous for further optimizing the efficacy–safety tradeoff, as recently shown by the SARS-CoV-2 global vaccination campaign ,51.Homo sapiens proteome. These sequences show an immunological potential by being embedded in epitopes that were experimentally validated in the human host, as shown in Hence, in order to design a safe and effective HTLV-1 vaccine, we applied the scientific rationale according to which vaccines based on peptides unique to the infectious pathogens and absent in the human host might avoid cross-reactivity phenomena and offer high potential for immunogenicity and self-adjuvanticity ,53. We fMessenger ribonucleic acid (mRNA) vaccines are the results of decades of studies on different mRNA intracellular delivery technologies and have come under the spotlight as one of the main tools of the global vaccination campaign against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) . DeliverThe epitope platform presented above can also be adopted in non-genetic peptide-based vaccine formulations, where the peptides are directly administered, possibly conjugated to a carrier . Such foWe presented here an epitope platform for effective HTLV-1 vaccination that minimizes the risks intrinsic to whole viral antigens, thus potentially reducing post-vaccination adverse events, cross-reactivity with human antigens and possible autoimmune pathologies .The short epitopes we identified are optimally suited for mRNA and nanoparticle vaccine technology, offering remarkable advantage in terms of production quality and stability of the final products, thus easing the currently challenging logistic aspects of storage and transportation. Moreover, the same epitope platform is a flexible resource that can also be used in peptide-based vaccines at lower cost, facilitating production and diffusion, for instance in developing countries.In sum, we presented an epitope set that offers remarkable real-world logistic advantages for a large-scale safe and effective prophylactic vaccination campaign against HTLV-1 that might be of cogent importance considering that no specific therapy exists.


Ulcerative colitis (UC) is a chronic and relapsing inflammatory bowel disorder in the colon and rectum leading to low life-quality and high societal costs. Ursolic acid (UA) is a natural product with pharmacological and biological activities. The studies are aimed at investigating the protective and treatment effects of UA against the dextran sulfate sodium- (DSS-) induced UC mouse model and its underlying mechanism. UA was orally administered at different time points before and after the DSS-induced model. Mice body weight, colon length, and histological analysis were used to evaluate colon tissue damage and therapeutic evaluation. Intestinal transcriptome and microbe 16 s sequencing was used to analyze the mechanisms of UA in the prevention and treatment of UC. The early prevention effect of UA could effectively delay mouse weight loss and colon length shorten. UA alleviated UC inflammation and lowered serum and colon IL-6 levels. Three classical inflammatory pathways: MAPKs, IL-6/STAT3, and PI3K were downregulated by UA treatment. The proportion of macrophages and neutrophils in inflammatory cell infiltration was reduced in UA treatment groups. UA could significantly reduce the richness of intestinal flora to avoid the inflammatory response due to the destruction of the intestinal epithelial barrier. The function of UA against UC was through reducing intestinal flora abundance and regulating inflammatory and fatty acid metabolism signaling pathways to affect immune cell infiltration and cytokine expression. Ulcerative colitis (UC) is a chronic and relapsing inflammatory bowel disorder in the colon and rectum, which can induce recurrent episodes of bloody diarrhea, abdominal pain, and even colorectal cancer . The unhβ, and TNF and reduced the high nuclear level of NFκB p65 [Ursolic acid (UA), purified from medicinal plants and foods such as lavender and apple peel, is a natural pentacyclic triterpenoid carboxylic acid with pharmacological and biological activities . The UA NFκB p65 –13. UA cNFκB p65 –16. MoreNFκB p65 , 18. TheNFκB p65 . MeanwhiNFκB p65 –22. The NFκB p65 , 24. TheNFκB p65 –27. The NFκB p65 –30.In our study, we hypothesized that UA may improve the microenvironment of intestinal flora and regulate the infiltration of immune cells to prevent ulcerative colitis. So, we used the DSS-induced mouse UC model to investigate the protective effects of UA against ulcerative colitis.Reagent-grade DSS salt ; UA ; Fast DNA Spin Kit for feces ; Qiagen RNeasy Kit ; BD™ Cytometric Beads Array (BD).n = 8) according to the experimental plan. In our experiment, all mice were randomly divided into four groups: control group (Con group), model group (DSS group), preventive treatment group (UA + DSS group), and treatment group (DSS + UA group)  were provided and fed a basal diet at 24°C and 55% humidity with 14 : 10 light-dark cycle in SPF laboratory animal facility according to the approval of the Animal Ethics Committee of Zhejiang Academy of Traditional Chinese Medicine (KTSC20200058). After a week of adaptation, mice were divided into 4 groups (A group) . The DSSγ, IL-17, IL-2, GM-CSF, and IL-4) levels were measured using the BD™ Cytometric Beads Array (CBA) according to manufacturers' protocol.The colonic tissue of mice was fixed with 4% formalin, embedded in paraffin, and then stained with hematoxylin and eosin (HE). The score of Nancy index was performed according to the Marchal-Bressenot method . The NanTotal RNA was isolated from 3 colonic tissues of each group using the Qiagen RNeasy kit following the manufacturers' protocol. RNA samples with good quality control  were sequenced using Hiseq-2500 by Novogene. The raw data of RNA-seq was inspected using FastQC and mapped to the reference genome (GRCm38). The read count of genes was calculated by Hisat2 . The exphttp://www.arb-silva.de/) [R software.The total DNA of the rectal feces was isolated using the Fast DNA Spin Kit for feces. The V4 region of the 16S rRNA gene was amplified and sequenced with the 515F/806R primer set by Illumina MiSeq platforms at Novogene. The operational taxonomic units (OTUs) and representative sequences for each OTU were obtained at 97% similarity by FLASH, QIIME, and UPARSE software –38. The lva.de/) . And theμg of extracted RNA was reverse transcribed (Applied Biosystems) and amplified using the SYBR green PCR master mix (Roche 480). The relative quantification of the gene expression was calculated with the 2 − ΔΔCt method referring to Gapdh.Briefly, 1 μg/ml aprotinin). The concentration of protein was determined using a BCA assay (Sangon Biotech), and 20 μg of protein per lane was added on an 8-12% SDS-polyacrylamide gel. The protein electrophoretically transferred to a nitrocellulose membrane (0.1-μM pore size). The proteins were detected using rabbit polyclonal antibodies against mouse Tgfb-1, Col1a1, Itga5, and Gapdh (Proteintech Group) as primary antibodies and peroxidase-conjugated anti-rabbit IgG (Proteintech Group) as a secondary antibody. Protein was detected by an enhanced chemiluminescence system (ECL) and exposure to X-ray film.The colonic tissues were washed with ice-cold PBS and lysed with lysis buffer  was able to alleviate DSS-induced weight loss and shortening the colon in mice . HoweverP value = 2e − 07). The genes of the black module were clustered into inflammation signaling pathways such as PI3K-Akt, MAPK, and cytokine interaction  and negatively correlated with the DSS group   and negatively correlated with the DSS group   by high throughput sequencing GSE150688). The Con and UA + DSS groups were clustered, while DSS and DSS + UA groups were individually separated according to transcriptomic data by PCA analysis . The spe50688. Theraction . The ECMd by DSS . The blu) Figure . The red) Figure . Interes) Figure . We also) Figure . Meanwhi) Figure . These rTo understand the composition of infiltrated immune cells in inflammatory microenvironments P value = 0.038) in the gut    and the = 0.319) . The maj= 0.319) .Inflammatory bowel disease (IBD) has been a global disease leading to low life-quality and high societal costs . UC and α, β, γ, δ) play a particularly important role in chemokine-mediated recruitment and activation of innate immune cells at sites of inflammation and B and T cell development, differentiation, and function [To further elaborate the mechanism of UA in the prevention and treatment of UC, the correlation between the transcriptome and the phenotypes of each treatment group was analyzed by WGCNA. Three classical inflammatory pathways: MAPKs, IL-6/STAT3, and PI3K were enriched into the black module which was significantly positively correlated with the DSS group  and 1S. function . UA coulfunction . In IBD function –46. AMP-function . The actfunction . In our κB activation in both intestinal epithelial cells and macrophages and attenuates experimental murine colitis [Immune cell infiltration is an important biomarker of inflammation. DSS feeding resulted in an increased production of macrophage-derived cytokines in BALB/c mice . We also colitis . However colitis . UA trea colitis . In our At present, there is no single agent that has been proven to cause IBD. The role of gut microbes has been suspected because of potential infectious, particularly when the intestinal epithelial barrier is destroyed . UA has In conclusion, we demonstrated that UA could prevent and ameliorate the DSS-induced UC mouse model. The function of UA against UC was through reducing intestinal flora abundance, regulating inflammatory and fatty acid metabolism signaling pathways to affect immune cell infiltration and cytokine expression . These r


Our results elucidate the binding of INSTIs to the HTLV-1 intasome and support their use for pre-exposure prophylaxis and possibly future treatment of HTLV-1 infection.Between 10 and 20 million people worldwide are infected with the human T-cell lymphotropic virus type 1 (HTLV-1). Despite causing life-threatening pathologies there is no therapeutic regimen for this deltaretrovirus. Here, we screened a library of integrase strand transfer inhibitor (INSTI) candidates built around several chemical scaffolds to determine their effectiveness in limiting HTLV-1 infection. Naphthyridines with substituents in position 6 emerged as the most potent compounds against HTLV-1, with XZ450 having highest efficacy in vitro. Using single-particle cryo-electron microscopy we visualised XZ450 as well as the clinical HIV-1 INSTIs raltegravir and bictegravir bound to the active site of the deltaretroviral intasome. The structures reveal subtle differences in the coordination environment of the Mg Human T-cell lymphotropic virus type 1 (HTLV-1) is an oncogenic deltaretrovirus. Here the authors provide structural characterization of the binding mechanism of novel integrase strand transfer inhibitor (INSTI) candidates to limit HTLV-1 infection. It is the causative agent of a severe form of blood cancer called adult T-cell leukaemia/lymphoma (ATLL)2 and a range of debilitating neuromuscular disorders—HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP)3, which often lead to paralysis. Although only 5–10% of the estimated 10–20 million people4 infected with HTLV-1 worldwide will develop ATLL2, a recent report showed that the vast majority of otherwise asymptomatic patients suffer from issues that severely impact their quality of life, including pain, discomfort and depression5. 12.7% of HAM/TSP patients reported their quality of life was worse than death5, based on the Euroqol five dimension questionnaire (EQ-5D).Human T-cell lymphotropic virus type 1 (HTLV-1) is a deltaretrovirus and one of the most oncogenic human viruses6. The prevailing dogma is that, following the initial infection, HTLV replication is primarily the result of the clonal proliferation of infected cells. However, recent data indicate there is ongoing viral replication, which leads to the generation of additional clones of HTLV-1 infected cells, and increases the proviral load. An increased viral load is correlated with an increased risk of developing HTLV-1-associated malignancy and inflammatory disease7. Hence, suppression of additional rounds of viral infection may reduce the risk of malignant transformation. Administering anti-retroviral agents may help prevent HTLV-1 transmission among drug users, serodiscordant couples, organ recipients and pregnant/breastfeeding mothers. Thus, strong arguments can be made for developing HTLV-targeted therapeutics8. Integration of a DNA copy of the viral genome is an essential step in the replication of all retroviruses. Integrase strand transfer inhibitors (INSTIs) are among the most effective anti-HIV drugs. Similarly, understanding the integrase (IN) enzyme of HTLV, and how it can be inhibited, will be a critical part of the effort to control HTLV infections. We recently reported that the first- and second-generation HIV INSTIs are effective at inhibiting HTLV-1 strand transfer in vitro and integration in tissue culture9. In this report, we have characterised several groups of INSTIs that have been reported to inhibit the replication of HIV11.HTLV research has largely remained in the shadow of HIV research, which has moved at a much faster pace. Like HIV-1, HTLV-1 is a blood-borne pathogen that is transmitted horizontally by contact with infected blood, sexual intercourse and vertically from mother to child during breastfeeding and labour. The likelihood of an HTLV-1 carrier developing ATLL is correlated with exposure early in life—most commonly during breastfeeding or child-birth13. Technical advances in cryogenic electron microscopy (cryo-EM), recently allowed the structural characterisation of HIV-114 and a closely related red-capped mangabey simian immunodeficiency virus (SIVrcm)15 intasomes in complexes with clinical and experimental INSTIs at near-atomic resolution. Currently, no structural information is available on the mode of INSTI binding to the deltaretroviral intasome. We recently determined the cryo-EM structure of the deltaretroviral simian T-cell lymphotropic virus type 1 (STLV-1) intasome bound to its host factor B56γ16. Here, in addition to characterising three compounds that block HTLV-1 transmission two- to fivefold more potently than raltegravir (RAL), we report three high-resolution cryo-EM structures of a deltaretroviral intasome bound to three different INSTIs. The 3D reconstructions allow for direct comparison between the binding modes of these inhibitors in lentiviral and deltaretroviral intasomes. Our findings have important implications for establishing therapies targeted at HTLV-1 and provides a framework for understanding the mechanism of INSTI action on the HTLV-1 intasome at the atomic scale.The target of the INSTIs is the IN active site engaged with viral DNA (vDNA) ends in the context of the nucleoprotein complex termed the intasome11. Long aliphatic or aromatic substituents at the 6- position are able to reach across the active site pocket, and associate with the protein backbone as shown in the crystal structures of PFV11, and cryo-EM reconstructions of HIV-114 intasomes. We also included naphthyridines designed to inhibit HIV RNase H that were not previously tested against IN, because the compounds also target a pair of magnesium ions in the active site, which is similar to INSTI binding17. Cross inhibition has previously been found with hydrazines and other INSTIs, which effectively blocked herpes simplex virus replication by inhibiting RNase H-like fold containing proteins18.We evaluated a library of 32 small molecules collectively comprising five classes of known and candidate INSTIs, including naphthyridines, pyridinones, diketo acids, oxoisoindoles and hydrazines  determination of the proviral load (PVL). In line with our in vitro data, XZ450 and XZ446 were most potent in inhibiting HTLV-1 infection of Jurkat T cells  compared with RAL (6.42 ± 4.24 nM)9. A block in the integration reaction was confirmed by Alu-qPCR experiments  is only about twofold more efficient in blocking HTLV-1 integrase strand transfer in vitro, BIC potently blocks HTLV-1 transmission in tissue culture, exhibiting a 20-fold improvement in EC50 (0.3 ± 0.173 nM)9 compared with RAL. We suspect this is owing to a more efficient uptake of BIC than RAL by Jurkat T cells9. Interestingly, XZ448, which was more potent than RAL in our in vitro strand transfer reactions exhibited ~20-fold lower activity in blocking HTLV-1 infection compared with RAL (see Discussion).Six of the most promising compounds were selected for assessment of their efficacy in inhibiting HTLV-1 viral infection. To this end, we employed an MT-2-Jurkat co-culture relying on cell-to-cell-based HTLV-1 transmission, which we successfully used in a previous study16. This is potentially explained by the stronger binding observed between STLV-1 IN and B56γ  harbour a Pro at this position, we thus investigated whether mutating Pro214 to a phosphomimetic Asp could increase the affinity between IN and B56γ. Moreover, a previous report showed that peptides in which additional acidic residues (Glu) were added immediately following the SLiM sequence, had significantly higher affinity for B5622. We thus mutated IN Ala219 into Glu. Approximately twofold more B56γ was recovered in pull-down assays when IN(A219E) was used as a bait compared with WT IN 16, a twofold symmetric IN dimer of dimers with the two B56γ molecules that laterally flank the intasome  grid support (the latter increasing both the particle concentration and the distribution in particle orientation) allowed us to obtain three STLV-1 intasome: INSTI complexes, with FDA-approved INSTIs RAL and BIC, and XZ450, which was the most promising INSTI candidate identified in our screen Fig. . Cryo-EMome Fig. . All 4 Iome Fig. . Putativ2+ ions. The metal ions are resolved in the STLV intasome:INSTI structures , which coordinates two catalytically essential Mgres Fig. . The bining Fig. .Fig. 3Cl15, INSTI binding is accompanied by a flip of the terminal vDNA 3’-adenosine, which vacates a pocket formed between a dC:dG basepair and a 310 helix (IN residues 151–155 in STLV and HTLV-1 INs). The pocket becomes occupied by the fluorobenzyl group of the drug, whereas the displaced adenosine base forms parallel π-π stacking interaction with the heterocyclic backbone of the drug  because the structure of the HIV-1 intasome with XZ450 bound is not available.Despite the fact that retroviral integrases, in general, share a well-conserved active site, previous structural research has shown that even small changes in the conformation of active site residues can significantly affect the binding and efficacy of INSTIs25 studies). STLV-1 IN residue Y149  exhibits the largest difference. The tyrosine side-chain twists, displacing the terminal oxygen atom of Y149 by 4 Å in the STLV-1 intasome structure compared with the HIV-1 structure. However, there are differences in the conformation of Y149 in different STLV intasome:INSTI complexes 11. The inhibitory potency of XZ446 for HIV-1 (2 ± 0.1 nM)10 was similar to that for HTLV-1 (2.57 ± 0.81 nM). This illustrates that small changes within the active site can have a significant effect on the binding and efficacy of inhibitors. XZ448 activity was much less potent in its ability to inhibit HIV-1 replication (263 ± 52 nM)11 similar to our results with HTLV-1. We suspect this could be due to the less efficient uptake of XZ448 by the cells.Having evaluated 32 compounds, we identified two small molecules, XZ450 and XZ446, which are two- to fivefold more potent than RAL in blocking HTLV-1 infection in viral culture. Interestingly, EC13. The binding of several classes of INSTIs to the PFV intasome has been structurally characterised and used as a model of INSTI binding to the HIV-1 intasome for the last 10 years30. However, it was recently reported by Lyumkis et al.14 that the binding of some INSTIs can differ between PFV and HIV. For example, the sulfonyl benzyl group of the 6-substituted naphthyridine compound XZ446 (4 f) is rotated by over 90° within the binding pocket of HIV intasome compared with that bound in PFV, resulting in association with a different set of IN residues14. This underlines the importance of structural characterisation of INSTI binding specifically for the clinically important virus HTLV-1.The architecture of the deltaretroviral intasome most closely resembles the structure of the PFV intasome, both are IN tetramers25. Although the STLV-1 structures show that there is a small change in conformation of residue Y149 depending on which INSTI is bound, this difference is owing to the way the oxadiazole ring of RAL and the 3-(dimethylamino)-3-oxopropyl substituent of XZ450 interact with the tyrosine ring. The shift of the tyrosine side chain is much more pronounced when STLV-1 and PFV intasome:RAL structures are compared . Single-point mutants of STLV-1 MarB43 IN were generated by two-step PCR splicing. The following primer pairs were used for cloning SLTV-1 IN mutants: R49Q , I147V , A219E , P214D . The PCR products were then spliced together using primers GNM770 and GNM769, digested with EcoRI/XhoI and ligated into similarly digested pET28(+)-SUMO-H6P9. For the expression of LEDGF/ΔIBD-B56γ, the previously described pET28a(+)SUMO-H6P-LEDGF/ΔIBD-PPP2R5C(11-380) construct was used9. For primer sequences, see Supplementary Table For expression of HTLV-1 IN, the previously described pET28a(+)-SUMO-H6P-STLV-1 MarB43 IN construct39. STLV-1 IN, its point mutants, and LEDGF/ΔIBD-B56γ were expressed and purified by affinity, ion exchange and size exclusion chormatography16.HTLV-1 IN was expressed and purified by affinity, ion exchange and size exclusion chromatographyp values were calculated in Prism 9.The donor DNA (vDNA) that mimics the 3′-processed LTR of STLV-1 DNA was preassembled by annealing Atto-680-labelled MarB43_U5_S30UP and MarB43_U5_S30B oligonucleotides  and 20 μg of LEDGF/ΔIBD-B56γ, in presence of 10 μg bovine serum albumin and pull-down buffer. The resin was subsequently washed five times with the pull-down buffer. After the last spin at 400 × g for 5 min, the supernatant was gently removed, and the beads boiled in 20 μl 1.5 × SDS loading buffer containing 200 mM imidazole. Samples were centrifuged and 10 μL of the supernatant was analysed on an 11% sodium dodecyl sulphate–polyacrylamide gel electrophoresis gel.Pull-down assays were conducted as follows2, 5.8 μM ZnCl2, 12.7 mM DTT, 0.53 μM donor DNA and 4 μg of HTLV-1 IN39. In brief, the reaction was incubated at RT for 15 min in the presence of an INSTI or dimethyl sulfoxide (DMSO) control and the reaction was started by the addition of 300 ng of pGEM-9Zf(-) supercoiled plasmid DNA. The reaction was incubated for 1 h at 37 °C and stopped by adding 0.5% SDS and 25 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0.The donor vDNA that mimics the 3′-processed LTR of HTLV-1 or STLV-1 DNA was preassembled by annealing the U5_S20Q_UP and U5_S20Q_B or MarB43_U5_S30UP or MarB43_U5_S30B oligonucleotides, respectively  supercoiled plasmid DNA and stopped after 15 min of 37 °C incubation by adding 0.5% SDS and 25 mM EDTA, pH 8.0.For STLV-1 IN, the reaction conditions were 25 mM BTP-HCl pH 6, 0.1 μM STLV-1 IN, 2 μM vDNA, 60 mM NaCl, 13.28 mM DTT, 10 mM MgClIC50 calculated as previously described9. In brief, the DNA band that corresponds to the concerted integration product was quantified by densitometry in ImageLab 4.1 (Bio-Rad). All experiments were done in triplicate. Quantified data points were fitted to dose–response curves in Prism 9. The cumulative standard deviation for each drug was calculated as an average of the upper limit and lower limit values:Proteins were degraded by incubation with 30 μg of proteinase K (Roche) at 37 °C for 1 h. DNA was precipitated at −20 °C overnight in the presence of ethanol supplemented with 20 μg of glycogen and then resuspended in 1.5× agarose loading dye. The DNA products were separated by electrophoresis through 1.5% agarose gel and stained with ethidium bromide and imaged using the BioRad GelDox XR + imager. The data were analysed and the Where SE denotes the standard error computed by Prism 9.g in a 30-kDa MWCO ultrafiltration device (Vivaspin) to a concentration of 0.2 mM. A mixture containing 0.7 mL 20 mM BTP-HCl pH 6, 10 mM CaCl2, 10 mM DTT, 10 μM ZnCl2 and 25 μM STLV-1 MarB_U5_S30 double-stranded vDNA  and 20 mM MgSO4, was incubated at RT for 45 min to allow for the intasome-INSTI complex formation40. For all complexes, UltraAuFoil R 1.2/1.3 Au 300-mesh grids (Electron Microscopy Sciences)41 were glow-discharged for 4 min at 45 mA on an Emitech K100X instrument (Electron Microscopy Sciences) and covered with a layer of GO 42 immediately before being used. In brief, to add the layer of GO, a solution of 0.2 mg/ml GO was made in ultrapure water, the suspension was centrifuged at 300 × g for 15 sec to remove aggregates. Following 1 min incubation at RT, the grids were carefully blotted, washed in three drops of water and allowed to air dry for 5 min before use. Four μL of STLV-1 intasome:INSTI complexes were spotted on the graphene side of the coated grid. The grids were incubated for 1 min at 22 °C and 95% humidity, blotted for 2.5 s prior to plunge-freezing in liquid ethane using a Vitrobot Mark IV instrument (Thermo Fisher Scientific).The STLV-1 intasome was assembled by mixing equimolar (0.03 mmol) quantities of recombinant STLV-1 IN(A219E) and LEDGF/ΔIBD-B56γ and dialysing overnight at 4 °C against 0.5 L of 25 mM Tris-HCl pH 7.4, 200 mM NaCl, 2 mM DTT. This sample was then concentrated at 4 °C, 1935 × 2 was fractionated across 41 movie frames. A 20-eV energy slit and a defocus range of −0.7 to −3.6 μm were used for all data collections. The number of micrograph movie stacks acquired for RAL, BIC and XZ450 complexes were 17744, 8502 and 9188, respectively.Cryo-EM data were collected on a 300-keV Titan Krios G3i cryo-electron microscope (Thermo Fisher Scientific) equipped with a Gatan GIF BioQuantum energy filter and a K3 Summit direct electron detector (Gatan). Micrographs were acquired in dose-fractionation mode, at a calibrated magnification corresponding to 1.1 Å per physical pixel (0.55 Å per super-resolution pixel) at the detector. A total electron exposure of 50 e/Å43. Contrast transfer function (CTF) parameters were estimated using Gctf-v1.0644. At this stage, images with crystalline ice contamination were discarded; 14,271 (intasome-RAL data set), 6129 (BIC) and 7719 (XZ450) micrographs were retained for further processing. Particles were picked with Gautomatch-v0.53 (http://www.mrc-lmb.cam.ac.uk/kzhang/) using STLV intasome 2D class averages16, low-pass filtered to 18 Å resolution, as templates. The particles, extracted in Relion-3.1 and binned to a pixel size of 4.4 Å, were subjected to two rounds of reference-free 2D classification in CryoSPARC-2. Particles belonging to well-defined 2D classes  within a 12 Å distance from the centre of the given INSTI were selected for the calculations.2 and maintained in Roswell Park Memorial Institute (RPMI) media supplemented with 10% fetal bovine serum (FBS), 100 U penicillin, 100 μg/mL streptomycin and 0.25 μg/mL fungizone.MT-2 and E6.1 Jurkat T-cell lines (ATCC) were used for the experiments. The cells were cultured at 37 °C with 5% CO9; Jurkat T cells were counted and diluted in complete RPMI to a final concentration of 0.25 × 106 cells/ml and pre-treated with INSTI drugs  or DMSO as a control for 24 h. MT-2 cells were resuspended in RPMI at a concentration of 2 × 106 cells/ml and subjected to a nonlethal γ-radiation dose of 400 Gy. Cells were resuspended at a concentration of 2 × 106 cells/ml in serum-free RPMI supplemented with the corresponding INSTI drug. Co-culture of 0.5 × 106 for each cell line was carried out for 16 h, after which cells were washed in PBS and resuspended in 1 ml of depletion buffer . MT-2 cells were depleted from the culture by means of anti-CD25 magnetic beads . Depletion efficiency was confirmed via flow cytometry as described previously9. Jurkat cells were cultured for 2 weeks in complete RPMI supplemented with the drugs and genomic DNA was extracted  and stored at −20 °C for subsequent analysis. All the experiments were done in triplicate.The HTLV-1 cell-to-cell infection was performed as previously described54. Jurkat genomic DNA was quantified and diluted to a concentration of 5 ng/μL. Real-time qPCR analysis were conducted on 20 ng of gDNA to measure the level of HTLV-1 tax and gapdh genes /(2×gapdh copy number)53, and normalised to the DMSO-treated control which was set to 100%. The PVL data were fitted to a dose–response curve in Prism 7 and the EC50 was calculated9. Alu-PCR was performed as previously described in ref. 55. In brief, the first PCR reaction was done with Phusion polymerase starting from 80 ng of gDNA using the ALU-specific primers (Alu-F/Gag-R). The nested PCR was run diluting the amplificated product 1:50. The reaction was done as a qPCR using Gag-specific primers (Gag-R/Gag-F) and the Luna SYBR dye reaction mix (New England Biolabs). The integrated provirus copy numbers were normalised to GAPDH and Jurkat cells treated with DMSO and infected with HTLV-1 were set to 100%. Experiments were done in triplicate.The PVL was measured as previously described57. Naphthyridine XZ420 was prepared by a modification of our previous methodology, which included the use of a key Suzuki coupling10. Detailed synthesis is presented in Supplementary Materials and Methods.Raltegravir and BIC were purchased from Adooq Bioscience (USA). The majority of the compounds shown in Supplementary Table Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary