Asymptomatic HTLV-1 Carriers Research Articles

The role of CREB and MAPK signaling pathways in ATLL patients

BackgroundHTLV-1 is a worldwide distribution retrovirus with 10–20 million infected individuals. ATLL is an Adult T-cell leukaemia lymphoma caused by aggressive T-cell proliferation that is infected by HTLV-1 and is associated with an inferior prognosis. The exact molecular pathogenesis has yet to be fully understood. CREB, a transcription factor, acts as a molecular switch that controls the expression of numerous genes in response to various extracellular signals. Its activation is primarily mediated through phosphorylation by multiple kinases, including MAPKs. MAPKs, a family of serine/threonine kinases, serve as crucial mediators of intracellular signaling cascades.Method and materialThis study investigated, 38 HTLV-I-infected individuals, including 18 HTLV-1 asymptomatic carriers (ACs) and 20 ATLL subjects. mRNA was extracted and converted to cDNA from Peripheral blood mononuclear cells (PBMCs), and then the expression of TAX, HBZ, CREB, and MAPK was analyzed by TaqMan qPCR. The genomic HTLV-1 Proviral loads were examined among the study group.ResultsThe data analysis showed a significant difference in the mean of CREB expression amongst study groups (ATLL and carriers, (p = 0.002). There is no statistical difference between the MAPK gene expression (p = 0.35). HBZ, TAX, and HTLV-1 proviral load weree significantly higher in ATLL subjects compared to ACs (p = 0.002, 0.000, and 0.000), respectively. Moreover, our results, demonstrated a direct positive correlation among HBZ, CREB, and TAX gene expression in ATLL patients (p = 0.001), whilst between the ACs, TAX gene expression had a positive significant correlation with HBZ and HTLV-1 proviral load (p = 0.007 and p = 0.004, respectively).ConclusionThe present study demonstrated that CREB gene expression was higher in the ATLL group than ACs, while there was no difference for MAPK. Therefore, this pathway may not strongly involve in the activation of CREB. The CREB may be a prognostic factor for the development of HTLV-I-associated diseases and can be used as a monitoring marker for the efficiency of the therapeutic regime and prognosis.

Correlation Between TWEAK Serum Level and HTLV-1 Proviral Load in HAM/TSP.

Human T-cell lymphotropic virus type-I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), the main neurological manifestation of HTLV-I, is a chronic inflammatory disease. Viral-host interaction and host genetics are two important contributors to the development of the HAM/TSP. This study was conducted to measure the serum level of tumor necrosis factor-alpha-like weak inducer of apoptosis (TWEAK) by ELISA method in three groups of participants including 34 HAM/TSP patients (HAM/TSP), 35 asymptomatic HTLV-1 carriers (ACs), and 20 healthy controls (HCs). Also, the titer of the proviral load in two groups of HAM/TSP and ACs was assessed by the real-time polymerase chain reaction (PCR). The statistical results showed that, there is no significant difference between the three groups in TWEAK cytokine level (p = 0.667). Also, there was no significant difference in proviral load titer between groups of HAM/TSP and ACs (p = 0.08). Furthermore, no significant difference was observed between proviral load and TWEAK cytokine concentration between groups of HAM/TSP and ACs. Our findings showed that despite the inflammatory nature of HAM/TSP disease, the expression level of TWEAK in HAM/TSP patients is not significantly different from the groups of ACs and HCs. Therefore, the involvement of other factors in causing HAM/TSP is not unexpected.

HLA-A*24 Increases the Risk of HTLV-1-Associated Myelopathy despite Reducing HTLV-1 Proviral Load.

Increased human T-cell leukemia virus type 1 (HTLV-1) proviral load (PVL) is a significant risk factor for HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). There is controversy surrounding whether HTLV-1-specific cytotoxic T lymphocytes (CTLs) are beneficial or harmful to HAM/TSP patients. Recently, HTLV-1 Tax 301-309 has been identified as an immunodominant epitope restricted to HLA-A*2402. We investigated whether HLA-A*24 reduces HTLV-1 PVL and the risk of HAM/TSP using blood samples from 152 HAM/TSP patients and 155 asymptomatic HTLV-1 carriers. The allele frequency of HLA-A*24 was higher in HAM/TSP patients than in asymptomatic HTLV-1 carriers (72.4% vs. 58.7%, odds ratio 1.84), and HLA-A*24-positive patients showed a 42% reduction in HTLV-1 PVL compared to negative patients. Furthermore, the PVL negatively correlated with the frequency of Tax 301-309-specific CTLs. These findings are opposite to the effects of HLA-A*02, which reduces HTLV-1 PVL and the risk of HAM/TSP. Therefore, we compared the functions of CTLs specific to Tax 11-19 or Tax 301-309, which are immunodominant epitopes restricted to HLA-A*0201 or HLA-A*2402, respectively. The maximum responses of these CTLs were not different in the production of IFN-γ and MIP-1β or in the expression of CD107a-a marker for the degranulation of cytotoxic molecules. However, Tax 301-309-specific CTLs demonstrated 50-fold higher T-cell avidity than Tax 11-19-specific CTLs, suggesting better antigen recognition at low expression levels of the antigens. These findings suggest that HLA-A*24, which induces sensitive HTLV-1-specific CTLs, increases the risk of HAM/TSP despite reducing HTLV-1 PVL.

HTLV-1-Associated Myelopathy (HAM) Incidence in Asymptomatic Carriers and Intermediate Syndrome (IS) Patients.

Several studies suggest that HTLV-1 infection may be associated with a wider spectrum of neurological and clinical manifestations that do not meet diagnostic criteria for HAM. These conditions may later progress to HAM or constitute an intermediate clinical form: intermediate syndrome (IS), a mid-point between asymptomatic HTLV-1 carriers and those with full myelopathy. Thus, we determined the incidence of HAM cases in the HTLV-1-asymptomatic and IS patients, and the clinical/laboratory associated markers. A total of 204 HTLV-1-positive patients were included in this study, divided into two groups: Group 1, including 145 asymptomatic HTLV-1 subjects (ASY), and Group 2, including 59 patients with inflammatory clinical symptoms in more than three systems and a high proviral load (PVL). During a 60-month follow-up time, with the age ranging from 47 to 79 years, ten patients of the fifty-nine initially diagnosed as IS developed HAM (iHAM), and two patients of the initial 145 ASY developed HAM directly. Women were more prevalent in all groups. For the iHAM patients, the age ranged from 20 to 72 years, with a mean of 53 (±15 SD). Older age was associated with the development of HAM, higher PVL and IS; however, there was no any specific symptom or clinical sign, that was associated with risk for iHAM. In conclusion, IS cases could be an early phase of development of HAM. These findings show the presence of higher incidence probabilities in our cohort than previously reported.

Single-Cell RNA Sequencing Revealed the YY1/EZH2/MLH1 Axis As a Possible Therapeutic Target of Intractable Adult T-Cell Leukemia

Background: Due to clinical heterogeneity of adult T-cell leukemia/lymphoma (ATL) and its diverse genetic abnormality, common therapeutic targets of ATL remains unclear. Whereas the YY1/EZH2 axis regulates global epigenetic modification of genes in ATL, its downstream target genes have not been fully elucidated. Meanwhile, DNA mismatch repair proteins may be targeted in ATL. Here we explored underlying molecular axes for ATL progression through comprehensive single-cell RNA-sequencing (sc-RNA-seq). Methods: Peripheral blood of 42 ATL patients and asymptomatic HTLV-1 carriers in addition to two cell lines of ATL43T and ED+ were subjected to the following analyses. Molecular markers associated with their clinical phenotypes were extracted through sc-RNA-seq analysis. Expression levels of DNA mismatch repair proteins (MLH1, MSH2) and target molecules were validated by flow cytometry. Finally, their function was verified through lentiviral shRNA-dependent knockdown and molecular-targeting reagents. Results: While the frequencies of CADM1+ cells among CD4+ T cells were generally correlated with HTLV-1 proviral loads (R = 0.76, p < 0.001), substantial CADM1+ cells were detected but did not increase in some long-remitted cases; thus, CADM1+ cells are biologically heterogeneous. sc-RNA-seq of CADM1+ cells of four ATL cases identified a cluster of CD48-deficient cells. in silico promoter analysis of the cluster extracted a transcription factor YY1 as a candidate for common master regulators. EZH2 and the putative driver genes (CELF2, PTPRC, CBLB, ATXN1, IKZF2) were identified as differentially expressed genes (DEGs) in the cluster. The EZH2-overexpressed cluster was increased in acute-type ATL compared with remitted ATL and smoldering-type ATL. Interestingly, the cluster increased moderately in the other smoldering-type ATL, which required UVB irradiation due to intractable skin lesion. Given that YY1 and EZH2 were up-regulated in acute-type ATL, the cluster may represent tumor aggressiveness. Furthermore, biphasic expression of YY1/EZH2 was observed in 2 acute-type ATL, which indicates that the EZH2 high ATL cells confer malignant signature. In-house analysis on microarray dataset GSE55851 specified the MLH1 associated with YY1/EZH2. MLH1 within CADM1+ fraction was down-regulated in ATL patients with remission (n = 7) but maintained in progressive ATL (n = 6) (p < 0.05). Lentiviral shYY1 knockdown resulted in down-regulation of MLH1 in both ATL43T and ED+ (p < 0.01). The EZH1/2 inhibitor valemetostat down-regulated EZH2 (p = 0.018) and MLH1 (p = 0.014) in primary ATL cells. Finally, knockdown of either YY1 or MLH1 suppressed proliferation of ATL43T and ED+ (p < 0.0001). Conclusions: Our findings indicate that down-regulation of MLH1 through YY1/EZH2 inhibition plays a key role in the treatment of aggressive ATL.

HTLV-1-associated myelopathy in Spain

BackgroundHTLV-1 infection is a neglected disease. Over 10 million people are infected worldwide, with hot spots of high endemicity across all continents. Roughly 5% of HTLV-1 carriers develop HTLV-1-associated myelopathy (HAM), a progressive subacute neurological disabling disease. MethodsWe report the main features of patients diagnosed with HAM up to date in Spain, a non-endemic country with a relatively high migrant flow from Latin America and Equatorial Africa, where HTLV-1 is endemic. ResultsA total of 451 cases of HTLV-1 had been recorded in Spain until the end of year 2022. HAM had been diagnosed in 58 (12.9%). The current incidence is of 2-3 new cases per year. Women represent 76%. Mean age at diagnosis is 49 years-old. Nearly 60% are Latin Americans. Although sexual transmission is the most likely route of HTLV-1 acquisition, up to 6 individuals had been infected following solid organ transplantation. Rapid onset myelopathy developed in all but one of these transplant recipients from three HTLV-1-positive donors. HTLV-1 subtype 1a transcontinental was the only variant recognized in HAM patients. HTLV-1 proviral load was significantly greater in HAM patients than in asymptomatic HTLV-1 carriers (677 vs 104 HTLV-1 DNA copies/104 PBMC; p=0.012). Symptom relief medications and physiotherapy have been the only treatment providing some benefit to HAM patients. Neither significant clinical nor virological efficacy was noticed using antiretrovirals in at least 9 HAM patients. Two thirds of HAM patients ended up in a wheelchair and with urinary/fecal sphincter incontinence. ConclusionHAM is the most frequent clinical manifestation of HTLV-1 infection in Spain, a non-endemic country. Middle aged women migrants from Latin America are the most frequently affected. Two thirds end up in a wheelchair despite using antiretroviral therapy.

Decoding dysregulated angiogenesis in HTLV-1 asymptomatic carriers compared to healthy individuals.

Human T-cell lymphotropic virus type 1 (HTLV-1) is the first identified human retrovirus responsible for two significant diseases: HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia/lymphoma (ATLL). Although the majority of infected individuals remain asymptomatic carriers, a small percentage may develop ATLL or HAM/TSP. In tumorigenesis, a crucial process is angiogenesis, which involves the formation of new blood vessels. However, the precise mechanism of HTLV-1 associated angiogenesis remains unclear. This study aims to investigate the gene regulation involved in the angiogenesis signaling pathway associated with HTLV-1 infection. The research enrolled 20 male participants, including asymptomatic carriers and healthy individuals. Blood samples were collected and screened using ELISA for HTLV-1 confirmation, and PCR was performed for both Tax and HBZ for validation. RNA extraction and cDNA synthesis were carried out, followed by RT-qPCR analysis targeting cellular genes involved in angiogenesis. Our findings indicate that gene expression related to angiogenesis was elevated in HTLV-1 ACs patients. However, the differences in gene expression of the analyzed genes, including HSP27, Paxillin, PDK1, PTEN, RAF1, SOS1, and VEGFR2 between ACs and healthy individuals were not statistically significant. This suggests that although angiogenesis-related genes may show increased expression in HTLV-1 infection, they might not be robust indicators of ATLL progression in asymptomatic carriers. The results of our study demonstrate that angiogenesis gene expression is altered in ACs of HTLV-1, indicating potential involvement of angiogenesis in the early stages before ATLL development. While we observed elevated angiogenesis gene expression in ACs, the lack of statistical significance between ACs and healthy individuals suggests that these gene markers may not be sufficient on their own to predict the development of ATLL in asymptomatic carriers.

Adult T-cell leukemia/lymphoma in HTLV-1 non-endemic regions

BackgroundHTLV-1 infection is a neglected disease, despite producing neurological and lymphoproliferative severe illnesses and affect over 10 million people worldwide. Roughly 5% of HTLV-1 carriers develop Adult T-cell leukemia/lymphoma (ATLL), one of the most aggressive hematological malignancies. MethodsA national HTLV-1 register exists since 1989 in Spain, a non-endemic country with a large migrant flow from Latin America and Equatorial Africa, where HTLV-1 is endemic. The main features of all patients diagnosed with ATLL in Spain up to date are reported. ResultsA total of 451 cases of HTLV-1 infection had been reported in Spain until the end of year 2022. ATLL had been diagnosed in 35 (7.8%). The current average incidence of ATLL in Spain is of two cases per year. Women represent 57% of ATLL patients. Mean age at diagnosis was 47 years-old. Roughly 57% were Latin Americans and 26% Africans. At diagnosis, the majority presented with acute or lymphoma clinical forms. Survival was shorter than one year in most of them.Mean HTLV-1 proviral load was significantly greater in ATLL patients than in asymptomatic HTLV-1 carriers (2,305 vs 104 copies/104 PBMC). HTLV-1 subtyping in 6 ATLL patients found the 1a transcontinental variant (n = 4) and the Japanese variant (n = 2). All ATLL patients were negative for HIV-1, did not develop HTLV-1-associated myelopathy and were not transplant recipients. ConclusionThe rate of ATLL is very low in Spain and mostly associated to migrants from HTLV-1 endemic regions. Given the poor clinical outcome of ATLL, HTLV-1 testing should be performed at least once in all migrants coming from HTLV-1 endemic countries and in natives who have lived in or had sex partners from such regions.

Pentraxin 3, a serum biomarker in human T-cell lymphotropic virus type-1-associated myelopathy patients and asymptomatic carriers.

Human T-cell lymphotropic virus type 1 (HTLV-1) can induce a neuroinflammatory condition that leads to myelopathy. Pentraxin 3 (PTX3) is an acute-phase protein that its plasma concentration increases during inflammation. We aimed to determine whether PTX3 serum level is elevated in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients and HTLV-1 asymptomatic carriers (ACs) and evaluate its association with proviral load and clinical features. The serum level of PTX3 was measured using an enzyme-linked immunosorbent assay in 30 HAM patients, 30 HTLV-1 ACs, and 30 healthy controls. Also, the HTLV-1 proviral load was determined via real-time PCR technique. The findings showed that PTX3 serum level was significantly higher in HAM patients than in both asymptomatic carriers and healthy controls (p values < 0.0001). No correlation between PTX3 and the proviral load was observed in HAM patients and asymptomatic carriers (r = -0.238, p = 0.205 and r = -0.078, p = 0.681, respectively). The findings showed that there was no significant correlation between PTX3 and motor disability grading (MDG) (r = -0.155, p = 0.41) nor urinary disturbance score (UDS) (r = -0.238, p = 0.20). Higher levels of PTX3 are associated with HTLV-1-associated myelopathy compared to asymptomatic carriers. This finding may support the idea that PTX3 has the potential as a diagnostic biomarker.

Cognitive deficits in HTLV-1 patients.

Human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus known to be associated with adult T-cell lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Previous researches and brain imaging techniques have suggested cognitive abnormalities as well as brain damage in individuals infected with this virus. Given the insufficient amount of studies on how this virus can impact the affected person's cognition, we aimed to assess and compare the cognitive abnormalities of HAM/TSP patients, asymptomatic HTLV-1 carriers, and healthy controls. This cross-sectional study was conducted on 51 patients divided into 3 groups; a group of HAM/TSP patients, a group of asymptomatic HTLV-1 carriers, and an uninfected control group. Each group contained 17 members. The cognitive state of the studied population was assessed using the Mini-Mental State Exam (MMSE), Symbol Digit Modalities Test (SDMT), Rey-Osterrieth complex figure test (ROCF), the "Verbal Fluency Test" and the "Trail Making Test" (TMT) components of the Delis-Kaplan executive function system (D-KEFS) test, the Rey Auditory Verbal Learning Test (RAVLT), and digit span memory test. Patients diagnosed with HAM/TSP received significantly lower scores on the SDMT, ROCF, TMT, RAVLT, digit span memory test, and the orientation, calculation, and recall component of the MMSE assessment (p-value < 0.001). In addition, the asymptomatic HTLV-1 carriers obtained lower scores on the SDMT, ROCF, digit span memory test, and the orientation, calculation, and recall component of the MMSE assessment compared to the control group (p-value < 0.001). Overall, the findings suggest that HAM/TSP, or an asymptomatic infection with HTLV-1 could lead to cognitive deficits in the affected individuals. This can further emphasize the importance of assessing the cognitive function and psychiatric abnormalities of those infected with this virus.

Prevalence of sexual dysfunction in HTLV-1 patients without spastic paraparesis and the association with psychiatric symptoms.

The findings of previous studies are inconclusive in terms of psychological abnormalities and sexual function in asymptomatic human lymphotropic virus type 1 (HTLV-1) carriers. This study aimed to evaluate the prevalence of sexual dysfunction and its relationship with psychological abnormalities in asymptomatic HTLV-1 carriers. This cross-sectional study was conducted on asymptomatic HTLV-1 patients who were referred to the Neurology Clinic of a tertiary hospital in Mashhad, Iran. Patients with spastic paraparesis, leukemia, and uveitis, and those with an expanded disability status scale (EDSS) score higher than 2 were excluded. Sexual function in male and female subjects was evaluated using the brief male sexual function inventory (BMSFI) and female sexual dysfunction index (FSFI) questionnaires, respectively. The severity of psychological symptoms was evaluated in all patients using the symptom checklist-90-revised (SCL-90-R) questionnaire. A total of 117 patients (61 males and 56 females) with a mean age of 35.3 ± 6.3 years were evaluated. Overall, 50.9% of males had a high and 39.3% of females had a good sexual function. Both male and female patients with poor sexual function were older and had more children compared to those with good sexual function (P < 0.05). There was no significant difference in the distribution pattern of SCL-90 domains between patients with high and low to moderate sexual function among male patients (P > 0.05). Depression, hostility, interpersonal sensitivity, paranoid ideation, and psychological abnormality were significantly more prevalent in female patients with poor sexual function compared to those with good sexual function (P < 0.05). The prevalence of psychological abnormalities was high in female with sexual dysfunction and these disorders might have a negative effect on various dimensions of sexual function.

TNF-induced metalloproteinase-9 production is associated with neurological manifestations in HTLV-1-infected individuals.

HTLV-1-infected individuals may develop a neurologic inflammatory condition known as HTLV-1-associated myelopathy (HAM/TSP), in which the high production of TNF is observed. These patients exhibit higher proviral loads, enhanced production of proinflammatory cytokines and lymphocyte proliferation in comparison to asymptomatic HTLV-1 carriers and those presenting overactive bladder (OAB-HTLV-infected). Metalloproteinases (MMPs) are known to degrade the components of the blood-brain barrier, favoring the migration of infected cells into the central nervous system. Moreover, the unbalanced production of MMPs and their inhibitors (TIMPs) has also been associated with tissue damage. The present work studied the production of MMP-9 and TIMPs in HTLV-1-infected individuals with and without neurological manifestations. HAM/TSP patients presented higher concentrations of MMP-9 in peripheral blood mononuclear cell (PBMC) culture supernatants, as well as a higher MMP-9/TIMP-3 ratio when compared to the other groups studied. MMP-9 levels positively correlated with proviral load and TNF in OAB-HTLV-infected individuals, and the in vitro neutralization of TNF significantly decreased MMP-9 levels in PBMC culture supernatants. Our findings indicate an association between MMP-9 production and the proinflammatory state associated with HTLV-1 infection, as well as HAM/TSP.

Lessons from the Cerebrospinal Fluid Analysis of HTLV-1-Infected Individuals: Biomarkers of Inflammation for HAM/TSP Development.

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease that leads to motor impairment due to a chronic inflammatory process in the central nervous system (CNS). However, the HAM/TSP pathogenesis is not completely clear, and biomarkers to define the disease prognosis are still necessary. Thus, we aimed to identify biomarkers for HAM/TSP and potential mechanisms involved in disease development. To that end, the concentrations of VILIP-1, BDNF, VEGF, β-NGF, TGF-β1, fractalkine/CX3CL1, IL-6, IL-18, and TNF-α, and the soluble forms of TREM-1, TREM-2, and RAGE, were assessed using a multiplex bead-based immunoassay in paired cerebrospinal fluid (CSF) and serum samples from HAM/TSP patients (n = 20), asymptomatic HTLV-1 carriers (AC) (n = 13), and HTLV-1-seronegative individuals (n = 9), with the results analyzed according to the speed of HAM/TSP progression. HAM/TSP patients had elevated fractalkine in the serum but not in the CSF, particularly those with low neuroinflammatory activity (CSF/serum ratio of neopterin <1 and of CXCL10 < 2). HAM/TSP patients with normal CSF levels of neurofilament light chain (NfL) showed elevated β-NGF in serum, and serum BDNF levels were increased in HTLV-1-infected individuals, particularly in HTLV-1 AC. Both HTLV-1 AC and HAM/TSP patients had lower TGF-β1 levels in CSF compared to uninfected individuals, and HAM/TSP patients with active CNS inflammation showed higher CSF levels of IL-18, which correlated with markers of inflammation, neuronal death, and blood–brain-barrier permeability. Although none of the factors evaluated were associated with the speed of HAM/TSP progression, reduced TGF-β1 levels in CSF suggest that suppressive responses to control subclinical and/or active neurodegeneration are impaired, while increased CSF IL-18 indicates the involvement of inflammasome-mediated mechanisms in HAM/TSP development.

Chitotriosidase 1 in the cerebrospinal fluid as a putative biomarker for HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) progression

Human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurodegenerative disease that affects motor, urinary, intestinal, and sensory functions. Typically, HAM/TSP is slowly progressive, but it may vary from limited motor disability after decades (very slow progression) to loss of motor function in a few years from disease onset (rapid). In this study, we aimed to identify prognostic biomarkers for HAM/TSP to support patient management. Thus, proteomic analysis of the cerebrospinal fluid (CSF) was performed with samples from HTLV-1 asymptomatic carriers (AC) (n=13) and HAM/TSP patients (n=21) with rapid, typical, and very slow progression using quantitative label-free liquid chromatography/tandem mass spectrometry. Enrichment analyses were also carried out to identify key biological processes associated with distinct neurological conditions in HTLV-1 infection. Candidate biomarkers were validated by ELISA in paired CSF and serum samples, and samples from HTLV-1-seronegative individuals (n=9) were used as controls. CSF analysis identified 602 proteins. Leukocyte/cell activation, immune response processes and neurodegeneration pathways were enriched in rapid progressors. Conversely, HTLV-1 AC and HAM/TSP patients with typical and very slow progression had enriched processes for nervous system development. Differential expression analysis showed that soluble vascular cell adhesion molecule 1 (sVCAM-1), chitotriosidase 1 (CHIT1), and cathepsin C (CTSC) were upregulated in HAM/TSP. However, only CHIT1 was significantly elevated after validation, particularly in HAM/TSP rapid progressors. In contrast, none of these biomarkers were altered in serum. Additionally, CSF CHIT1 levels in HAM/TSP patients positively correlated with the speed of HAM/TSP progression, defined as points in the IPEC-2 HAM/TSP disability scale per year of disease, and with CSF levels of phosphorylated neurofilament heavy chain, neopterin, CXCL5, CXCL10, and CXCL11. In conclusion, higher CSF levels of CHIT1 were associated with HAM/TSP rapid progression and correlated with other biomarkers of neuroinflammation and neurodegeneration. Therefore, we propose CHIT1 as an additional or alternative CSF biomarker to identify HAM/TSP patients with a worse prognosis.

Association of HTLV-1 infection prevalence with TLR7 single nucleotide polymorphisms (rs179008 & rs179009) in asymptomatic blood donors in Khorasan Province of Iran

BackgroundHuman T-lymphotropic virus type 1 (HTLV-1) cause two diseases in humans such as adult T-cell leukemia/lymphoma (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). About, 15–20 million persons are infected with HTLV-1 in worldwide. Single nucleotide polymorphisms (SNPs) are a genetic variation that occurs in >1% of the general population; they can alter promoter activities, gene expression, mRNA stability, and protein structure and function.Our aim in this study was to investigate the association between SNPs within Toll like receptor 7 (TLR7) (rs179008 and rs179009) and susceptibility to HTLV-1 infection among asymptomatic blood donors in Iran. MethodsA total of 100 asymptomatic HTLV-1 carriers and 118 healthy blood donors were studied. After extracting genomic DNA from all samples, amplification was performed using specific primers. Then, the restriction fragmentation length polymorphism (RFLP) method was used to evaluate the SNPs within TLR7. Data analysis was administered using SPSS version 22. ResultsThe frequency of the AA, AT, TT genotypes of TLR7 (rs178008) was 73%, 22%, and 5% in the patient group; while for those in the control group the values were 50%, 40.7%, and 9.3%, respectively. Moreover, the frequency of the TLR7 rs179008 A allele was 84% in the HTLV-1 patients group and 69.6 % in the control group. Also, the two groups were significantly different concerning the frequency distribution of the TLR7 (rs178008) genotypes and alleles (p = 0.001). ConclusionThere was a significant association between TLR7 (rs178008) SNP and HTLV-1 infection and the T allele of TLR7 (rs178008) SNP might be considered as a protective factor against HTLV-1 virus infection although additional evidence is needed.

Late presentation of human T-lymphotropic virus type 1 infection in Spain reflects suboptimal testing strategies

ObjectivesAlthough only 10% of persons infected with human T-lymphotropic virus type 1 (HTLV-1) may develop virus-associated illnesses over their lifetime, missing the earlier diagnosis of asymptomatic carriers frequently leads to late presentation. MethodsA nationwide HTLV-1 register was created in Spain in 1989. We examined the main demographics and clinical features at the time of the first diagnosis for more than three decades. ResultsA total of 428 individuals infected with HTLV-1 had been reported in Spain until the end of 2021. Up to 96 (22%) individuals presented clinically with HTLV-1-associated conditions, including subacute myelopathy (57%), T-cell lymphoma (34%), or Strongyloides stercoralis infestation (8%). Since 2008, HTLV-1 diagnosis has been made at blood banks (44%) or clinics (56%). Native Spaniards and Sub-Saharan Africans are overrepresented among patients presenting with HTLV-1-associated illnesses suggesting that poor epidemiological and/or clinical suspicion, which led to the late presentation are more frequent in them than carriers from Latin America (LATAM) (31.7% vs 20.4%, respectively; P = 0.015). ConclusionHTLV-1 infection in Spain is frequently diagnosed in patients presenting with characteristic illnesses. Although screening in blood banks mostly identifies asymptomatic carriers from LATAM, a disproportionately high number of Spaniards and Africans are diagnosed too late at the time of clinical manifestations. Expanding testing to all pregnant women and clinics for sexually transmitted infections could help to unveil HTLV-1 asymptomatic carriers.

Possible deterioration of Apolipoproteins expression by HTLV-1 infection in favor of infected leukemic cells in adult T-cell leukemia/lymphoma (ATLL)

BackgroundIt has been suggested that human T-cell lymphotropic virus type 1 (HTLV-1) infection can affect the lipid profile. Therefore, in this study, the main apolipoproteins and HTLV-1 regulatory factors were assessed in infected subjects. Methods and resultsA cross-sectional study was performed on 12 adult T-cell leukemia/lymphoma (ATLL) and 10 HAM/TSP patients, and 10 HTLV-1 asymptomatic carriers (ACs). The peripheral blood mononuclear cells (PBMCs) were assessed for proviral load (PVL) measurement and the expression of HBZ, ABCA, APOA, and APOE, using the qRT-PCR, Taqman method.The ABCA1 expression level in the ATLL group was significantly higher than ACs and HAM/TSP, (P = 0.034 and P = 0.019, respectively). The APOA1 expression, in ATLL (0.89 ± 0.30) and HAM/TSP patients (0.31 ± 0.25), was lower than HTLV-1 ACs (1.15 ± 0.63); but not significant. The APOE expression in the ACs' group (22.66 ± 7.88) was strongly higher than ATLL (0.003 ± 0.0004) and HAM/TSP (1.81 ± 0.60) subjects (P = 0.007 and P = 0.012, respectively); ATLL and HAM/TSP groups had no significant differences (P = 0.957). All of the ATLL patients expressed HBZ, but not Tax, on the other hand more than 70% of HAM/TSPs and ACs expressed Tax but only one AC and one HAM/TSP patient expressed HBZ. The PVL median in ATLL patients was higher (2983.87 ± 1797.07copies/104PBMCs), than ACs (17.00 ± 10.00copies/104) (P = 0.016), and HAM/TSP (314.00 ± 185.00) (P = 0.05). ConclusionsThe HBZ probably altering the expression of ABCA1 and APOE, which particularly, can deteriorate the lipid profile in ATLL patients. The importance of APOE in healthy condition and its negative correlation with PVL in ATLL patients could be a novel target in therapy.

Cognitive screening in HTLV-1-infected people using a self-perceived memory score and auditory P300.

The HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is the most common neurological manifestation associated with human T-cell lymphotropic virus type-1 (HTLV-1) infection. Although cognitive impairment has been highlighted in the spectrum of HTLV-1 neurological manifestations, it may go unnoticed in those who do not spontaneously report it. We aimed at evaluating the applicability of a self-perceived memory score (SMS) and the cognitive event-related potential (P300) for early detection of cognitive impairment in HTLV-1-infected people. The SMS was measured by a 0-10 point numeric scale combined with a sad-happy face rating scale. The higher the number, the better was the SMS. The P300 was obtained through an oddball paradigm with a mental counting task. The participants were 15 (21.4%) individuals with HAM/TSP, 20 (28.6%) HTLV-1 asymptomatic carriers, and 35 (50%) seronegative controls. We found that SMS (p < 0.001) and P300 latency (p < 0.001) got progressively worse from the seronegative controls to the asymptomatic carriers and then to the HAM/TSP. The results that indicated cognitive impairment were SMS < 7.2 points and P300 latency > 369.0ms. The HAM/TSP group showed the highest prevalence of altered P300 (80%) and SMS (87%). Interestingly, the asymptomatic group also presented significantly higher prevalence of altered SMS (60%) and P300 (35%) when compared to controls (< 10%). The frequency of cognitive impairment was 16 times higher in the HTLV-1 asymptomatic group and 69 times higher in the HAM/TSP group when compared to controls. The use of SMS in the medical consultation was a useful and easy-to-apply method to screen HTLV-1-infected subjects for everyday memory complaints.

Immune Senescence-Related Gene Expression Profile in CD4+ T-Lymphocytes of HTLV-1 Asymptomatic Carriers and Patients with Adult T-Cell Leukemia/Lymphoma (ATLL): A Brazilian Preliminary Study

Introduction: Adult T-cell leukemia/lymphoma (ATLL) is a rare and aggressive neoplasm caused by the human T-lymphotropic virus type 1 (HTLV-1). It is estimated, worldwide, that at least 5-10 million people carry HTLV-1 and 2-5% out of them will develop ATLL. Previously, our group demonstrated an increase of cells in G0/G1 phase of cell cycle and aneuploidy in CD4+ T-cells in HTLV-1 asymptomatic carriers [1]. These findings may reflect an adverse intracellular environment, caused by genetic stress due to viral particles inserted into host DNA. Intracellular mechanisms aiming to control and prevent replication of cells carrying genetic aberrations in genes involved in cell cycle regulation, DNA repair and apoptosis could be activated to hold cell division while DNA damage is repaired. Based on this hypothesis, delay of cell cycle in G0/G1 may be a step in the process of oncogenesis [1]. Herein, we did a pilot study in order to analyze the pattern of expression of a set of genes involved in cell cycle control and senescence in CD4+ T-lymphocytes of HTLV-1 infected individuals searching for additional genetic abnormalities in this setting.Methods: Peripheral blood samples were tested obtained from five HTLV-1 asymptomatic carriers and four ATLL patients for this pilot study. T-CD4+ cells were isolated in magnetic column followed by RNA extraction. Subsequently, it was converted into cDNA for the assays of the real-time quantitative polymerase chain reaction (qRT-PCR) in microplates of 96 wells previously customized with 44 senescence related genes pursued from ThermoFischer Scientific. A pool of five samples from healthy individuals (control group) was used as a calibrator. RNA transcription was measured using 7500 Fast real time PCR system (Applied Biosystems) and data were collected by the 7500 software v2.0.5 (Applied Biosystems). The expression levels of the target genes were calculated using the Livak and Schmittgen (2001) method [2]. The mRNA expression was normalized using the β-glucuronidase (GUSB) gene (Applied Biosystems, cod. Hs99999908_m1) and those genes with expression ≥ 2x or ≤ 2x in comparison to control group were considered as differentially expressed and were chosen to be validated in a secondary cohort of thirty HTLV-1 infected individuals.Results: In HTLV-1 asymptomatic carriers the median age was 55 years (37-62 years) and 20% (1/5) of them were males, in ATLL patients: 45 years (38-66 years) and 100% (4/4) were females, while the control group had median age of 43 years (22-62 years) and 60% (3/5) were males. Among the ATLL group, 50% (2/4) were classified as smoldering, 25% (1/4) were acute and 25% (1/4) were lymphomatous, according to Shimoyama classification [3]. COL3A1, SPARC and TWIST1 genes were overexpressed and CDKN1A, ID1, IFNG and TERT were suppressed in HTLV-1 asymptomatic carriers when compared to healthy controls. HTLV-1 infected and ATLL patients showed TWIST1 and COL3A1 genes overexpressed and IFNG, CDKN1A and TERT repressed (Figure 1 and 2). The ALDH1, FN1, NOX4 and COL1A1 genes did not amplified.Conclusion: These preliminaries results showed for the first time that HTLV-1 asymptomatic carriers present a set of genes differentially expressed in T-CD4+ cells, especially SPARC, TWIST1, COL3A1, CDNK1A and IFNG. These results will be confirmed in a validation cohort. We expected that this data shed some light on the comprehension of the cell microenvironment of the T-CD4+ lymphocyte in HTLV-1 asymptomatic carriers. Furthermore, we will be able to understand potential mechanisms associated with leukemogenesis in chronic infection by this virus, explaining eventual progression to ATLL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Single-Cell RNA Sequencing Unveils the Hallmarks of Populations at Different Stages of HTLV-1 Infection

BackgroundAdult T-cell leukemia-lymphoma (ATL) is an aggressive mature T-cell neoplasm caused by human T -cell leukemia virus type 1 (HTLV-1). Up to 5% of infected individuals develop to ATL. HTLV-1 preferentially infects CD4 + T cells, and stimulates cell proliferation and prevents cell death by apoptosis. The viral oncogene-encoded proteins, Tax and HBZ, play important roles in viral infection and cell immortalization. However, the host factor of developing from carrier to patient is not clear.ResultsTo characterize the heterogeneity of ATL patients, we performed single-cell RNA-sequencing (10x Genomics) analysis on single cell suspensions isolated from PBMCs of nine samples, including three ATL patients, three HTLV-1 asymptomatic carriers as well as three healthy donors (HD). We acquired 82977 high-quality cells with a median of 1718 genes detected per cell. Unsupervised clustering using Seurat followed by visualization in t-Stochastic Neighbor Embedding (t-SNE) identified 29 distinct cell clusters (Figure 1A). The single cell profiling of ATL patients were significantly different from that of carriers and healthy donors, while the latter two had little difference (Figure 1B). Based on singleR packages and marker genes of each cluster, 4 major cell populations (T cells, NK cells, B cells and myeloid cells) and other rare cell types were annotated, such as erythrocyte cluster and eosinophils cluster.We observed an enrichment of CD4 + T cell from patients in 4 cluster (Figure 1C), which proportion of cells was higher than that of carriers and healthy donors. According to cell type annotation, cells from cluster 11 were CD4 + CD25 + Foxp3 + Treg cells. Based on the increasing proportion of cluster 11 in healthy people, carriers and patients, without significant statistical differences, we assumed that Foxp3 + Treg cells were involved in the evolution of ATL tumor cells. That was identical with published literatures.To investigate the differences between tumor and normal CD4 + T cell, the gene expression was compared among 7 clusters of CD4 + T cell from three groups. Using a threshold of p value < 0.05 and | fold change| >2. Through integrated analysis, we identified 26 commonly upregulated genes (gene expression level: patients > carriers > HD) and 9 downregulated genes (gene expression level: patients < carriers < HD. To further analyze the biological function of the common DEGs, gene ontology (GO) analysis showed that these genes could be mainly categorized into plasma membrane and protein binding.Subsequently, we validated the mRNA expression level of upregulated common DEGs among three groups by qRT-PCR. The isolated CD4 + T cell using CD4 microbeads of a total of 6 patients, 3 carriers and 9 normal specimens were included. The result showed that the mRNA expression levels of gene CADM1 and RGS13 in patients were higher than those in carriers and healthy donors, although there was no statistical difference between patients and carriers, and the expression levels of carriers tended to be higher than those in normal people (Figure 1D and E). Previously, CADM1 has been revealed to be highly expressed in HTLV‐1‐infected CD4 + T cells. Our study confirmed this result by single-cell profiling. RGS13, a member of the regulators of G protein signaling (RGS) family, participates in cellular communication. The role of RGS13 in ATL needs to be investigated.ConclusionsThis study is the first time to analyze the single-cell RNA level of ATL patients, HTLV-1 virus carriers and normal people. The peripheral blood cell composition of the patient is significantly different from that of the carriers and healthy donors, while it is similar between carriers and normal people. CD4 + T cells are the main cell population of patients. The proportion of CD4 + CD25 + Foxp3 + Treg cells increased gradually in healthy people, carriers and patients. DEGs analysis showed that CADM1 and RGS13 were highly expressed in CD4 + T cells of patients, followed by carriers, validated by 18 clinical samples. However, the molecular mechanism of RGS13 in the process from HTLV-1 infection to ATL needs to be further studied. [Display omitted] DisclosuresHu: Astellas Pharma, Inc.: Research Funding.