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An elevated human T cell lymphotropic virus 1 (HTLV)-1 proviral load (PVL) is the main risk factor for developing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in HTLV-1 infected subjects, and a high cerebrospinal fluid (CSF) to peripheral blood mononuclear cell (PBMC) PVL ratio may be diagnostic of the condition. However, the...
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Proviral load (PVL) is one of the determining factors for the pathogenesis and clinical progression of the human T-lymphotropic virus type I (HTLV-1) infection. In the present study, we optimized a sensitive multiplex real-time PCR for the simultaneous detection and quantification of HTLV-1 proviral load and beta-globin gene as endogenous control....
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... These quantitative expression data are also consistent with ELISA findings (Fig. 2F). These findings were validated by Intra-and Inter-Assay Variability factor by Evaluating variation within (intra-assay) and between (inter-assay) different runs or experiments 49 . As low Coefficient of Variation (CV) values indicate that repeated measurements of the same sample yield consistent results of RT-qPCR and ELISA and these method show high reproducibility based on low CV etc. lastly Linearity and Accuracy was also analyzed to validate the results by Constructing standard curves using known standards or controls with a high correlation coefficient (R-squared) these results also conducted measurements with an appropriate number of replicates to ensure statistical significance and robustness of these findings through t-tests, ANOVA, or regression analysis, by considering significant p-values 50 . ...
Glioblastoma multiforme (GBM) IDH-wildtype is the most prevalent brain malignancy in adults. However, molecular mechanisms, which leads to GBM have not been completely elucidated. Granulocyte colony-stimulating factor (GCSF), Granulocyte colony-stimulating factor receptor GCSFR, and Signal transducers and activators of transcription 3 (STAT3) have been involved in the occurrence and development of various cancers, but their role in GBM is little known. Herein, we have investigated the gene and protein expression of GCSF, GCSFR, and STAT3 in 21 tissue biopsy samples and also in tumor associated normal tissue (TANT) samples derived from glioblastoma patients, which revealed significantly differential expression of these genes. To validate our findings, we performed a comprehensive integrated analysis of transcriptomic and proteomic profiling of respective genes by retrieving GBM RNA-sequence data from Genome Atlas Databases. GO and KEGG analysis revealed enrichment in disease-related pathways, such as JAK/STAT pathway activation, which were associated with GBM progression. We further performed computational docking analysis of potential drug candidate Nisin against GCSF, and the results were validated in vitro through cytotoxic activity assay using a human glioblastoma cell line SF-767 in a dose-dependent manner. Our comprehensive analysis reveals that GCSF augments glioma progression, and its blockade with anticancer bacteriocin peptide Nisin can potentially inhibit the growth and metastasis of GBM.
... In AC hPVL and HAM groups, there was a 2-fold higher frequency of Tax expression in DP cells as compared with CD4 + SP T cells (6.65% and 8.84% Tax + cells in CD4 + SP T cells in AC hPVL and HAM, respectively, compared with 14.1% and 18.3% Tax + cells in DP T cells, respectively). Although Tax is expressed dynamically by infected cells (1,31), in our experimental setting, Tax expression in T cells after 12 hours in culture strongly correlated with the PVL of sorted CD4 + SP, CD8 + SP, DP, and DN T cells measured by digital droplet PCR (ddPCR; Figure 1E) (32); this allowed us to use Tax expression at 12 hours as a reliable readout for PVL and infection rate. Given that Tax expression correlated with PVL in all T cell populations, the high levels of Tax expression in DP T cells were not due to different Tax expression dynamics in DP T cells ( Figure 1E). ...
Human T cell leukemia virus type 1 (HTLV-1) is a retrovirus with preferential CD4+ T cell tropism that causes a range of conditions spanning from asymptomatic infection to adult T cell leukemia and HTLV-1-associated myelopathy (HAM), an inflammatory disease of the CNS. The mechanisms by which HTLV-1 induces HAM are poorly understood. By directly examining the ex vivo phenotype and function of T cells from asymptomatic carriers and patients with HAM, we show that patients with HAM have a higher frequency of CD4+CD8+ double-positive (DP) T cells, which are infected with HTLV-1 at higher rates than CD4+ T cells. Displaying both helper and cytotoxic phenotypes, these DP T cells are highly proinflammatory and contain high frequencies of HTLV-1-specific cells. Mechanistically, we demonstrate that DP T cells arise by direct HTLV-1 infection of CD4+ and CD8+ T cells. High levels of CD49d and CXCR3 expression suggest that DP T cells possess the ability to migrate to the CNS, and when cocultured with astrocytes, DP T cells induce proinflammatory astrocytes that express high levels of CXCL10, IFN-γ, and IL-6. These results demonstrate the potential of DP T cells to directly contribute to CNS pathology.
... Hence unlike qPCR assay, dPCR assay is a direct quantification method and, it does not rely on an external calibration curve. Moreover, dPCR assay has a higher precision and sensitivity in detecting and quantifying target nucleic acid in samples with low target copies (Brunetto et al., 2014;Campomenosi et al., 2016;Renault et al., 2022;Tang et al., 2016) and it is comparatively less susceptible to potential PCR inhibitors Huggett et al., 2008;Rački et al., 2014). ...
... However, these methods may not always detect mutations at very low frequencies: i.e., when drug-resistant viruses are present as a small proportion of the total virus population in a clinical isolate (22,23). Technologies such as droplet digital PCR (ddPCR) and next-generation sequencing (NGS) may provide a more sensitive means for detection of viral mutations associated with drug resistance in clinical specimens and isolates (22,24,25). NGS has been deployed to detect nucleotide mutations encoding PA I38F/M/S/T substitutions that may exist as minor variants in clinical isolates (26)(27)(28). ...
Baloxavir marboxil (baloxavir) is a recently FDA-approved influenza virus polymerase acidic (PA) endonuclease inhibitor. Several PA substitutions have been demonstrated to confer reduced susceptibility to baloxavir; however, their impacts on measurements of antiviral drug susceptibility and replication capacity when present as a fraction of the viral population have not been established. We generated recombinant A/California/04/09 (H1N1)-like viruses (IAV) with PA I38L, I38T, or E199D substitutions and B/Victoria/504/2000-like virus (IBV) with PA I38T. These substitutions reduced baloxavir susceptibility by 15.3-, 72.3-, 5.4-, and 54.5-fold, respectively, when tested in normal human bronchial epithelial (NHBE) cells. We then assessed the replication kinetics, polymerase activity, and baloxavir susceptibility of the wild-type:mutant (WT:MUT) virus mixtures in NHBE cells. The percentage of MUT relative to WT virus necessary to detect reduced baloxavir susceptibility in phenotypic assays ranged from 10% (IBV I38T) to 92% (IAV E199D). While I38T did not alter IAV replication kinetics or polymerase activity, IAV PA I38L and E199D MUTs and the IBV PA I38T MUT exhibited reduced replication levels and significantly altered polymerase activity. Differences in replication were detectable when the MUTs comprised ≥90%, ≥90%, or ≥75% of the population, respectively. Droplet digital PCR (ddPCR) and next-generation sequencing (NGS) analyses showed that WT viruses generally outcompeted the respective MUTs after multiple replication cycles and serial passaging in NHBE cells when initial mixtures contained ≥50% of the WT viruses; however, we also identified potential compensatory substitutions (IAV PA D394N and IBV PA E329G) that emerged and appeared to improve the replication capacity of baloxavir-resistant virus in cell culture. IMPORTANCE Baloxavir marboxil, an influenza virus polymerase acidic endonuclease inhibitor, represents a recently approved new class of influenza antivirals. Treatment-emergent resistance to baloxavir has been observed in clinical trials, and the potential spread of resistant variants could diminish baloxavir effectiveness. Here, we report the impact of the proportion of drug-resistant subpopulations on the ability to detect resistance in clinical isolates and the impact of substitutions on viral replication of mixtures containing both drug-sensitive and drug-resistant variants. We also show that ddPCR and NGS methods can be successfully used for detection of resistant subpopulations in clinical isolates and to quantify their relative abundance. Taken together, our data shed light on the potential impact of baloxavir-resistant I38T/L and E199D substitutions on baloxavir susceptibility and other biological properties of influenza virus and the ability to detect resistance in phenotypic and genotypic assays.
... Clonal proliferation of HTLV-1-infected cells is crucial for disease development. Indeed, patients with HAM/TSP have a higher proviral load (PVL) in the cerebrospinal fluid (CSF) as well as in the peripheral blood (4)(5)(6), and infected cells aggregate in the spinal cord lesion (7,8). These HTLV-1-infected T cells express viral proteins that lead to the activation and expansion of HTLV-1-specific CD8 + T lymphocytes (CTLs) (9,10), which subsequently produce proinflammatory cytokines and chemokines and may cause bystander damage to neurons and glial cells in patients with HAM/TSP. ...
... TCR repertoire analysis can detect long-lived, proliferating HTLV-1-infected clones and may be a surrogate marker of disease monitoring and treatment response. The PVL is higher in the CSF than in peripheral blood (5,6), and accumulation of infected cells has been identified in the spinal cord lesion in patients with HAM/TSP (7,8). HTLV-1-infected cells produce INF-γ that stimulates astrocytes to secret the chemokine CXCL10, which recruits more CXCR3 + T cells -including infected cells -into the CNS, leading to an inflammatory response and neural damage (36). ...
Human T lymphotropic virus type 1-assoicated (HTLV-1-associated) myelopathy/tropical spastic paraparesis (HAM/TSP) is a neuroinflammatory disease caused by the persistent proliferation of HTLV-1-infected T cells. Here, we performed a T cell receptor (TCR) repertoire analysis focused on HTLV-1-infected cells to identify and track the infected T cell clones that are preserved in patients with HAM/TSP and migrate to the CNS. TCRβ repertoire analysis revealed higher clonal expansion in HTLV-1-infected cells compared with noninfected cells from patients with HAM/TSP and asymptomatic carriers (ACs). TCR clonality in HTLV-1-infected cells was similar in patients with HAM/TSP and ACs. Longitudinal analysis showed that the TCR repertoire signature in HTLV-1-infected cells remained stable, and highly expanded infected clones were preserved within each patient with HAM/TSP over years. Expanded HTLV-1-infected clones revealed different distributions between cerebrospinal fluid (CSF) and peripheral blood and were enriched in the CSF of patients with HAM/TSP. Cluster analysis showed similarity in TCRβ sequences in HTLV-1-infected cells, suggesting that they proliferate after common antigen stimulation. Our results indicate that exploring TCR repertoires of HTLV-1-infected cells can elucidate individual clonal dynamics and identify potential pathogenic clones expanded in the CNS.
... 8,9 A high proviral load (PVL) and a PVL greater in the CSF than in the blood are risk factors of developing HAM. 10 The PVL and the host's immune response are affected by factors such as the host's human leukocyte antigen genotype and singlenucleotide polymorphisms. 11 The CSF concentrations of the cytokines neopterin and CXCL10 (IP-10) correlate with the rate of disease progression in HAM. ...
Purpose of Review
Human T-cell lymphotropic virus type 1 (HTLV-1)–associated myelopathy (HAM) is a well-recognized neurologic complication of HTLV-1. Beyond HAM, several other neurologic manifestations are increasingly recognized, including acute myelopathy, encephalopathy, and myositis. The clinical and imaging features of these presentations are less well understood and potentially underdiagnosed. In this study, we summarize the imaging features of HTLV-1–related neurologic disease, providing both a pictorial review and pooled series of the less well-recognized presentations.
Recent Findings
35 cases of acute/subacute HAM and 12 cases of HTLV-1–related encephalopathy were found. In subacute HAM, cervical and upper thoracic longitudinally extensive tranverse myelitis was noted, while in HTLV-1–related encephalopathy, confluent lesions in the frontoparietal white matter and along the corticospinal tracts were the most prevalent finding.
Summary
There are varied clinical and imaging presentations of HTLV-1–related neurologic disease. Recognition of these features aids early diagnosis where therapy may have the greatest benefit.
... The pathophysiology of HAM is not well understood; nonetheless, previous studies reported that host genetic and immunological factors can be correlated with the disease. PVLs are commonly higher among HAM patients than in asymptomatic individuals, and a high PVL suggests a higher risk of inflammatory diseases (Brunetto et al, 2014;Nagai et al, 2001;Primo et al, 2009). Regarding the presence of the polymorphism in the promoter region of FOXP3, in this study, carriers of the GG genotype had higher PVLs. ...
Human T lymphotropic virus 1 (HTLV-1) is a retrovirus associated with inflammatory diseases, including HTLV-1-associated myelopathy (HAM), and host genetic factors may be involved in disease evolution. The forkhead Box P3 (FOXP3) transcription factor is linked to homeostasis of the immune system, and the presence of polymorphisms in the promoter region of the FOXP3 gene should reflect its expression levels and consequent activation of regulatory T cells, which may contribute to severe inflammatory disorders, such as HAM. This study evaluated the rs2232365 polymorphism (-924 A/G) located in the promoter region of the FOXP3 gene and its association with HAM. Forty DNA samples from asymptomatic carriers and 25 samples from HAM patients were used, in addition to 130 control samples. The polymorphism was genotyped by conducting real-time polymerase chain reaction (PCR) (quantitative PCR [qPCR]) on extracted DNA. The proviral loads (PVLs) and CD4+ and CD8+ T lymphocyte counts were determined by qPCR and FACSCalibur flow cytometry, respectively. The PVLs, CD4+ T lymphocyte concentrations, and tumor necrosis factor-α dosages were considered predictive factors of the clinical profiles of HTLV-1 infection, all of which had higher levels in the HAM group. Carriers of the GG genotype for the polymorphism rs2232365 had high PVLs and CD4+ T lymphocyte concentrations.
... primer sets, can only target a limited number of pathogens, and have a high demand for sample volume. 128,129 Compared to the above methods, metagenomic next-generation sequencing(mNGS) is an unbiased high-throughput sequencing technique that can, in principle, detect all pathogens in clinical samples in a short period of time. 130 It can also provide information on antibiotic resistance by comparing sequenced genes in microorganisms against antibiotic resistance databases. ...
Ocular lesions due to Brucella infection are uncommon and easily overlooked in clinical management, but must be differentiated from non-infectious eye diseases and treated promptly to protect the patient’s vision. We reviewed the relevant literature and identified 47 patients with ocular complications of Brucella infection. Among them, 28 showed ocular neuropathy, 15 presented with uveitis, and four patients displayed other ocular symptoms. Ocular symptoms accompanying Brucella infection require prompt diagnosis and treatment. The main methods of diagnosis are intraocular fluid tests and blood tests. Early diagnosis and treatment with suitable antibiotics are central to protecting the patient’s vision. Notably, in terms of mechanism of injury, Brucella infection is chronic and cannot be eliminated by phagocytes, and can cause damage to the eye by inducing autoimmune reactions, antigen-antibody complex production, release of endogenous and exogenous toxins, and bacterial production of septic thrombi in the tissues. In this review, we summarize the ocular symptoms, diagnosis, treatment and prognosis of Brucella infection, and discuss the mechanisms of Brucella in ocular lesions, providing a reference for the diagnosis and treatment of Brucella ocular lesions.
... HAM/TSP is a progressive, chronic inflammatory myelopathy of the central nervous systems (CNS) (28). HTLV-1 PVL in PBMCs is higher in HAM/TSP than in asymptomatic carriers (29) and has been shown to be significantly elevated in HAM/TSP cerebrospinal fluid (CSF) cells than in PBMCs (30,31). These observations have led to the hypothesis that an increased HTLV-1 PVL is associated with an increased risk of HAM/TSP disease progression. ...
Human T lymphotropic virus 1 (HTLV-1) is a human retrovirus identified as the causative agent in adult T-cell leukemia/lymphoma (ATL) and chronic-progressive neuroinflammatory disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 is estimated to infect between 5-20 million people worldwide, although most infected individuals remain asymptomatic. HTLV-1 infected persons carry an estimated lifetime risk of approximately 5% of developing ATL, and between 0.25% and 1.8% of developing HAM/TSP. Most HTLV-1 infection is detected in CD4⁺ T cells in vivo which causes the aggressive malignancy in ATL. In HAM/TSP, the increase of HTLV-1 provirus induces immune dysregulation to alter inflammatory milieu, such as expansion of HTLV-1-specific CD8⁺ T cells, in the central nervous system of the infected subjects, which have been suggested to underlie the pathogenesis of HAM/TSP. Factors contributing to the conversion from asymptomatic carrier to disease state remain poorly understood. As such, the identification and tracking of HTLV-1-specific T cell biomarkers that may be used to monitor the progression from primary infection to immune dysfunction and disease are of great interest. T cell receptor (TCR) repertoires have been extensively investigated as a mechanism of monitoring adaptive T cell immune response to viruses and tumors. Breakthrough technologies such as single-cell RNA sequencing have increased the specificity with which T cell clones may be characterized and continue to improve our understanding of TCR signatures in viral infection, cancer, and associated treatments. In HTLV-1-associated disease, sequencing of TCR repertoires has been used to reveal repertoire patterns, diversity, and clonal expansions of HTLV-1-specific T cells capable of immune evasion and dysregulation in ATL as well as in HAM/TSP. Conserved sequence analysis has further been used to identify CDR3 motif sequences and exploit disease- or patient-specificity and commonality in HTLV-1-associated disease. In this article we review current research on TCR repertoires and HTLV-1-specific clonotypes in HTLV-1-associated diseases ATL and HAM/TSP and discuss the implications of TCR clonal expansions on HTLV-1-associated disease course and treatments.
... [18][19][20] More recently, digital droplet PCR has been added to the quantification techniques, but it is not widely available. [21][22][23] In Japan, a study was conducted to replace the WB assay with LIA, which improved specificity, but this possibility needs further evaluation, especially for costeffectiveness. 24 Although a commercial HTLV PCR assay is available for qualitative assessment, in most cases, these assays are developed in house, making comparisons between laboratories difficult, which is compounded by the lack of standards and external quality assurance. ...
HTLV-1-associated myelopathy (HAM) is a chronic disabling inflammatory disorder of the spinal cord caused by HTLV-1 infection. The diagnosis of HAM is based on clinical and laboratorial findings. The disease is characterized by the presence of spastic paraparesis associated with the demonstration of anti-HTLV-1 antibodies or HTLV-1 genomes in blood and cerebrospinal fluid (CSF). New inflammatory markers have been proposed for the diagnosis and assessment of the prognosis of HAM. We reviewed the laboratory diagnostic and potential surrogate markers for HAM. The serological screening tests for detection of anti-HTLV-1/2 antibodies are highly sensitive and specific, but confirmation and typing of HTLV-1 or HTLV-2 infection by other serology or molecular methods is essential. Detection of intrathecal anti-HTLV-1 antibodies and/or quantification of HTLV-1 provirus in CSF provide additional evidence for diagnosis especially in atypical cases or where alternative causes of neuroinflammation cannot be excluded. Chemokine ligand 10 (CXCL10) and neopterin in serum and CSF are now emerging as inflammatory markers with prognostic value and for HAM monitoring management. In addition, measures of neurodegeneration such as neurofilament light chain in CSF and blood may also contribute to the HAM prognosis. This review is useful for clinicians and researchers evaluating potential benefits and limitations of each biomarker in the clinical practice. The advent of new markers makes it necessary to update the criteria on the best evidence-based approach and worldwide consensus to the use of diagnostic and surrogate markers for HAM.
