To perform a systematic review to describe the available findings on clinical outcomes in HIV-1 and HTLV-1/HTLV-2 co-infected individuals since 1995.This Systematic Review used PECO criteria follow by PRISMA reporting guidelines and registered as CRD42021279062 (Prospero database). The Newcastle-Ottawa Scale assessed the methodological quality of included studies.A systematical search in PubMed/MEDLINE, Embase, Web of Sciences databases for cross-sectional, case-control, or cohort studies design to identify clinical and laboratorial outcomes related to HIV-1 and HTLV-1/2 coinfection. Search strategy: [(“HIV-1” AND “HTLV-1” OR “HTLV-2”) AND (“Coinfection”) AND (1990/01/01:2021/12/31[Date- Publication])].A total of 15 articles were included on this systematic review describing data of 2,566 mono and coinfected patients, 58% male, with mean age was 35.7 ± 5.7 years. HIV-1 and HTLV-1 coinfected patients were more likely to had shorter survival and faster progression to death or mortality than monoinfected ones. Coinfected had higher CD4 cell counts and less likelihood of ART use. In addition, higher frequency of diseases like ichthyosis (22.2 vs. 6.8%), scabies (18.6 vs. 0%), candidiasis (42 vs. 12%), Strongyloidiasis (15.4 vs. 2%) and neurological manifestations like encephalopathy, peripheral neuropathy and HAM/TSP were more frequently reported in coinfected patients.HIV-1 and HTLV-1 coinfection and HIV-1 and HTLV-1 /2 triple coinfection were related to shorter survival, higher mortality rate, and faster progression to death, while coinfection by HIV-1/HTLV-2 seems to have neutral association with longer survival, slower AIDS progression, and lower mortality rate. The available evidence indicates an urgent need for prevention and control measures, including screening, diagnosis, and treatment of HIV-1 and HTLV-1/2 coinfected patients. Test-and-treat strategy for patients living with HIV in areas endemic for HTLV infection is mandatory, to avoid the risks of delayed therapy and death for coinfected patients.https://www.crd.york.ac.uk/prospero/, identifier: CRD42021279062. Human T-cell lymphotropic virus (HTLV) was the first human retrovirus described . There aHIV-1 and HTLV-1 infect the same cells but have different biological characteristics . This caSome studies have identified that HIV-1 subjects coinfected by HTLV are at higher risk of developing neurological complications, especially HAM/TSP , Adult T-cell leukemia (ATL), neuropathies, opportunistic infections, accelerated progression of HIV, and shorter survival , 8. On tPreferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) reporting guidelines were followed , and ourStudies considered for inclusion were those with a cross-sectional, case-control, or cohort design reporting clinical outcomes: AIDS progression, death, mortality, survival, comorbidities, and opportunistic diseases in a population of HIV-1 and HTLV-1 or HTLV-2 coinfected individuals of all ages, regardless gender, race or ethnicity.A systematical search in Pubmed/MEDLINE, Embase, Web of Sciences databases was done in August 2021, for articles published from 1990 to 2021, without language restriction. Our search strategy included three principal clusters of terms, one related to HIV-1 infection, another one to HTLV-1 or HTLV-2 infection and the last one about coinfection. The following terms to search PubMed/MEDLINE were:AND (“Human T-lymphotropic virus 1”[Mesh] OR “HTLV-I Infections”[Mesh] OR “HTLV-II Infections”[Mesh] OR “HTLV-1”[Title/Abstract] OR “HTLV-2”[Title/Abstract] OR “HTLV-I”[Title/Abstract] OR “HTLV-II”[Title/Abstract]) AND (“Coinfection*”[Mesh] OR “Coinfection*”[Title/Abstract] OR “Co-infection*”[Title/Abstract] OR “coinfected”[Title/Abstract]) AND (1990/01/01:2021/12/31[Date - Publication])).((“HIV”[Mesh] OR “HIV Infections”[Mesh] OR “HIV-1”[Mesh] OR “HIV-1”[Title/Abstract] OR “HIV-I”[Title/Abstract]) n = 33) were read in full and 15 studies were eligible for this review. Any divergence between the researchers was resolved by sending the conflicting article to a fourth reviewer, and disagreements were solved by discussion among the reviewers. The elimination of duplicates and the full text read stage were done using the Mendeley reference manager. Evaluation of the methodological quality of the studies was assessed by The Newcastle-Ottawa Scale (NOS) and one version adapted for cross-sectional studies  criteria , to iden studies .n = 2), 324 articles were eligible for title and abstract reading. By using PECO criteria as described in A total of 326 articles were identified by the search strategy as possibly relevant for the systematic review. After duplicates were removed , 6 , and 6.5 (cohort studies).The data of 2,566 mono and coinfected patients were described, 58% male. Mean age was 35.7 ± 5.7 years, only one study included children (2–16 years). Two studies did not report mean age of patients but informed that most participants were aged >35 or <40 years. A total of 581 individuals were coinfected, as follows: 319 (HIV-1/HTLV-1), 178 (HIV-1/ HTLV-2), and 84 (HIV-1/ HTLV-1/2), as shown in Six articles describing survival were identified , 23, 26.p = 0.54)  .p = 0.038   vs. 46/357 (12.8%)  , 25, cen = 0.04) , periphe (12.8%) , 25 or mSP, 2/12 % vs. 46/atoderma .p = 0.023) (p = 0.04) (p = 0.04) (p = 0.58) (p = 0.5)  . Surviva = 0.04) . ART use = 0.04) , 16/30 ( = 0.04) , 5 vs. 0 = 0.58) , 44 vs. p = 0.5) , but in p = 0.5) .The aim of this systematical review was to describe the published information about clinical outcomes in HIV-1 and HTLV-1/HTLV-2 co-infected individuals from 1990 to October 2021. The available studies are controversial, methodologically heterogeneous and fail to identify the factors driving the outcomes observed in HIV-HTLV coinfected population. Although the present review lacks important information due to design of included studies, we were able to detect some relevant findings associated to coinfection.p = 0.06), despite describing that AIDS clinical stage  and lack of antiretroviral therapy , were associated to a higher risk of dying in HIV-1 and HTLV-1 coinfected ones  related to HIV-1/HTLV-1 coinfection) and peripheral neuropathy . HAM/TSP like manifestations was also described among HIV-1/HTLV-2 coinfection . Such fiThe rate of HIV disease progression may be affected by many factors, like the infecting viral strain, host susceptibility and immune function, as well as, to exogenous influences such as access to healthcare and presence of coinfections , 32. OurSeveral reports concluded that HIV/HTLV-1 co-infected patients show an increase in CD4+T cells count in comparison with HIV mono-infected ones, although there is no clear benefit in terms of immune response , 24, 35.This review has some limitations: the findings described by the selected articles were observed in only 5 countries , which limits the generalization to other populations in prevalent regions like, Australia and Japan. Most studies included middle-aged, and only one study was focused on a pediatric population limiting any conclusion related to this age group . FinallyHIV-1 and HTLV-1 coinfection and HIV-1 and HTLV-1/2 triple infection are related to lower survival rate, death, and faster progression to death, while coinfection by HIV-1/HTLV-2 seems have neutral associations with higher survival rate, slower AIDS progression, and lower mortality rate. AIDS defining conditions, opportunistic and neurological manifestations were more frequently described on HIV-HTLV-1 coinfected subjects, which indicates AIDS condition as the main cause of death for them. The implementation of test-and-treat strategy for patients living with HIV in areas endemic for HTLV infection is mandatory, to avoid the risks of delayed therapy and death for coinfected patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.IM-C, CM, and CS: conception and design of the study, acquisition of data, analysis and interpretation of data, and drafting the article. CB: conception and design of the study, analysis and interpretation of data, drafting the article, and final approval of the version to be submitted. All authors agree to be accountable for the content of the work. All authors contributed to the article and approved the submitted version.IM-C has a CAPES  Ph.D. scholarship, finance Code 001. CM has a CNPq  Ph.D. scholarship, process number: 140187/2021-9. CB is a CNPq researcher, process number: 311095/2020-8. The funder had no role in study design, data collection, data analysis, data interpretation, and writing of the report.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.


Pathogens MDPI, it was the year where a special edition on Human T-cell Leukemia Virus Type 1 (HTLV-1) took centre stage, and for people living with HTLV-1 (PLHTLV), it was the year that the World Health Organisation adopted HTLV-1 as a health topic.The years 2020 and 2021 will remain memorable years for many reasons. For This sexually transmitted virus was identified in 1980 ,3,4.Pathogen’s Special Issue on HTLV-1 Disease aims to collate recent advances in basic science, clinical and epidemiological research enhancing our understanding of this virus’s pathogenicity. It is hard to believe, but it is true that 41 years since the discovery of HTLV-1, we still have no antiretroviral treatment that can cure this virus, prevent its transmission or avert disease from developing.HTLV-1 infection is associated with the development of disease in approximately 5% of infected individuals. Although the vertical acquisition of HTLV-1 through breastfeeding has mostly been associated with the development of adult T-cell leukaemia/lymphoma (ATL), epidemiological data support the notion that vertical HTLV-1 transmission is also associated with HTLV-1-associated myelopathy (HAM). Strengthening this notion, Schwalb et al. provide data on the early-onset of HAM in Peru [The natural history of HTLV-1 infection requires further investigation focusing on different populations. Marcusso et al. report on a 20-year longitudinal cohort study of PLHTLV-1 in Sao Paulo, Brazil, and confirm that HAM disease is a significant predictor of early mortality . In asymStrongyloides stercoralis co-infection [S. stercoralis is an intestinal parasite highly prevalent in HTLV-1 endemic regions, known to cause hyperinfestation in PLHTLV. The review explains how HTLV-1 infection dysregulates the immune response increasing host susceptibility and increasing S. stercoralis pathogenicity. The strongyloides infestation seems to support higher HTLV-1 pro-viral load levels, maybe through an upregulation of oligoclonal proliferation, which is a risk factor for ATL development.Two review articles also focus on the impacts of co-infections on the outcomes of HTLV-1 disease. Abelardo Araujo summarises eloquently the neurological sequelae of HTLV-1 or -2 with HIV-1 co-infection, where a potentially higher risk of HAM development or HAM-like myelopathy in co-infected individuals has been described . Howevernfection . S. sterMarino-Merlo et al. provide an update on antiretroviral therapies tested in preclinical and clinical studies . As a reThree review articles focus on the molecular aspects of HTLV-1 biology. Mohanty and Harhaj review how multiple signaling pathways modulated by the viral transactivator Tax protein synergize in the process of cell transformation . They alfunction . Prochasfunction . This reWe will recall the year 2020 as the year where our lives were affected by one virus in one way or other. Araujo and Martin complement this Special Issue with an editorial on HTLV-1 infection and COVID-19, in which they stress the lack of clinical data on PLHTLV exposed to SARS-CoV-2 infection . HTLV-1


Wnts are highly-conserved lipid-modified secreted proteins that activate multiple signaling pathways. These pathways regulate crucial processes during various stages of development and maintain tissue homeostasis in adults. One of the most fascinating aspects of Wnt protein is that despite being hydrophobic, they are known to travel several cell distances in the extracellular space. Research on Wnts in the past four decades has identified several factors and uncovered mechanisms regulating their expression, secretion, and mode of extracellular travel. More recently, analyses on the importance of Wnt protein gradients in the growth and patterning of developing tissues have recognized the complex interplay of signaling mechanisms that help in maintaining tissue homeostasis. This review aims to present an overview of the evidence for the various modes of Wnt protein secretion and signaling and discuss mechanisms providing precision and robustness to the developing tissues. Wnt proteins are secreted signaling molecules present in all metazoans. Signaling pathways activated by Wnt proteins play a crucial role in governing various aspects of development, including cell fate determination, body axis patterning, cell migration, cell proliferation, tissue maintenance and tissue regeneration . DysreguDrosophila melanogaster. Here, we aim to review the progress made in understanding the mechanisms of Wnt protein secretion, spreading and the mechanisms of signaling in developing tissues.Analysis of Wnt-mediated processes during development have revealed Wnt proteins to possess morphogen-like activity. According to the classical definition, morphogens are diffusible molecules, which form a gradient across a field of cells and activate the target gene expression in a concentration-dependent manner, thereby establishing tissue patterns via differential gene expression . HoweverDrosophila led to the identification of an X-ray-induced dominant mutation called Glazed, which showed a narrow and smooth eyed phenotype (1 (wg1)wingless was identified, which as the name suggests, showed a loss of adult Drosophila wing structures  called int-1 (wnt (wnt (int1) encoded protein showed a cysteine-rich domain (CRD) and a signal sequence, indicating that Wnts are secreted proteins (Drosophila Wnt proteins have been identified (Wnt homepage1), all of which share a common signature of 23-24 highly conserved cysteine residues at the N-terminus of their peptide  . HoweverDrosophila)  . These rsophila) . This insophila) .Drosophila, the PCP pathway consists of six core components; Fz, Dsh, Prickle (Pk), Strabismus/Van Gogh (Vang) or Vang-like (Vangl) in vertebrates, Flamingo (Fmi) and Diego (Dgo), which are conserved amongst the metazoans  pathway. Initially identified in etazoans . In varietazoans , at the etazoans . These cetazoans . For exaetazoans . Moreoveetazoans .Drosophila, the role of Wnt ligands in the activation of PCP remains controversial. A previous study showed that localized mis-expression of Wnts in the developing fly wing epithelium can modulate the global orientation of the cellular asymmetry  pathway , are lipid-modified in the lumen of the endoplasmic reticulum (ER)  . This oclum (ER) . Earlierlum (ER) . Howeveride bond . Palmitoide bond . Besideside bond . The patide bond . HoweverFollowing the transport and modification of Wnt proteins in the ER, they are transferred to a cargo-receptor protein Wntless /MIG-14/Gpr177, referred to as Evi hereafter), a multipass transmembrane protein which is an essential component of the Wnt secretory pathway . Recent Drosophila cells . The retrieval of Evi from endosomes to the TGN is mediated by retromer complex proteins VPS26, VPS29 and VPS35  and the An important and largely unresolved question in the process of Wnt secretion is how and where Wnt proteins separate from Evi. Studies on murine Wnt3a have shown that acidification of secretory vesicles facilitates the dissociation of the Wnt-Evi complex . HoweverWnt proteins also play an important role in the development of tissues made of polarized epithelial cells. Generation of a functional extracellular pool of the ligand by these cells requires polarized secretion, either from the apical or basolateral side. Studies have shown that Wnt proteins take a specific route for secretion from the polarized cells, which is believed to regulate their signaling abilities. For example, in polarized Madin–Darby canine kidney (MDCK) epithelial cells Wnt11 and Wnt3a are secreted preferentially from the apical or basolateral side, respectively. This polarized sorting of Wnt11 and Wnt3a is decided by their differential glycosylation patterns and the complexity of the glycans . InteresDrosophila epithelial cells. For instance, analysis of Wg in Drosophila embryonic epidermal cells showed that both wg mRNA and Wg protein are localized apically , may also bestow different signaling abilities to the Wnt ligands.Drosophila Wg  which is also observed for other secreted hydrophobic proteins, like Hedgehog . This olIn vitro studies in Drosophila identified a lipocalin family protein known as Swim (Secreted Wingless Interacting Molecule), which binds Wg  in the Drosophila wing epithelium, and depletion of lipophorins led to the impaired spreading of Wg and concomitant defects in long-range signaling  can also interact with lipid-modified Wnts and act as a vehicle for their extracellular transport . This haal fluid . Howeverignaling . On the ignaling , arguingDrosophila neuromuscular junctions (NMJ)  . For exans (NMJ) . A recenns (NMJ) . Similarns (NMJ) .Drosophila wing epithelium suggested that “argosomes,” which were believed to be extracellular vesicles, could carry Wg in the extracellular space  to the producing cells in order to trap Wg from the wing imaginal disc  showed localized expression of several Wnt genes in C. elegans  boundary  is also detected across the wing disc primordium (Drosophila embryo, the range of Wg is restricted to only a few adjacent cells from the producing cells (One of the best-known examples for long-range action of Wnt protein, and interestingly also the strongest contradiction has been observed with the boundary . Besidesimordium . Moreoveimordium . Furtherimordium , highligng cells , indicatNRT-wg flies emerged as normal appearing adults although with smaller but normally patterned wings and with developmental delay (Dll and fz3 in NRT-wg discs was comparable to that of the wildtype (NRT-wg but not the endogenous wg led to the reduction in target gene expression in the receiving cells (The paradigm of long-range Wg signaling has been further complicated by the dynamic changes in the Wg expression during the development of wing discs. Studies have shown that Wg expression pattern changes gradually from a broader expression in the entire wing pouch region during early larval stages to a narrow stripe of cells at the DV boundary, at later stages . This leal delay . Also, twildtype . This inng cells . This suNRT-wg flies do have other defects, for example in the proximo-distal patterning in the Drosophila renal tube (Drosophila intestinal epithelial and muscle tissues (In any case, the observation of a broader gene expression in the absence of a long-range gradient has revealed that there are compensatory mechanisms that could allow maintenance of target gene expression. This could provide developmental robustness to the growing tissues. However, these mechanisms may work in a tissue-specific manner. For instance, recent studies have shown that the nal tube  and patt tissues , which aIt is somewhat easier to envisage how a morphogen gradient could directly pattern an unchanging field of cells, however, in rapidly developing tissues, cells are rarely stationary. Thus, a challenging problem in developing tissues is how cells balance signaling levels, while experiencing a constant change in the levels of extracellular ligands. It is now well-accepted that numerous signaling pathways use positive and negative feedback mechanisms to ensure balanced signaling and maintain developmental robustness. Positive feedback is required for the amplification of a weak signal whereas negative feedback dampens signaling. Moreover, signaling pathways regulate the expression of these feedback regulators, whereby the expression of positive regulators is generally suppressed and negative regulators are activated by high-level signaling .Drosophila, Naked cuticle on the other hand is highly conserved (The canonical Wnt signaling pathway has highly diverse and complex feedback regulations, which allows it to modulate signaling in various developmental contexts. For example, in vertebrates, a Wnt signaling induced protein Axin2 acts as a cytoplasmic inhibitor of Wnt signaling , which donserved . Besidesonserved .Rnf43 and Znrf3 inhibits Wnt signaling at the receptor level by either interacting with the co-receptor LRP5/6 (Drosophila, the Fz receptors are part of both positive and negative feedback regulations. For instance, the expression of fz3 receptor is activated by the canonical Wg signaling and loss of fz3 was shown to rescue the morphological defects in wg hypomorphic mutants, suggesting that it acts as a negative regulator of the pathway (A common mechanism of feedback regulation is by modulating signaling at the level of surface receptors. For example, in vertebrate a set of Wnt target genes, including Dickkopf-1, r LRP5/6  or reducr LRP5/6 . In Dros pathway . In cont pathway . In the  pathway . Similar pathway . Togethe pathway .Drosophila leg imaginal disc is activated by the transient action of Wg and Dpp while at later stages of development this expression is maintained in the absence of Wg via cis-regulatory elements (Drosophila wing epithelium removal of Wg at later stages of development showed persisted expression of low-threshold targets, Dll and Vg (Another mechanism to achieve this developmental robustness could work at the level of target gene expression. While it is commonly understood that the expression of target genes depends on the signal activation directly by the ligand-receptor complexes, studies have also suggested that once activated, expression of certain target genes can persist in the absence of the extracellular ligand, which is generally referred to as “cellular memory.” For example, the expression of Wg target gene in the elements . Furtherl and Vg . As discl and Vg . Howeverl and Vg . TherefoFor example, one possible mechanism could be via ligand-independent signaling by the Frizzled receptors. A recent study showed that the apparent normal appearing long-range expression of Wg target genes in the membrane-tethered NRT-Wg expressing wing imaginal discs, is mediated via the Fz2 receptors . TherefoXenopus Fz receptors was shown to be sufficient for the activation of Wnt target gene expression in Xenopus embryo (Xenopus Fz3 receptor leads to its dimerization, which can mediate ligand-independent signal activation (While the mechanism of ligand-independent receptor activation of canonical signaling remains poorly understood, some insight came from the overexpression studies with vertebrate Fz receptors. For example, the overexpression of rat and s embryo . Studiestivation . Furthertivation  and Wnt tivation . Therefotivation .Drosophila wing epithelium, but loss of Fz2 and not Fz1 affects the maintenance of target gene expression (Xenopus embryo (The family of Frizzled receptors have several members but do all the receptors possess the ability to maintain canonical Wnt signaling? As mentioned above Fz2 acts redundantly along with Fz1 to activate canonical Wg signaling in pression . Similars embryo . These fThe processes and mechanisms related to Wnts and their signaling have been extensively studied due to their involvement in numerous cellular processes covering various developmental aspects. Using different model organisms, studies reviewed here have shown that the mechanisms regulating steps like Wnt protein expression and modification, their polarized secretion and modes of extracellular transport, their reception and signaling activity in the receiving cells, are diverse and context-dependent. However, the identification of specific and well-conserved regulatory proteins involved at various steps also indicate that the Wnt-related processes are tightly regulated.An unanswered question is how Wnt proteins utilize these multiple non-redundant functional routes, to finally converge at a particular signaling outcome and fate of the cell. Further, fine dissection of each route will be necessary to ascertain its functional specificity and to identify the regulatory processes directing the Wnt protein toward a particular functional route. In parallel to this, studies focusing on analyzing the contribution of feedback regulations and ligand-independent signaling mechanisms will provide a better understanding of the processes facilitating developmental robustness.SM, SH, and VC conceptualized and wrote the manuscript. SM and SH contributed equally. All authors read and approved the final manuscript.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.


A subgroup comparison (between UA-IL/UA-L and UA-M) was also performed. The clinical parameters of the patients were also analysed from files for clinicopathological correlation with recurrence. Results: sub-epithelial hyalinisation (SEH) significantly correlated with the recurrence of UA-L and UA-M (p = 0.001). When the histologic types (UA-L and UA-IL vs. UA-M) were grouped and the correlation of SOH with recurrence was checked, it was observed that both groups (p = 0.001) showed strong statistical correlation. UA-M lesions with multilocular radiolucency (p = 0.001) also showed significant correlation with recurrence. SOH can be a reliable histological predictor of recurrence and of aggressive biologic behaviour in UA. The present study shows a significant association of hyalinisation with the biologic behaviour of UA. Further studies with immunohistochemical investigations could validate the presence of hyalinisation and identify the origin of the hyalinised product in UAs. The inductive effect of hyalinisation and its influence on the biologic behaviour of ameloblastoma variants represent a scarcely researched domain of oral pathology. The complexity of the induction effects within the odontogenic apparatus, with the involvement of both ectodermal and mesodermal tissues, is responsible for diverse histopathological characteristics, hyalinisation being the major feature. The present study aims to deduce for the first time the correlation between the severity of hyalinisation (SOH) and recurrence in three unicystic ameloblastoma (UA) variants, namely, intra-luminal (UA-IL), luminal (UA-L) and mural (UA-M). Retrospectively diagnosed archival cases of UA-IL ( Hyalinisation is a condition in which normal tissue deteriorates into a homogeneous, translucent material. The phenomenon of hyalinisation can be physiological or pathological . HyaliniOdontogenic tumours (OTs) are a diverse group of lesions varying from innocuous benign lesions, including hamartomas, to aggressive malignant tumours . The varThe current study tests the hypothesis that the presence of SEH favours recurrence in UA. The UA-L and UA-IL variants are assumed to be less aggressive with non-recurrent potential.  However,The present study aims to assess the possible correlation between SEH and recurrence in three histologic variants of UA with clinicopathological correlation. The clinicopathological correlations obtained in this study could help predict the biologic behaviour of UA variants. This will ensure better treatment outcomes for patients by enabling the surgeon to rationally choose the best management option available.n = 08), UA-L (n = 22) and UA-M (n = 30) from the Department of Oral Pathology and Microbiology, Faculty of Dental Sciences. Clinical data comprising the biological parameters and patient details were retrieved and documented from the outpatient department. The retrieved tissues were heated to 55 °C for 10 min to facilitate deparaffinisation, followed by a xylene dip. The sections were stained with H&E stain for analysis. Ethical clearance was procured from the University Ethics Committee for Human and Animal Trials at MS Ramaiah University of Applied Sciences (MSRUAS), bearing number EC-2021/F/052. The study was performed on FFPE-diagnosed archival cases of UA, UA-IL  to evaluate the SOH; in ambiguous cases, a third pathologist (S.L.) assessed the histopathological features of hyalinisation and its severity. The histologic type of UA was also noted and tabulated. The correlation between SOH and recurrence in UA was statistically analysed. A subgroup comparison  was also performed.The clinical parameters of size , jaw involved (maxilla/mandible), site, radiographic features (multilocular/unilocular), cortical expansion and root resorption were analysed for statistical correlation with recurrence. The SOH was noted in the cases of UA and scored . Photomicrographs of the slides were captured at ×100 magnification using a research microscope. The histopathological and clinicopathological parameter scores were analysed for statistical significance by employing the chi-square test .n = 60) are shown in The descriptive features of UAs (p = 0.001)  .p = 0.001), It was observed that both variants (UA-IL/UA-L vs. UA-M) showed strong statistical correlation between SOH and recurrence (This indicates that the SOH influences the biologic behaviour of UA irrespective of the histologic subtype. p = 0.001) showed strong statistical correlation with recurrence  has emerged as a new, independent prognostic determinant in several cancers, such as colorectal and pancreatic cancers. Desmoplastic reaction is considered mature or immature based on whether the stroma is myxoid, and whether it contains or does not contain keloid such as collagen . HyaliniCottom et al., in 2012, demonstrated a strong correlation between SEH and recurrent odontogenic keratocysts. The authors concluded that the presence of subepithelial hyalinisation may be used as an additional histopathological feature to predict a greater tendency for the recurrence of OKCs. This was the first report on the presence of hyalinisation and recurrence of the OKC . Similarp = 0.001), The current study considered three histologic subtypes of UA and attempted to draw an association between hyalinisation and recurrence aided by clinicopathological correlation. The SOH was compared between the subtypes of UA  between SOH and recurrence in UA-L type, we suggest that this be overlooked, since only 4 recurrent UA-L cases were available compared to 18 nonrecurrent UA-L cases in the present study. The smaller sample of recurrent UA-L is a limitation of the present study. Even though only four UA-L were recurrent, it is of considerable interest to note that the SOH was intense in all four recurrent cases of UA-L. Although the current study shows a statistical correlation , followed by curettage with cryotherapy (40%), with the remainder (15%) being treated with curettage alone. UA-IL and UA-L were treated conservatively by enucleation with curettage or cryotherapy ,23. SMA When the clinicopathological features of size, jaw involved, site, radiographic features, cortical expansion and root resorption were correlated with UA-IL, UA-L and UA-M, no correlation was observed in the UA-IL and UA-L category. However, UA-M showed significant statistical correlation between the presence of multilocular radiolucency and recurrence. Lesions that are multilocular represent different growth sites of an infiltrative lesion. Despite the fact that UA compares favourably with SMA in terms of clinical behaviour and response to therapy, the subsets of multilocular lesions, UA-mural type and UA-L with intense SOH, could have a high risk of recurrence. The role of integrins and the mechanism of cross-talk induced to favour hyalinisation is a future area to investigate in odontogenic tumours. The presence of intense hyalinisation in subtypes of UA should be considered as an indicator of aggressive behaviour in UAs when determining the prognosis. This rational consideration by surgeons can help in deciding on the best therapeutic option to advocate to the patient.


Adenoid hypertrophy among orthodontic patients may be detected in lateral cephalograms. The study investigates the aerodynamic characteristics within the upper airway (UA) by means of computational fluid dynamics (CFD) simulation. Furthermore, airflow features are compared between subgroups according to the adenoidal nasopharyngeal (AN) ratios.ms), maximum wall shear stress (Pws), and minimum wall static pressure (Pw).This retrospective study included thirty-five patients aged 9–15 years having both lateral cephalogram and cone beam computed tomography (CBCT) imaging that covered the UA region. The cases were divided into two subgroups according to the AN ratios measured on the lateral cephalograms: Group 1 with an AN ratio < 0.6 and Group 2 with an AN ratio ≥ 0.6. Based on the CBCT images, segmented UA models were created and the aerodynamic characteristics at inspiration and expiration were simulated by the CFD method for the two groups. The studied aerodynamic parameters were pressure drop (ΔP), maximum midsagittal velocity (Vms exhibits nearly 30% increases in Group 2 at both inspiration (p = 0.013) and expiration (p = 0.045) compared to Group 1. For the other aerodynamic parameters such as ΔP, the maximum Pws, and minimum Pw, no significant difference is found between the two groups.The maximum Vms seems to be the most sensitive aerodynamic parameter for the groups of cases. An AN ratio of more than 0.6 measured on a lateral cephalogram may associate with a noticeably increased maximum Vms, which could assist clinicians in estimating the airflow features in the UA.The maximum V Hans Wilhelm Meyer first described the clinical condition of nasal obstruction caused by adenoid hypertrophy (AH) in 1868 , 3. ManyLateral cephalograms, cone beam computed tomography (CBCT), computed tomography (CT), and magnetic resonance imaging have been investigated to evaluate the size, shape, and location of the adenoid –12. AmonThe adenoid size and patency of the surrounding nasopharynx could be presented in terms of the absolute dimensions of adenoid thickness and nasopharyngeal width , percentComputational fluid dynamics (CFD) simulations may be the solution to link the UA morphology and airflow characteristics. CFD simulation is a well-established method for simulating the flow of gases or fluids and their interactions with the surrounding surfaces, as defined by boundary conditions. It has been widely used in the industry to predict the dynamic characteristics of the targeted flow. However, the application of CFD in dentistry was nevertheless sparse and had mainly been applied in evaluating the outcome of mandibular advancement devices in sleep-disordered breathing –24. CFD The study aims to investigate the aerodynamic characteristics within UA among orthodontic patients by CFD simulation. Furthermore, airflow features are compared between subgroups classified according to the AN ratios.Maximum midsagittal velocity of the airflow in the UA is considered to be the primary outcome variable based on a previous study by Feng et al. . A samplThis cross-sectional study is a subset of a longitudinal prospective study performed at Dalian Stomatological Hospital between 2015 and 2017, in which 2D and 3D images were compared for tracing anatomic landmarks before and after orthodontic treatment. The study was approved by the regional ethics review boards in Dalian, China (DLKQLL201604) and Bergen, Norway (2018/1547 REK Vest). Informed consent was obtained from all patients or their legal guardians. The baseline images from 2015 were retrospectively collected and employed in the current study. The inclusion criteria were individuals aged 9 to 15 years who had had both a lateral cephalogram and CBCT scan examined within one week. For CBCT images, the field of view was required to cover the UA regions, including the nasal cavity, nasopharynx, and oropharynx. The exclusion criteria were severe maxillofacial abnormalities and previous surgery on skeletal and soft tissue related to respiration. In the present study, ninety-two cases were initially included. X.F previewed all the CBCT scans and lateral cephalograms. Fifty-seven cases were excluded, of which 53 did not cover the UA, 3 scans had motion artefact and 1 showed suboptimal patient positioning. Eventually, thirty-five cases were recruited. All the cases were divided into two groups: Group 1 with AN ratios < 0.6 (n = 25) and Group 2 with AN ratios ≥ 0.6 (n = 10).The AN ratios were measured and calculated on the lateral cephalograms captured by a digital pan/ceph system  at 73 kVp and 15 mA with exposure times of 9.4 s and a contrast resolution of 16-bit depth. A is defined as a perpendicular distance between the point of maximal convexity of the adenoid to the anterior margin of the basiocciput. N is the distance between the posterosuperior edge of the hard palate and the anteroinferior edge of the spheno-occipital synchondrosis [All CBCT scans were obtained by 3D eXam . The following parameters were used: a field of view (FOV) of 16 × 13 cm, tube voltage of 120 kV, tube current of 5 mA, scanning time of 14.7 s, voxel size of 0.2 mm, and contrast resolution of 14-bit depth.The CBCT images were imported in the digital imaging and communications in medicine (DICOM) format to MIMICS software  for later analysis. 3D renderings of the CBCT scans were oriented with axial planes parallel to the Frankfurt horizontal plane; the midsagittal planes intersected the nasion and anterior nasal spine, and the coronal plane was adjusted to the level of the porions. For each case, a mask was reconstructed, making sure the integrity of UA was displayed correctly. CFD simulation was then conducted on the 3D model within the mask region. The superior boundary of the studied UA was defined as a vertical plane in the nasal cavity, passing through the most posterior point of the middle turbinate, whereas the inferior boundary was a horizontal plane, in the pharynx, in line with the most anterior-inferior point of cervical vertebra 4. Each end of the boundary was extended by 20 mm to avoid flow reversing during the simulating process. The inlet and outlet of UA were set on the extended planes. A surface model was then created according to the extended 3D model for mesh generation. We chose tetrahedral and prismatic cells to construct the main body and boundary layer of the UA mesh . The SST κ-ω model was used to calculate the aerodynamic characteristics of UA by applying ANSYS Fluent . The wall of the UA was defined as no-slip, stationary, and rigid. The temperature and density of air were set as fixed. At inspiration, the inlet was set with the pressure 0 Pa and the outlet at a flow rate of -200 mL/s . The corThe aerodynamic parameters applied and computed are listed in Table Two experienced operators performed CFD simulations, one on all cases and one on ten randomly selected cases. The first operator repeated the measurements one month later on the ten selected cases.Data were processed using IBM-SPSS, version 25.0 . Significance was set at p-values less than 0.05. The assumption of normal distribution for all variables was tested. An independent-samples T-test or Mann–Whitney U test was applied to compare the aerodynamic parameters between subgroups and between genders. The Intraclass Correlation Coefficient (ICC) was applied to test intra- and inter-observer reliability on the selected ten cases using a random number generator.ms in Group 2 exhibits a statistically significant increase of nearly 30% (p < 0.05) at both inspiration and expiration in contrast to Group 1. None of the other aerodynamic parameters, including ΔP, maximum wall shear stress (Pws), and minimum wall static pressure (Pw) were significantly different between the two groups at both inspiration and expiration.Fig. The mean age of cases was 12.03 ± 1.42 . AN ratios ranged between 0.33 and 0.80 with a mean and standard deviation of 0.54 ± 0.15. We did not find any statistically significant difference between females and males in terms of AN ratio and aerodynamic characteristics. Figure Regarding the measurement precision, intra- and interobserver agreement ranged between 0.872 and 0.997 for various aerodynamic parameters.ms might be the most sensitive aerodynamic parameter that had demonstrated a significant difference between the two groups [ms significantly increased at both inspiration and expiration in cases with an AN ratio of more than 0.6. This finding may imply that clinicians could initially estimate airflow alteration in UA by measuring the AN ratio in a lateral cephalogram. However, caution is needed in extrapolating our results due to the relatively low number of patients, particularly in Group 2. The statistical efficiency would have been higher if there had been an equal number of patients in the two groups.The present study bridges UA morphology presented as AN ratio and UA function in terms of aerodynamic characteristics by applying CFD simulation. Among all the tested aerodynamic parameters, maximum Vo groups . CFD simo groups , which sBesides patients having enlarged adenoid, other conditions such as enlarged tonsils, patients undergo mandibular setback surgery, and obstructive sleep apnea syndrome (OSAS) patients may also cause UA aerodynamic insufficiency. OSAS patients had been mostly studied in previous CFD studies showing significantly different aerodynamic characteristics in UA compared to the healthy subjects. Chen et al.  reportedAs compared to medical CT, CBCT is more cost-effective and has demonstrated a lower radiation dose . NeverthOwing to the retrospective study design, a limited number of cases fulfilled the inclusion criteria of having both lateral cephalogram and CBCT among the orthodontic patients. Further prospective research on aerodynamics characteristics involving more cases having enlarged adenoids is warranted. The application of CFD in dentistry is still in the exploration stage of scientific research. One reason is the requirement of 3D imaging to generate a 3D model of UA, which entails automatically higher patient dose as compared to conventional lateral cephalograms. In addition, performing CFD on a large number of cases is challenging since it is highly dependent on the skillfulness of the operator, and the simulation procedure is complicated and time-consuming. CBCT-based aerodynamics simulation is partially performed manually, which may lead to inconsistent reliability for inexperienced operators. Future studies should investigate UA examination using low dose CBCT and the association between UA morphology, respiratory function, and clinical symptoms to better manage children with AH.ms seems to be the most sensitive aerodynamic parameter for the groups of cases. An AN ratio of more than 0.6 measured on a lateral cephalogram may associate with a noticeably increased maximum Vms, which could assist clinicians in estimating the airflow features in the UA.The maximum V


This secondary step in apoptosis induction can be prevented by inhibition of aquaporins, inhibition of NOX1 and decomposition of H2O2. Pretreatment with inhibitors of glutathione synthase or the cysteine-glutamine antiporter (xC transporter) abrogate the requirement for aquaporin/H2O2-mediated glutathione depletion, thus demonstrating that intracellular glutathione is the target of intruding H2O2. These data allow definition of mechanistic interactions between ROS/RNS signaling after inhibition of membrane-associated catalase, the sensitizing effects of aquaporins/H2O2 and the counteraction of the xC transporter/glutathione synthase system. Knowledge of these mechanistic interactions is required for the understanding of selective apoptosis induction in tumor cells through reestablishment of apoptosis-inducing ROS/RNS signaling.The human gastric carcinoma cell line MKN-45 is a prototype of bona fide tumor cells, as it is protected from the NADPH oxidase-1 (NOX-1)-driven HOCl- and nitric oxide (NO)/peroxynitrite apoptosis-inducing signaling pathways by a membrane-associated catalase. The use of inhibitors/scavengers shows that inhibition of membrane-associated catalase is sufficient for the activation of NO/peroxynitrite or HOCl signaling. However, this signaling is not sufficient for apoptosis induction, as intracellular glutathione peroxidase/glutathione counteracts these signaling effects. Therefore, intrusion of extracellular tumor cell-derived H This finding opened the path for the elucidation of numerous physiological and pathophysiological roles of H2O2, as well as its control by enzymes such as catalase, peroxidase and others ]bis [6-methoxyphenolato-κO]]]-manganese was a product of Cayman  and was obtained from Biomol . Photofrin  was obtained from Meduna Arzneimittel GmbH . Nω-Hyroxy-nor-L-arginine.acetate (NOR-NOHA) was obtained from Axxora/Enzo Life Sciences, Lörrach, Germany. Detailed information on inhibitors has been previously published [The NOX1 inhibitor 4-(2-Aminoethyl)benzenesulfonyl fluoride (AEBSF), the aquaporin inhibitor AgNOublished ,54,55,56The human gastric adenocarcinoma cell line MKN-45 (ACC 409) (established from the poorly differentiated adenocarcinoma of the stomach (medullary type) of a 62-year-old woman), was purchased from DSMZ, Braunschweig, Germany.MKN-45 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS). MKN-45 cells show active membrane-associated NOX-1, resulting in the generation of extracellular superoxide anions and are protected against intercellular ROS/RNS-dependent apoptosis-inducing signaling through expression of membrane-associated catalase ,31. MKN-6/mL.Fetal bovine serum  was heated for 30 min at 56 °C prior to use. Medium was supplemented with penicillin (40 U/mL), streptomycin (50 µg/mL), neomycin (10 µg/mL), moronal (10 U/mL) and glutamine (280 µg/mL). Care was taken to avoid cell densities below 300,000/mL and above 102, that allowed intercellular ROS-mediated apoptosis induction, the percentage of apoptotic cells was determined by inverted phase contrast microscopy based on classical criteria for apoptosis as described below.Autocrine apoptosis induction by intercellular ROS/RNS signaling. Tumor cells in complete medium were seeded in 96-well tissue culture clusters at a density of 12,500 cells/100 µL. Reactivation of intercellular apoptosis-inducing ROS/RNS signaling required inactivation of membrane-associated catalase of tumor cells. Assays received the indicated concentrations of the catalase inhibitor 3-AT or other compounds, as indicated in the figures. In inhibitor studies, inhibitors were applied 10 min before 3-AT, or as indicated in the respective legends. In all experiments, assays were performed in duplicate. After the indicated time of incubation at 37 °C and 5% CO2, the percentage of apoptotic cells was determined by inverted phase contrast microscopy based on the classical criteria for apoptosis, i.e., nuclear condensation/fragmentation or membrane blebbing [Determination of the percentage of apoptotic cells. After the indicated time of incubation at 37 °C and 5% COblebbing ,56,57,58blebbing ,56,59,60blebbing ,47,59,60blebbing . The Supp < 0.01 = significant; p < 0.001 = highly significant).Statistical analysis. In all experiments, assays were performed in duplicate and standard deviations were calculated. Absence of standard deviation bars indicates that the standard deviation was too small to be reported by the graphic program. Standard deviations demonstrate reproducibility in parallel assays but do not allow statistical analysis of variance. The experiments were always performed with duplicate assays and were repeated at least twice. The Yates continuity corrected chi-square test was used for the statistical determination of significances  were added to all assays. This regime allowed focus explicitly on direct H2O2-mediated apoptosis induction [2O2-generating glucose oxidase (GOX). As shown in 2O2 up to a concentration of 1 mU/mL GOX. However, when their membrane-associated catalase was inhibited by neutralizing antibodies, apoptosis was induced dependent on the concentration of GOX, and finally resulting in a plateau-type of curve. GOX-mediated apoptosis induction after inhibition of catalase was prevented when aquaporins were inhibited by Ag+ prior to the addition of GOX. When the aquaporin inhibitor was added 30 min after, cells were no longer protected against the apoptosis-inducing effect of 0.25 mU/mL GOX and higher concentrations. However, there was still substantial protection against apoptosis induction at lower concentrations of GOX. These findings show (i) that an influx of H2O2 into the cells was required for apoptosis induction under the conditions of this experiment, (ii) that membrane-associated catalase controls the effective H2O2 level and (iii) that aquaporins control the influx of H2O2  inhibitor L-NAME and the peroxynitrite decomposition catalyst FeTPPS, whereas apoptosis induction at high concentrations of 3-AT was inhibited by the peroxidase inhibitor ABH and the HOCl scavenger taurine. These findings demonstrate that 3-AT induced NO/peroxynitrite signaling at low concentrations, and HOCl signaling seemed to follow and to replace NO/peroxynitrite signaling at higher concentrations of 3-AT. Both signaling pathways were finally executed by caspase-9 and caspase-3, indicating the action of the mitochondrial pathway of apoptosis. The presence of the aquaporin inhibitor Ag+ or remained free of inhibitor. Control experiments confirmed that the NO donor exclusively induced NO/peroxynitrite signaling, which was inhibited by FeTPPS, but not by taurine. In contrast, MPO established HOCl signaling that was inhibited by taurine but not by FeTPPS. +.In order to focus the analysis rigidly on defined individual signaling pathways, the tumor cells were confronted with exogenously added defined signaling compounds such as the NO donor DEA NONOate or MPO at the respective optimal cell density. This approach specifically enhances NO/PON signaling or HOCl signaling . The relatively low cell density and the presence of the peroxidase inhibitor ABH thereby prevented the establishment of HOCl signaling, whereas the applied relatively high concentrations of GOX ensured direct apoptosis induction by H2O2. Intracellular catalase, which was not reached by the cell-impermeable antibodies obviously played no protective role towards exogenous H2O2, in line with a previous report [2O2 was strictly dependent on the parallel inhibition of membrane-associated catalase, the dominant control of H2O2 by membrane-associated catalase was confirmed [2O2 into the cells was also ensured. These data show the tight connection between the control function of membrane-associated catalase and aquaporins, resulting in striking cooperativity: membrane-associated catalase controlled the concentration of available H2O2, whereas aquaporins ensured the entry of H2O2 into the cells. The differential effect of silver ions added 10 min before or 30 min past GOX, sheds light on the dynamics and concentration dependencies of the underlying processes. The results obtained through approach A also confirmed the analytical strength of catalase neutralizing antibodies and of the aquaporin inhibitor Ag+. This was the basis for the inhibitor-based analysis of the more complex intercellular NO/peroxynitrite and HOCl signaling and its control by membrane-associated catalase and intracellular glutathione/glutathione peroxidase.Approach A was performed by abrogating the resistance of tumor cells towards exogenous Hs report . As, in onfirmed ,31. As p2O2 into the cells is ensured in parallel. This is seen by the prevention of apoptosis induction after application of the aquaporin inhibitor Ag+. This aquaporin-mediated effect is strictly depending on the preceding inhibition of membrane-associated catalase. It seems to comprise a H2O2-dependent sensitization step for subsequent ROS/RNS signaling. This sensitization step seems to be completed within less than 1 h. As intracellular catalase is also inhibited by 3-AT, the intracellular resistance towards exogenous ROS/RNS signaling seems to be mediated by an enzyme system that is different from catalase. Glutathione peroxidase is the most likely candidate for this effect. The interdependency between intercellular ROS/RNS signaling and counteracting aquaporin action is reflected (i) in the lag phase of the apoptosis kinetics after catalase inhibition and (ii) in the restriction of the inhibitory effect of Ag+ exactly to this lag phase.Approach B utilized the reactivation of intercellular NO/peroxynitrite or HOCl signaling after inhibition of tumor cell membrane-associated catalase by 3-AT ,31. Both2O2 intruding through aquaporins seemed to originate from the dismutation of NOX1-generated extracellular superoxide anions, as its effect was inhibited by the NOX1 inhibitor AEBSF and the catalase mimetic EUK-134. Importantly, H2O2 intruding through aquaporins was not the direct cause of apoptosis induction. Rather, it triggered a sensitizing step that allowed extracellular ROS/RNS signaling to become effective. The necessity for extracellular signaling is particularly demonstrated by strong and immediate inhibition of apoptosis induction through addition of the HOCl scavenger taurine even at late time points. If there had been a direct apoptosis-inducing effect of intruding H2O2, scavenging of HOCl by taurine would not have been sufficient to block of the apoptosis kinetics.H2O2 has a function as substrate for extracellular HOCl synthesis, but also for the establishment of a second intracellular sensitizing effect. This sensitizing step seems to be mediated by aquaporins, as it is effectively prevented by inhibition of aquaporins. Both of these cooperating effects, HOCl signaling and aquaporin-mediated influx of H2O2, are efficiently controlled by membrane-associated catalase. Inhibition of catalase is therefore necessary to allow both of these interacting steps to become effective. NO/peroxynitrite signaling  also requires the sensitizing intracellular effect of H2O2, mediated by aquaporins. The balance between aquaporin-mediated sensitizing effects of H2O2 that allow NO/peroxynitrite signaling, and H2O2-dependent interference with NO/peroxynitrite signaling is delicate. With increasing inhibition of catalase and resultant increase in free extracellular H2O2, NO/peroxynitrite signaling is suppressed on the expense of reactivated HOCl signaling. This suppression is mediated by complex iron-driven reactions [In total, these findings show that tumor cell-derived extracellular Heactions .2O2 that has passed the cell membrane through aquaporins can be completely mimicked through pretreatment of the tumor cells with (i) BSO, an inhibitor of glutathione synthetase or (ii) with sulfasalazine, an inhibitor of the xC transporter. Each one of these pretreatments leads to massive depletion of intracellular glutathione [2O2 influx through aquaporins can be fully explained as well as substituted by the depletion of the intracellular glutathione level. In line with this conclusion, the kinetics of apoptosis induction in BSO- or sulfasalazine-pretreated tumor cells after addition of the catalase inhibitor started immediately, without showing the lag phase that is typical for control cells that had not been pretreated with BSO or sulfasalazine.The sensitizing effect of Htathione ,64. Pret2O2, leading to strong inhibition of the GSH/GPx system [It is conceivable that glutathione depletion also allows inactivation of glutathione peroxidase through Hx system .2O2 under these conditions was not sufficient to deplete glutathione to a level that would sensitize the cells for NO/peroxynitrite signaling. However, when GSH depletion was reached through BSO- or sulfasalazine pretreatment, the efficiency even of a low degree of catalase inhibition became overt. This interaction bears a chance for synergistic effects during tumor therapy, based on interactive glutathione depletion and catalase inhibition.In glutathione-depleted cells, but not in control cells, very low concentrations of 3-AT led to apoptosis induction through the NO/peroxynitrite pathway. This finding demonstrates that very low concentrations of the catalase inhibitor were sufficient for reactivation of NO/peroxynitrite signaling, but that the parallel influx of H2O2 or inhibition of glutathione synthesis through diminishment of the necessary building block cysteine or inhibition of glutathione synthase, seems to abrogate this negative interference with extracellular signaling.The sensitizing effect of lowering the glutathione level for the effects of extracellular ROS/RNS signaling can be best explained by the negative effect on the action of glutathione peroxidase 4. This enzyme uses glutathione to repair lipid oxidation in the membrane and thus has the potential to counteract the final step of intercellular RNS/ROS signaling . GlutathThese findings allow the drawing of a hierarchical scheme of interactions between membrane-associated NOX1, catalase, aquaporins, xC transporters and intracellular glutathione peroxidase 4/glutathione .2O2. In parallel, the xC transporter ensures optimal synthesis of intracellular glutathione , lipid peroxidation is no longer counteracted by glutathione peroxidase/glutathione and triggers the onset of the mitochondrial pathway of apoptosis  from intercellular HOCl signaling and (ii) from the aquaporin-mediated influx of NOX1/SOD-derived Htathione A. Inhibitathione B abrogatpoptosis C. The mu+ under these conditions. Conversely, this interplay allows the conclusion that strong overexpression of xC transporters or glutathione synthase might prolong the lag phase between catalase inhibition and onset of apoptosis. Eventually this might even lead to resistance towards apoptosis induction by exogenous ROS/RNS attack. The recent findings by Bekuschus et al. [The outstanding role of the GSH/GPx system is summarized in s et al.  might po2O2, catalase can also oxidize NO and decompose peroxynitrite [2O2 has caused an abrogation of the protective effect of GPx/GSH.For simplicity, so far, the discussion had been focused solely on reactivation of HOCl signaling after inhibition of membrane-associated catalase. However, the situation in reality is more complex, as catalase acts as a multifunctional enzyme A. In addynitrite ,66,67,68The dependency of apoptosis induction by intercellular RNS/ROS signaling on a dominant cooperative action of aquaporins was not only shown after direct inhibition of catalase by 3-AT, but also after treatment of compounds that cause catalase inactivation through different regimes. These were direct singlet oxygen generated by an illuminated photosensitizer, as well as modulation of cell-derived NO concentrations through the NOD inhibitors cyanidin and taxol, or the arginase inhibitor NOR-NOHA. Modulation of the NO level has been shown to lead to singlet oxygen generation . SingletIt is also obvious that the hierarchy of interactions established for MKN-45 cells is specifically relevant for tumor cells and has no impact on nonmalignant cells, as these lack the key features of active NOX1 and membrane-associated catalase.2O2 through aquaporins. ROS/RNS signaling, as well as aquaporin action, are under the control of membrane-associated catalase.Inactivation of membrane-associated catalase of tumor cells reactivates extracellular NOX1-driven ROS/RNS signaling through the NO/peroxynitrite or the HOCl signaling pathway. The apoptosis-inducing effect of extracellular ROS/RNS signaling is counteracted by intracellular glutathione/glutathione peroxidase. Apoptosis induction, therefore, requires abrogation of intracellular glutathione/glutathione peroxidase action through an influx of extracellular, tumor cell-derived H


Furthermore, we investigated the effect of UA in vivo using a full-thickness skin wound model. Subsequently, we found that UA promoted the regeneration of new blood vessels, which is consistent with the results of accelerated angiogenesis in vitro experiments. After UA treatment, the blood vessels in the wound are rapidly formed, and the deposition and remodeling process of the collagen matrix is also accelerated, which ultimately promotes the effective wound healing. Mechanistic studies have shown that UA promotes angiogenesis by inhibiting the PI3K/AKT pathway. Our study provides evidence that UA can promote angiogenesis and skin regeneration in chronic wounds, especially ischemic wounds.The treatment of chronic wound is an important topic of current clinical issue. Neovascularization plays a crucial role in skin wound healing by delivering fresh nutrients and oxygen to the wound area. The aim of this study was to investigate the mechanisms of urolithin A (UA) in angiogenesis during wound healing. The results of  Skin wounds remain a major global public health problem due to the increasing number of burns, trauma, and chronic diseases and can result in pain, infection, and even amputation . Wound hAngiogenesis is a key factor in wound healing caused by chronic and ischemic injuries . New bloUrolithin A (UA) is one of the metabolites of ellagitannins and ellagic acid extracted from pomegranates and other fruits and nuts, which have a wide range of functions including anti-inflammatory Y.  and antiIn the present study, we hypothesized that UA could activate the angiogenic capacity of HUVECs and accelerate wound healing. The mechanism of pro-angiogenic UA in HUVECs was investigated. A full-thickness skin wound model was used to study the therapeutic efficacy of UA.All experiments were carried out under the guidelines of the Ethics Committee of Sir Run Run Shaw Hospital and in compliance with the Guidelines for Care and Use of Laboratory Animals published by the National Institutes of Health.Urolithin A (purity ≥98%) was purchased from MCE . Crystal violet and DAPI were purchased from Beyotime . DMSO was purchased from Sigma-Aldrich . Cell Counting Kit-8 (CCK-8) was purchased from MCE .HUVECs were obtained from iCell Bioscience . We maintained cells at 37°C in RMPI 1640 containing 10% fetal bovine serum. HUVECs were cultured at 37°C in a humidifying incubator under 5% CO2 atmosphere. The first 20 passages of cells were used for the experiment.4 cells in the upper chamber. An inverted light microscope  was used to generate representative images.The Transwell chamber (BD Biosciences) was used for Transwell migration. For the migration assay, a one-day incubation process was performed in 100 μL serum-free medium. 500 μL of complete medium (containing FBS and 1640 medium) was placed in the lower chamber of the Transwell chamber and 5 × 103 cells in the 96-well plates for 48 h. The cells were added with 100 μL RMPI 1640 including 10% tetrazolium substrate and incubated for 1 h. Then, we use a spectrophotometer to measure the absorbance value at 450 nm.The cell viability exhibited by HUVECs with different doses of UA (0–40 μM) was detected by the CCK-8 assay. Briefly, HUVECs were incubated with 5 × 10A tube-forming experiment was performed on Matrigel glue to evaluate the effect of UA on the morphogenesis and tube-forming ability of HUVECs. Simply put, the matrix gel solution was melted overnight at 4°C, then placed in a µ-slide, and placed in a cell incubator for 1 h to solidify. 5,000 HUVECs pretreated with different concentrations of UA  were seeded on the pre-coated µ-slide of the matrix. Observing the formation of the tube, the average of five independent fields was counted under a Nikon inverted optical microscope.Firstly, HUVECs were plated in a six-well plate and cultured at 40–60% density. After HUVECs were fully attached, they were exposed to different treatments for 48 h. Next, we use radioimmunoprecipitation analysis (RIPA) lysis buffer which contains protease inhibitors and phosphatase inhibitors to lyse HUVECs for 30 min. The lysate is collected and centrifuged for 15 min at 14000 g at 4°C. The supernatant is collected and mixed with 5× loading buffer. It is put in a metal bath at 100°C for 10 min. Next, we perform WB experiments. SDS-PAGE (10%) gel is used to separate the proteins and transfer them to 0.45-μm PVDF membranes. 5% skimmed milk powder is used to seal the PVDF membrane for 1 h at room temperature. The membrane is cut according to the corresponding molecular weight, and then the specific primary antibody is used: anti-AKT , anti-p-AKT , anti-PI3K , and anti-p-PI3K , to incubate overnight at 4°C. The next day, the PVDF membrane was washed three times with TBST buffer and then incubated with the corresponding secondary antibody for 1 h at room temperature. Then, the PVDF membrane was washed three times again with TBST buffer. We use the GE imaging system to observe and store protein bands.Immunofluorescence was used to evaluate protein expression levels. The cells were fixed with paraformaldehyde and permeabilized with 0.3% Triton X-100 and incubated with the CD31 antibody (1:200) at 4°C. Then, a fluorescein-conjugated secondary antibody was added to the cells, which were incubated for 1 h at 4°C in the dark, followed by a counterstaining process of 15 min with DAPI at room temperature in the dark to stain the nuclei. A Nikon A1 microscope  with a digital camera was used to capture fluorescence images.EdU assay was used EdU kit (Beyotime) according to the protocol. Firstly, we seeded HUVECs in 12-well plates. After adherence, HUVECs were starved for 8 h with FBS-free 1640 to synchronize the cell cycle. Afterward, the cells were treated with different doses of UA for 24 h. The cell culture medium was then replaced with fresh 1640 containing EdU and incubated for 2 h. Next, we fix the cells with 4% paraformaldehyde for 15 min and then treat them with 0.3% Triton X-100 for 20 min. After that, the medium of each well was replaced with fresh 1640 containing 10 μM EdU and cultured at 37°C for 2 h. Then, 4% paraformaldehyde was used to fix the cells for 15 min, and the fixed cells received 20 min of incubation with 0.3% Triton X-100. Afterward, they were incubated for 25 min in Click reaction buffer in the dark. The nucleus is stained with DAPI. A Nikon A1 microscope  with a digital camera was used to capture fluorescence images.https://www.rcsb.org/) and docked. AutoDockTools (version 1.5.6) was used for protein–ligand docking analysis. This tool can provide the flexibility of binding the ligand to the residues of the binding pocket. The final image is generated by UCSF PyMoL.We choose AKT (PDB ID: 3QKK) and PI3K (PDB ID: 5ITD) for molecular docking research . Both prTwenty-four Sprague Dawley (SD) rats were anesthetized with pentobarbital and placed in the prone position. After the rat’s back hair was shaved and disinfected, two full-thickness wounds with a diameter of approximately 20 mm were made on both sides by using a 20 mm diameter mold and scissors. The UA group was intragastrically administered at a dose of 25 mg/kg/d until 21 days after surgery . All ratTo prepare the animal tissue samples, we embedded the formalin-fixed tissue samples in paraffin, cut them into 5 μm sections, and placed them on slides. For immunohistochemical staining, the slides were dewaxed in xylene and rehydrated with graded alcohol. Then, the slides were incubated in 3% hydrogen peroxide to impede endogenous peroxidase activity. The slides were boiled for 30 min in 10 mM sodium citrate (pH 6.0) for antigen retrieval, blocked with 5% normal goat serum for 15 min, and then incubated with the indicated antibodies: collagen I (1:300) or anti-collagen III (1:300), overnight at 4°C in a humid room. The next day, the slides were incubated with the secondary antibody for 1 h at room temperature after the PBS washing process. A Metal Enhanced DAB Substrate Kit  was used to detect immunoreactivity.Masson’s trichrome staining: The slices were dewaxed. Next, we stained slices with the Weigert iron hematoxylin staining solution for 5 min. Subsequently, we stained slices with Masson blue liquid for 5 min and with Ponceau red fuchsin staining for 5 min and aniline blue staining for 2 min. Finally, the slices were dehydrated and mounted. Hematoxylin and eosin staining: Firstly, the slices were dewaxed and stained with hematoxylin for 2 min. The slices were soaked in the differentiation solution for 3 min. Next, the slices were dyed with eosin dye solution for 1 min.p-value less than 0.05.Prism 8  was adopted for statistical analysis. The data were analyzed using either one-way ANOVA when necessary or Student’s t-test. The results were expressed as mean ± SD. Statistically significant differences were identified at a in vitro angiogenesis experiments were used to study the tube-forming ability of HUVECs. As illustrated in The targeted migration of endothelial cells plays a crucial role in the initial stage of angiogenesis . Next, wIn vitro experiments showed that the expression of P-PI3K and P-AKT proteins decreased in a concentration-dependent manner after UA treatment (in vitro.To investigate whether UA has a direct affinity with PI3K and AKT, we performed molecular docking analysis of the molecular structure of UA with the protein structure of PI3K and AKT, according to respective binding pockets of the antagonist. Through the docking model, we discovered that UA formed some good connections and docked well in the binding sites of PI3K and AKT . Moreovereatment . Collectin vitro, and we supposed that UA may promote skin wound healing. Therefore, a full-thickness skin wound model was used to investigate whether UA has the ability of promoting wound repair. As shown in UA has the effect of promoting angiogenesis Next, we used Masson’s trichrome staining and type I and type III collagen immunohistochemical staining to evaluate collagen deposition and remodeling. Strong blue staining could be illustrated in the UA treatment group, showing regularly arranged regenerated collagen deposits . Moreovein vivo. On day 21, hair follicles have grown in the UA group. To evaluate wound angiogenesis, CD31 was used to characterize wound blood vessels. The number of new blood vessels on the seventh day in the UA group was significantly higher than that in the control group (As shown in ol group . CollectRapid regenerative wound healing has always been a clinical challenge due to the high demands and complexity of the healing process, especially in chronic wounds . In the in vitro.To ensure adequate nutrition and oxygen supplementation for large-area wound defects and prevent cell death in ischemic areas, rapid and continuous vascularization is required, which is essential for effective skin wound regeneration . The vasIt has been demonstrated that the PI3K/AKT pathway is an important cellular signaling pathway involved in a variety of cellular activities, including cell migration, proliferation, and survival W. . PreviouTo further elucidate the role of UA in wound repair, we used a full-thickness skin wound model. Immunohistochemistry and Masson staining showed that, in the process of wound healing, UA promoted collagen deposition and remodeling, which was characterized by a large number of collagen fibers arranged under the wound . During In summary, our research provides evidence that UA promotes angiogenesis by inhibiting the PI3K/AKT signaling pathway. In addition, UA increases blood vessel formation, collagen deposition, and remodeling, all of which accelerate skin wound healing. These results indicate that UA has great therapeutic potential for endothelial injury–mediated vascular diseases, especially for patients with skin wounds.


Human T-lymphotropic virus (HTLV) infection is endemic in indigenous populations of the Americas. We describe herein the prevalence of HTLV-1 and HTLV-2 infection among Warao indigenous refugees from Venezuela living in Belém, Pará, Brazil.env region (630 bp) followed by enzymatic digestion with XhoI was performed to identify the molecular subtype of HTLV-2, in addition to sequencing analysis of the 5'LTR-I and 5′-LTR-II regions.In total, 101 individuals of both sexes (43 men and 58 women) between 18 and 77 years of age were investigated. Blood samples were collected and separated into plasma and leukocytes. Serological screening was performed using an enzyme-linked immunosorbent assay , and seropositive samples were submitted to proviral DNA extraction followed by real-time polymerase chain reaction (qPCR). A nested PCR of the pol and tax genes confirmed the HTLV-1 infection in a 55-year-old woman and HTLV-2 infection in a man (68 years old) and a woman (23 years old). HTLV-2 strains were defined by enzymatic digestion as belonging to the HTLV-2b subtype. The sequencing of the 5′LTR regions confirmed the presence of subtype 2b and identified HTLV-1 as belonging to subtype 1A (Cosmopolitan) and the Transcontinental subgroup. Among the infected patients, it was possible to conduct medical interviews with two individuals after delivery of the result. One patient with HTLV-2 reported symptoms such as joint pain, foot swelling, frequent headache, dizziness and lower back pain. The HTLV-1-positive woman was diagnosed with a tumor, dementia, urinary incontinence, felt body pain, and had spots on her body. The presence of the HTLV-2b subtype highlights the prevalence of this molecular variant among indigenous South Americans, as well as the presence of HTLV-1 Transcontinental, which has a worldwide distribution.Of the 101 individuals analyzed, 3 (3.0%) were seropositive. Molecular analysis of the These results reveal a high prevalence of HTLV-1/2 infection among Warao immigrants, suggesting migratory flow as a virus spread mechanism among human populations and alert public authorities to the need to create epidemiological surveillance programs, public social and health policies aimed at welcoming immigrants in the Brazilian territory. Retroviridae family that infect humans and have numerous similarities in biological and molecular properties (Human T-lymphotropic viruses 1 and 2 (HTLV-1 and HTLV-2) are members of the HTLV-1 infection has endemic characteristics in well-defined geographic regions such as southern Japan, the Caribbean, and Australia , 3, wherHTLV-2 infection has an endemic distribution in Amerindian populations –7. In NoMolecular studies demonstrate the circulation of three HTLV-2 molecular subtypes among these different indigenous peoples of the Americas, with HTLV-2a and HTLV-2b  being prvia the Bering Strait, with a subsequent descent toward Central and South America .Collections were carried out from September 2020 to January 2021. From all participants, 4.5 mL of venous blood was collected in a vacuum tube containing EDTA (ethylenediamine tetraacetic acid) as an anticoagulant. Then, the samples were transported to the Virology Laboratory of the Federal University of Pará in an insulated box. The samples were submitted to plasma and leukocyte separation by means of centrifugation at 8,000 rpm for 15 min, followed by storage at −20°C until analysis.The samples were submitted to serological screening for the detection of anti-HTLV-1 and anti-HTLV-2 antibodies using an enzyme-linked immunosorbent assay .ELISA-positive samples were subjected to DNA extraction using a QIAamp DNA Mini and Blood Mini Handbook Kit .qPCR was used to confirm and differentiate HTLV-1 and HTLV-2 infection. In each sample, the human albumin gene was used as an endogenous control, and the viral gene regions pol (186 bp) and tax (75 bp)  were amp2O), 0.5 μL of each primer, 0.5 μL of each probe and 5.0 μL of DNA were used, resulting in a total volume of 25 μL. The following temperature cycles were used: 95°C for 10 min, followed by 45 cycles of 95°C for 15 s and 60°C for binding of primers and probes for 1 min.The samples were tested in triplicate using Applied Biosystems Step One Plus Real-Time PCR equipment. For each reaction, 12.5 μL of TaqMan Universal PCR Master Mix (2X), 6.0 μL of ultrapure water (HThe primers used were 5′-CCCTACAATCCAACCAGCTCAG-3′ (HTLV-1F), 5′-GTGGTGAAGCTGCCATCGGGTTTT-3′ (HTLV-1R), 5′-CGATTGTGTACAGGCCGATTG-3′(HTLV-2F), 5′-CAGGAGGGCATGTCGATGTAG-3′ (HTLV-2R), 5′-GCTGTCATCTCTTGTGGGCTGT-3′ (Albumin F), and 5′-AAACTCATGGGAGCTGCTGGTT-3′ (Albumin R), and the probe sequences were FAM-5′-CTTTACTGACAAACCCGACCTACCCATGGA-3′-MGB (HTLV-1), FAM-5′-TGTCCCGTCTCAGGTGGTCTATGTTCCA-3′-MGB (HLTV-2) and FAM-5′-CCTGTCATGCCCACACAAATCTC-3′-MGB (Albumin) .Samples confirmed by qPCR as HTLV-2 positive were subjected to molecular subtype identification through a nested PCR of the env region (630 bp), followed by enzymatic digestion using the restriction enzyme XhoI, which generates two fragments (430 and 180 bp). The first reaction consisted of 29.0 μL of water (H2O), 5.0 μL of buffer (10X), 1.5 μL of MgCl2 (50 mM), 0.5 μL of primer (E2) 5′CTGCAGAAGCTAGCAGGTCTA-3′ (20 pmol), 0.5 μL primer (E5) 5′-AGCCAAGTGTCCCTTCGACTA-3′ (20 pmol), 8.0 μL of dNTPs (1.25 mM), 0.5 μL of Taq (1 U/μL), and 5.0 μL of DNA.The second reaction followed the same protocol, using the primers (E1) 5′-CTGCAACAACTCCATTATCCT-3′ and (E2) 5′CTGCAGAAGCTAGCAGGTCTA-3′ and 5.0 μL of DNA from the first reaction . Both re2O), 2.0 μL of RE Buffer (10X), 0.2 μL of BSA, 10.0 μL of DNA from the second PCR and 0.5 μL of the XhoI enzyme were incubated for 4 h at 37°C. The products were submitted to 3% agarose gel electrophoresis at a voltage of 100 V for 40 min and visualized with ethidium bromide staining.For enzymatic digestion with XhoI, 7.3 μL of water (H2O), 1.25 μL of buffer (10X), 3.0 μL of MgCl2 (50 mM), 6.0 μL of dNTPs (10 mM), 0.5 μL of each primer (20 pmol), 0.3 μL of Taq (1 U/μL) and 2.0 μL of DNA were combined. The following HTLV-1 primer sequences were used: (LTR-I.01) 5′-TGACAATGACCATGAGCCCCAA-3′, (LTR-I.02) 5′-CGCGGAATAGGGCTAGCGCT-3′, (LTR-I.03) 5′-GGCTTAGAGCCTCCCAGTGA-3′, and (LTR-I.04) 5′-GCTAGGGAATAAAGGGGCGC-3′. For HTLV-2, (F-IILTR) 5′-TCGCGATGACAATGGCGACTAGCCTC-3′, (Long-Gag) 5′-GGGGGCTTTGGGTATTGGAGTTGGG-3′, (Mo16) 5′-GCCTCCCAAGCCAGCCAC-3′, and (MSW-Gag) 5′- GGGAAAAGCCCGTGGATTTGCCCCAT-3′ were used. The following temperature cycle was used: 94°C for 5 min; 35 cycles at 94°C for 40 s, 62°C for 30 s and 72°C for 40 s; ending at 72°C for 10 min.The sample confirmed for HTLV-1 was subjected to amplification of the 5′LTR-1 region, with the aim of characterizing the molecular subtype. First, 11.45 μL of ultrapure water (HAfter purification of the PCR product (5'LTR region), the amplified DNA was sequenced using the Sanger method with a BigDye Terminator v3.1 Cycle Sequencing Kit  using Genetic Analyzer 3130xl equipment (Applied Biosystems) .Sequence alignment of 430 bp for HTLV-1 and 534 bp for HTLV-2 of the 5′LTR region was performed using the Clustal W program implemented in BioEdit software v7.1.9 . The phyAmong the 101 samples analyzed, the mean age of the participants was 36 years, ranging between 18 and 77 years. Three individuals (3.0%) tested positive by ELISA. Afterward, the infection was confirmed by characterizing of the viral type in the samples by means of qPCR analysis for the pol and tax genes.HTLV-1 infection was confirmed in a 55-year-old woman (1%), and HTLV-2 was present in a man (68 years old) and in a woman (23 years old) (2.0%). Of the three positive samples, it was possible to amplify the 5′LTR regions in only two for subsequent sequencing and phylogenetic analysis.The sample characterized as HTLV-1 positive showed, in the phylogenetic tree, 100% grouping with samples of the Cosmopolitan subtype (HTLV-1a), Transcontinental subgroup (A) .The HTLV-2 detected in one of the samples grouped in the phylogenetic tree, with 76% support, with samples belonging to the HTLV-2b subtype , formingAs part of the Screening and Counseling Program for People Living with HTLV implemented by the UFPA Virology Laboratory, the results of the serological screening and molecular confirmation were delivered to all participants.The three individuals positive for the infection were evaluated by a physician from the Belém Municipal Health Department for individual monitoring by the Unified Health System (SUS), at which time a consultation and interview were carried out to obtain information on risk factors and signs/symptoms associated with HTLV infection .The individual is female, 55 years old, and single; has lived in Brazil for 4 years; is illiterate and unemployed; and earns less than half the national minimum wage per month (U$200). She did not have tattoos or piercing, reported not knowing about receiving blood transfusions, and reported having 25 children who were breastfed for more than 6 months. She was breastfed as a child. She refused to answer whether she had had sex with a sex worker during her lifetime or if she had been sexually abused. She stated that she did not use condoms during sexual intercourse and did not remember the age of first sexual intercourse.The patient had a central nervous system tumor identified by CT at the health service, reported going to the bathroom more frequently than usual, felt pain in her body, was progressing to dementia, and had spots on her arm.The individual was male and 68 years old, lived together with his partner and had resided in Brazil for 3 years. He could read and write Spanish, but he had incomplete basic education, was unemployed, received emergency aid from the government, and earned ~1 national minimum wage per month. He reported having been a smoker but had quit a long time ago. He did not drink alcohol, had a tattoo located on his arm, reported that he had never received a blood transfusion, had never used illegal drugs, was breastfed as a child, had his first sexual intercourse at age 20, had a steady sexual partner, did not use a condom during sexual relations, had never had sex with a sex worker and reported never having been sexually abused.During the clinical evaluation, he reported having dizziness, frequent headache, joint pain, lower back pain and a swollen foot with spots for more than 1 year.The individual was female and 23 years old, had lived in Brazil for 1 year, lived with her partner, knew how to read and write in Spanish, had incomplete basic education, and was unemployed with a family income between half and one national minimum wage per month.Upon delivery of the result and interview with the physician for information about the presence of signs and symptoms, the patient refused to receive the result.In the present study, we identified the presence of HTLV-1 and HTLV-2 infections in Warao immigrant refugees in Belém, capital of the State of Pará. There are few studies on the prevalence of HTLV-1/2 infection in Venezuela, with emphasis by León et al.  on bloodThe first description of HTLV-2 in Venezuelan indigenous peoples occurred in 1993 in the Pumé ethnic group, with 5% of the samples being positive for HTLV-2. Perez et al.  found thOn the other hand, the description of the presence of HTLV-1 in the present study diverges from the three previous studies that investigated the seroprevalence for HTLV-1/2 in Venezuela –48. NotaStudies revealing a high prevalence of HIV-1 (9.55%) in Warao have also indicated a high probability of HTLV infection in this population since transmission routes and risk factors are common for these and other sexually transmitted infections (STIs) , 49.The Warao refugee population in Brazil is looking for better living conditions, such as work and access to the education and health system. However, they ultimately live in precarious situations, living in makeshift shelters and facing situations of great social and economic vulnerability, commonly becoming victims of racism, other forms of discrimination and the absence of public policies that are culturally sensitive to their needs as indigenous people and refugees , 50. AllIn our study, the occurrence of HTLV-1 and HTLV-2 in two females is perhaps associated with a greater exposure of women to a situation of risk for STIs due to the living conditions faced by these indigenous communities, where women are subordinate to men, resulting in cases of sexual abuse, family violence and risky sexual practices .To date, the results described here are unprecedented and reinforce the findings of the high prevalence of HTLV-2 in indigenous peoples, as already demonstrated by our group in populations from the Brazilian Amazon , 30, 51.One of the hypotheses to explain the presence of HTLV-2b in the Warao people would be the proximity of their place of origin to the Guahibo people, in addition to the similar life habits . During The sexual route being the main HTLV transmission route in the Warao population was supported by reports of not using condoms during sexual intercourse, increasing the susceptibility to STIs. Additionally, this hypothesis is reinforced by the precarious situation in which the Warao live, reporting lack of food, child malnutrition, and cases of pneumonia, tuberculosis, measles, coronavirus disease 2019 (COVID-19) and other diseases such as HIV and other STIs , 54, 55.HTLV-1 infection is associated with neurological symptoms that can affect between 1 and 5% of those infected, especially affecting middle-aged women . In the Finally, our results reveal for the first time the circulation of HTLV-1 and HTLV-2 in the Warao ethnic group from Venezuela, who are refugees in the State of Pará (northern Brazil). The presence of the HTLV-2b subtype reinforces that this molecular variant is prevalent among the indigenous peoples of South America and highlights migratory flow as an important means of introduction and dispersion of HTLV in human populations, especially those in the Brazilian Amazon. Due to the economic, political, environmental and humanitarian crises that several countries in Latin America are experiencing, resulting in intense migratory flows that mainly affect the most vulnerable groups, national states need to plan and implement culturally sensitive public health policies to welcome and care for traditional peoples in their territories, as well as guarantee adequate epidemiological surveillance and health services (among others) for immigrants, following multilateral legal provisions and the principles of humanitarian assistance and international solidarity.vallinoto@ufpa.br).The datasets presented in this article are not readily available for reasons related to data confidentiality and participant privacy. Requests to access the datasets should be directed to Prof. Antonio Carlos R. Vallinoto . The patients/participants provided their written informed consent to participate in this study.AV, SC-B, and HS conceived and designed the study. IA, CL, FL, FF, and MS performed the laboratory experiments. IA, CL, MF, LO, and AB carried out sample collection. VL conducted field medical monitoring. IA, AV, and HS wrote the manuscript. All authors read and approved the final manuscript.This study was funded by the National Council for Scientific and Technological Development (CNPQ #442522/2019-3 and #301869/2017-0), the Federal University of Pará (PAPQ/2021), and the Amazon Foundation for Studies and Support Research in Pará (FAPESPA).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. 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