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J Oral Pathol Med (2010) 39: 753-760 Background: There are several kinds of oral soft tissue lesions that are common manifestations observed in human immunodeficiency virus (HIV)-infected children; for example, linear gingival erythema (LGE) that is a distinctive fiery red band along the margin of the gingivae. The etiology and pathogenesis of LGE are questionable, but a candidal origin has been suggested. Proteases are key virulence attributes produced by a variety of pathogenic fungi, including Candida. The objective of the present study is to identify the protease production in Candida species including, C. albicans (n = 5), C. dubliniensis (n = 1) and C. tropicalis (n = 1), isolated directly from typical LGE lesions observed in six HIV-positive children, and also to test the effect of a serine protease inhibitor on the interaction of Candida spp. and epithelial cells in vitro. Methods: The ability of Candida strains to release proteases in the culture supernatant fluids was visualized by gelatin-SDS-PAGE. Gel strips containing 30-fold concentrated supernatant (1.5 x 10(8) yeasts) were incubated at 37°C for 48 h in 50 mM sodium phosphate buffer, pH 5.5. The concentrated supernatants were also incubated with fibronectin, laminin, immunoglobulin G, bovine serum albumin and human serum albumin. The effect of serine protease inhibitor on the interaction of Candida spp. and epithelial cells (MA 104) was measured after pre-treatment of fungi with the inhibitor (phenylmethylsulphonyl fluoride, PMSF). Results: All the extracellular proteases were completely inhibited by PMSF, identifying these activities as serine-type proteases. Interestingly, a common 62-kDa serine protease was observed in all Candida strains. The culture supernatants, rich in serine protease activities, cleaved several soluble proteinaceous substrates. Additionally, we demonstrated that pre-treatment of C. albicans, C. dubliniensis and C. tropicalis with PMSF diminished the interaction with epithelial cells. Conclusions: Collectively, our results show that Candida spp. isolated from LGE lesions produced and secreted serine proteases and these enzymes may be involved in the initial colonization events. [ABSTRACT FROM AUTHOR]

Background: Recent data have shown that HTLV-1 is prevalent among HIV positive patients in Mozambique, although the impact of HTLV-1 infection on HIV disease progression remains controversial. Our aim was to determine the phenotypic profile of T lymphocytes subsets among Mozambican patients co-infected by HIV and HTLV-1.Methods: We enrolled 29 patients co-infected by HTLV-1 and HIV (co-infected), 59 patients mono-infected by HIV (HIV) and 16 healthy controls (HC), respectively.For phenotypic analysis, cells were stained with the following fluorochrome-labeled anti-human monoclonal antibodies CD4-APC, CD8-PerCP, CD25-PE, CD62L-FITC, CD45RA-FITC. CD45RO-PE, CD38-PE; being analysed by four-colour flow cytometry.Results: We initially found that CD4+ T cell counts were significantly higher in co-infected, as compared to HIV groups. Moreover, CD4+ T Lymphocytes from co-infected patients presented significantly higher levels of CD45RO and CD25, but lower levels of CD45RA and CD62L, strongly indicating that CD4+ T cells are more activated under HTLV-1 plus HIV co-infection.Conclusion: Our data indicate that HTLV-1/HIV co-infected patients progress with higher CD4+ T cell counts and higher levels of activation markers. In this context, it is conceivable that in co-infected individuals, these higher levels of activation may account for a faster progression to AIDS. [ABSTRACT FROM AUTHOR]

The functional restoration of a damaged cardiac tissue relies on a synchronized contractile capacity of exogenous and/or endogenous cardiomyocytes, which is challenging to achieve. Here, we explored the potential of the short glycopeptide diphenylalanine glucosamine-6-sulfate (FFGlcN6S) conjugated with an aromatic moiety, namely fluorenylmethoxycarbonyl (Fmoc), to enhance cardiac tissue regeneration. At physiological conditions, Fmoc-FFGlcN6S assembles into nanofibrous hydrated meshes, i.e., matrix mimicking hydrogels. These hydrogels can be further micropatterned allowing co-existence of hierarchical structures at different lenght. The patterned hydrogels support the culture of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and promote their alignment. The cultured iPSC-CMs exhibit anisotropic synchronized contractions, indicating maturation and electrical interconnectivity. Moreover, the cultures express specific cardiac markers including, connexin-43 and sarcomeric-α-actinin, confirming enhanced cell-cell crosstalk, spontaneous contractility, and efficient transmission of electrical signals. Our results showcase the potential of short amphiphilic glycopeptides to mimic physical and biochemical cues that are essential for cardiomyocytes functionality and thus, these conjugates can be used in cardiac tissue engineering and regeneration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Viral encephalitis is a growing public health threat with limited diagnostic and treatment options. Simian immunodeficiency virus (SIV)-infected macaques are an established model for human immunodeficiency virus (HIV), and approximately 60% of untreated pigtail macaques rapidly progress to characteristic SIV encephalitis (SIVE). The immune responses of SIV-infected macaques are investigated in plasma, cerebrospinal fluid (CSF), and brain tissue to determine correlates with SIVE pathology. Macaques with SIVE show myeloid-dominant brain lesions with inflammasome activation in infected and bystander cells, as assessed by interleukin (IL)-1β, IL-18, and apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and elevations in monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, and tumor necrosis factor alpha (TNF-α). SIV-specific immunoglobulin (Ig)G in plasma and CSF is predictive of SIVE as early as 21 days post-inoculation; animals with SIVE continue to show negligible seroconversion 3 months after infection. This dichotomy in immune responses, wherein some macaques fail to initiate robust IgG responses and subsequently develop SIVE, provides insight into the pathogenesis and heterogeneous outcomes in viral encephalitis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

This study assessed the impact of phytoremediation on reducing the residual concentration of metolachlor in soil treated with doses of 530.7 and 1061.4 g/ha and its effect on microbial biodiversity in contaminated areas. For the plant species Avena sativa and Medicago sativa , a significant efficacy of 54.5 and 36.4% was observed in the dissipation of the herbicide, especially at higher doses. Although metolachlor application reduced soil microbial biodiversity, phytoremediating plants, especially M. sativa , promoted greater richness and distribution of microbial species, mitigating the negative effects of the herbicide. Principal component analysis revealed the influence of these plants and metolachlor on the composition of the microbial community. These results highlight the importance of phytoremediation in promoting soil biodiversity and reducing herbicide contamination, providing crucial insights for remediation strategies in contaminated areas.

Competing Interests: 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.

Background: Brain tissue-derived extracellular vesicles (bdEVs) act locally in the central nervous system (CNS) and may indicate molecular mechanisms in human immunodeficiency virus (HIV) CNS pathology. Using brain homogenate (BH) and bdEVs from a simian immunodeficiency virus (SIV) model of HIV disease, we identified RNA networks in SIV infection and neuroinflammation., Methods: Postmortem occipital cortex samples were obtained from uninfected controls and SIV-infected subjects (acute and chronic phases with or without CNS pathology [SIV encephalitis]). bdEVs were separated and characterized per international consensus guidelines. RNAs from bdEVs and BH were sequenced and quantitative polymerase chain reaction (qPCR)-amplified to detect levels of small RNAs (sRNAs, including microRNAs [miRNAs]) and longer RNAs including messenger RNAs (mRNAs) and circular RNAs (circRNAs)., Results: Dysregulated RNAs in BH and bdEVs were identified in acute and chronic infection with pathology groups, including mRNAs, miRNAs, and circRNAs. Most dysregulated mRNAs in bdEVs reflected dysregulation in source BH. These mRNAs are disproportionately involved in inflammation and immune responses. Based on target prediction, several circRNAs that were differentially abundant in source tissue might be responsible for specific differences in sRNA levels in bdEVs during SIV infection., Conclusions: RNA profiling of bdEVs and source tissues reveals potential regulatory networks in SIV infection and SIV-related CNS pathology., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The dermal papilla (DP), a specialized compartment within the hair follicle, regulates hair growth. However, human DP cells rapidly lose their inductivity in 2D-culture given the loss of positional and microenvironmental cues. Spheroids have been capable of recreating the 3D intercellular organization of DP cells, however, DP cell-matrix interactions are poorly represented. Considering the specific nature of the DP's extracellular matrix (ECM), we functionalized gellan gum (GG) with collagen IV-(HepIII) or fibronectin-(cRGDfC) derived peptide sequences to generate a 3D environment in which the phenotype and physiological functions of DP cells are restored. We further tuned the stiffness of the microenvironments by varying GG amount. Biomimetic peptides in stiffer hydrogels promoted the adhesion of DP cells, while each peptide and amount of polymer independently influenced the type and quantity of ECM proteins deposited. Furthermore, although peptides did not seem to have an influence, stiffer hydrogels improved the inductive capacity of DP cells after short term culture. Interestingly, independently of the peptide, these hydrogels supported the recapitulation of basic hair morphogenesis-like events when incorporated in an organotypic human skin in vitro model. Our work demonstrates that tailored GG hydrogels support the generation of a microenvironment in which both cell-ECM and cell-cell interactions positively influence DP cells towards the creation of an artificial DP., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

The development of persistent cellular reservoirs of latent human immunodeficiency virus (HIV) is a critical obstacle to viral eradication since viral rebound takes place once anti-retroviral therapy (ART) is interrupted. Previous studies show that HIV persists in myeloid cells (monocytes and macrophages) in blood and tissues in virologically suppressed people with HIV (vsPWH). However, how myeloid cells contribute to the size of the HIV reservoir and what impact they have on rebound after treatment interruption remain unclear. Here we report the development of a human monocyte-derived macrophage quantitative viral outgrowth assay (MDM-QVOA) and highly sensitive T cell detection assays to confirm purity. We assess the frequency of latent HIV in monocytes using this assay in a longitudinal cohort of vsPWH (n = 10, 100% male, ART duration 5-14 yr) and find half of the participants showed latent HIV in monocytes. In some participants, these reservoirs could be detected over several years. Additionally, we assessed HIV genomes in monocytes from 30 vsPWH (27% male, ART duration 5-22 yr) utilizing a myeloid-adapted intact proviral DNA assay (IPDA) and demonstrate that intact genomes were present in 40% of the participants and higher total HIV DNA correlated with reactivatable latent reservoirs. The virus produced in the MDM-QVOA was capable of infecting bystander cells resulting in viral spread. These findings provide further evidence that myeloid cells meet the definition of a clinically relevant HIV reservoir and emphasize that myeloid reservoirs should be included in efforts towards an HIV cure., (© 2023. The Author(s).)

Introduction: Antiretroviral treatment regimens can effectively control HIV replication and some aspects of disease progression. However, molecular events in end-organ diseases such as central nervous system (CNS) disease are not yet fully understood, and routine eradication of latent reservoirs is not yet in reach. Brain tissue-derived extracellular vesicles (bdEVs) act locally in the source tissue and may indicate molecular mechanisms in HIV CNS pathology. Regulatory RNAs from EVs have emerged as important participants in HIV disease pathogenesis. Using brain tissue and bdEVs from the simian immunodeficiency virus (SIV) model of HIV disease, we profiled messenger RNAs (mRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs), seeking to identify possible networks of RNA interaction in SIV infection and neuroinflammation., Methods: Postmortem occipital cortex tissue were collected from pigtailed macaques: uninfected controls and SIV-infected subjects (acute phase and chronic phase with or without CNS pathology). bdEVs were separated and characterized in accordance with international consensus standards. RNAs from bdEVs and source tissue were used for sequencing and qPCR to detect mRNA, miRNA, and circRNA levels., Results: Multiple dysregulated bdEV RNAs, including mRNAs, miRNAs, and circRNAs, were identified in acute infection and chronic infection with pathology. Most dysregulated mRNAs in bdEVs reflected dysregulation in their source tissues. These mRNAs are disproportionately involved in inflammation and immune responses, especially interferon pathways. For miRNAs, qPCR assays confirmed differential abundance of miR-19a-3p, let-7a-5p, and miR-29a-3p (acute SIV infection), and miR-146a-5p and miR-449a-5p (chronic with pathology) in bdEVs. In addition, target prediction suggested that several circRNAs that were differentially abundant in source tissue might be responsible for specific differences in small RNA levels in bdEVs during SIV infection., Conclusions: RNA profiling of bdEVs and source tissues reveals potential regulatory networks in SIV infection and SIV-related CNS pathology., Competing Interests: Conflicts of interest The authors report no conflicts of interest.

Objectives: Latent infection by HIV hinders viral eradication despite effective antiretroviral treatment (ART). Among proposed contributors to viral latency are cellular small RNAs that have also been proposed to shuttle between cells in extracellular vesicles. Thus, we profiled extracellular vesicle small RNAs during different infection phases to understand the potential relationship between these extracellular vesicle associated small RNAs and viral infection., Design: A well characterized simian immunodeficiency virus (SIV)/macaque model of HIV was used to profile extracellular vesicle enriched blood plasma fractions harvested during preinfection, acute infection, latent infection/ART treatment, and rebound after ART interruption., Methods: Measurement of extracellular vesicle concentration, size distribution, and morphology was complemented with qPCR array for small RNA expression, followed by individual qPCR validations. Iodixanol density gradients were used to separate extracellular vesicle subtypes and virions., Results: Plasma extracellular vesicle particle counts correlated with viral load and peaked during acute infection. However, SIV gag RNA detection showed that virions did not fully explain this peak. Extracellular vesicle microRNAs miR-181a, miR-342-3p, and miR-29a decreased with SIV infection and remained downregulated in latency. Interestingly, small nuclear RNA U6 had a tight association with viral load peak., Conclusion: This study is the first to monitor how extracellular vesicle concentration and extracellular vesicle small RNA expression change dynamically in acute viral infection, latency, and rebound in a carefully controlled animal model. These changes may also reveal regulatory roles in retroviral infection and latency., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Introduction: Most cancers occur in older individuals, who are more vulnerable due to functional impairment, multiple comorbidities, cognitive impairment, and lack of socio-familial support. These can undermine patients' sense of dignity. This study seeks to compare dignity scores in older patients with advanced cancer on sociodemographic and clinical variables and analyze the predictive value of anxiety, depression, functional limitations, and social support on dignity scores., Methods: A prospective, multicenter, observational study conducted with participation of 15 hospitals in Spain from February 2020 to October 2021. Patients with newly-diagnosed, advanced cancer completed the dignity (PPDS), anxiety and depression (BSI), Social Support (Duke-UNC-11), and functional limitations (EORTC-C30) scales. Lineal regression analyses explored the effects of anxiety, depression, functional status, and social support on dignity, adjusting for sociodemographic and clinical variables., Results: A total of 180 subjects participated in this study. The results of the correlation analysis revealed that dignity correlated negatively with anxiety, depression, and sex, and positively with social support, functional status, and longer estimated survival. Thus, women, and more anxious and depressed individuals scored lower on the dignity scale, whereas patients with more social support, fewer functional limitations, and longer estimated survival scored higher., Conclusion: In conclusion, being female, having a lower educational level, lower estimated survival, depression, anxiety, less social support, and limited functionality are correlated with less dignity in the elderly with advanced cancer. It is a priority to manage both physical and psychological symptoms in patients with unresectable advanced cancer to mitigate psychological distress and increase their sense of dignity., (© 2022. The Author(s).)

Germ cell tumors are the most frequent neoplasia in young males. The aims of this study is to describe a case in which a postpuberal teratoma suffers a transformation to choriocarcinoma and metastasize to stomach. We have made a systematic review in PubMed and consensus documents to study this mismatch between the tumour, metastasis and the exception of gastric metastatic affectation. We describe three options to explain this discordance: a mixed germ cells tumour, a burned out tumour or a germ cells tumour derived from a malignant germ cell tumour precursor or different clonal strains. After made a thorough investigation we conclude that the most truly option is the last one as we extensive explain below. Once the gastric metastatic lesions are extremely rare and reach to <5%, but there are not conclusive assessments., (Copyright © 2020 Sociedad Española de Anatomía Patológica. Publicado por Elsevier España, S.L.U. All rights reserved.)

This study examines the mediating role of social support between anxious preoccupation and resilience in patients with cancer during COVID-19. NEOetic&#95;SEOM is a prospective, multicenter study involving individuals with advanced, unresectable cancer who completed the following scales: Resilience (BCRS), Social Support (Duke-UNC-11), and anxious preoccupation subscale of the Mini-Mental Adjustment to Cancer (M-MAC) before starting antineoplastic treatment. Between March 2020 and July 2021, 507 patients (55% male; mean age, 65) were recruited. No differences in resilience were observed based on sociodemographic or clinical characteristics. Social support in people with advanced, unresectable cancer promotes both decreased anxious preoccupation and greater resilience.

Monocytes play a major role in the initial innate immune response to SARS-CoV-2. Although viral load may correlate with several clinical outcomes in COVID-19, much less is known regarding their impact on innate immune phenotype. We evaluated the monocyte phenotype and mitochondrial function in severe COVID-19 patients (n = 22) with different viral burden (determined by the median of viral load of the patients) at hospital admission. Severe COVID-19 patients presented lower frequency of CD14 + CD16- classical monocytes and CD39 expression on CD14 + monocytes, and higher frequency of CD14 + CD16 + intermediate and CD14-CD16 + nonclassical monocytes as compared to healthy controls independently of viral load. COVID-19 patients with high viral load exhibited increased GM-CSF, PGE-2 and lower IFN-α as compared to severe COVID-19 patients with low viral load (p < 0.05). CD14 + monocytes of COVID-19 patients with high viral load presented higher expression of PD-1 but lower HLA-DR on the cell surface than severe COVID-19 patients with low viral load. All COVID-19 patients presented decreased monocyte mitochondria membrane polarization, but high SARS-CoV-2 viral load was associated with increased mitochondrial reactive oxygen species. In this sense, higher viral load induces mitochondrial reactive oxygen species generation associated with exhaustion profile in CD14 + monocytes of severe COVID-19 patients. Altogether, these data shed light on new pathological mechanisms involving SARS-CoV-2 viral load on monocyte activation and mitochondrial function, which were associated with COVID-19 severity., (Copyright © 2022. Published by Elsevier B.V.)

An important hallmark in cancer research is the discovery of suitable features capable to reliably predict tumor invasiveness, and consequently, their metastatic potential at an early stage. Current methods are based on molecular biomarker screening and imaging that may not reveal the altered properties of tumor cells, being also labor-intensive and costly. Biophysical-based methodologies provide a new framework assessing-and even predicting-the invasion potential of tumors with improved accuracy. In particular, the stochastic fluctuations of cancer invasive protrusions can be used as a tumor-specific biophysical indicator of its aggressiveness. In this methodology, tumor micro-spheroids with different metastatic capabilities were employed as in vitro models to analyze protrusion activity. It is described the procedure for extracting the descriptive biophysical parameters characteristic of protrusion activity, which magnitude depends on the invasion capability of tumors. Next, a simple mathematical approach is employed to define a predictive index that correlates with tumor invasiveness. Overall, this innovative approach may provide a simple method for unveiling cancer invasiveness and complement existing diagnosis methodologies., Supplementary Information: The online version contains supplementary material available at 10.1007/s44164-022-00020-1., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s), under exclusive licence to Springer Nature Switzerland AG 2022.)

Extracellular vesicles (EVs) are small lipidic particles packed with proteins, DNA, messenger RNA and microRNAs of their cell of origin that act as critical players in cell-cell communication. These vesicles have been identified as pivotal mediators in cancer progression and the formation of metastatic niches. Hence, their isolation and analysis from circulating biofluids is envisioned as the next big thing in the field of liquid biopsies for early non-invasive diagnosis and patient follow-up. Despite the promise, current benchtop isolation strategies are not compatible with point-of-care testing in a clinical setting. Microfluidic platforms are disruptive technologies capable of recovering, analyzing, and quantifying EVs within clinical samples with limited volume, in a high-throughput manner with elevated sensitivity and multiplexing capabilities. Moreover, they can also be employed for the controlled production of synthetic EVs and effective drug loading to produce EV-based therapies. In this review, we explore the use of microfluidic platforms for the isolation, characterization, and quantification of EVs in cancer, and compare these platforms with the conventional methodologies. We also highlight the state-of-the-art in microfluidic approaches for EV-based cancer therapeutics. Finally, we analyze the currently active or recently completed clinical trials involving EVs for cancer diagnosis, treatment or therapy monitoring and examine the future of EV-based point-of-care testing platforms in the clinic and EV-based therapy production by the industry.

Background: Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target genes by molecular methods has been chosen as the main approach to identify individuals with Coronavirus disease 2019 (COVID-19) infection., Objectives: In this study, we developed an open-source RNA standard-based real-time quantitative RT-PCR (RT-qPCR) assay for quantitative diagnostics of SARS-CoV-2 from nasopharynx, oropharynx, saliva and plasma samples., Methods and Findings: We evaluated three SARS-CoV-2 target genes and selected the RNA-dependent RNA polymerase (RdRp) gene, given its better performance. To improve the efficiency of the assay, a primer gradient containing 25 primers forward and reverse concentration combinations was performed. The forward and reverse primer pairs with 400 nM and 500 nM concentrations, respectively, showed the highest sensitivity. The LOD95% was ~60 copies per reaction. From the four biological matrices tested, none of them interfered with the viral load measurement. Comparison with the AllplexTM 2019-nCoV assay (Seegene) demonstrated that our test presents 90% sensitivity and 100% specificity., Main Conclusions: We developed an efficient molecular method able to measure absolute SARS-CoV-2 viral load with high replicability, sensitivity and specificity in different clinical samples.

The tumor microenvironment (TME) is like the Referee of a soccer match who has constant eyes on the activity of all players, such as cells, acellular stroma components, and signaling molecules for the successful completion of the game, that is, tumorigenesis. The cooperation among all the "team members" determines the characteristics of tumor, such as the hypoxic and acidic niche, stiffer mechanical properties, or dilated vasculature. Like in soccer, each TME is different. This heterogeneity makes it challenging to fully understand the intratumor dynamics, particularly among different tumor subpopulations and their role in therapeutic response or resistance. Further, during metastasis, tumor cells can disseminate to a secondary organ, a critical event responsible for approximately 90% of the deaths in cancer patients. The recapitulation of the rapidly changing TME in the laboratory is crucial to improve patients' prognosis for unraveling key mechanisms of tumorigenesis and developing better drugs. Hence, in this chapter, we provide an overview of the characteristic features of the TME and how to model them, followed by a brief description of the limitations of existing in vitro platforms. Finally, various attempts at simulating the TME using microfluidic platforms are highlighted. The chapter ends with the concerns that need to be addressed for designing more realistic and predictive tumor-on-a-chip platforms., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Microfluidics and biosensors have already demonstrated their potential in cancer research. Typical applications of microfluidic devices include the realistic modeling of the tumor microenvironment for mechanistic investigations or the real-time monitoring/screening of drug efficacy. Similarly, point-of-care biosensing platforms are instrumental for the early detection of predictive biomarkers and their accurate quantification. The combination of both technologies offers unprecedented advantages for the management of the disease, with an enormous potential to contribute to improving patient prognosis. Despite their high performance, these methodologies are still encountering obstacles for being adopted by the healthcare market, such as a lack of standardization, reproducibility, or high technical complexity. Therefore, the cancer research community is demanding better tools capable of boosting the efficiency of cancer diagnosis and therapy. During the last years, innovative microfluidic and biosensing technologies, both individually and combined, have emerged to improve cancer theranostics. In this chapter, we discuss how these emerging-and in some cases unconventional-microfluidics and biosensor technologies, tools, and concepts can enhance the predictive power of point-of-care devices and the development of more efficient cancer therapies., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Early cancer screening and effective diagnosis is the most effective form to diminish the number of cancer-related deaths. Liquid biopsy constitutes an attractive alternative to tumor biopsy due to its non-invasive nature and sample accessibility, which permits effective screening and patient monitoring. Within the plethora of biomarkers present in circulation, liquid biopsy has mainly been performed by analyzing circulating tumor cells, and more recently, extracellular vesicles. Tracking these biological particles could provide valuable insights into cancer origin, progression, treatment efficacy, and patient prognosis. Microfluidic devices have emerged as viable solutions for point-of-care cancer screening and monitoring due to their user-friendly operation, low operation costs, and capability of processing, quantifying, and analyzing these bioparticles in a single device. However, the size difference between cells and exosomes (micrometer vs nanometer) requires an adaptation of microfluidic isolation approaches, particularly in label-free methodologies governed by particle and fluid mechanics. This chapter will explore the theory behind particle isolation and sorting in different microfluidic techniques necessary to guide researchers into the design and development of such devices., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

The role of CD4+ T cells in HIV infection and the latent reservoir, that is, latently infected cells that harbor replication competent virus, has been rigorously assessed. We have previously reported a quantitative viral outgrowth assay (QVOA) for SIV that demonstrated the frequency of latently infected CD4+ T cells is approximately 1 in a million cells, similar to that of HIV infected individuals on ART. However, the frequency of productively infected monocytes in blood and macrophages in tissues has not been similarly studied. Myeloid cells are infected during acute HIV and SIV infection; however, unlike lymphocytes, they are resistant to the cytopathic effects of the virus. Moreover, tissue-resident macrophages have the ability to self-renew and persist in the body for months to years. Thus, tissue macrophages, once infected, have the characteristics of a stable viral reservoir. A better understanding of the number of productively infected macrophages is critical to understanding the role of infected myeloid cells as a viral reservoir. In order to assess the functional latent reservoir. we have developed specific QVOAs for monocytes in blood, and macrophages in spleen, BAL and brain, which are described in detail in this chapter., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Despite significant achievements in the understanding and treatment of cancer, it remains a major burden. Traditional therapeutic approaches based on the 'one-size-fits-all' paradigm are becoming obsolete, as demonstrated by the increasing number of patients failing to respond to treatments. In contrast, more precise approaches based on individualized genetic profiling of tumors have already demonstrated their potential. However, even more personalized treatments display shortcomings mainly associated with systemic delivery, such as low local drug efficacy or specificity. A large amount of effort is currently being invested in developing precision medicine-based strategies for improving the efficiency of cancer theranostics and modelling, which are envisioned to be more accurate, standardized, localized, and less expensive. To this end, interdisciplinary research fields, such as biomedicine, material sciences, pharmacology, chemistry, tissue engineering, and nanotechnology, must converge for boosting the precision cancer ecosystem. In this regard, precision biomaterials have emerged as a promising strategy to detect, model, and treat cancer more efficiently. These are defined as those biomaterials precisely engineered with specific theranostic functions and bioactive components, with the possibility to be tailored to the cancer patient needs, thus having a vast potential in the increasing demand for more efficient treatments. In this review, we discuss the latest advances in the field of precision biomaterials in cancer research, which are expected to revolutionize disease management, focusing on their uses for cancer modelling, detection, and therapeutic applications. We finally comment on the needed requirements to accelerate their application in the clinic to improve cancer patient prognosis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The generation of invasive fluctuating protrusions is a distinctive feature of tumor dissemination. During the invasion, individual cancer cells modulate the morphodynamics of protrusions to optimize their migration efficiency. However, it remains unclear how protrusion fluctuations govern the invasion of more complex multi-cellular structures, such as tumors, and their correlation with the tumor metastatic potential. Herein, a reductionist approach based on 3D tumor cell micro-spheroids with different invasion capabilities is used as a model to decipher the role of tumor-associated fluctuating protrusions in cancer progression. To quantify fluctuations, a set of key biophysical parameters that precisely correlate with the invasive potential of tumors is defined. It is shown that different pharmacological drugs and cytokines are capable of modulating protrusion activity, significantly altering protrusion fluctuations, and tumor invasiveness. This correlation is used to define a novel quantitative invasion index encoding the key biophysical parameters of fluctuations and the relative levels of cell-cell/matrix interactions, which is capable of assessing the tumor's metastatic capability solely based on its magnitude. Overall, this study provides new insights into how protrusion fluctuations regulate tumor cell invasion, suggesting that they may be employed as a novel early indicator, or biophysical signature, of the metastatic potential of tumors., (© 2021 Wiley-VCH GmbH.)

The microlepidoptera, Bedellia somnulentella (Zeller), is an important pest of sweetpotato, Ipomoea batatas (L.) Lam. Damage by B. somnulentella occurs in the larval stage and when consuming the foliar mesophyll of I. batatas make the leaves brown, wrinkled, and reducing the photosynthetic area and the yield. The detection and management of this pest depends on knowing its biological cycle and identifying its natural enemies. The objectives of this study were to determine the life history of B. somnulentella feeding on I. batatas leaves and to survey parasitoids of this pest in the field. The duration and viability of B. somnulentella egg, larva, prepupa, pupa, and adult stages were evaluated under laboratory conditions. Cephalic capsule width was measured to determine the number of B. somnulentella instars, based on the Dyar rule and analyzed by the Akaike statistical model (AIC). The developmental period of B. somnulentella was 32.5 ± 0. 21 d with a viability of 75, 84, 100, and 84% for the egg, larva, prepupa, and pupa stages, respectively. The identification of this pest on the plants is possible from the third instar and in the pupal and adult stages. The parasitoid Conura sp. (Hymenoptera: Chalcididae) was identified parasitizing pupae of B. somnulentella and could be considered a potential natural enemy for the integrated management of this pest., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Human dermal papilla (DP) cells and melanocytes (hMel) are central players in hair growth and pigmentation, respectively. In hair follicles (HFs), oxygen (O 2 ) levels average 5%, being coupled with the production of reactive oxygen species (ROS), necessary to promote hair growth., Materials and Methods: DP cell and hMel proliferation and phenotype were studied under physiological (5%O 2 , physoxia) or atmospheric (21%O 2 , normoxia) oxygen levels. hMel-DP cells interactions were studied in indirect co-culture or by directly co-culturing hMel with DP spheroids, to test whether their interaction affected the response to physoxia., Results: Physoxia decreased DP cell senescence and improved their secretome and phenotype, as well as hMel proliferation, migration, and tyrosinase activity. In indirect co-cultures, physoxia affected DP cells' alkaline phosphatase (ALP) activity but their signalling did not influence hMel proliferation or tyrosinase activity. Additionally, ROS production was higher than in monocultures but a direct correlation between ROS generation and ALP activity in DP cells was not observed. In the 3D aggregates, where hMel are organized around the DP, both hMel tyrosinase and DP cells ALP activities, their main functional indicators, plus ROS production were higher in physoxia than normoxia., Conclusions: Overall, we showed that the response to physoxia differs according to hMel-DP cells interactions and that the microenvironment recreated when in direct contact favours their functions, which can be relevant for hair regeneration purposes., (© 2021 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

The hair follicle (HF) is an exquisite skin appendage endowed with cyclical regenerative capacity; however, de novo follicle formation does not naturally occur. Consequently, patients suffering from extensive skin damage or hair loss are deprived of the HF critical physiological and/or aesthetic functions, severally compromising skin function and the individual's psychosocial well-being. Translation of regenerative strategies has been prevented by the loss of trichogenic capacity that relevant cell populations undergo in culture and by the lack of suitable human-based in vitro testing platforms. Here, we provide a comprehensive overview of the major difficulties associated with HF regeneration and the approaches used to overcome these drawbacks. We describe key cellular requirements and discuss the importance of the HF extracellular matrix and associated signaling for HF regeneration. Finally, we summarize the strategies proposed so far to bioengineer human HF or hair-bearing skin models and disclose future trends for the field., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

BACKGROUNDIdentifying a quantitative biomarker of neuropsychiatric dysfunction in people with HIV (PWH) remains a significant challenge in the neuroHIV field. The strongest evidence to date implicates the role of monocytes in central nervous system (CNS) dysfunction in HIV, yet no study has examined monocyte subsets in blood as a correlate and/or predictor of neuropsychiatric function in virally suppressed PWH.METHODSIn 2 independent cohorts of virologically suppressed women with HIV (vsWWH; n = 25 and n = 18), whole blood samples were obtained either in conjunction with neuropsychiatric assessments (neuropsychological [NP] test battery, self-report depression and stress-related symptom questionnaires) or 1 year prior to assessments. Immune cell subsets were assessed by flow cytometry.RESULTSA higher proportion of intermediate monocytes (CD14+CD16+) was associated with lower global NP function when assessing monocytes concurrently and approximately 1 year before (predictive) NP testing. The same pattern was seen for executive function (mental flexibility) and processing speed. Conversely, there were no associations with monocyte subsets and depression or stress-related symptoms. Additionally, we found that a higher proportion of classical monocytes was associated with better cognition.CONCLUSIONAlthough it is widely accepted that lentiviral infection of the CNS targets cells of monocyte-macrophage-microglial lineage and is associated with an increase in intermediate monocytes in the blood and monocyte migration into the brain, the percentage of intermediate monocytes in blood of vsWWH has not been associated with neuropsychiatric outcomes. Our findings provide evidence for a new, easily measured, blood-based cognitive biomarker in vsWWH.FUNDINGR01-MH113512, R01-MH113512-S, P30-AI094189, R01-MH112391, R01-AI127142, R00-DA044838, U01-AI35004, and P30-MH075673.

Rheumatoid arthritis (RA) is an autoimmune and chronic inflammatory disease characterized by joint inflammation. Since the inflammatory condition plays an important role in the disease process, it is important to develop and test new therapeutic approaches that specifically target and treat joint inflammation. In this study, a human 3D inflammatory cartilage-on-a-chip model was established to test the therapeutic efficacy of anti-TNFα mAb-CS/PAMAM dendrimer NPs loaded-Tyramine-Gellan Gum in the treatment of inflammation. The results showed that the proposed therapeutic approach applied to the human monocyte cell line (THP-1) and human chondrogenic primary cells (hCH) cell-based inflammation system revealed an anti-inflammatory capacity that increased over 14 days. It was also possible to observe that Coll type II was highly expressed by inflamed hCH upon the culture with anti-TNF α mAb-CS/PAMAM dendrimer NPs, indicating that the hCH cells were able maintain their biological function. The developed preclinical model allowed us to provide more robust data on the potential therapeutic effect of anti-TNF α mAb-CS/PAMAM dendrimer NPs loaded-Ty-GG hydrogel in a physiologically relevant model.

Cilia are microtubule-based organelles that carry out a wide range of critical functions throughout the development of higher animals. Regardless of their type, all cilia rely on a motor-driven, bidirectional transport system known as intraflagellar transport (IFT). Of the many components of the IFT machinery, IFT20 is one of the smallest subunits. Nevertheless, IFT20 has been shown to play critical roles in the assembly of several types of mammalian cilia. Here we show that the IFT20 homolog in Caenorhabditis elegans , IFT-20, is also important for correct cilium assembly in sensory neurons. Strikingly, however, we find that IFT-20-deficient animals are able to assemble short, vestigial cilia. In spite of this, we show that practically all IFT-20-deficient animals fail to respond to environmental cues that are normally detected by cilia to modulate their behavior. Altogether, our results indicate that IFT-20 is critical for both the correct assembly and function of cilia in C. elegans ., (Copyright: © 2021 by the authors.)

Liquid-gated Graphene Field-Effect Transistors (GFET) are ultrasensitive bio-detection platforms carrying out the graphene's exceptional intrinsic functionalities. Buffer and dilution factor are prevalent strategies towards the optimum performance of the GFETs. However, beyond the Debye length (λD), the role of the graphene-electrolytes' ionic species interactions on the DNA behavior at the nanoscale interface is complicated. We studied the characteristics of the GFETs under different ionic strength, pH, and electrolyte type, e.g., phosphate buffer (PB), and phosphate buffer saline (PBS), in an automatic portable built-in system. The electrostatic gating and charge transfer phenomena were inferred from the field-effect measurements of the Dirac point position in single-layer graphene (SLG) transistors transfer curves. Results denote that λ D is not the main factor governing the effective nanoscale screening environment. We observed that the longer λ D was not the determining characteristic for sensitivity increment and limit of detection (LoD) as demonstrated by different types and ionic strengths of measuring buffers. In the DNA hybridization study, our findings show the role of the additional salts present in PBS, as compared to PB, in increasing graphene electron mobility, electrostatic shielding, intermolecular forces and DNA adsorption kinetics leading to an improved sensitivity.

Background: Hair follicle (HF) development and growth are dependent on epithelial-mesenchymal interactions (EMIs). Dermal papilla (DP) cells are recognized as the key inductive mesenchymal player, but the ideal source of receptive keratinocytes for human HF regeneration is yet to be defined. We herein investigated whether human interfollicular epidermal keratinocytes with stem-like features (EpSlKCs), characterized by a α6 bri /CD71 dim expression, can replace human hair follicular keratinocytes (HHFKCs) for the recreation of the HF epithelium and respective EMIs., Methods: The α6 bri /CD71 dim cellular fraction was selected from the whole interfollicular keratinocyte population through fluorescence-activated cell sorting and directly compared with follicular keratinocytes in terms of their proliferative capacity and phenotype. The crosstalk with DP cells was studied in an indirect co-culture system, and EpSlKC hair forming capacity tested in a hair reconstitution assay when combined with DP cells., Results: EpSlKCs exhibited a phenotypic profile similar to follicular keratinocytes and were capable of increasing DP cell proliferation and, for short co-culture times, the number of alkaline phosphatase-active cells, suggesting an improvement of their inductivity. Moreover, the recreation of immature HFs and sebaceous glands was observed after EpSlKC and DP cell co-grafting in nude mice., Conclusions: Our results suggest that EpSlKCs are akin to follicular keratinocytes and can crosstalk with DP cells, contributing to HF morphogenesis in vivo, thus representing an attractive epithelial cell source for hair regeneration strategies.

The relevance of monocyte and macrophage reservoirs in virally suppressed people with HIV (vsPWH) has previously been debatable. Macrophages were assumed to have a moderate life span and lack self-renewing potential. However, recent studies have challenged this dogma and now suggest an important role of these cell as long-lived HIV reservoirs. Lentiviruses have a long-documented association with macrophages and abundant evidence exists that macrophages are important target cells for HIV in vivo. A critical understanding of HIV infection, replication, and latency in macrophages is needed in order to determine the appropriate method of measuring and eliminating this cellular reservoir. This review provides a brief discussion of the biology and acute and chronic infection of monocytes and macrophages, with a more substantial focus on replication, latency and measurement of the reservoir in cells of myeloid origin., (Copyright © 2021. Published by Elsevier Ltd.)

Engineering complex tissues requires the use of advanced biofabrication techniques that allow the replication of the tissue's 3D microenvironment, architecture and cellular interactions. In the case of skin, the most successful strategies to introduce the complexity of hair follicle (HF) appendages have highlighted the importance of facilitating direct interaction between dermal papilla (DP) cells and keratinocytes (KCs) in organotypic skin models. In this work, we took advantage of microscopy-guided laser ablation (MGLA) to microfabricate a fibroblast-populated collagen hydrogel and create a subcompartment that guides the migration of KCs and lead their interaction with DP cells to recreate follicular structures. Upon definition of the processing parameters (laser incidence area and power), MGLA was used to create 3D microchannels from the surface of a standard organotypic human skin model up to the aggregates containing DP cells and KCs, previously incorporated into the dermal-like fibroblast-collagen layer. Analysis of the constructs showed that the fabricated microfeatures successfully guided the fusion between epidermal and aggregates keratinocytes, which differentiated into follicular-like structures within the organotypic human skin model, increasing its functionality. In summary, we demonstrate the fabrication of a highly structured 3D hydrogel-based construct using MGLA to attain a complex skin model bearing folliculoid structures, highlighting its potential use as an in vitro platform to study the mechanisms controlling HF development or for the screening of bioactive substances., Competing Interests: The authors declare that there is no conflict of interest., (© 2020 The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

Introduction: The dermal papilla (DP) represents the major regulatory entity within the hair follicle (HF), inducing hair formation and growth through reciprocal interactions with epithelial cells. However, human DP cells rapidly lose their hair inductive ability when cultured in an epithelium-deficient environment., Objectives: To determine if the conditioned medium collected from interfollicular keratinocytes (KCs-CM) is capable of improving DP cell native properties and inductive phenotype., Methods: DP cells were cultured with KCs-CM both in 2D and 3D culture conditions (spheroids). Further, the hair-inductive capacity of DP cells precultured with KCs-CM was tested in a hair reconstitution assay, after co-grafting with human keratinocytes in nude mice., Results: We demonstrate that KCs-CM contributes to restore the inductivity of cultured human DP cells in a more effective mode than the conventional 3D-cultures. This is supported by the higher active alkaline phosphatase (ALP) levels in DP cells, the improved self-aggregative capacity and the reduced expression of α-SMA and the V1-isoform of versican. Moreover, DP cells cultured with KCs-CM displayed a secretome profile (VEGF, BMP2, TGF- β1, IL-6) that matches the one observed during anagen. KCs-CM also enhanced DP cell proliferation, while preventing cells to undergo morphological changes characteristic of high passage cells. In opposition, the amount of collagenous and non-collagenous proteins deposited by DP cells was lower in the presence of KCs-CM. The improvement in ALP activity was maintained in 3D spheroidal cultures, even after KCs-CM retrieval, being superior to the effect of the gold-standard culture conditions. Moreover, DP cells cultured with KCs-CM and grafted with human keratinocytes supported the formation of HF- and sebaceous gland-like structures in mice., Conclusion: The proposed strategy encourages future cell-based strategies for HF regeneration not only in the context of hair-associated disorders, but also in the management of wounds to aid in restoring critical skin regulatory appendages., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors.)

Bladder cancer is the most common malignancy of the urinary tract, having one of the highest recurrence rates and progression from non-muscle to muscle invasive bladder cancer that commonly leads to metastasis. Cystoscopy and urine cytology are the standard procedures for its detection but have limited clinical sensitivity and specificity. Herein, a microfluidic device, the UriChip, was developed for the enrichment of urothelial exfoliated cells from fresh and frozen urine, based on deformability and size, and the cancer-associated glycan Sialyl-Tn explored as a putative bladder cancer urinary biomarker. Spiking experiments with bladder cancer cell lines showed an isolation efficiency of 53%, while clinical sample analyses revealed retention of cells with various morphologies and sizes. in situ immunoassays demonstrated significantly higher number of Sialyl-Tn-positive cells in fresh and frozen voided urine from bladder cancer patients, compared to healthy individuals. Of note, urothelial exfoliated cells from cryopreserved urine sediments were also successfully isolated by the UriChip, and found to express significantly high levels of Sialyl-Tn. Remarkably, Sialyl-Tn expression is correlated with tumor stage and grade. Overall, our findings demonstrate the potential of UriChip and Sialyl-Tn to detect urothelial bladder cancer cells in follow-up and long-term retrospective studies., (Copyright © 2020 Carvalho, Abreu, Ferreira, Lima, Ferreira, Santos, Ribeiro, Grenha, Martínez-Fernández, Duenas, Suárez-Cabrera, Paramio, Diéguez, Freitas and Oliveira.)

In vitro fertilization (IVF) is the most common assisted reproductive technology used to treat infertility. Embryo selection for transfer in IVF cycles relies on the morphological evaluation by embryologists, either by conventional microscopic assessment or more recently by time-lapse imaging systems. Despite the introduction of time-lapse imaging improvements in IVF success rates have failed to materialize, therefore alternative approaches are needed. Recent studies have shown that embryos resulting in successful pregnancy differ in their secretome and metabolism compared to embryos that fail to implant, suggesting that molecular analysis of embryo culture medium could assist in non-invasive single embryo selection. However, this approach has yet to be adopted clinically due to the lack of appropriate highly sensitive screening technologies needed to assess volume-limited samples. Here we report the detection of hCGβ, IL-8 and TNFα from conditioned culture media of single human embryos using electrochemical impedance spectroscopy. The impedimetric immunosensors revealed that morphologically non-viable embryos produce higher levels of IL-8 and TNFα, associated with abnormal cell division and cell death, respectively. More importantly, hCGβ detection was able to discriminate apparently morphologically identical viable embryos. This work brings an objective dimension to embryo selection, which could overcome the major limitations of morphology-based embryo selection for implantation. Future work should include the validation of these biomarkers in a large patient cohort., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

DNA methylation and histone deacetylation are key epigenetic processes involved in normal cellular function and tumorigenesis. Therapeutic strategies based on DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors are currently in use and under development for the treatment of cancers. Genome-wide DNA methylation profiling has been proposed for use in disease diagnosis, and histone modification profiling for disease stratification will follow suit. However, whether epigenome sequencing technologies will be feasible for rapid clinic diagnosis and patient treatment monitoring remains to be seen, and alternative detection technologies will almost certainly be needed. Here we used electrochemical impedance spectroscopy (EIS) employing a graphene-based screen-printed electrode system to directly measure global DNA methylation and histone H3 acetylation to compare non-cancer and breast cancer cell lines. We demonstrated that whilst global methylation was not useful as a differential marker in the cellular systems tested, histone H3 acetylation was effective at higher chromatin levels. Using breast and endometrial cancer cell models, EIS was then used to monitor cellular responses to the DNMT and HDAC inhibitors 5-Aza-2'-deoxycytidine and suberoylanilide hydroxamic acid in vitro, and proved very effective at detecting global cellular responses to either treatment, indicating that this approach could be useful in following treatment response to epigenetic drugs. Moreover, this work reports the first combined analysis of two epigenetic markers using a unified graphene-based biosensor platform, demonstrating the potential for multiplex analysis of both methylation and acetylation on the same sample., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Human immunodeficiency virus (HIV) eradication or long-term suppression in the absence of antiretroviral therapy (ART) requires an understanding of all viral reservoirs that could contribute to viral rebound after ART interruption. CD4 T cells (CD4s) are recognized as the predominant reservoir in HIV type 1 (HIV-1)-infected individuals. However, macrophages are also infected by HIV-1 and simian immunodeficiency virus (SIV) during acute infection and may persist throughout ART, contributing to the size of the latent reservoir. We sought to determine whether tissue macrophages contribute to the SIVmac251 reservoir in suppressed macaques. Using cell-specific quantitative viral outgrowth assays (CD4-QVOA and MΦ-QVOA), we measured functional latent reservoirs in CD4s and macrophages in ART-suppressed SIVmac251-infected macaques. Spleen, lung, and brain in all suppressed animals contained latently infected macrophages, undetectable or low-level SIV RNA, and detectable SIV DNA. Silent viral genomes with potential for reactivation and viral spread were also identified in blood monocytes, although these cells might not be considered reservoirs due to their short life span. Additionally, virus produced in the MΦ-QVOA was capable of infecting healthy activated CD4s. Our results strongly suggest that functional latent reservoirs in CD4s and macrophages can contribute to viral rebound and reestablishment of productive infection after ART interruption. These findings should be considered in the design and implementation of future HIV cure strategies. IMPORTANCE This study provides further evidence that the latent reservoir is comprised of both CD4 + T cells and myeloid cells. The data presented here suggest that CD4 + T cells and macrophages found throughout tissues in the body can contain replication-competent SIV and contribute to rebound of the virus after treatment interruption. Additionally, we have shown that monocytes in blood contain latent virus and, though not considered a reservoir themselves due to their short life span, could contribute to the size of the latent reservoir upon entering the tissue and differentiating into long-lived macrophages. These new insights into the size and location of the SIV reservoir using a model that is heavily studied in the HIV field could have great implications for HIV-infected individuals and should be taken into consideration with the development of future HIV cure strategies., (Copyright © 2019 Abreu et al.)

Understanding the cellular and anatomical sites of latent virus that contribute to human immunodeficiency virus (HIV) rebound is essential for eradication. In HIV-positive patients, CD4 + T lymphocytes comprise a well-defined functional latent reservoir, defined as cells containing transcriptionally silent genomes able to produce infectious virus once reactivated. However, the persistence of infectious latent virus in CD4 + T cells in compartments other than blood and lymph nodes is unclear. Macrophages (Mϕ) are infected by HIV/simian immunodeficiency virus (SIV) and are likely to carry latent viral genomes during antiretroviral therapy (ART), contributing to the reservoir. Currently, the gold standard assay used to measure reservoirs containing replication-competent virus is the quantitative viral outgrowth assay (QVOA). Using an SIV-macaque model, the CD4 + T cell and Mϕ functional latent reservoirs were measured in various tissues using cell-specific QVOAs. Our results showed that blood, spleen, and lung in the majority of suppressed animals contain latently infected Mϕs. Surprisingly, the numbers of CD4 + T cells, monocytes, and Mϕs carrying infectious genomes in blood and spleen were at comparable frequencies (∼1 infected cell per million). We also demonstrate that ex vivo viruses produced in the Mϕ QVOA are capable of infecting activated CD4 + T cells. These results strongly suggest that latently infected tissue Mϕs can reestablish productive infection upon treatment interruption. This study provides the first comparison of CD4 + T cell and Mϕ functional reservoirs in a macaque model. It is the first confirmation of the persistence of latent genomes in monocytes in blood and Mϕs in the spleen and lung of SIV-infected ART-suppressed macaques. Our results demonstrate that transcriptionally silent genomes in Mϕs can contribute to viral rebound after ART interruption and should be considered in future HIV cure strategies. IMPORTANCE This study suggests that CD4 + T cells found throughout tissues in the body can contain replication-competent SIV and contribute to rebound of the virus after treatment interruption. In addition, this study demonstrates that macrophages in tissues are another cellular reservoir for SIV and may contribute to viral rebound after treatment interruption. This new insight into the size and location of the SIV reservoir could have great implications for HIV-infected individuals and should be taken into consideration for the development of future HIV cure strategies., (Copyright © 2019 Abreu et al.)