Clonally expanded CD8 T cells patrol the cerebrospinal fluid in Alzheimer's disease.

Alzheimer's disease is an incurable neurodegenerative disorder in which neuroinflammation has a critical function1. However, little is known about the contribution of the adaptive immune response in Alzheimer's disease2. Here, using integrated analyses of multiple cohorts, we identify peripheral and central adaptive immune changes in Alzheimer's disease. First, we performed mass cytometry of peripheral blood mononuclear cells and discovered an immune signature of Alzheimer's disease that consists of increased numbers of CD8+ T effector memory CD45RA+ (TEMRA) cells. In a second cohort, we found that CD8+ TEMRA cells were negatively associated with cognition. Furthermore, single-cell RNA sequencing revealed that T cell receptor (TCR) signalling was enhanced in these cells. Notably, by using several strategies of single-cell TCR sequencing in a third cohort, we discovered clonally expanded CD8+ TEMRA cells in the cerebrospinal fluid of patients with Alzheimer's disease. Finally, we used machine learning, cloning and peptide screens to demonstrate the specificity of clonally expanded TCRs in the cerebrospinal fluid of patients with Alzheimer's disease to two separate Epstein-Barr virus antigens. These results reveal an adaptive immune response in the blood and cerebrospinal fluid in Alzheimer's disease and provide evidence of clonal, antigen-experienced T cells patrolling the intrathecal space of brains affected by age-related neurodegeneration.

Extracellular lipidosomes containing lipid droplets and mitochondria are released during melanoma cell division.

BACKGROUND: The incidence of melanoma is increasing worldwide. Since metastatic melanoma is highly aggressive, it is important to decipher all the biological aspects of melanoma cells. In this context, we have previously shown that metastatic FEMX-I melanoma cells release small (< 150 nm) extracellular vesicles (EVs) known as exosomes and ectosomes containing the stem (and cancer stem) cell antigenic marker CD133. EVs play an important role in intercellular communication, which could have a micro-environmental impact on surrounding tissues. RESULTS: We report here a new type of large CD133+ EVs released by FEMX-I cells. Their sizes range from 2 to 6 µm and they contain lipid droplets and mitochondria. Real-time video microscopy revealed that these EVs originate from the lipid droplet-enriched cell extremities that did not completely retract during the cell division process. Once released, they can be taken up by other cells. Silencing CD133 significantly affected the cellular distribution of lipid droplets, with a re-localization around the nuclear compartment. As a result, the formation of large EVs containing lipid droplets was severely compromised. CONCLUSION: Given the biochemical effect of lipid droplets and mitochondria and/or their complexes on cell metabolism, the release and uptake of these new large CD133+ EVs from dividing aggressive melanoma cells can influence both donor and recipient cells, and therefore impact melanoma growth and dissemination.

Triazole derivatives inhibit the VOR complex-mediated nuclear transport of extracellular particles: Potential application in cancer and HIV-1 infection.

Extracellular vesicles (EVs) appear to play an important role in intercellular communication in various physiological processes and pathological conditions such as cancer. Like enveloped viruses, EVs can transport their contents into the nucleus of recipient cells, and a new intracellular pathway has been described to explain the nuclear shuttling of EV cargoes. It involves a tripartite protein complex consisting of vesicle-associated membrane protein-associated protein A (VAP-A), oxysterol-binding protein (OSBP)-related protein-3 (ORP3) and late endosome-associated Rab7 allowing late endosome entry into the nucleoplasmic reticulum. Rab7 binding to ORP3-VAP-A complex can be blocked by the FDA-approved antifungal drug itraconazole. Here, we design a new series of smaller triazole derivatives, which lack the dioxolane moiety responsible for the antifungal function, acting on the hydrophobic sterol-binding pocket of ORP3 and evaluate their structure-activity relationship through inhibition of VOR interactions and nuclear transfer of EV and HIV-1 cargoes. Our investigation reveals that the most effective compounds that prevent nuclear transfer of EV cargo and productive infection by VSV-G-pseudotyped HIV-1 are those with a side chain between 1 and 4 carbons, linear or branched (methyl) on the triazolone region. These potent chemical drugs could find clinical applications either for nuclear transfer of cancer-derived EVs that impact metastasis or viral infection.

Bridging the generational gap between nurses and nurse managers: a qualitative study from Qatar.

BACKGROUND: The nursing workforce comprises multiple generations, each with unique values, beliefs, and expectations that can influence communication, work ethic, and professional relationships. In Qatar, the generational gap between nurses and nurse managers poses challenges to effective communication and teamwork, impacting job satisfaction and patient outcomes. AIM: This study investigates the generational gap between nurses and nurse managers in Qatar, aiming to identify strategies to enhance collaboration and create a positive work environment. METHODS: A qualitative research design was used, involving semi-structured interviews with 20 participants, including frontline nurses and senior nurse managers. Participants were purposively sampled to represent different generations. Data were collected through face-to-face and virtual interviews, then transcribed and thematically analyzed. FINDINGS: Four key themes emerged: Optimizing the Work Environment: Older generations preferred transformational and situational leadership, while younger nurses valued respect, teamwork, accountability, and professionalism. Strengthening Work Atmosphere through Communication and values: Older nurses favored face-to-face communication, while younger nurses preferred digital tools. Cultivating Respect and Empathy: Younger nurses emphasized fairness in assignments and promotions, while older nurses focused on empathy and understanding. Dynamic Enhancement of Healthcare Systems: Younger nurses were more adaptable to technology and professional development, while older nurses prioritized clinical care and patient outcomes. CONCLUSION: The study reveals significant generational differences in leadership preferences, communication styles, and adaptability to technology. Addressing these gaps through effective leadership, ongoing education, and open communication can improve job satisfaction and patient care.

Telling our stories: describing the experiences and contributions of African migrants living with HIV.

HIV in the UK is concentrated in a few key populations, and African migrants are among them. To date, there has been no documented record of the personal experiences of this group to accompany the significant amount of epidemiological data on these communities. There is no record celebrating the contribution, resilience and lived experience of Africans living with HIV in the UK, their allies and their response to the epidemic. A group of African women who are long-standing HIV activists and advocates, much respected for their leadership within the HIV community, considered that it was important to capture and tell these stories to ensure they were accurately recorded in the history of HIV. Their experience spans the story of the African community's experience of HIV in the UK. They formed a steering group and the project aimed to showcase 40 stories to coincide with the 40th anniversary of HIV in 2021.

Factors influencing self-care management in adult hemodialysis patients: An integrative review.

Background: End-stage renal disease (ESRD) poses a significant health challenge, with hemodialysis (HD) being the most prevalent therapy. Patients undergoing HD must comply with a strict therapeutic regimen, including dietary control, fluid restriction, and medication adherence. Successful disease management and improved outcomes rely on patients' involvement and participation in their care. Aim: To identify the factors that hinder or facilitate self-care management (SCM) in HD patients. Methodology: This review followed Whittemore and Knafl's integrative review framework. A comprehensive literature search of articles published between 2017 and 2022 was conducted in CINAHL, Medline, and PubMed using the keywords end-stage renal disease, hemodialysis, self-care management, self-care, and self-management. This search yielded 21 suitable articles for review. Results: SCM is influenced by three main factors: facilitators, barriers, and outcomes. Facilitators of SCM include self-care management interventions, patient knowledge, socio-demographic factors, family support, healthcare professionals, peer support, and psychological factors. Barriers encompass psychological and physical conditions. Outcomes include both physiological and psychological aspects. Conclusion: Understanding the factors influencing SCM in HD patients is vital for developing reliable and effective self-care strategies and interventions to enhance both physical and psychological outcomes.

Colorectal cancer-derived small extracellular vesicles induce TGFß1-mediated epithelial to mesenchymal transition of hepatocytes.

BACKGROUND: Metastatic disease is the major cause of cancer-related deaths. Increasing evidence shows that primary tumor cells can promote metastasis by preparing the local microenvironment of distant organs, inducing the formation of the so-called "pre-metastatic niche". In recent years, several studies have highlighted that among the tumor-derived molecular components active in pre-metastatic niche formation, small extracellular vesicles (sEVs) play a crucial role. Regarding liver metastasis, the ability of tumor-derived sEVs to affect the activities of non-parenchymal cells such as Kupffer cells and hepatic stellate cells is well described, while the effects on hepatocytes, the most conspicuous and functionally relevant hepatic cellular component, remain unknown. METHODS: sEVs isolated from SW480 and SW620 CRC cells and from clinical samples of CRC patients and healthy subjects were used to treat human healthy hepatocytes (THLE-2 cells). RT-qPCR, Western blot and confocal microscopy were applied to investigate the effects of this treatment. RESULTS: Our study shows for the first time that TGFß1-carrying CRC_sEVs impair the morphological and functional properties of healthy human hepatocytes by triggering their TGFß1/SMAD-dependent EMT. These abilities of CRC_sEVs were further confirmed by evaluating the effects elicited on hepatocytes by sEVs isolated from plasma and biopsies from CRC patients. CONCLUSIONS: Since it is known that EMT of hepatocytes leads to the formation of a fibrotic environment, a well-known driver of metastasis, these results suggest that CRC_sEV-educated hepatocytes could have an active and until now neglected role during liver metastasis formation.

Conserved and cell type-specific transcriptional responses to IFN-γ in the ventral midbrain.

Dysregulated inflammation within the central nervous system (CNS) contributes to neuropathology in infectious, autoimmune, and neurodegenerative disease. With the exception of microglia, major histocompatibility complex (MHC) proteins are virtually undetectable in the mature, healthy central nervous system (CNS). Neurons have generally been considered incapable of antigen presentation, and although interferon gamma (IFN-γ) can elicit neuronal MHC class I (MHC-I) expression and antigen presentation in vitro, it has been unclear whether similar responses occur in vivo. Here we directly injected IFN-γ into the ventral midbrain of mature mice and analyzed gene expression profiles of specific CNS cell types. We found that IFN-γ upregulated MHC-I and associated mRNAs in ventral midbrain microglia, astrocytes, oligodendrocytes, and GABAergic, glutamatergic, and dopaminergic neurons. The core set of IFN-γ-induced genes and their response kinetics were similar in neurons and glia, but with a lower amplitude of expression in neurons. A diverse repertoire of genes was upregulated in glia, particularly microglia, which were the only cells to undergo cellular proliferation and express MHC classII (MHC-II) and associated genes. To determine if neurons respond directly via cell-autonomous IFN-γ receptor (IFNGR) signaling, we produced mutant mice with a deletion of the IFN-γ-binding domain of IFNGR1 in dopaminergic neurons, which resulted in a complete loss of dopaminergic neuronal responses to IFN-γ. Our results demonstrate that IFN-γ induces neuronal IFNGR signaling and upregulation of MHC-I and related genes in vivo, although the expression level is low compared to oligodendrocytes, astrocytes, and microglia.

Anti-human CD9 antibody Fab fragment impairs the internalization of extracellular vesicles and the nuclear transfer of their cargo proteins.

The intercellular communication mediated by extracellular vesicles (EVs) has gained international interest during the last decade. Interfering with the mechanisms regulating this cellular process might find application particularly in oncology where cancer cell-derived EVs play a role in tumour microenvironment transformation. Although several mechanisms were ascribed to explain the internalization of EVs, little is our knowledge about the fate of their cargos, which are crucial to mediate their function. We recently demonstrated a new intracellular pathway in which a fraction of endocytosed EV-associated proteins is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into the nucleoplasmic reticulum. Silencing tetraspanin CD9 both in EVs and recipient cells strongly decreased the endocytosis of EVs and abolished the nuclear transfer of their cargos. Here, we investigated whether monovalent Fab fragments derived from 5H9 anti-CD9 monoclonal antibody (referred hereafter as CD9 Fab) interfered with these cellular processes. To monitor the intracellular transport of proteins, we used fluorescent EVs containing CD9-green fluorescent protein fusion protein and various melanoma cell lines and bone marrow-derived mesenchymal stromal cells as recipient cells. Interestingly, CD9 Fab considerably reduced EV uptake and the nuclear transfer of their proteins in all examined cells. In contrast, the divalent CD9 antibody stimulated both events. By impeding intercellular communication in the tumour microenvironment, CD9 Fab-mediated inhibition of EV uptake, combined with direct targeting of cancerous cells could lead to the development of novel anti-melanoma therapeutic strategies.

VAMP-associated protein-A and oxysterol-binding protein-related protein 3 promote the entry of late endosomes into the nucleoplasmic reticulum.

The endocytic pathway plays an instrumental role in recycling internalized molecules back to the plasma membrane or in directing them to lysosomes for degradation. We recently reported a new role of endosomes-the delivery of components from extracellular vesicles (EVs) to the nucleoplasm of recipient cells. Using indirect immunofluorescence, FRET, immunoisolation techniques, and RNAi, we report here a tripartite protein complex (referred to as the VOR complex) that is essential for the nuclear transfer of EV-derived components by orchestrating the specific localization of late endosomes into nucleoplasmic reticulum. We found that the VOR complex contains the endoplasmic reticulum-localized vesicle-associated membrane protein (VAMP)-associated protein A (VAP-A), the cytoplasmic oxysterol-binding protein-related protein 3 (ORP3), and late endosome-associated small GTPase Rab7. The silencing of VAP-A or ORP3 abrogated the association of Rab7-positive late endosomes with nuclear envelope invaginations and, hence, the transport of endocytosed EV-derived components to the nucleoplasm of recipient cells. We conclude that the VOR complex can be targeted to inhibit EV-mediated intercellular communication, which can have therapeutic potential for managing cancer in which the release of EVs is dysregulated.

Extracellular Vesicles from Thyroid Carcinoma: The New Frontier of Liquid Biopsy.

The diagnostic approach to thyroid cancer is one of the most challenging issues in oncology of the endocrine system because of its high incidence (3.8% of all new cancer cases in the US) and the difficulty to distinguish benign from malignant non-functional thyroid nodules and establish the cervical lymph node involvement during staging. Routine diagnosis of thyroid nodules usually relies on a fine-needle aspirate biopsy, which is invasive and often inaccurate. Therefore, there is an urgent need to identify novel, accurate, and non-invasive diagnostic procedures. Liquid biopsy, as a non-invasive approach for the detection of diagnostic biomarkers for early tumor diagnosis, prognosis, and disease monitoring, may be of particular benefit in this context. Extracellular vesicles (EVs) are a consistent source of tumor-derived RNA due to their prevalence in circulating bodily fluids, the well-established isolation protocols, and the fact that RNA in phospholipid bilayer-enclosed vesicles is protected from blood-borne RNases. Recent results in other types of cancer, including our recent study on plasma EVs from glioblastoma patients suggest that information derived from analysis of EVs from peripheral blood plasma can be integrated in the routine diagnostic tumor approach. In this review, we will examine the diagnostic and prognostic potential of liquid biopsy to detect tumor-derived nucleic acids in circulating EVs from patients with thyroid carcinoma.

The Impact of Diabetes on Outcomes After Acute Ischemic Stroke: A Prospective Observational Study.

BACKGROUND: Stroke in diabetics may delay recovery and increases the risk of early recurrence of stroke. We compared the outcomes of patients (with and without diabetes) admitted with an acute ischemic stroke (AIS) in the state of Qatar. PATIENTS AND METHODS: We prospectively compared the clinical presentation, complications, discharge outcome, and stroke recurrence at 90 days in patients with and without diabetes. RESULTS: Five thousand two hundred twenty-eight stroke patients were admitted between January 2014 and December 2017. Two thousand nine hundred sixty-one had confirmed AIS, 1695 (57.2%) had diabetes, 429 (14.5%) had prediabetes and 873 (29.5%) had no diabetes. Comparing diabetic patients to prediabetic and nondiabetics, they were significantly older (58.5 ± 11.9 versus 54.0 ± 12.9 versus 49.5 ± 13.8, P = .0001), had higher rates of hypertension (80.8% versus 67.4% versus 59.2%), previous stroke (18.0% versus 5.4% versus 6.2%), and coronary artery disease (12.9% versus 5.6% versus 5.0%; P = .001 for all). The percentage of patients with modified Rankin scale 3-6 at discharge (39.7% versus 32.6% versus 30.2%; P = .0001) and 90 days (26.7% versus 18.8% versus 21.4%, P = .001); 90-day mortality (6.2% versus 2.2% versus 5.2%; P = .03) and stroke recurrence (4.2% versus .7% versus 2.2%; P = .005) was significantly higher in diabetic patients. CONCLUSIONS: Patients with diabetes and AIS have more in-hospital complications, worse discharge outcomes, higher mortality and stroke recurrence at 90 days, compared to prediabetes and no diabetes.

Corneal Confocal Microscopy Detects Corneal Nerve Damage in Patients Admitted With Acute Ischemic Stroke.

BACKGROUND AND PURPOSE: Corneal confocal microscopy can identify corneal nerve damage in patients with peripheral and central neurodegeneration. However, the use of corneal confocal microscopy in patients presenting with acute ischemic stroke is unknown. METHODS: One hundred thirty patients (57 without diabetes mellitus [normal glucose tolerance], 32 with impaired glucose tolerance, and 41 with type 2 diabetes mellitus) admitted with acute ischemic stroke, and 28 age-matched healthy control participants underwent corneal confocal microscopy to quantify corneal nerve fiber density, corneal nerve branch density, and corneal nerve fiber length. RESULTS: There was a significant reduction in corneal nerve fiber density, corneal nerve branch density, and corneal nerve fiber length in stroke patients with normal glucose tolerance (P<0.001, P<0.001, P<0.001), impaired glucose tolerance (P=0.004, P<0.001, P=0.002), and type 2 diabetes mellitus (P<0.001, P<0.001, P<0.001) compared with controls. HbA1c and triglycerides correlated with corneal nerve fiber density (r=-0.187, P=0.03; r=-0.229 P=0.01), corneal nerve fiber length (r=-0.228, P=0.009; r=-0.285; P=0.001), and corneal nerve branch density (r=-0.187, P=0.033; r=-0.229, P=0.01). Multiple linear regression showed no independent associations between corneal nerve fiber density, corneal nerve branch density, and corneal nerve fiber length and relevant risk factors for stroke. CONCLUSIONS: Corneal confocal microscopy is a rapid noninvasive ophthalmic imaging technique that identifies corneal nerve fiber loss in patients with acute ischemic stroke.

Stroke Thrombolysis Protocol Shortens "Door-to-Needle Time" and Improves Outcomes-Experience at a Tertiary Care Center in Qatar.

BACKGROUND AND PURPOSE: To assess the effect of acute thrombolysis protocol on "door-to-needle time" (DTN) and improvement in outcome following acute stroke (AS). METHODS: The charts of all patients receiving intravenous (IV) thrombolysis for AS between January 2008 and June 2015 were reviewed for DTN, complications, and clinical outcome. Good prognosis was defined as modified Rankin Scale (mRS) score of less than 2 at 90 days. In January 2014, a protocol for faster DTN was introduced. We reviewed the prognosis before and after the introduction of the new protocol. RESULTS: Up to 204 patients received IV recombinant tissue plasminogen activator (r-tPA) (mean age 52.5 ± 12.4 years). Mean door-to-CT time improved from 42.5 ± 41.1 to 27.1 ± 26.3 minutes (P < .001); DTN improved from 83.26 ± 47.7 to 47.09 ± 25.7 minutes (P < .001). Complications were reduced from 15.7% to 8.8% (P = .14). The mRS score of less than or equal to 2 improved from 47.1% to 73.3% at 90 days (P = .001). After implementing new protocol, thrombolysis rate increased to 11.8% in 2014 (before 3.3% in 2011, 4.9% in 2012, and 4.4% in 2013), P < .0001. NIHSS (National Institutes of Health Stroke Scale) score at admission (P = .002), hypodensity on initial CT brain (P = .041), protocol implementation (P = .014), and reduced length of stay (P = .004) were associated with outcome at 90 days (mRS score ≤2). CONCLUSION: Implementation of specific protocols to reduce DTN in patients receiving IV r-tPA leads to reduction in complications and improves outcome.

Prolonged Stay of Stroke Patients in the Emergency Department May Lead to an Increased Risk of Complications, Poor Recovery, and Increased Mortality.

BACKGROUND AND AIM: Following an acute stroke (AS), patients are at an increased risk of developing complications that may affect prognosis. With overcrowding in the emergency department (ED), patients stay longer hours to days before transfer to a proper stroke ward. The aim of this study was to evaluate the effect of increasing length of stay (LOS) in the ED on the risk of stroke-related complications. METHODS: We analyzed data from our stroke registry of patients admitted with AS during 2014. Stay in ED was divided into 2 groups: less than 8 hours and more than 8 hours. Data regarding demographics, stroke type, severity of stroke, ED (LOS) in hours, total LOS in hospital, number and types of complications, and prognosis were collected. RESULTS: Mean age was 54.8 years and 78.9% were males (total n = 894). Prior to ward admission, 265 (29.5%) patients remained in the ED for less than 8 hours and 629 (70.4%) remained for more than 8 hours. There was no significant difference in comorbidities or the severity of stroke at admission between the 2 groups. An ED LOS of less than 8 hours was associated with reduced risk of complications (14.3% versus 19.2%, P = .06), reduced LOS in hospital, better prognosis at discharge (72.5% versus 57.6% had modified Rankin Scale of ≤2, P = .001) and at 90 days (89% versus 78.8%, P = .007) and lower in-hospital mortality (1.5% versus 5.4 %, P = .004). CONCLUSION: Delays in transferring AS patients from the ED may lead to an increase in complications resulting in an increased LOS and slower recovery.

Beneficial Effects of Implementing Stroke Protocols Require Establishment of a Geographically Distinct Unit.

BACKGROUND AND PURPOSE: Usefulness of multidisciplinary stroke units in acute stroke patients is well established. There is extensive western literature on usefulness of stroke units in outcome, but limited evidence from the rest of the world. We aim to evaluate the impact of establishing a stroke unit on outcome in patients presenting to a tertiary care facility. METHODS: This is a retrospective study of 1003 patients with acute stroke admitted to Hamad General Hospital, Qatar, between January 2014 and February 2015. Patients directly admitted to intensive care unit (132) were excluded. We compared outcomes of pre- and poststroke ward (SW) establishment and in SW patients versus those of general medical wards. RESULTS: Before the establishment of the SW, 175 patients were admitted to the hospital. From April 2014 to February 2015, 696 patients were admitted (SW, 545; medical ward, 151). There was a significant reduction in length of stay from 14.7±27.7 to 6.2±20.2 days (P=0.0001) and incidence of complications (23.6% versus 6.4%, P=0.0001) after implementation of stroke-specific protocols. Prognosis at discharge (modified Rankin Scale 0-2 in 56.0% versus 70.4%, P=0.001) and at 90 days (modified Rankin Scale 0-2 in 70.6% versus 95.0%, P=0.001) also significantly improved. Compared with medical ward patients, outcome was significantly better in SW patients with fewer complications (10.9% versus 5.0%, P=0.013) and shorter length of stay (8.9±30.7 versus 5.4±16.1 days, P=0.05). CONCLUSIONS: Establishing a distinct SW is essential for achieving full benefits of stroke protocols implementation. SW patients have significantly fewer complications and better prognosis when compared with patients in medical wards.

Comparative analysis of innate immune system function in metastatic breast, colorectal, and prostate cancer patients with circulating tumor cells.

In recent years, circulating tumor cells (CTCs) in metastatic cancer patients have been found to be a promising biomarker to predict overall survival and tumor progression in these patients. A relatively high number of CTCs has been correlated with disease progression and poorer prognosis. This study was designed to assess innate immune system function, known to be responsible for the immune defense against developing neoplasms, in metastatic cancer patients with CTCs. Our aim is to provide a link between indication of poorer prognosis, represented by the number of CTCs to the cytotoxic activity of natural killer cells, an important component of the innate immune system, and to represent a promising expanded approach to management of metastatic cancer patients with CTCs. Seventy-four patients, with metastatic breast, colorectal, or prostate cancer, were recruited for this study. Using a flow cytometric assay, we measured natural killer (NK) cell cytotoxicity against K562 target cells; and CTCs were enumerated using the CellSearch System. Toll-like receptors 2 and 4 expression was also determined by flow cytometry. We found that within each of our three metastatic cancer patient groups, NK cell cytotoxic activity was decreased in patients with a relatively high number of CTCs in peripheral blood compared to patients with a relatively low number of CTCs. In the breast and prostate cancer group, patients with CTCs greater than 5 had decreased NK cell cytotoxicity when compared to patients with less than 5 CTCs. In the colorectal cancer group, we found that 3 or more CTCs in the blood was the level at which NK cell cytotoxicity is diminished. Additionally, we found that the toll-like receptors 2 and 4 expression was decreased in intensity in all the metastatic cancer patients when compared to the healthy controls. Furthermore, within each cancer group, the expression of both toll-like receptors was decreased in the patients with relatively high number of CTCs, i.e. greater than 5 for the breast and prostate cancer group and greater than 3 for the colorectal cancer group, compared to the patients with relatively low number, i.e. less than 5 or 3, respectively. Treatment options to increase NK cell cytotoxic activity should be considered in patients with relatively high numbers of CTCs.

Toll-like receptor (TLR) expression of immune system cells from metastatic breast cancer patients with circulating tumor cells.

The risk posed by breast cancer represents a complex interaction among factors affecting tumor immunity of the host. Toll-like receptors (TLRs) are members of the innate immune system and generally function to attract host immune cells upon activation. However, the good intentions of TLRs are sometimes not transferred to positive long-term effects, due to their involvement in exacerbating inflammatory effects and even contributing to continued inflammation. Chronic inflammatory states are considered to favor an increased predisposition to cancer, with continuous activation of inflammatory cytokines and other hallmarks of inflammation exerting a deleterious effect. Circulating tumor cells (CTCs) are neoplastic cells present in the peripheral blood circulation that have been found to be an indicator of disease progression and long-term survival. In the present study, we examined the expression of TLRs on dendritic cells, which play a major role in eliciting anti-tumor immunity, in metastatic breast cancer patients with CTCs. Flow cytometric data showed significant differences between circulating tumor cell (CTC) positive patients and CTC negative patients in their expression of TLR2 by CD8 positive cytotoxic T cells and TLR2, TLR4, TLR3, and TLR8 by CD11c positive dendritic cells (p<0.05). Expression of TLR2, TLR4, and TLR8 was increased in CTC positive patients, whereas TLR3 expression was decreased in the dendritic cell population.

Extracellular vesicle isolation methods identify distinct HIV-1 particles released from chronically infected T-cells.

The current study analyzed the intersecting biophysical, biochemical, and functional properties of extracellular particles (EPs) with the human immunodeficiency virus type-1 (HIV-1) beyond the currently accepted size range for HIV-1. We isolated five fractions (Frac-A through Frac-E) from HIV-infected cells by sequential differential ultracentrifugation (DUC). All fractions showed a heterogeneous size distribution with median particle sizes greater than 100 nm for Frac-A through Frac-D but not for Frac-E, which contained small EPs with an average size well below 50 nm. Synchronized and released cultures contained large infectious EPs in Frac-A, with markers of amphisomes and viral components. Additionally, Frac-E uniquely contained EPs positive for CD63, HSP70, and HIV-1 proteins. Despite its small average size, Frac-E contained membrane-protected viral integrase, detectable only after SDS treatment, indicating that it is enclosed in vesicles. Single particle analysis with dSTORM further supported these findings as CD63, HIV-1 integrase, and the viral surface envelope (Env) glycoprotein (gp) colocalized on the same Frac-E particles. Surprisingly, Frac-E EPs were infectious, and infectivity was significantly reduced by immunodepleting Frac-E with anti-CD63, indicating the presence of this protein on the surface of infectious small EPs in Frac-E. To our knowledge, this is the first time that extracellular vesicle (EV) isolation methods have identified infectious small HIV-1 particles (smHIV-1) that are under 50 nm. Collectively, our data indicate that the crossroads between EPs and HIV-1 potentially extend beyond the currently accepted biophysical properties of HIV-1, which may have further implications for viral pathogenesis.

HIV-1-induced nuclear invaginations mediated by VAP-A, ORP3, and Rab7 complex explain infection of activated T cells.

The mechanism of human immunodeficiency virus 1 (HIV-1) nuclear entry, required for productive infection, is not fully understood. Here, we report that in HeLa cells and activated CD4+ T cells infected with HIV-1 pseudotyped with VSV-G and native Env protein, respectively, Rab7+ late endosomes containing endocytosed HIV-1 promote the formation of nuclear envelope invaginations (NEIs) by a molecular mechanism involving the VOR complex, composed of the outer nuclear membrane protein VAP-A, hyperphosphorylated ORP3 and Rab7. Silencing VAP-A or ORP3 and drug-mediated impairment of Rab7 binding to ORP3-VAP-A inhibited the nuclear transfer of the HIV-1 components and productive infection. In HIV-1-resistant quiescent CD4+ T cells, ORP3 was not hyperphosphorylated and neither VOR complex nor NEIs were formed. This new cellular pathway and its molecular players are potential therapeutic targets, perhaps shared by other viruses that require nuclear entry to complete their life cycle.