MAdCAM-1 costimulation in the presence of retinoic acid and TGF-ß promotes HIV infection and differentiation of CD4+ T cells into CCR5+ TRM-like cells.

CD4+ tissue resident memory T cells (TRMs) are implicated in the formation of persistent HIV reservoirs that are established during the very early stages of infection. The tissue-specific factors that direct T cells to establish tissue residency are not well defined, nor are the factors that establish viral latency. We report that costimulation via MAdCAM-1 and retinoic acid (RA), two constituents of gut tissues, together with TGF-ß, promote the differentiation of CD4+ T cells into a distinct subset α4ß7+CD69+CD103+ TRM-like cells. Among the costimulatory ligands we evaluated, MAdCAM-1 was unique in its capacity to upregulate both CCR5 and CCR9. MAdCAM-1 costimulation rendered cells susceptible to HIV infection. Differentiation of TRM-like cells was reduced by MAdCAM-1 antagonists developed to treat inflammatory bowel diseases. These finding provide a framework to better understand the contribution of CD4+ TRMs to persistent viral reservoirs and HIV pathogenesis.

HIV vaccine candidate efficacy in female macaques mediated by cAMP-dependent efferocytosis and V2-specific ADCC.

The development of an effective vaccine to protect against HIV acquisition will be greatly bolstered by in-depth understanding of the innate and adaptive responses to vaccination. We report here that the efficacy of DNA/ALVAC/gp120/alum vaccines, based on V2-specific antibodies mediating apoptosis of infected cells (V2-ADCC), is complemented by efferocytosis, a cyclic AMP (cAMP)-dependent antiphlogistic engulfment of apoptotic cells by CD14+ monocytes. Central to vaccine efficacy is the engagement of the CCL2/CCR2 axis and tolerogenic dendritic cells producing IL-10 (DC-10). Epigenetic reprogramming in CD14+ cells of the cyclic AMP/CREB pathway and increased systemic levels of miRNA-139-5p, a negative regulator of expression of the cAMP-specific phosphodiesterase PDE4D, correlated with vaccine efficacy. These data posit that efferocytosis, through the prompt and effective removal of apoptotic infected cells, contributes to vaccine efficacy by decreasing inflammation and maintaining tissue homeostasis.

Lumbar surgical drains do not increase the risk of infections in patients undergoing spine surgery.

PURPOSE: The aim of this study was to characterize if the use of surgical drains or length of drain placement following spine surgery increases the risk of post-operative infection. METHODS: Records of patients undergoing elective spinal surgery at a tertiary care center were collected between May 5, 2016 and August 16, 2018. Pre-operative baseline characteristics were recorded including patient's demographics and comorbidities. Intraoperative procedure information was documented related to procedure type, blood loss, and antibiotics used. Following surgery, patients were then further subdivided into two groups: patients who were discharged with a spinal surgical site drain and patients who did not receive a drain. Post-operative surgical variables included length of stay (LOS), drain length, number of antibiotics given, and type of post-operative infection. Univariate and multivariate statistical analysis was conducted. RESULTS: A total of 671 patients were included in the current study, 386 (57.5%) with and 285 (42.5%) without the drain. The overall infection rate was 5.7% with 6.22% among patients with the drain compared to 4.91% in patients without drain. The univariate analysis identified the following variables to be significantly associated with the infection: total number of surgical levels, spinal region, blood loss, redosing of antibiotics, length of stay, length of drain placement, and number of antibiotics (P < 0.05). However, the multivariate analysis none of the predictors was significant. CONCLUSIONS: The current study shows that the placement of drain does not increase rate of infection, irrespective of levels, length of surgery, or approach.

CD47 interactions with exportin-1 limit the targeting of m7G-modified RNAs to extracellular vesicles.

CD47 is a marker of self and a signaling receptor for thrombospondin-1 that is also a component of extracellular vesicles (EVs) released by various cell types. Previous studies identified CD47-dependent functional effects of T cell EVs on target cells, mediated by delivery of their RNA contents, and enrichment of specific subsets of coding and noncoding RNAs in CD47+ EVs. Mass spectrometry was employed here to identify potential mechanisms by which CD47 regulates the trafficking of specific RNAs to EVs. Specific interactions of CD47 and its cytoplasmic adapter ubiquilin-1 with components of the exportin-1/Ran nuclear export complex were identified and confirmed by coimmunoprecipitation. Exportin-1 is known to regulate nuclear to cytoplasmic trafficking of 5'-7-methylguanosine (m7G)-modified microRNAs and mRNAs that interact with its cargo protein EIF4E. Interaction with CD47 was inhibited following alkylation of exportin-1 at Cys528 by its covalent inhibitor leptomycin B. Leptomycin B increased levels of m7G-modified RNAs, and their association with exportin-1 in EVs released from wild type but not CD47-deficient cells. In addition to perturbing nuclear to cytoplasmic transport, transcriptomic analyses of EVs released by wild type and CD47-deficient Jurkat T cells revealed a global CD47-dependent enrichment of m7G-modified microRNAs and mRNAs in EVs released by CD47-deficient cells. Correspondingly, decreasing CD47 expression in wild type cells or treatment with thrombospondin-1 enhanced levels of specific m7G-modified RNAs released in EVs, and re-expressing CD47 in CD47-deficient T cells decreased their levels. Therefore, CD47 signaling limits the trafficking of m7G-modified RNAs to EVs through physical interactions with the exportin-1/Ran transport complex.

CD63+ and MHC Class I+ Subsets of Extracellular Vesicles Produced by Wild-Type and CD47-Deficient Jurkat T Cells Have Divergent Functional Effects on Endothelial Cell Gene Expression.

T cells and endothelial cells engage in bidirectional communication that regulates angiogenesis and T cell transmigration. Extracellular vesicles (EVs) mediate intercellular communication by the transfer of bioactive molecules including RNAs. EVs produced by a given cell type are heterogeneous in their RNA content, but it is unclear how specific EV surface markers relate to their functional effects on target cells. Our previous work established that Jurkat T cell EVs bearing CD63, MHC-I, or CD47 surface markers contain distinct noncoding RNA populations. The present study reveals that CD63+ and MHC-I+ EVs from CD47-deficient Jurkat T cells are enriched in small non-coding RNAs relative to EVs from wild-type Jurkat T cells. CD47-deficient Jurkat T cells secrete more CD63+ and MHC-I+ EVs, but MHC-I+ EVs are selectively taken up more by human umbilical vein endothelial cells. Transcriptomics analysis of endothelial cells treated with CD63+ or MHC-I+ EVs showed surface marker- and CD47-dependent changes in gene expression in the target cells. Gene set enrichment analysis identified CD47-dependent, and surface marker-dependent effects of T cell EVs on VEGF and inflammatory signaling, cell cycle, and lipid and cholesterol metabolism. Thus, subsets of T cell EVs differentially regulate endothelial cell metabolism and inflammatory and angiogenic responses.

Anti-V2 antibodies virus vulnerability revealed by envelope V1 deletion in HIV vaccine candidates.

The efficacy of ALVAC-based HIV and SIV vaccines in humans and macaques correlates with antibodies to envelope variable region 2 (V2). We show here that vaccine-induced antibodies to SIV variable region 1 (V1) inhibit anti-V2 antibody-mediated cytotoxicity and reverse their ability to block V2 peptide interaction with the α4ß7 integrin. SIV vaccines engineered to delete V1 and favor an α helix, rather than a ß sheet V2 conformation, induced V2-specific ADCC correlating with decreased risk of SIV acquisition. Removal of V1 from the HIV-1 clade A/E A244 envelope resulted in decreased binding to antibodies recognizing V2 in the ß sheet conformation. Thus, deletion of V1 in HIV envelope immunogens may improve antibody responses to V2 virus vulnerability sites and increase the efficacy of HIV vaccine candidates.

Extracellular vesicles released from the filarial parasite Brugia malayi downregulate the host mTOR pathway.

We have previously shown that the microfilarial (mf) stage of Brugia malayi can inhibit the mammalian target of rapamycin (mTOR; a conserved serine/threonine kinase critical for immune regulation and cellular growth) in human dendritic cells (DC) and we have proposed that this mTOR inhibition is associated with the DC dysfunction seen in filarial infections. Extracellular vesicles (EVs) contain many proteins and nucleic acids including microRNAs (miRNAs) that might affect a variety of intracellular pathways. Thus, EVs secreted from mf may elucidate the mechanism by which the parasite is able to modulate the host immune response during infection. EVs, purified from mf of Brugia malayi and confirmed by size through nanoparticle tracking analysis, were assessed by miRNA microarrays (accession number GSE157226) and shown to be enriched (>2-fold, p-value<0.05, FDR = 0.05) for miR100, miR71, miR34, and miR7. The microarray analysis compared mf-derived EVs and mf supernatant. After confirming their presence in EVs using qPCR for these miRNA targets, web-based target predictions (using MIRPathv3, TarBAse and MicroT-CD) predicted that miR100 targeted mTOR and its downstream regulatory protein 4E-BP1. Our previous data with live parasites demonstrated that mf downregulate the phosphorylation of mTOR and its downstream effectors. Additionally, our proteomic analysis of the mf-derived EVs revealed the presence of proteins commonly found in these vesicles (data are available via ProteomeXchange with identifier PXD021844). We confirmed internalization of mf-derived EVs by human DCs and monocytes using confocal microscopy and flow cytometry, and further demonstrated through flow cytometry, that mf-derived EVs downregulate the phosphorylation of mTOR in human monocytes (THP-1 cells) to the same degree that rapamycin (a known mTOR inhibitor) does. Our data collectively suggest that mf release EVs that interact with host cells, such as DC, to modulate host responses.

Impact of a Perioperative Protocol on Length of ICU and Hospital Stay in Complex Spine Surgery.

BACKGROUND: In an attempt to improve patient care, a perioperative complex spine surgery management protocol was developed through collaboration between spine surgeons and neuroanesthesiologists. The aim of this study was to investigate whether implementation of the protocol in 2015 decreased total hospital and intensive care unit (ICU) length of stay (LOS) and complication rates after elective complex spine surgery. MATERIALS AND METHODS: A retrospective cohort study was conducted by review of the medical charts of patients who underwent elective complex spine surgery at an academic medical center between 2012 and 2017. Patients were divided into 2 groups based on the date of their spine surgery in relation to implementation of the spine surgery protocol; before-protocol (January 2012 to March 2015) and protocol (April 2015 to March 2017) groups. Outcomes in the 2 groups were compared, focusing on hospital and ICU LOS, and complication rates. RESULTS: A total of 201 patients were included in the study; 107 and 94 in the before-protocol and protocol groups, respectively. Mean (SD) hospital LOS was 14.8±10.8 days in the before-protocol group compared with 10±10.7 days in the protocol group (P<0.001). The spine surgery protocol was the primary factor decreasing hospital LOS; incidence rate ratio 0.78 (P<0.001). Similarly, mean ICU LOS was lower in the protocol compared with before-protocol group (4.2±6.3 vs. 6.3±7.3 d, respectively; P=0.011). There were no significant differences in the rate of postoperative complications between the 2 groups (P=0.231). CONCLUSION: Implementation of a spine protocol reduced ICU and total hospital LOS stay in high-risk spine surgery patients.

Macrophage-derived HIV-1 carries bioactive TGF-beta.

Infected T cells and macrophages are the main producers of HIV-1 in infected individuals. Upon release from infected cells, HIV-1 incorporates various cellular membrane proteins, some of which are specific for these cells. However, the functions of cell-encoded proteins in virions remain largely unknown. We performed flow virometry to identify, in plasma of HIV-infected individuals, macrophage- and T-cell-derived HIV-1 virions, using cell-specific markers CD36 and CD27, respectively. Using four different methods, we demonstrated that CD36 on virions binds the immunosuppressive cytokine transforming growth factor beta (TGF-ß) through a ligand, thrombospondin one (TSP-1). Flow virometry of individual virions showed that TGF-ß was present on CD36+ virions (average, 28.2% ± 6.6% (n = 3)) but not on CD27+ virions (average, 1% ± 0.1% (n = 3)). TGF-ß molecules present on captured CD36+ virions were biologically active, as evaluated with a reporter cell line. Delivery of TGF-ß on HIV virions to HIV target cells may affect them, playing a significant role in viral pathogenesis.

Syncytin 1 dependent horizontal transfer of marker genes from retrovirally transduced cells.

Retroviral transduction is routinely used to generate cell lines expressing exogenous non-viral genes. Here, we show that human cells transduced to stably express GFP transfer GFP gene to non-transduced cells. This horizontal gene transfer was mediated by a fraction of extracellular membrane vesicles that were released by the transduced cells. These vesicles carried endogenous retroviral envelope protein syncytin 1 and essentially acted as replication-competent retroviruses. The ability to transfer the GFP gene correlated with the levels of syncytin 1 expression in the transduced cells and depended on the fusogenic activity of this protein, substantiating the hypothesis that endogenous syncytin 1 mediates fusion stage in the delivery of extracellular vesicle cargo into target cells. Our findings suggest that testing for replication-competent retroviruses, a routine safety test for transduced cell products in clinical studies, should be also carried out for cell lines generated by retroviral vectors in in vitro studies.

The DOWN Questionnaire: A Novel Screening Tool for Cervical Spondylotic Myelopathy.

STUDY DESIGN: Case-control study. OBJECTIVES: Cervical spondylotic myelopathy (CSM) is the most common cause of spinal cord injury in adults aged over 55 years. However, since the onset is typically insidious, accurately diagnosing CSM can be challenging, often requiring referral to a subspecialist and advanced imaging. To help identify patients at risk for CSM, this case-control study compared responses to a series of 4 questions (DOWN questionnaire) in myelopathic and non-myelopathic patients. METHODS: Ninety-two patients, 46 with and 46 without myelopathy, were recruited for the study. Each patient answered 4 questions encompassing common symptoms associated with CSM. Responses between patient groups were compared, and Cohen's κ was used to assess for agreement between responses and the diagnosis of myelopathy. RESULTS: We found a sensitivity of 91% and a κ of 0.54 to 3 positive responses and a sensitivity of 72% and a κ of 0.61 to 4 positive responses. CONCLUSIONS: Positive responses to 3 or more DOWN questions has high sensitivity and moderate agreement with the diagnosis of myelopathy based on history, physical exam, and review of advanced imaging by an orthopedic or neurological surgeon. The DOWN questionnaire is a potentially useful screening tool to identify patients at risk for CSM.

Experiences of an outpatient infusion center with intravenous magnesium therapy for status migrainosus.

OBJECTIVES: Exploratory study to investigate the effectiveness of intravenous magnesium as an abortive for status migrainosus in an outpatient infusion center, and characterize the patients who benefit from the therapy. PATIENTS & METHODS: Retrospective analysis of 234 migraine patients who received IV magnesium as a headache abortive, at the headache clinic of University of Southern California. Additional intramuscular (IM) injections for nausea (prochlorperazine, odansetron, metoclopramide) or for refractory pain (ketorolac, dexamethasone, sumatriptan, dihydroergotamine), were administered as necessary. Immediately before and after treatment, self-reported pain levels were recorded using an 11-point numeric pain rating scale (0-10). RESULTS: Our patient sample has a mean age of 44 years and was predominantly female (79%). 36 (19%) had migraine with aura. Overall, pain score decreased from 5.46±2.39 to 3.56 ± 2.75 (P < 0.001) after magnesium infusion. One hundred twenty-seven (54%) patients had clinically significant pain reduction, as defined by pain decrease ≥ 30%. One hundred and four patients (44%) received IV magnesium and did not require additional intramuscular (IM) medications for pain. In patients who did not receive additional IM medications for pain, pain score decreased from 4.76 ± 2.41 to 2.95 ± 2.70 (p < 0.001), and 61 out of 104 (59%) experienced ≥ 30% pain reduction. Patients with less severe pain tended to have a better response than patients with more severe pain, as patients with ≥30% pain reduction had a significantly lower pre-treatment pain score (p = 0.018). CONCLUSION: For a subset of patients with status migrainosus, IV magnesium therapy results in clinically significant pain relief without the need for intramuscular pain medications. Therefore, IV magnesium may be useful as a cost-effective first-line parental therapy for status migrainosus, especially for patients who initially present with lower pain intensity.

Recurrent Rathke's Cleft Cysts: Incidence and Surgical Management in a Tertiary Pituitary Center over 2 Decades.

BACKGROUND: Limited data exist pertaining to outcomes following surgery for recurrent Rathke's cleft cysts (RCC). OBJECTIVE: To determine treatment outcomes in patients undergoing reoperation for recurrent or residual RCCs. METHODS: A retrospective analysis of 112 consecutive RCC operations in 109 patients between 1995 and 2017 was conducted. RESULTS: Eighteen patients underwent 21 RCC reoperations with a mean follow-up of 58 mo. Patient symptoms prior to reoperation included headaches (14, 66.7%) and vision loss (12, 57.1%). Thirteen of 18 patients (72.2%) required hormone supplementation prior to reoperation including 5 with diabetes insipidus (DI). Mean RCC diameter was 16 mm and 76% had suprasellar extension. Compared to index RCC cases, intraoperative cerebrospinal fluid leak repair was more common in reoperation cases (15/21, 71% vs 43/91, 47%, P = .05). There was 1 carotid artery injury without neurological sequelae, and 2 postoperative cerebrospinal fluid (CSF) leaks (9.5%). Rates of transient hyponatremia (3/10, 30% vs 4/91, 4.4%, P = .04) and transient DI (5/10, 50% vs 17/91, 18.7%, P = .04) were higher in the reoperation vs index group. Improved headaches and vision were reported in 4/12 (33%) and 8/12 (61.5%) of RCC reoperation patients, respectively. Two patients developed new permanent DI. A higher proportion of reoperation patients had RCC squamous metaplasia (24% vs 5.4%, P = .02) or wall inflammation (42.9% vs 2.2%, P < .001) on pathological examination. CONCLUSION: Reoperation for RCCs is generally safe at tertiary pituitary centers and often results in improved vision. Hypopituitarism is less likely to improve following reoperation for recurrent RCCs. Several histopathological features may help characterize "atypical RCCs" with a higher likelihood of recurrence/progression.

Monocytes of Different Subsets in Complexes with Platelets in Patients with Myocardial Infarction.

Acute myocardial infarction (AMI) is associated with activation of various cells, including platelets that form monocyte-platelet complexes (MPCs). Here, we analysed MPC in vivo and in vitro and investigated the abilities of different monocyte subclasses to form MPC, the characteristics of the cells involved in MPC formation and MPC changes in AMI. We identified MPC by co-staining for platelet antigen CD41a and monocyte antigens CD14 and CD16. Platelet activation was evaluated from expression of phosphatidylserine as revealed by annexin V. Our results confirm published data and provide new information regarding the patterns of MPC in AMI patients. We found that the patterns of platelet aggregation with monocytes were different in AMI patients and controls: (1) in AMI patients, MPC formed by intermediate monocytes carry more platelets whereas in healthy controls more platelets aggregated with classical monocytes; (2) the numbers of MPC in AMI patients, being already higher than in controls, were further increased if these patients suffered various in-hospital complications; (3) on the basis of the CD41a fluorescence of the antibody-stained MPC, some of the aggregates seem to consist of monocytes and platelet-derived extracellular vesicles (EVs); (4) aggregation of monocytes with platelet EV occurred in in vitro experiments; and (5) these experiments demonstrated that monocytes from AMI patients aggregate with both platelets and platelet EVs more efficiently than do monocytes from controls. MPC in AMI patients may play an important role in this pathology.

Human cytomegalovirus-infected cells release extracellular vesicles that carry viral surface proteins.

Extracellular vesicles (EVs) released by virus-infected cells typically incorporate host and viral components inside the vesicles (cargo molecules). Here, we investigated if human cytomegalovirus (HCMV) proteins are incorporated in EV outer membrane released by HCMV-infected cells. We separated EVs from HCMV using an iodixanol step-gradient and found that the separated vesicles carried EV markers such as the tetraspanin CD63 and Rab27A. Flow analysis of individual EVs demonstrated that on average, 15 ±â€¯3.7% of EVs were positive for gB, 5.3 ±â€¯2.3% were positive for gH and 3.74 ±â€¯1.5% were positive for both gB and gH. In light of previous findings demonstrating HIV envelope proteins in EV membranes, the presence of viral protein at the surface of EVs released by HCMV-infected cells indicated that viral membrane proteins incorporated in EVs released by virus-infected cells may be a general phenomenon.

Select gp120 V2 domain specific antibodies derived from HIV and SIV infection and vaccination inhibit gp120 binding to α4ß7.

The GI tract is preferentially targeted during acute/early HIV-1 infection. Consequent damage to the gut plays a central role in HIV pathogenesis. The basis for preferential targeting of gut tissues is not well defined. Recombinant proteins and synthetic peptides derived from HIV and SIV gp120 bind directly to integrin α4ß7, a gut-homing receptor. Using both cell-surface expressed α4ß7 and a soluble α4ß7 heterodimer we demonstrate that its specific affinity for gp120 is similar to its affinity for MAdCAM (its natural ligand). The gp120 V2 domain preferentially engages extended forms of α4ß7 in a cation -sensitive manner and is inhibited by soluble MAdCAM. Thus, V2 mimics MAdCAM in the way that it binds to α4ß7, providing HIV a potential mechanism to discriminate between functionally distinct subsets of lymphocytes, including those with gut-homing potential. Furthermore, α4ß7 antagonists developed for the treatment of inflammatory bowel diseases, block V2 binding to α4ß7. A 15-amino acid V2 -derived peptide is sufficient to mediate binding to α4ß7. It includes the canonical LDV/I α4ß7 binding site, a cryptic epitope that lies 7-9 amino acids amino terminal to the LDV/I, and residues K169 and I181. These two residues were identified in a sieve analysis of the RV144 vaccine trial as sites of vaccine -mediated immune pressure. HIV and SIV V2 mAbs elicited by both vaccination and infection that recognize this peptide block V2-α4ß7 interactions. These mAbs recognize conformations absent from the ß- barrel presented in a stabilized HIV SOSIP gp120/41 trimer. The mimicry of MAdCAM-α4ß7 interactions by V2 may influence early events in HIV infection, particularly the rapid seeding of gut tissues, and supports the view that HIV replication in gut tissue is a central feature of HIV pathogenesis.

The evaluation of lumbar paraspinal muscle quantity and quality using the Goutallier classification and lumbar indentation value.

PURPOSE: The cross-sectional area and fat infiltration are accepted as standard parameters for quantitative and qualitative evaluation of muscle degeneration. However, they are time-consuming, which prevents them from being used in a clinical setting. The aim of this study was to analyze the relationship between lumbar muscle degeneration and spinal degenerative disorders, using lumbar indentation value (LIV) as quantitative and Goutallier classification as qualitative measures. METHODS: This is a retrospective analysis of kinematic magnetic resonance images (kMRI). Two-hundred and thirty patients with kMRIs taken in weight-bearing positions were selected randomly. The LIV and Goutallier classification were evaluated at L4-5. The correlation of these two parameters with patients' age, gender, lumbar lordosis (LL), range of motion, disc degeneration, disc height, and Modic change were analyzed. RESULTS: There was no significant trend of LIV among the different grades of Goutallier classification (p = 0.943). There was a significant increase in age with higher grades of Goutallier classification (p < 0.001). In contrast, there was no correlation between LIV and age (p = 0.799). The Goutallier classification positively correlated with LL (r = 0.377) and severe disc degeneration (r = 0.249). The LIV positively correlated with LL (r = 0.476) and degenerative spondylolisthesis (r = 0.184). Multinomial logistic regression analysis showed that age (p = 0.026), gender (p = 0.003), and LIV (p < 0.001) were significant predictors for patients with low LL (< 10°). CONCLUSION: Lumbar muscle quantity and quality showed specific correlation with age and spine disorders. Additionally, LL can be predicted by the muscle quantity, but not the quality. These time-saving evaluation tools potentially accelerate the study of lumbar muscles. These slides can be retrieved under Electronic Supplementary Material.

CD63, MHC class 1, and CD47 identify subsets of extracellular vesicles containing distinct populations of noncoding RNAs.

Extracellular vesicles (EVs) mediate the intercellular transfer of RNAs, which alter gene expression in target cells. EV heterogeneity has limited progress towards defining their physiological functions and utility as disease-specific biomarkers. CD63 and MHC1 are widely used as markers to purify EVs. CD47 is also present on EVs and alters their effects on target cells, suggesting that specific surface markers define functionally distinct EVs. This hypothesis was addressed by comparing Jurkat T cell EVs captured using CD47, CD63, and MHC1 antibodies. These EV subsets have similar sizes but divergent RNA contents. Apart from differences in numbers of nonannotated transcripts, CD63+, MHC1+, and CD47+ EVs have similar overall contents of most noncoding RNA classes, but the relative enrichment of specific RNAs differs. The enrichment of micro-RNAs is highly divergent, and some including miR320a are selectively concentrated in CD47+ EVs. Small nucleolar RNAs including SNORD116@ and SNHG10 are also selectively enriched in CD47+ EVs, whereas no small nuclear RNAs are enriched in CD47+ EVs. Conversely, MHC1+ EVs are selectively enriched in a subset of tRNAs including TRE-CTC and TRR-CCG. This heterogeneity in RNA composition suggests multiple sorting mechanisms that direct specific RNAs into subsets of EVs that express specific surface markers.

A function-blocking CD47 antibody modulates extracellular vesicle-mediated intercellular signaling between breast carcinoma cells and endothelial cells.

Tumor cells release extracellular vesicles (EVs) into the tumor microenvironment that may facilitate malignant progression and metastasis. Breast carcinoma EVs express high levels of the thrombospondin-1 and signal regulatory protein-α receptor CD47, which is the target of several experimental therapeutics currently in clinical trials. We analyzed changes in gene expression and function in human umbilical vein endothelial cells (HUVEC) induced by treatment with EVs derived from breast carcinoma cells and the effects of the function-blocking CD47 antibody B6H12 on the resulting intercellular communication. CD47+ EVs exhibited greater uptake by HUVEC compared to CD47- EVs, but the CD47 antibody did not inhibit their uptake. Global and targeted analyses of transcripts demonstrated that treatment of HUVEC with EVs derived from MDA-MB-231 breast carcinomas cells altered pathways associated with tumor necrosis factor-α signaling, angiogenesis, lymphangiogenesis, endothelial-mesenchymal transition, and extracellular matrix. EVs from triple-negative MDA-MB-231 cells were more active than EVs from less metastatic breast carcinoma cell lines. Treatment with MDA-MB-231 EVs down-regulated VEGFR2 mRNA expression and tyrosine phosphorylation while enhancing phosphorylation of the tyrosine phosphatase SHP2. VEGFR2 expression and phosphorylation in HUVEC was further inhibited by the CD47 antibody. Consistent with the observed changes in endothelial-mesenchymal transition genes and SHP2, treatment with MDA-MB-231-derived EVs decreased Zeb1 protein levels in HUVEC, whereas the CD47 antibody increased Zeb1 levels. The induction of E-selectin and other known targets of tumor necrosis factor-α signaling by EVs was also enhanced by the CD47 antibody, and E-selectin was the most up-regulated transcript following CD47 antibody treatment alone. These studies reveal several mechanisms by which therapeutics targeting CD47 could modulate tumor growth by altering the cross talk between cancer-derived EVs and nonmalignant cells in the tumor stroma.

A Novel Lumbar Motion Segment Classification to Predict Changes in Segmental Sagittal Alignment After Lateral Interbody Fixation.

STUDY DESIGN: Retrospective cohort study. OBJECTIVES: Lateral interbody fixation is being increasingly used for the correction of segmental sagittal parameters. One factor that affects postoperative correction is the resistance afforded by posterior hypertrophic facet joints in the degenerative lumbar spine. In this article, we describe a novel preoperative motion segment classification system to predict postoperative correction of segmental sagittal alignment after lateral lumbar interbody fusion. METHODS: Preoperative computed tomography scans were analyzed for segmental facet osseous anatomy for all patients undergoing lateral lumbar interbody fusion at 3 institutions. Each facet was assigned a facet grade (min = 0, max = 2), and the sum of the bilateral facet grades was the final motion segment grade (MSG; min = 0, max = 4). Preoperative and postoperative segmental lordosis was measured on standing lateral radiographs. Postoperative segmental lordosis was also conveyed as a percentage of the implanted graft lordosis (%GL). Simple linear regression was conducted to predict the postoperative segmental %GL according to MSG. RESULTS: A total of 36 patients with 59 operated levels were identified. There were 19 levels with MSG 0, 14 levels with MSG 1, 13 levels with MSG 2, 8 levels with MSG 3, and 5 levels with MSG 4. Mean %GL was 115%, 90%, 77%, 43%, and 5% for MSG 0 to 4, respectively. MSG significantly predicted postoperative %GL (P < .01). Each increase in MSG was associated with a 28% decrease in %GL. CONCLUSIONS: We propose a novel facet-based motion segment classification system that significantly predicted postoperative segmental lordosis after lateral lumbar interbody fusion.