SMR peptide antagonizes Staphylococcus aureus biofilm formation.

The emergence and international dissemination of multi-drug resistant Staphylococcus aureus (S. aureus) strains challenge current antibiotic-based therapies, representing an urgent threat to public health worldwide. In the U.S. alone, S. aureus infections are responsible for 11,000 deaths and 500,000 hospitalizations annually. Biofilm formation is a major contributor to antibiotic tolerance and resistance-induced delays in empirical therapy with increased infection severity, frequency, treatment failure, and mortality. Developing novel treatment strategies to prevent and disrupt biofilm formation is imperative. In this article, we test the Secretion Modification Region (SMR) peptides for inhibitory effects on resistant S. aureus biofilm-forming capacity by targeting the molecular chaperone DnaK. The dose effect of SMR peptides on biofilm formation was assessed using microtiter plate methods and confocal microscopy. Interaction between the antagonist and DnaK was determined by immune precipitation with anti-Flag M2 Affinity and Western blot analysis. Increasing SMR peptide concentrations exhibited increasing blockade of S. aureus biofilm formation with significant inhibition found at 18 µM, 36 µM, and 72 µM. This work supports the potential therapeutic benefit of SMR peptides in reducing biofilm viability and could improve the susceptibility to antimicrobial agents.IMPORTANCEThe development of anti-biofilm agents is critical to restoring bacterial sensitivity, directly combating the evolution of resistance, and overall reducing the clinical burden related to pervasive biofilm-mediated infections. Thus, in this study, the SMR peptide, a novel small molecule derived from the HIV Nef protein, was preliminarily explored for anti-biofilm properties. The SMR peptide was shown to effectively target the molecular chaperone DnaK and inhibit biofilm formation in a dose-dependent manner. These results support further investigation into the mechanism of SMR peptide-mediated biofilm formation and inhibition to benefit rational drug design and the identification of therapeutic targets.

Anti-human immunodeficiency virus-1 activity of MoMo30 protein isolated from the traditional African medicinal plant Momordica balsamina.

BACKGROUND: Plants are used in traditional healing practices of many cultures worldwide. Momordica balsamina is a plant commonly used by traditional African healers as a part of a treatment for HIV/AIDS. It is typically given as a tea to patients with HIV/AIDS. Water-soluble extracts of this plant were found to contain anti-HIV activity. METHODS: We employed cell-based infectivity assays, surface plasmon resonance, and a molecular-cell model of the gp120-CD4 interaction to study the mechanism of action of the MoMo30-plant protein. Using Edman degradation results of the 15 N-terminal amino acids, we determined the gene sequence of the MoMo30-plant protein from an RNAseq library from total RNA extracted from Momordica balsamina. RESULTS: Here, we identify the active ingredient of water extracts of the leaves of Momordica balsamina as a 30 kDa protein we call MoMo30-plant. We have identified the gene for MoMo30 and found it is homologous to a group of plant lectins known as Hevamine A-like proteins. MoMo30-plant is distinct from other proteins previously reported agents from the Momordica species, such as ribosome-inactivating proteins such as MAP30 and Balsamin. MoMo30-plant binds to gp120 through its glycan groups and functions as a lectin or carbohydrate-binding agent (CBA). It inhibits HIV-1 at nanomolar levels and has minimal cellular toxicity at inhibitory levels. CONCLUSIONS: CBAs like MoMo30 can bind to glycans on the surface of the enveloped glycoprotein of HIV (gp120) and block entry. Exposure to CBAs has two effects on the virus. First, it blocks infection of susceptible cells. Secondly, MoMo30 drives the selection of viruses with altered glycosylation patterns, potentially altering their immunogenicity. Such an agent could represent a change in the treatment strategy for HIV/AIDS that allows a rapid reduction in viral loads while selecting for an underglycosylated virus, potentially facilitating the host immune response.

Moving Morehouse School of Medicine Translation Tx Research through MDTTs-Multidisciplinary Translational Teams.

Morehouse School of Medicine (SOM) works to achieve its vision of advancing health equity through conducting transformational, translation science (Tx). Tx describes our translational research continuum, symbolizing a method and scientific philosophy that intentionally promotes and supports convergence of interdisciplinary approaches and scientists to stimulate exponential advances for the health of diverse communities. Morehouse SOM actualizes Tx through multidisciplinary translational teams (MDTTs). We chronicle the identification of MDTTs by documenting formation, composition, functioning, successes, failures, and sustainability. Data and information were collected through key informant interviews, review of research documents, workshops, and community events. Our scan identified 16 teams that meet our Morehouse SOM definition of an MDTT. These team science workgroups cross basic science, clinical, and public health academic departments, and include community partners and student learners. We present four MDTTs, in various stages of progress, at Morehouse SOM and how they are advancing translational research.

Identification of a Novel Anti-HIV-1 Protein from Momordica balsamina Leaf Extract.

Our lab investigates the anti-HIV-1 activity in Momordica balsamina (M. balsamina) leaf extract. Traditional Senegalese healers have used M. balsamina leaf extract as a part of a plant-based treatment for HIV/AIDS infections. Our overall goal is to define and validate the scientific basis for using M. balsamina leaf extract as a part of the traditional Senegalese treatment. As an initial characterization of this extract, we used activity-guided fractionation to determine the active ingredient's solubility and relative size. We found that M. balsamina leaf extract inhibits HIV-1 infection by >50% at concentrations of 0.02 mg/mL and above and is not toxic over its inhibitory range (0-0.5 mg/mL). We observed significantly more antiviral activity in direct water and acetonitrile extractions (p ≤ 0.05). We also observed significantly more antiviral activity in the aqueous phases of ethyl acetate, chloroform, and diethyl ether extractions (p ≤ 0.05). Though most of the antiviral activity partitioned into the aqueous layers, some antiviral activity was present in the organic layers. We show that the active agent in the plant extracts is at least 30 kD in size. Significantly more antiviral activity was retained in 3, 10, and 30 kD molecular weight cutoff filters (p ≤ 0.05). In contrast, most of the antiviral activity passed through the 100 kD filter (p ≤ 0.05). Because the active anti-HIV-1 agent presented as a large, amphiphilic molecule we ran the purified extract on an SDS-page gel. We show that the anti-HIV-1 activity in the leaf extracts is attributed to a 30 kDa protein we call MoMo30. This article describes how MoMo30 was determined to be responsible for its anti-HIV-1 activity.

Novel secretion modification region (SMR) peptide exhibits anti-metastatic properties in human breast cancer cells.

Breast cancer is the second leading cause of cancer-related mortality in women worldwide, with nearly 90% attributed to metastatic progression. Exosomes containing epithelial-mesenchymal transition (EMT) 'programs' transmit pro-metastatic phenotypes. Our group discovered and developed a novel anti-cancer SMR peptide that antagonizes breast cancer cell exosome release resulting in cell cycle arrest and tumor growth suppression. This study aims to evaluate the anti-metastatic capabilities of the SMR peptide, focusing on exosomes and EMT. Breast cancer cell lines MDA-MB-231 and MCF-7 were treated with the SMRwt peptide, and the following assays were performed: cell wound-healing, migration, invasion. The SMRwt peptide consists of the following amino acid sequence VGFPVAAVGFPVDYKDDDDK and contains the SMR domain (66VGFPV70) of the HIV-1 Nef protein. Western blot analysis detected epithelial and mesenchymal markers to evaluate EMT progression. Extracellular vesicle type and quantity were assessed through NanoSight analysis. Mortalin and Vimentin knockdown was achieved through antibody targeting and miRNAs. Data gathered demonstrated that the SMR peptide interacts with Mortalin and Vimentin to inhibit pro-EMT exosome release and induce EMT tumor suppressor protein expression. Specifically, SMRwt treatment reduced mesenchymal markers Mortalin and Vimentin expression, while the epithelial marker E-cadherin expression was increased in breast cancer cells and breast cancer-derived exosomes. The SMR peptide specificity was identified as no effect was observed for MCF-10A exosome release or function. Direct Mortalin knockdown paralleled the results of SMR peptide treatment with an effective blockade of breast cancer cell migration. Conversely, the invasion assay differed between breast cancer cell lines with invasion blocked for in MCF-7 but not in MDA-MB-231. These results reinforce the therapeutic value of targeting breast cancer exosome release and reinforce Mortalin and Vimentin as critical regulators and therapeutic targets in breast cancer cell progression, EMT, and metastatic potential. A greater understanding of the SMR peptide mechanism of action will benefit the therapeutic design of anti-metastatic agents.

Improved Aitongxiao prescription (I-ATXP) induces apoptosis, cell cycle arrest and blocks exosomes release in hepatocellular carcinoma (HCC) cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is the second most common malignancy globally, after lung cancer, accounting for 85-90% of primary liver cancer. Hepatitis B virus (HBV) infection is considered the leading risk factor for HCC development in China. HCC is a highly malignant cancer whose metastasis is primarily influenced by the tumor microenvironment. The role of exosomes in cancer development has become the focus of much research due to the many newly described contents of exosomes, which may contribute to tumorigenesis. However, the possible role exosomes play in the interactions between HCC cells and their surrounding hepatic milieu is mainly unknown. We discovered an Improved Aitongxiao Prescription (I-ATXP): an 80% alcohol extract from a mix of 15 specific plant and animal compounds, which had been shown to have an anticancer effect through inducing apoptosis and cell cycle arrest and blocking exosomes release in HCC cells. However, the anticancer mechanism of I-ATXP on human liver carcinoma is still unclear. OBJECTIVE: Due to its inhibitory effects on chemical carcinogenesis and inflammation, I-ATXP has been proposed as an effective agent for preventing or treating human liver carcinoma. In this study, we aimed to explore the effect of I-ATXP on proliferation, apoptosis, and cell cycles of different HCC cell lines. We investigated the impact of I-ATXP on exosomes' secretion derived from these HCC cells. METHODS: The inhibitory effect of I-ATXP on proliferation and cytotoxicity of HepG2, SMMC7721, HKCL-C3 HCC cell lines, and MIHA immortalized hepatocyte cell line was assessed by CCK-8 assay. The cell cycle distribution and cell apoptosis were determined by flow cytometry using Annexin V-FITC/PI staining. The expression of Alix and CD63 of exosome marker proteins was detected by western blotting. The exosome protein concentration was measured by a fluorescent plate reader. The exosome-specific enzyme activity was measured by acetylcholinesterase (AchE) assay, and exosome morphological characteristics were identified by transmission electron microscopy (TEM). RESULTS: I-ATXP inhibited the growth of HCC cells in a dose and time-dependent manner. Flow cytometry analysis showed that I-ATXP induced G0/G1 phase arrest and cell apoptosis. The I-ATX reduced HepG2, SMMC7721, and HKCI-C HCC cell lines exosomes release and low-dose I-ATXP significantly enhanced the growth inhibition induced by 5-Fu. Western blot analysis shows that after HCC cell lines were treated with various concentrations of I-ATXP (0.125-1 mg/ml) for 24 h, exosomes derived from three different HCC cells expressed exosome-specific proteins Alix and CD63. Compared with the untreated group, with the increment of the concentration of I-ATXP, the expression of exosome-specific proteins Alix and CD63 were reduced. These results suggest that I-ATXP can inhibit the release of exosomes with Alix and CD63 protein from HCC cells. CONCLUSIONS: I-ATXP is a traditional Chinese medicine that acts as an effective agent for preventing or treating human liver carcinoma. (i) I-ATXP can effectively inhibit cell proliferation of different HCC cells in a time and dose-dependent manner. Compared with 5-Fu, I-ATXP exhibited more selective proliferation inhibition in HCC cells, displaying traditional Chinese medicine advantages on tumor therapy and providing the experimental basis for I-ATXP clinical application. (ii) I-ATXP can induce apoptosis and cell cycle arrest in HCC cells. The CCK-8 assay results indicated that I-ATXP could inhibit HCC cell proliferation mediated by apoptosis and cell cycle arrest. (iii) I-ATXP can inhibit both the exosome releases and expression of CD63, and Alix derived from HCC cells, but the exosomes derived from liver cancer cells affect liver cancer cells' biological properties such as proliferation, invasion, and migration. These suggest that I-ATXP may affect HCC cells via regulation of exosomes of HCC cells, further indicating the potential clinical values of I-ATXP for the prevention or treatment of human liver carcinoma.

Macrophage M2 polarization induced by exosomes from adipose-derived stem cells contributes to the exosomal proangiogenic effect on mouse ischemic hindlimb.

BACKGROUND: M2 macrophages and exosomes from adipose-derived stem cells (ASCs) are both reported to promote angiogenesis. However, the possible synergistic effects between exogenous exosomes and endogenous M2 macrophages are poorly understood. METHODS: Exosomes were isolated from conditioned medium of normoxic and hypoxic ASCs using the combined techniques of ultrafiltration and size-exclusion chromatography and were identified with nanoparticle tracking analysis and immunoblotting for exosomal markers. Macrophages were collected from the mouse peritoneal cavity. M1 and M2 macrophages were detected by immunoblotting for the intracellular markers inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1) and by flow cytometry for the surface markers F4/80, CD86, and CD206. Murine models of Matrigel plug and hindlimb ischemia were employed as in vivo angiogenic assays. RESULTS: When M1 macrophages were treated with exosomes from normoxic ASCs (Nor/Exo), and particularly from hypoxic ASCs (Hyp/Exo), the expression of the M1 marker iNOS decreased, and the M2 marker Arg-1 increased in a time- and dose-dependent manner. Additionally, a decrease in the M1 surface marker CD86 and an increase in the M2 surface marker CD206 were observed, which suggested that M1 macrophages were polarized to an M2-like phenotype. Conditioned medium from these M2-like macrophages presented lower levels of proinflammatory cytokines and higher levels of proangiogenic factors and promoted endothelial cell proliferation, migration, and tube formation. Furthermore, M2 polarization and angiogenesis were induced upon the administration of exosomes in mouse Matrigel plug and hindlimb ischemia (HLI) models. Interestingly, these exosomal effects were attenuated by using a colony stimulating factor 1 receptor (CSF-1R) inhibitor, BLZ945, in vitro and in vivo. Downregulation of microRNA-21 (miR-21) in hypoxic ASCs reduced the exosomal effects on M2 polarization, Akt phosphorylation, and CSF-1 secretion. A similar reduction in exosomal activity was also observed when exosomes were administered along with BLZ945. CONCLUSION: Our findings provide evidence that exosomes from ASCs polarize macrophages toward an M2-like phenotype, which further enhances the exosomal proangiogenic effects. Exosomal delivery of miR-21 and positive feedback of secreted CSF-1 may be involved in macrophage polarization.

SMR peptide antagonizes mortalin promoted release of extracellular vesicles and affects mortalin protection from complement-dependent cytotoxicity in breast cancer cells and leukemia cells.

Background: Mortalin/GRP-75/mt-hsp70 is a mitochondrial chaperone protein, found in the cytoplasm, endoplasmic reticulum and cytoplasmic vesicles. It functions in many cellular processes such as mitochondrial biogenesis, intracellular trafficking, cell proliferation, signaling, immortalization and tumorigenesis. Thus, inhibition of mortalin is a promising avenue for cancer therapy. Previous studies in our lab have suggested that mortalin contributes to breast cancer development and progression. We showed that tumor extracellular vesicle secretion was decreased by knockdown of mortalin expression using HIV-1 Nef SMR peptides. Specifically, these peptides can block extracellular vesicle secretion and mediate cell cycle arrest in MDA-MB-231 and MCF-7 breast cancer cells. Aims: This study aims to investigate further the function and mechanism of interaction of PEG-SMR-CLU and SMR-CPP peptides with the chaperone protein mortalin and to explore the effect of SMR-derived peptides and mortalin expression on extracellular vesicle release and complement dependent cell toxicity in human breast cancer and leukemia cell lines. Results: Our results demonstrated additional effects reversing the tumorigenicity of these cells. First, the modified SMRwt peptides reduced the expression of the mesenchymal marker vimentin (VIM). Second, exposure to the SMRwt peptide inhibited mortalin and complement C9 expression in MDA-MB-231, MCF-7 breast cancer cells and K562 leukemia cells as measured by the Western blot analysis. Third, the SMRwt peptides blocked the cancer cells' ability to release extracellular vesicles, which we observed blocked extracellular vesicle-mediated release of complement, re-establishing complements mediated cell death in those peptide-treated cells. Methods: We developed a series of peptides derived from the Secretion Modification Region (SMR) of HIV-1 Nef protein, modified by the addition of either a cell-penetrating peptide (CPP), a positively charged arginine-rich peptide derived from HIV-1 regulatory protein Tat, or a Clusterin-binding peptide (CLU), a molecular chaperone involved in protein secretion. Both CPP and CLU peptide sequences were added at the C-terminus of the Nef SMR peptide. The CLU-containing peptides were also modified with polyethylene glycol (PEG) to enhance solubility. After treatment of cells with the peptides, we used the MTT cell viability and complement-mediated cytotoxicity assays to confirm the inhibitory role of modified SMRwt peptides on the proliferation of MDA-MB-231 and MCF-7 breast cancer cells and K562 leukemia cells. Flow cytometry was used to determine complement mediated cell apoptosis and death. Western blot analysis was used to track SMR peptides impact on expression of mortalin, vimentin and complement C9 and to measure the expression of extracellular vesicle proteins. NanoSight analysis and acetylcholinesterase (AChE) assay were used for measuring extracellular vesicles particle size and concentration and acetylcholinesterase. Conclusions: Mortalin promotes cell proliferation, metastasis, angiogenesis, downregulate apoptotic signaling. Thus, mortalin is a potential therapeutic target for cancer immunotherapy. The novel SMRwt peptides antagonize the functions of mortalin, blocking tumor extracellular vesicle release and extracellular vesicle-mediated release of complement. This leads to decreases in breast cancer cell metastasis and allows standard treatment of these late stage tumor cells, thus having important clinical implications for late stage breast cancer chemotherapy. These findings support further investigation into the therapeutic value of the SMR peptide in cancer metastasis.

The Research Centers in Minority Institutions (RCMI) Translational Research Network: Building and Sustaining Capacity for Multi-Site Basic Biomedical, Clinical and Behavioral Research.

The Research Centers in Minority Institutions (RCMI) program was established by the US Congress to support the development of biomedical research infrastructure at minority-serving institutions granting doctoral degrees in the health professions or in a health-related science. RCMI institutions also conduct research on diseases that disproportionately affect racial and ethnic minorities (ie, African Americans/Blacks, American Indians and Alaska Natives, Hispanics, Native Hawaiians and Other Pacific Islanders), those of low socioeconomic status, and rural persons. Quantitative metrics, including the numbers of doctoral science degrees granted to underrepresented students, NIH peer-reviewed research funding, peer-reviewed publications, and numbers of racial and ethnic minorities participating in sponsored research, demonstrate that RCMI grantee institutions have made substantial progress toward the intent of the Congressional legislation, as well as the NIH/NIMHD-linked goals of addressing workforce diversity and health disparities. Despite this progress, nationally, many challenges remain, including persistent disparities in research and career development awards to minority investigators. The continuing underrepresentation of minority investigators in NIH-sponsored research across multiple disease areas is of concern, in the face of unrelenting national health inequities. With the collaborative network support by the RCMI Translational Research Network (RTRN), the RCMI community is uniquely positioned to address these challenges through its community engagement and strategic partnerships with non-RCMI institutions. Funding agencies can play an important role by incentivizing such collaborations, and incorporating metrics for research funding that address underrepresented populations, workforce diversity and health equity.

Characterization of Exosomes in Plasma of Patients with Breast, Ovarian, Prostate, Hepatic, Gastric, Colon, and Pancreatic Cancers.

Detection of circulating tumor-specific DNA, RNA or proteins can be difficult due to relative scarcity. Exosomes are extracellular vesicles, 30 - 150 nm in diameter derived from fusion of multivesicular bodies with the plasma membrane. They are composed of a lipid bilayer membrane and contain proteins, mRNA and miRNA. Exosomes are secreted by multiple cell types, including cancer cells. However, there is a relative lack of information concerning the contents of exosomes secreted by various tumor cell types. To examine exosomes in cancer, we collected blood plasma samples from patients with breast, ovarian, prostate, hepatic, gastric, colon, and pancreatic cancers. Exosomes were isolated from plasma and confirmed by AchE assay, transmission electron microscopy and expression of the CD63 exosomal marker. Expression of AFP, CA724, CA153, CEA, CA125, CA199 and PSA antigens were determined using an automated electro-chemiluminescence assay. Expression of the tumor-related chaperone protein, mortalin, was determined by Western blot analysis. Levels of exosome secretion were variable among the different tumor types. Both exosome levels and mortalin expression within tumor cell exosomes were higher than in healthy donors, except in pancreatic carcinoma, where exosomes were elevated but mortalin expression was not significantly different from healthy donors. Exosomes provide unique opportunities for the enrichment of tumor-specific materials and may be useful as biomarkers and possibly as tools of cancer therapies. Mortalin, which has been linked to cell proliferation and induction of epithelial-mesenchymal transition of cancer cells, may be useful as a prognostic bio-marker and as a possible therapeutic target.

Small molecule CXCR4 antagonists block the HIV-1 Nef/CXCR4 axis and selectively initiate the apoptotic program in breast cancer cells.

The chemokine receptor CXCR4 plays an integral role in the development of highly metastatic breast cancer and in the pathogenesis of chronic HIV infection. In this study, we compared the effects of CXCR4 antagonists on apoptosis induction in hematopoietic cells and in tumor cells. We incubated cells expressing CXCR4 with a series of CXCR4 antagonists and subsequently exposed the cultures to a pro-apoptotic peptide derived from the HIV-1 Nef protein (NefM1). The NefM1 peptide contains residues 50-60 of Nef and was previously shown to be the sequence necessary for Nef to initiate the apoptotic program through CXCR4 signaling. We found that several of the compounds studied potently blocked Nef-induced apoptosis in Jurkat T-lymphocyte cells. Interestingly, many of the same compounds selectively triggered apoptosis in MDA-MB-231 breast cancer cells, in some cases at sub-nanomolar concentrations. None of the compounds were toxic to lymphocyte, monocyte or macrophage cells, suggesting that aggressive breast cancer carcinomas may be selectively targeted and eliminated using CXCR4-based therapies without additional cytotoxic agents. Our results also demonstrate that not all CXCR4 antagonists are alike and that the observed anti-Nef and pro-apoptotic effects are chemically tunable. Collectively, these findings suggest our CXCR4 antagonists have promising clinical utility for HIV or breast cancer therapies as well as being useful probes to examine the link between CXCR4 and apoptosis.

Proceedings of the 2017 ISEV symposium on "HIV, NeuroHIV, drug abuse, & EVs".

Despite the success of combination antiretroviral therapy (cART), there is increased prevalence of HIV-associated neurocognitive disorders (HAND) in HIV-1-infected individuals on cART, which poses a major health care challenge. Adding further complexity to this long-term antiretroviral use is the comorbidity with drugs of abuse such as morphine, cocaine, and methamphetamine, which can in turn, exacerbate neurologic and cognitive deficits associated with HAND. Furthermore, HIV proteins, such as the transactivator of transcription (Tat) and the envelope protein (gp120), as well as antiretrovirals themselves can also contribute to the progression of neurodegeneration underlying HAND. In the field of NeuroHIV and drug addiction, EVs hold the potential to serve as biomarkers of cognitive dysfunction, targets of therapy, and as vehicles for therapeutic delivery of agents that can ameliorate disease pathogenesis. Based on the success of a previous Satellite Symposium in 2015 at the ISEV meeting in Washington, experts again expanded on their latest research findings in the field, shedding light on the emerging trends in the field of Extracellular Vesicle (EV) biology in NeuroHIV and drug abuse. The satellite symposium sought to align experts in the fields of NeuroHIV and drug abuse to share their latest insights on the role of EVs in regulating neuroinflammation, neurodegeneration, peripheral immune response, and HIV latency in HIV-infected individuals with or without the comorbidity of drug abuse.

Proceedings of the ISEV symposium on "HIV, NeuroAIDS, drug abuse & EVs".

Extracellular vesicles (EVs) are globular, membrane bound nanovesicles (30-100 nm range) that are shed both during normal cellular functioning and under pathological conditions by most cell types. In recent years, there has been significant interest in the study of these vesicles as conduits for the delivery of information between cells from both analogous and disparate tissues. Their ability to carry specialised cargo including signalling mediators, proteins, messenger RNA and miRNAs characterises these vesicles as primary facilitators of cell-to-cell communication and regulation. EVs have also been demonstrated to play important roles in the field of cancer biology and metastasis. However, significant knowledge gaps exist in the role these vesicles play in the context of HIV infection and drug abuse. To foster discussion in this area a satellite symposium on "HIV, NeuroAIDS, Drug Abuse & EVs", was held in conjunction with the annual meeting of the International Society for Extracellular Vesicles (ISEV) in Bethesda, in April 2015. Experts in HIV and drug abuse fields were invited to share their findings on the role of EVs in HIV-1 infection and drug addiction. Additional discussion included current areas of research in EV biology in HIV infection and drug abuse.

Secretion modification region-derived peptide blocks exosome release and mediates cell cycle arrest in breast cancer cells.

PURPOSE: Discovery and development of a novel anticancer PEG-SMR-Clu peptide to prevent breast cancer metastasis. How breast cancer cells and primary mammary epithelial cells interact and communicate with each other to promote tumorigenesis and how to prevent tumor metastasis has long been a concern of researchers. Cancer cells secrete exosomes containing proteins and RNA. These factors can influence tumor development by directly targeting cancer cells and tumor stroma. In this study, we determined the effects of a peptide as an inhibitor of exosome secretion on breast tumors. We developed a peptide derived from the Secretion Modification Region (SMR) of HIV-1 Nef protein that was modified with PEG on the N-terminus and with a Clusterin (Clu)-binding peptide on the C-terminus. Attachment of PEG to the SMR peptide, termed PEGylation, offers improved water solubility and stability as well as reduced clearance through the kidneys, leading to a longer circulation time. The 12-mer Clu-binding peptide plays multiple roles in tumor development and metastasis. The Clu peptide can be detected by antibody in vivo, thus it has the potential to be used to monitor tumor status and treatment efficacy in animal studies and eventually in cancer patients. RESULTS: PEG-SMRwt-Clu and PEG-SMRwt peptides inhibited the growth of both of MCF-7 (estrogen responsive, ER+) and MDA-MD-231 (estrogen non-responsive, ER-) human breast cancer cells in a dose and time-dependent manner, without inducing cytotoxic effects. The SMRwt peptide, combined with paclitaxel, induced G2/M phase cell cycle arrest on MCF-7 and MDA-MB-231 cells but did not promote apoptosis. PEG-SMRwt-Clu peptide treatment blocked exosome release from both MCF-7 and MDA-MB-231 cells. This effect was blocked by knockdown of the chaperone protein mortalin by either antibody or siRNA. MATERIALS AND METHODS: MCF-7 and MDA-MB-231 breast tumor cells were treated with PEG-SMR-Clu peptide alone and in combination with paclitaxel and cisplatin. Cell proliferation and viabilty were determined via cell cycle analysis using Cellometer imaging cytometry, Annexin V and MTT assays. The effects of the PEG-SMR-Clu peptide on tumor exosome release were determined by testing isolated exosome fractions, for (i) expression of CD63 and Alix proteins by Western blotting, (ii) NanoSight nanoparticle tracking analysis (NTA 10) to measure exosomes size and concentration, and (iii) measurement of acetylcholinesterase (AchE) for exosome specific enzyme activity. CONCLUSIONS: PEG-SMRwt-CLU peptides inhibited the growth of human breast cancer cells and blocked tumor exosome release in vitro. The peptide alone did not cause increased cytotoxicity or apoptosis induction, but did cause cell cycle G2/M phase arrest in both estrogen responsive and non-responsive breast cancer cells. These data suggest a potential therapeutic value of SMR to prevent breast cancer metastasis and as an adjuvant for the chemotherapeutic treatment of human breast cancer.

Adipose-Derived Stem Cells Induce Angiogenesis via Microvesicle Transport of miRNA-31.

UNLABELLED: Cell secretion is an important mechanism for stem cell-based therapeutic angiogenesis, along with cell differentiation to vascular endothelial cells or smooth muscle cells. Cell-released microvesicles (MVs) have been recently implicated to play an essential role in intercellular communication. The purpose of this study was to explore the potential effects of stem cell-released MVs in proangiogenic therapy. We observed for the first time that MVs were released from adipose-derived stem cells (ASCs) and were able to increase the migration and tube formation of human umbilical vein endothelial cells (HUVECs). Endothelial differentiation medium (EDM) preconditioning of ASCs upregulated the release of MVs and enhanced the angiogenic effect of the released MVs in vitro. RNA analysis revealed that microRNA was enriched in ASC-released MVs and that the level of microRNA-31 (miR-31) in MVs was notably elevated upon EDM-preconditioning of MV-donor ASCs. Further studies exhibited that miR-31 in MVs contributed to the migration and tube formation of HUVECs, microvessel outgrowth of mouse aortic rings, and vascular formation of mouse Matrigel plugs. Moreover, factor-inhibiting HIF-1, an antiangiogenic gene, was identified as the target of miR-31 in HUVECs. Our findings provide the first evidence that MVs from ASCs, particularly from EDM-preconditioned ASCs, promote angiogenesis and the delivery of miR-31 may contribute the proangiogenic effect. SIGNIFICANCE: This study provides the evidence that microvesicles (MVs) from adipose-derived stem cells (ASCs), particularly from endothelial differentiation medium (EDM)-preconditioned ASCs, promote angiogenesis. An underlying mechanism of the proangiogenesis may be the delivery of microRNA-31 via MVs from ASCs to vascular endothelial cells in which factor-inhibiting HIF-1 is targeted and suppressed. The study findings reveal the role of MVs in mediating ASC-induced angiogenesis and suggest a potential MV-based angiogenic therapy for ischemic diseases.

Nef exosomes isolated from the plasma of individuals with HIV-associated dementia (HAD) can induce Aß(1-42) secretion in SH-SY5Y neural cells.

In the era of combined antiretroviral therapy (CART), many of the complications due to HIV-1 infection have diminished. One exception is HIV-associated neurocognitive disorder (HAND). HAND is a spectrum of disorders in cognitive function that ranges from asymptomatic disease to severe dementia (HAD). The milder form of HAND has actually remained the same or slightly increased in prevalence in the CART era. Even in individuals who have maintained undetectable HIV RNA loads, viral proteins such as Nef and Tat can continue to be expressed. In this report, we show that Nef protein and nef messenger RNA (mRNA) are packaged into exosomes that remain in circulation in patients with HAD. Plasma-derived Nef exosomes from patients with HAD have the ability to interact with the neuroblastoma cell line SH-SY5Y and deliver nef mRNA. The mRNA can induce expression of Nef in target cells and subsequently increase expression and secretion of beta-amyloid (Aß) and Aß peptides. Increase secretion of amyloid peptide could contribute to cognitive impairment seen in HAND.

Characterizing the HIV/AIDS Epidemic in the United States and China.

The HIV/AIDS data from the national surveillance systems of China and the United States from 1985 to 2014 were compared to characterize the HIV/AIDS epidemic in both countries. The current estimated national HIV prevalence rate in China and the United States are 0.0598% and 0.348%, respectively. In the United States, the annual number of new HIV infections has remained relatively stable (~50,000 each year) and has shown a downward trend in recent years. The Chinese national HIV prevalence is still low, and new HIV infections have been contained at a low level (50,000-100,000 each year). However, the epidemic has showed an increasing trend since 2012. By risk group, in both countries, men who have sex with men (MSM), heterosexual sex, and injection drug use (IDU) are the most common modes of transmission of new HIV infections. However, in the United States, MSM is the dominant transmission route, accounting for >60% of new infections; whereas in China, heterosexual sex has now become the dominant route, also accounting for >60% of new infections. A rapid increase in the proportion of HIV cases that were attributed to MSM and an obvious decrease in the proportion of HIV cases attributed to IDU in China in recent years imply that the China's epidemic is still evolving, to some extent, copying what was experienced in the United States. By age group, the proportions of HIV cases that were attributed to the age group 25-59 were comparable between the two countries. However, the United States had a higher proportion of cases that were attributed to age groups 15-19 and 20-24 than China, indicating that youth account for more infections in the United States. One other fact worth noting: in China there is a significant increase in the number of HIV new infections in individuals over 50 years of age, which results in much higher proportion of cases that were attributed to age groups 60-64 and over 65 in China than those in the United States. By race/ethnicity, in the United States, Blacks/African Americans continue to experience the most severe HIV burden, followed by Hispanics/Latinos. In China, no official data on race/ethnicity disparities are currently available. Thus, region, risk group, age are important factors in the HIV epidemics in both countries.

Micro RNA in Exosomes from HIV-Infected Macrophages.

Exosomes are small membrane-bound vesicles secreted by cells that function to shuttle RNA and proteins between cells. To examine the role of exosomal micro RNA (miRNA) during the early stage of HIV-1 infection we characterized miRNA in exosomes from HIV-infected macrophages, compared with exosomes from non-infected macrophages. Primary human monocytes from uninfected donors were differentiated to macrophages (MDM) which were either mock-infected or infected with the macrophage-tropic HIV-1 BaL strain. Exosomes were recovered from culture media and separated from virus particles by centrifugation on iodixanol density gradients. The low molecular weight RNA fraction was prepared from purified exosomes. After pre-amplification, RNA was hybridized to microarrays containing probes for 1200 miRNA species of known and unknown function. We observed 48 miRNA species in both infected and uninfected MDM exosomes. Additionally, 38 miRNAs were present in infected-cell exosomes but not uninfected-cell exosomes. Of these, 13 miRNAs were upregulated in exosomes from HIV-infected cells, including 4 miRNA species that were increased by more than 10-fold. Though numerous miRNA species have been identified in HIV-infected cells, relatively little is known about miRNA content in exosomes from these cells. In the future, we plan to investigate whether the upregulated miRNA species we identified are increased in exosomes from HIV-1-positive patients.

Hallmarks of HIV-1 pathogenesis are modulated by Nef's Secretion Modification Region.

CD4+ T cell depletion and immune activation are hallmarks of HIV infection. Despite extensive studies, the mechanisms underlying immune modulation remain elusive. HIV-1 Nef protein is secreted in exosomes from infected cells and is abundant in the plasma of HIV+ individuals. Exosomal Nef (exNef) was also shown to induce apoptosis in bystander CD4+ T cells. We hypothesized that exNef contributes to HIV pathogenesis. A HIV-1 NL4-3 virus containing alanine substitutions in the secretion modification region (SMR; amino acids 66 to 70; HIVNefsmr5a) was developed. Nef protein containing this modified SMR was shown to be deficient in exNef secretion in nef-transfected cells. Using both HIV-1 NL4-3 wild type (HIVwt) and HIVNefsmr5a, correlates of pathogenesis were evaluated in cell-lines, human peripheral blood mononuclear cells, and humanized NOD-RAG1-/- IL2r-/- double mutant (NRG) mice. Disruption of the SMR did not affect viral replication or exNef secretion from infected cell cultures as compared with nef-transfected cells. However, T cell apoptosis was reduced in HIVNefsmr5a infected cell cultures and CD4+ T cell depletion was reduced in the spleen and peripheral blood of similarly infected NRG mice. Inflammatory cytokine release was also decreased in the sera of HIVNefsmr5a infected mice relative to HIVwt infected controls. These findings demonstrate the importance of Nef and the SMR motif in HIV pathogenesis and suggest a potential role for exNef in HIV-driven immune modulation.

Nef-M1, a peptide antagonist of CXCR4, inhibits tumor angiogenesis and epithelial­to­mesenchymal transition in colon and breast cancers.

The Nef-M1 peptide competes effectively with the natural ligand of CXC chemokine receptor 4 (CXCR4), stromal cell-derived factor 1-alpha, to induce apoptosis and inhibit growth in colon cancer (CRC) and breast cancer (BC). Its role in tumor angiogenesis, and epithelial-to-mesenchymal transition (EMT) regulation, key steps involved in tumor growth and metastasis, are unknown. We evaluated the angioinhibitory effect of Nef-M1 peptide and examined its role in the inhibition of EMT in these cancers. Colon (HT29) and breast (MDA-MB231) cancer cells expressing CXCR4 were studied in vitro and in xenograft tumors propagated in severe combined immunodeficient mice. The mice were treated intraperitoneally with Nef-M1 or scrambled amino acid sequence of Nef-M1 (sNef-M1) peptide, a negative control, starting at the time of tumor implantation. Sections from tumors were evaluated for tumor angiogenesis, as measured by microvessel density (MVD) based on immunostaining of endothelial markers. In vitro tumor angiogenesis was assessed by treating human umbilical vein endothelial cells with conditioned media from the tumor cell lines. A BC cell line (MDA-MB 468) which does not express CXCR4 was used to study the actions of Nef-M1 peptide. Western blot and immunofluorescence analyses assessed the effect of Nef-M1 on tumor angiogenesis and EMT in both tumors and cancer cells. Metastatic lesions of CRC and BC expressed more CXCR4 than primary lesions. It was also found that tumors from mice treated with sNef-M1 had well established vascularity, while Nef-M1 treated tumors had very poor vascularization. Indeed, the mean MVD was lower in tumors from Nef-M1 treated mice than in sNef-M1 treated tumors. Nef-M1 treated tumor has poor morphology and loss of endothelial integrity. Although conditioned medium from CRC or BC cells supported HUVEC tube formation, the conditioned medium from Nef-M1 treated CRC or BC cells did not support tube formation. Western blot analyses revealed that Nef-M1 effectively suppressed the expression of VEGF-A in CRC and BC cells and tumors. This suggests that Nef-M1 treated CRC and BC cells are more consistent with E-cadherin signature, and thus appears more epithelial in nature. Our data indicate that Nef-M1 peptide inhibits tumor angiogenesis and the oncogenic EMT process. Targeting the chemokine receptor, CXCR4, mediated pathways using Nef-M1 may prove to be a novel therapeutic approach for CRC and BC.