C-C Chemokine Receptor Type 5 Expression Research Articles

High Expression of C-C Chemokine Receptor Type 5 in Urothelial Carcinoma When Compared to Other Common Malignancies Based on a Novel, Quantitative Immunohistochemistry Assay and Scoring System From FFPE Tissue not Requiring Antigen Retrieval

Purpose: To compare C-C chemokine receptor type 5 (CCR5) expression in urothelial carcinoma tissue to other common malignancies using a specific CCR5 inhibitor-based assay. Methods: We used an immunohistochemistry (IHC) assay to compare the expression of CCR5 in urothelial carcinoma to different types of common cancer. The IHC assay was based on labeled PRO140 (PRO140 CCR5 Hu IgG4a), a CCR5 inhibitor with potential therapeutic uses. The expression levels were compared using Percent Scores and H-Score methods. Results: Quantification of CCR5 expression utilized a panel of 63 evaluable samples from 9 cancer indications as follows: 6 UC, 7 non-small cell lung (NSCL) adenocarcinoma, 9 NSCL squamous cell carcinoma, 5 triple negative breast cancer, 5 breast cancer, 7 pancreatic cancer, 9 colorectal cancer, 7 head, and neck cancer, and 8 sarcoma. Overall, most of the 63 cases evaluated tended to have either H-Scores >175 (highly reactive) (24/63) or <100 (low or non-reactive) (32/63). Urothelial cancer had the highest expression (H score=183.3), while sarcoma exhibited the most moderate expression of CCR5 (H score =23.6). Conclusions: We show, for the first time, a high expression of CCR5 in the tissue of urothelial carcinoma using an assay based on a safe and effective receptor inhibitor. Our findings may have therapeutic implications if validated in more extensive studies.

CCR5 as a Prognostic Factor in Lower-Grade Glioma is Involved in the Remodeling of the Tumor Microenvironment.

Background: Lower-grade gliomas (LGGs) carry a high risk of malignant transformation, leading to severe neurologic deterioration and ultimately, death. The tumor microenvironment (TME) plays an essential role in tumor maintenance, progression, and immunotherapy resistance. Therefore, the LGG TME deserves comprehensive exploration for a novel therapeutic target. Methods: The ESTIMATE algorithm was used to estimate infiltrating stromal and immune cells of LGG patients obtained from the Cancer Genome Atlas (TCGA) database. Kaplan–Meier analysis was performed to classify survival differences. TME-related differentially expressed genes were identified between the low- and high-immune/stromal groups. Hub genes were screened by constructing protein–protein interaction networks and performing the Cox regression analysis. Differential analysis, survival analysis, gene set enrichment analysis, and clinical relevance analysis specific to hub genes were evaluated by using the TCGA and the Chinese Glioma Genome Atlas datasets, and the results were validated by qRT-PCR, Western blotting, and immunohistochemistry in tissues from LGG patients. Results: The immune and stromal components in TME were negatively related to patient prognosis. Differentially expressed genes sharing immune score and stromal score were mainly involved in the immune response. C-C chemokine receptor type 5 (CCR5), as only a hub gene, was significantly higher in LGG patients than normal patients and negatively correlated with the prognosis of patients. High-expression CCR5 was positively related to immune-related and tumor progression pathways. CCR5 protein expression was higher in LGG with isocitrate dehydrogenase wildtype. Validated results showed that CCR5 was upregulated in LGG tissues at mRNA and protein levels and could affect immune cell infiltration. These results suggested that CCR5 was a potential indicator for the status of TME. Conclusion: Glioma cells remodel the immune microenvironment through the high expression of CCR5 and lead to a poor prognosis in patients with LGG. The inhibition of CCR5 may contribute to the efficacy of LGG immunotherapy.

CCR5 closes the temporal window for memory linking.

Real-world memories are formed in a particular context and are often not acquired or recalled in isolation1-5. Time is a key variable in the organization of memories, as events that are experienced close in time are more likely to be meaningfully associated, whereas those that are experienced with a longer interval are not1-4. How the brain segregates events that are temporally distinct is unclear. Here we show that a delayed (12-24 h) increase in the expression of C-C chemokine receptor type 5 (CCR5)-an immune receptor that is well known as a co-receptor for HIV infection6,7-after the formation of a contextual memory determines the duration of the temporal window for associating or linking that memory with subsequent memories. This delayed expression of CCR5 in mouse dorsal CA1 neurons results in a decrease in neuronal excitability, which in turn negatively regulates neuronal memory allocation, thus reducing the overlap between dorsal CA1 memory ensembles. Lowering this overlap affects the ability of one memory to trigger the recall of the other, and therefore closes the temporal window for memory linking. Our findings also show that an age-related increase in the neuronal expression of CCR5 and its ligand CCL5 leads to impairments in memory linking in aged mice, which could be reversed with a Ccr5 knockout and a drug approved by the US Food and Drug Administration (FDA) that inhibits this receptor, a result with clinical implications. Altogether, the findings reported here provide insights into the molecular and cellular mechanisms that shape the temporal window for memory linking.

Discovery of Novel CCR5 Ligands as Anticolorectal Cancer Agents by Sequential Virtual Screening.

C-C chemokine receptor type 5 (CCR5) is a member of the G protein-coupled receptor. CCR5 and its interaction with chemokine ligands have been crucial for understanding and tackling human immunodeficiency virus (HIV)-1 entry into target cells. In recent years, the change in CCR5 expression has been related to the progression of different cancer types. Patients treated with the CCR5 ligand, maraviroc (MVC), showed a deceleration in tumor development especially for metastatic colorectal cancer. Based on the crystal structure of CCR5, we herein describe a multistage virtual screening protocol including pharmacophore screening, molecular docking, and protein–ligand interaction fingerprint (PLIF) postdocking filtration for discovery of novel CCR5 ligands. The applied virtual screening protocol led to the identification of four hits with binding modes showing access to the major and minor pockets of the MVC binding site. Compounds 2–4 showed a decrease in cellular proliferation upon testing on the metastatic colorectal cancer cell line, SW620, displaying 12, 16, and 4 times higher potency compared to MVC, respectively. Compound 3 induced apoptosis by arresting cells in the G0/G1 phase of the cell cycle similar to MVC. Further in vitro assays showed compound 3 drastically decreasing the CCR5 expression and cellular migration 48 h post treatment, indicating its ability to inhibit metastatic activity in SW620 cells. The discovered hits represent potential leads for the development of novel classes of anticolorectal cancer agents targeting CCR5.

The impact of intradialytic exercise on immune cells expressing CCR5+ in patients with chronic kidney disease: A cross-over trial.

The C-C chemokine receptor type 5 (CCR5) plays a role in the immunopathogenesis of chronic kidney disease (CKD). Exercise has anti-inflammatory properties that may contribute to the rehabilitation of CKD patients. To date, the impact of the intradialytic exercise on CCR5 expression in monocytes and lymphocytes of CKD patients is unknown. We aimed to evaluate the effects of an acute intradialytic moderate-intensity exercise on CD4+CCR5+ T-cells and CD14+CCR5+ monocytes of elderly individuals with Chronic Kidney Disease (CKD). Eight CKD elderly patients performed a single bout of 20 min intradialytic exercise and a control hemodialysis (HD) session. Blood samples were collected at baseline, during and immediately after the trials. HD therapy increased the peripheral frequency of CD4+CCR5+ T-cells. The systemic CCL5 levels and the peripheral CD14+CCR5+ proportions increased during and after HD therapy. No significant alterations in CD4+CCR5+ and CD14+CCR5+ proportions or CCL5 levels were identified in CKD patients during and after intradialytic exercise. A negative correlation between the peripheral frequency of CD14+CCR5+ and the creatinine levels was identified in the intradialytic exercise session. A single moderate-intensity intradialytic exercise imposes an immunomodulatory impact in CKD elderly patients, preventing an excessive inflammatory response induced by hemodialysis.

The role of CCR5 polymorphism in colorectal cancer and liver metastasis in the Tunisian population

Chemokines and their receptors are involved in cancer initiation and progression, including colorectal cancer (CRC) and liver metastasis formation. Our aim was to elucidate C-C chemokine receptor type 5 (CCR5) gene polymorphism (CCR5?32) impact on CRC and colorectal cancer liver metastases (CRLM) occurrence risk. We analyzed the CCR5 gene mutational status in 108 primary CRC cases, 35 CRLM and 248 healthy individuals, and evaluated CCR5 expression in healthy tissue and tumors. Rare allele ??32? was more frequent in controls (7.2% vs 2.8% in CRC). All 35 metastases had wild-type CCR5. Our analysis showed that CCR5 wild type has a significant risk of 2.73-fold (95% CI=1.22-7.31) to cause CRC while ?32 reduced the risks 0.36-fold (95% CI=0.13-0.82). For CRC, CCR5 correlated with left-sided tumors and liver metastases (P=0.040 and P= 0.039 respectively). As for CRLM, no correlation was found. Immunohistochemical profile analysis of CCR5 revealed a significant association with the male gender (P=0.049) and non-mucinous carcinomas (P< 0.001) in primary CRC. CCR5 expression revealed an association with the degree of tumor differentiation for both CRC and CRLM (P < 0.001). CCR5?32 might be a protective factor against CRC development and dissemination.

HIV Protease Inhibitor Ritonavir Impairs Endothelial Function Via Reduction in Adipose Mass and Endothelial Leptin Receptor-Dependent Increases in NADPH Oxidase 1 (Nox1), C-C Chemokine Receptor Type 5 (CCR5), and Inflammation.

BackgroundCardiovascular disease is currently the leading cause of death in patients with human immunodeficiency virus on combination antiretroviral therapy. Although the use of the protease inhibitor ritonavir has been associated with increased prevalence of cardiovascular disease, the underlying mechanisms remain ill‐defined. Herein, we tested the hypothesis that ritonavir‐mediated lipoatrophy causes endothelial dysfunction via reducing endothelial leptin signaling.Methods and ResultsLong‐term (4 weeks) but not short‐term (3 days) treatment with ritonavir reduced body weight, fat mass, and leptin levels and induced endothelial dysfunction in mice. Moreover, ritonavir increased vascular NADPH oxidase 1, aortic H2O2 levels as well as interleukin‐1β, GATA3 (GATA binding protein 3), the macrophage marker (F4/80), and C‐C chemokine receptor type 5 (CCR5) expression. Reactive oxygen species scavenging with tempol restored endothelial function, and both NADPH oxidase 1 and CCR5 deletion in mice protected from ritonavir‐mediated endothelial dysfunction and vascular inflammation. Remarkably, leptin infusion markedly improved endothelial function and significantly reduced vascular NADPH oxidase 1, interleukin‐1β, GATA3, F4/80, and CCR5 levels in ritonavir‐treated animals. Selective deficiency in endothelial leptin receptor abolished the protective effects of leptin infusion on endothelial function. Conversely, selective increases in endothelial leptin signaling with protein tyrosine phosphatase deletion blunted ritonavir‐induced endothelial dysfunction.ConclusionsAll together, these data indicate that ritonavir‐associated endothelial dysfunction is a direct consequence of a reduction in adiposity and leptin secretion, which decreases endothelial leptin signaling and leads to a NADPH oxidase 1–induced, CCR5‐mediated reduction in NO bioavailability. These latter data also introduce leptin deficiency as an additional contributor to cardiovascular disease and leptin as a negative regulator of CCR5 expression, which may provide beneficial avenues for limiting human immunodeficiency virus infection.

Antineuropathic pain actions of Wu-tou decoction resulted from the increase of neurotrophic factor and decrease of CCR5 expression in primary rat glial cells

Wu-tou decoction (WTD), a classic Traditional Chinese medicine formula, has been extensively used in the treatment of neuropathic pain (NP) such as chronic inflammatory pain, trigeminal neuralgia, and cancer-induced pain. Our previous studies have shown that the severity of mechanical allodynia and thermo hypersensitivity in NP rats are reduced by WTD, of which analgesic candidates are paeoniflorin (Pae) and liquiritin (Liq). The aim of this study was to clarify the molecular mechanisms of WTD, Pae and Liq against NP based on the primary rat glial cells in vitro. The gene expression levels of neurotrophic factors such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and Artemin and C-C chemokine receptor type 5 (CCR5) were augmented by inflammatory cytokines, while chemokines increased only CCR5 gene expression. The constitutive and cytokine-augmented neurotrophic factor gene expression was enhanced by WTD, Pae, and Liq through PI3K- and PKA-dependent pathways in rat glial cells, leading to the increase of NGF and BDNF production. Furthermore, the CCR5 gene expression under basal and chemokine-treated conditions was suppressed by these reagents, in which signal pathway(s) was independent on the activation of PI3K and PKA. Moreover, there was no cytotoxicity in the WTD, Pae, and Liq treatments in glial cells. Thus, these results provide a novel evidence that WTD may exert the anti-NP actions by predominantly increasing the production of neurotrophic factors through PI3K- and PKA-signaling pathways in rat glial cells. Furthermore, Pae and Liq may play as analgesic candidates in WTD-mediated NP management.

CCR5-mediated Recruitment of NK Cells to the Kidney Is a Critical Step for Host Defense to Systemic Candida albicans Infection.

C-C chemokine receptor type 5 (CCR5) regulates the trafficking of various immune cells to sites of infection. In this study, we showed that expression of CCR5 and its ligands was rapidly increased in the kidney after systemic Candida albicans infection, and infected CCR5−/− mice exhibited increased mortality and morbidity, indicating that CCR5 contributes to an effective defense mechanism against systemic C. albicans infection. The susceptibility of CCR5−/− mice to C. albicans infection was due to impaired fungal clearance, which in turn resulted in exacerbated renal inflammation and damage. CCR5-mediated recruitment of NK cells to the kidney in response to C. albicans infection was necessary for the anti-microbial activity of neutrophils, the main fungicidal effector cells. Mechanistically, C. albicans induced expression of IL-23 by CD11c+ dendritic cells (DCs). IL-23 in turn augmented the fungicidal activity of neutrophils through GM-CSF production by NK cells. As GM-CSF potentiated production of IL-23 in response to C. albicans, a positive feedback loop formed between NK cells and DCs seemed to function as an amplification point for host defense. Taken together, our results suggest that CCR5-mediated recruitment of NK cells to the site of fungal infection is an important step that underlies innate resistance to systemic C. albicans infection.

Recent Advances Targeting CCR5 for Cancer and Its Role in Immuno-Oncology.

Experiments of nature have revealed the peculiar importance of the G-protein-coupled receptor, C-C chemokine receptor type 5 (CCR5), in human disease since ancient times. The resurgence of interest in heterotypic signals in the onset and progression of tumorigenesis has led to the current focus on CCR5 as an exciting new therapeutic target for metastatic cancer with clinical trials now targeting breast and colon cancer. The eutopic expression of CCR5 activates calcium signaling and thereby augments regulatory T cell (Treg) differentiation and migration to sites of inflammation. The misexpression of CCR5 in epithelial cells, induced upon oncogenic transformation, hijacks this migratory phenotype. CCR5 reexpression augments resistance to DNA-damaging agents and is sufficient to induce cancer metastasis and "stemness". Recent studies suggest important cross-talk between CCR5 signaling and immune checkpoint function. Because CCR5 on Tregs serves as the coreceptor for human immunodeficiency virus (HIV) entry, CCR5-targeted therapeutics used in HIV, [small molecules (maraviroc and vicriviroc) and a humanized mAb (leronlimab)], are now being repositioned in clinical trials as cancer therapeutics. As CCR5 is expressed on a broad array of tumors, the opportunity for therapeutic repositioning and the rationale for combination therapy approaches are reviewed herein.

Bone marrow-derived mesenchymal stem cells promote colorectal cancer progression via CCR5

Mesenchymal stem cells (MSCs) are recruited from BM to the stroma of developing tumors, where they serve as critical components of the tumor microenvironment by secreting growth factors, cytokines, and chemokines. The role of MSCs in colorectal cancer (CRC) progression was controversial. In this study, we found that C-C chemokine receptor type 5 (CCR5) ligands (i.e., C-C motif chemokine ligand 3 (CCL3), CCL4, and CCL5) were highly produced from MSCs using a chemokine array screening with conditioned media from the cultured human MSCs. A relatively strong CCR5 expression could be detected within the cytoplasm of several CRC cell lines. Regarding the effect of MSC, we found that the xenografts in which CCR5-overexpressing HCT116 cells were inoculated into immunocompromised mice were highly promoted in vivo by a mixture with MSCs. Notably, the CCR5 inhibitor, maraviroc, significantly abolished the MSC-induced tumor growth in vivo. In human clinical specimens (n = 89), 20 cases (29%) were high for CCR5, whereas 69 cases (71%) were low. Statistical analyses indicated that CCR5 expression in primary CRC was associated with CRC patients’ prognosis. Especially, stage III/IV patients with CCR5-high CRCs exhibited a significantly poorer prognosis than those with CCR5-low CRCs. Furthermore, we investigated the effects of preoperative serum CCR5 ligands on patients’ prognosis (n = 114), and found that CRC patients with high serum levels of CCL3 and CCL4 exhibited a poorer prognosis compared to those with low levels of CCL3 and CCL4, while there was no association between CCL5 and prognosis. These results suggest that the inhibition of MSC–CRC interaction by a CCR5 inhibitor could provide the possibility of a novel therapeutic strategy for CRC, and that serum levels of CCL3 and CCL4 could be predictive biomarkers for the prognosis of CRC patients.

Induction of Antihuman C-C Chemokine Receptor Type 5 Antibodies by a Bovine Herpesvirus Type-4 Based Vector.

Bovine herpesvirus 4 (BoHV-4) is a promising vector for the delivery and intracellular expression of recombinant antigens and can thus be considered as a new prototype vaccine formulation system. An interesting, and actively pursued, antigen in the context of human immunodeficiency virus (HIV) infection prophylaxis (and therapy) is the C–C chemokine receptor type 5 (CCR5) co-receptor, whose blockage by specific antibodies has been shown to inhibit both viral entry and cell-to-cell transmission of the virus. Building on our previous work on the BoHV-4 vector system, we have engineered and tested a replication-competent derivative of BoHV-4 (BoHV-4-CMV-hCCR5ΔTK) bearing a human CCR5 (hCCR5) expression cassette. We show here that CCR5 is indeed expressed at high levels in multiple types of BoHV-4-CMV-hCCR5ΔTK-infected cells. More importantly, two intravenous inoculations of CCR5-expressing BoHV-4 virions into rabbits led to the production of anti-CCR5 antibodies capable of reacting with the CCR5 receptor exposed on the surface of HEK293T cells through specific recognition of the amino-terminal region (aa 14–34) of the protein. Given the growing interest for anti-CCR5 immunization as an HIV control strategy and the many advantages of virus-based immunogen formulations (especially for poorly immunogenic or self-antigens), the results reported in this study provide preliminary validation of BoHV-4 as a safe viral vector suitable for CCR5 vaccination.

Host and Viral Factors in HIV-Mediated Bystander Apoptosis.

Human immunodeficiency virus (HIV) infections lead to a progressive loss of CD4 T cells primarily via the process of apoptosis. With a limited number of infected cells and vastly disproportionate apoptosis in HIV infected patients, it is believed that apoptosis of uninfected bystander cells plays a significant role in this process. Disease progression in HIV infected individuals is highly variable suggesting that both host and viral factors may influence HIV mediated apoptosis. Amongst the viral factors, the role of Envelope (Env) glycoprotein in bystander apoptosis is well documented. Recent evidence on the variability in apoptosis induction by primary patient derived Envs underscores the role of Env glycoprotein in HIV disease. Amongst the host factors, the role of C-C Chemokine Receptor type 5 (CCR5), a coreceptor for HIV Env, is also becoming increasingly evident. Polymorphisms in the CCR5 gene and promoter affect CCR5 cell surface expression and correlate with both apoptosis and CD4 loss. Finally, chronic immune activation in HIV infections induces multiple defects in the immune system and has recently been shown to accelerate HIV Env mediated CD4 apoptosis. Consequently, those factors that affect CCR5 expression and/or immune activation in turn indirectly regulate HIV mediated apoptosis making this phenomenon both complex and multifactorial. This review explores the complex role of various host and viral factors in determining HIV mediated bystander apoptosis.

CCR5 Is Involved in Interruption of Pregnancy in Mice Infected with Toxoplasma gondii during Early Pregnancy.

Toxoplasmosis can cause abortion in pregnant humans and other animals; however, the mechanism of abortion remains unknown. C-C chemokine receptor type 5 (CCR5) is essential for host defense against Toxoplasma gondii infection. To investigate the relationship between CCR5 and abortion in toxoplasmosis, we inoculated wild-type and CCR5-deficient (CCR5-/-) mice with T. gondii tachyzoites intraperitoneally on day 3 of pregnancy (embryonic day 3 [E3]). The pregnancy rate decreased as pregnancy progressed in infected wild-type mice. Histopathologically, no inflammatory lesions were observed in the fetoplacental tissues. Although wild-type mice showed a higher parasite burden at the implantation sites than did CCR5-/- mice at E6 (3 days postinfection [dpi]), T. gondii antigen was detected only in the uterine tissue and not in the fetoplacental tissues. At E8 (5 dpi), the embryos in infected wild-type mice showed poor development compared with those of infected CCR5-/- mice, and apoptosis was observed in poorly developed embryos. Compared to uninfected mice, infected wild-type mice showed increased CCR5 expression at the implantation site at E6 and E8. Furthermore, analyses of mRNA expression in the uterus of nonpregnant and pregnant mice suggested that a lack of the CCR5 gene and the downregulation of tumor necrosis factor alpha (TNF-α) and CCL3 expression at E6 (3 dpi) are important factors for the maintenance of pregnancy following T. gondii infection. These results suggested that CCR5 signaling is involved in embryo loss in T. gondii infection during early pregnancy and that apoptosis is associated with embryo loss rather than direct damage to the fetoplacental tissues.

C-C Chemokine Receptor Type 5 (CCR5)-Mediated Docking of Transferred Tregs Protects Against Early Blood-Brain Barrier Disruption After Stroke.

BackgroundDespite recent evidence demonstrating a potent protective effect of adoptively transferred regulatory T cells (Tregs) in ischemic stroke, the mechanism for Treg mobilization and activation in the ischemic brain is, remarkably, unknown. This study determines the role of C‐C chemokine receptor type 5 (CCR5) in mediating the docking and activation of transferred Tregs in their protection of early blood‐brain barrier disruption after stroke.Methods and ResultsAdoptive transfer of CCR5−/− Tregs failed to reduce brain infarct or neurological deficits, indicating an indispensable role of CCR5 in Treg‐afforded protection against cerebral ischemia. Two‐photon live imaging demonstrated that CCR5 was critical for Treg docking at the injured vessel wall, where they interact with blood‐borne neutrophils/macrophages after cerebral ischemic injury. CCR5 deficiency on donor Tregs deprived of their early protection against blood‐brain barrier damage. Using flow cytometry, real‐time polymerase chain reaction, and immunostaining, we confirmed that the expression of CCL5, a CCR5 ligand, was significantly elevated on the injured endothelium after cerebral ischemia, accompanied by CCR5 upregulation on circulating Tregs. In a Treg‐endothelial cell coculture, CCR5 expression was induced on Tregs on their exposure to ischemia‐injured endothelial cells. Furthermore, CCR5 induction on Tregs enhanced expression of the inhibitory molecule programmed death ligand 1, which in turn inhibited neutrophil‐derived matrix metallopeptidase 9.ConclusionsThese results suggest that CCR5 is a critical molecule for Treg‐mediated blood‐brain barrier protection and a potential target to optimize Treg therapy for stroke.

Comparison of CCR5 Expression on T Lymphocytes between the Bone Marrow and Peripheral Blood Grafts after Mobilization

To compare the expression of C-C chemokine receptor type 5 (CCR5) on T cells between bone marrow grafts (G-BM) and peripheral blood grafts (G-PB) nobilized by recombinant human granulocyte colony-stimulating factor (rhG-CSF), and to analyze the correlation of CCR5+ T lymphocyte expression in the grafts with the occurrence of acute GVHD. Forty-six healthy donor and their recipient pairs of related allogeneic hematopoietic stem cell transplantation (allo-HSCT) were enrolled in this study. All the recipients were received the infusion of G-BM and G-PB. The relative proportion and quantity of CCR5+ T cell subset in G-BM and G-PB were detected and compared. Then the correlation of the quantity of infused CCR5+ T cells with the occurrence of acute GVHD was analyzed. After mobilization, the proportions of CD4+ CCR5+ and CD8+ CCR5+ T cells occupying T cells in G-PB were both lower than those in G-BM. However, the absolute counts in G-PB were 15-25 times more than those in the bone marrow. And the absolute counts could not predict the occurrence of acute GVHD after transplantation (P>0.05). The difference of CCR5+ subsets between G-PB and G-BM may partially explain that grafts from different sources have different immunologic characteristics. Besides, the quantity of CCR5+ T cells in the grafts are not related with the occurrence of acute GVHD. However, the relative proportion of CCR5+ T cell subset in the grafts may be predictive of acute GVHD.

580. In Vivo Analyses of Aptamers and siRNA Therapeutics Against HIV-1 in Humanized Mouse Model

Nucleic acid-based therapeutics are quickly emerging as a strong alternative or co-therapy to the chemical antiviral agents currently used to treat HIV-1/AIDS. Systemic delivery of siRNAs typically requires a carrier to prevent renal clearance, minimize the inflammatory response, and enhance the cellular uptake. This is particularly true for siRNAs against HIV, which require systemic intravenous injection to access the cells and tissues that are sites of HIV infection. Human CCR5 (C-C chemokine receptor type 5), a protein expressed by T-cells and macrophages, is an important coreceptor for macrophage-tropic virus, including HIV-1 R5 isolates. Variations in CCR5 are associated with resistance or susceptibility to HIV-1. As an essential factor for viral entry, CCR5 has represented an attractive cellular target for the treatment of HIV-1. We therefore sought to develop CCR5 directed RNA aptamers to target HIV-1 susceptible cells, and specifically regulate both gene silencing of HIV-1 and the blockage of CCR5 which is required for HIV-1 to enter cells. We have successfully identified several 2’- Fluoropyrimidine modified RNA aptamers directed to human CCR5 and G-3 aptamer has turned out to be the potential candidate for further studies.In order to assess the potential of the aptamer in HIV prophylaxis we transplanted neonatal NOD/SCID/gc null (NSG) mice with human CD34+ HSC cells (1E6 cells/pup). The tissues and peripheral blood was processed and analyzed for expression of HIV-1 permissible cells and cell surface expression of CCR5. An ex vivo HIV-1 protection assay was performed and the culture supernatants were collected for HIV-1 p24 ELISA. The sorted CD4+ T-cells were pre-treated with G-3 aptamer and subsequently were challenged ex vivo by R5 stains (HIV-1 JR-FL or Bal virus) or X4 strains (HIV-1 NL4-3 virus). HIV-1 JR-FL or Bal replication was inhibited in human CD4+ T-cells from humanized mice which showed about 50≈250 nM of IC50. The data indicate that the selected CCR5 aptamer (G-3) inhibits HIV-1 p24 production and provided protection from HIV infection by R5 viruses.In another approach, we have also developed two different RNA aptamers that can be multiplexed with anti-latency and/or cytotoxic siRNAs. One binds with high affinity and specificity to the HIV gp120 protein expressed on the surface of HIV-infected cells, while the other binds to the CD7 receptor on latent CD4+ T cells and macrophages. We are in the process of validating the efficacy of these aptamers in reactivating and eradicating latent infections in the humanized NSG model of HIV-1 infection.

CCR5 small interfering RNA ameliorated joint inflammation in rats with adjuvant-induced arthritis

Rheumatoid arthritis (RA) is a systemic inflammatory disease. C-C chemokine receptor type 5 (CCR5) is found in inflamed synovium of RA patients and is necessary for formation of RA. We aimed to check whether delivery of CCR5-specific small interfering RNA (siRNA) via electroporation suppresses local inflammation in arthritis rats. Vectors encoding siRNA that target CCR5 or negative control siRNA were constructed for gene silencing and the silencing effects of suppressing CCR5 expression in synovium examined by western blot. The vector with strongest effect was delivered into the knee joint of adjuvant-induced arthritis (AIA) rats by the in vivo electroporation method 7, 10, 13, and 16 days after immunization with Complete Freund's adjuvant. During an observation of 28 days, behavior, paw swelling, arthritis and histopathologic scoring were estimated. The expression level of CCR5 in synovium was evaluated by western blot and real-time PCR. Anti-CCR5 D1 siRNA was effectively inhibited CCR5 expression in vitro. Moreover, delivery of the siRNA into inflammatory joint also suppressed the expression of CCR5 in vivo and markedly suppressed paw swelling and inflammation. Local electroporation of anti-CCR5 siRNA into the left inflamed joints could achieve the silencing of CCR5 gene and alleviate local inflammation just in the knee joint injected with siRNA other than the opposite joint. Inhibition of CCR5 expression may provide a potential for treatment of RA.

DT-13, a saponin of dwarf lilyturf tuber, exhibits anti-cancer activity by down-regulating C-C chemokine receptor type 5 and vascular endothelial growth factor in MDA-MB-435 cells

AimTo investigate the anticancer activity of DT-13 under normoxia and determine the underlying mechanisms of action. MethodsMDA-MB-435 cell proliferation, migration, and adhesion were performed to assess the anticancer activity of DT-13, a saponin from Ophiopogon japonicus, in vitro. In addition, the effects of DT-13 on tumor growth and metastasis in vivo were evaluated by orthotopic implantation of MDA-MB-435 cells into nude mice; mRNA levels of vascular endothelial growth factor (VEGF), C-C chemokine receptor type 5 (CCR5) and hypoxia-inducible factor 1α (HIF-1α) were evaluated by real-time quantitative PCR; and CCR5 protein levels were detected by Western blot assay. ResultsAt 0.01 to 1 μmol·L−1, DT-13 inhibited MDA-MB-435 cell proliferation, migration, and adhesion significantly in vitro. DT-13 reduced VEGF and CCR5 mRNAs, and decreased CCR5 protein expression by down-regulating HIF-1α. In addition, DT-13 inhibited MDA-MB-435 cell lung metastasis, and restricted tumor growth slightly in vivo. ConclusionDT-13 inhibited MDA-MB-435 cell proliferation, adhesion, and migration in vitro, and lung metastasis in vivo by reducing VEGF, CCR5, and HIF-1α expression.

CCR5‐Delta32: implications in SLE development

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease with strong genetic and environmental components. Previous studies have shown increased levels of several chemokines in active SLE. C-C chemokine receptor type 5 (CCR5) is involved in the recruitment of inflammatory cells into tissues, and mechanisms modulating CCR5 expression and function may interfere in SLE development, influencing the clinical course of the disease. The aim of this study was to evaluate the possible association between the CCR5∆32 base-pair deletion polymorphism and SLE disease in a group of Portuguese patients. A total of 219 patients with SLE and 205 healthy individuals were studied. The frequency of CCR5/∆32 heterozygotes was lower in patients with SLE than in controls (8% vs. 15% OR = 0.5162; P = 0.0319), suggesting a protective association between CCR5∆32 allele and SLE. These results highlight the protective role of Th1 cells that express CCR5 in SLE pathogenesis.