Curcumae Radix Extract Decreases Mammary Tumor-Derived Lung Metastasis via Suppression of C-C Chemokine Receptor Type 7 Expression.

Curcumae radix is the dry root of Curcuma longa L. (turmeric) that can be used either as a spice or traditional medicine. The aim of this study was to investigate the survival benefits and the anti-metastatic activity of curcumae radix extract (CRE) in MCF7 cells and in MMTV-PyMT transgenic mice-a mouse model of breast cancer metastasis. In vitro wound scratch assay revealed that CRE treatment inhibited cell motility and cell migration in a dose-dependent manner. To investigate the effect of CRE in breast cancer metastasis, MMTV-PyMT transgenic female virgin mice were used and randomly divided into two groups. For survival curve analysis, CRE was administered in a dose of 50 mg/kg to 8⁻20-week-old mice. Interestingly, CRE treatment significantly increased the median and prolonged survival of MMTV-PyMT mice. Furthermore, CRE treatment decreased tumor burden and inhibited cell proliferation in primary breast tumor, and also suppressed mammary tumor-derived lung metastasis. The size of the lung metastases substantially decreased in the CRE-treated group compared with the ones in the control group. Curcumae radix extract showed anti-metastatic activity through regulating the expression of metastasis markers including C-C Chemokine Receptor Type 7, Matrix Metalloproteinase 9 and the proto-oncogenes c-fos and c-jun. We demonstrated that these metastatic regulators were decreased when CCR7 expression was suppressed in MCF7 cells transfected with CCR7 siRNA. The results of this study show that curcumae radix exerts antitumor and anti-metastatic activities, and we suggest that curcumae radix might be a potential supplement for the treatment and prevention of breast cancer metastasis.

Ectopic expression of CC chemokine receptor 7 promotes prostate cancer cells metastasis via Notch1 signaling.

There currently exists no satisfactory treatment for patients with prostate cancer with local evolution and distant metastasis. Previous studies have confirmed the importance of CC chemokine receptor 7 (CCR7) in the invasion and metastasis of prostate cancer. And increasing evidence prove that Notch1 can play diametrically opposite roles in the development and progression of different tumors. To demonstrate the correlation between CCR7 and Notch1, PC-3 cells were transfected with pcDNA3.1-CCR7 or CCR7 si-RNA, respectively. Then Western blot analysis was used to detect the expressions of Notch1, ERK, P38, JNK, NF-κB, MMP-9, and epithelial-mesenchymal transition (EMT)-related proteins. Moreover, matrigel invasion assays were performed to assess the migratory and invasive activities of PC-3 cells. PcDNA3.1-CCR7 increased the expression of Notch1, phospho-MAPK, phospho-P65, MMP-9, N-cadherin, and Snail in PC-3 cells, but decreased the expression of E-cadherin. PcDNA3.1-CCR7 also promoted the migration and invasion of PC-3 cells. However, CCR7 si-RNA reversed the effect of pcDNA3.1-CCR7 in PC-3 cells. And MAPK and NF-κB pathway inhibitors were used to testify that activation of Notch1 induces EMT through MAPK and NF-κB pathway. All these results indicate that upregulation of Notch1 by CCR7 can accelerate the evolution of EMT and develop the invasion and metastasis in prostate cancer cells by activating MAPK and NF-κB signaling pathways in prostate cancer cells, which provides a new molecular evidence for targeted therapy in metastatic prostate cancer.

Airway Epithelial Cell-Derived Colony Stimulating Factor-1 Promotes Allergen Sensitization.

Airway epithelial cells (AECs) secrete innate immune cytokines that regulate adaptive immune effector cells. In allergen-sensitized humans and mice, the airway and alveolar microenvironment is enriched with colony stimulating factor-1 (CSF1) in response to allergen exposure. In this study we found that AEC-derived CSF1 had a critical role in the production of allergen reactive-IgE production. Furthermore, spatiotemporally secreted CSF1 regulated the recruitment of alveolar dendritic cells (DCs) and enhanced the migration of conventional DC2s (cDC2s) to the draining lymph node in an interferon regulatory factor 4 (IRF4)-dependent manner. CSF1 selectively upregulated the expression of the chemokine receptor CCR7 on the CSF1R+ cDC2, but not the cDC1, population in response to allergen stimuli. Our data describe the functional specification of CSF1-dependent DC subsets that link the innate and adaptive immune responses in T helper 2 (Th2) cell-mediated allergic lung inflammation.

Autoimmune Retinopathy: An Immunologic Cellular-Driven Disorder.

Autoimmune retinopathy (AIR) was often mistaken for retinitis pigmentosa (RP), due to an overlap of clinical findings, but increasingly has been recognized as a unique entity in the last decade. AIR has distinctive features: sudden onset of photopsias and scotomata in patients with no family history of RP, followed by visual field and central vision loss. Initially, retina exams are normal with no sign of pigment deposits or retinal degeneration. A family history of autoimmune diseases (all types) occurs in 60% of patients. One hallmark of AIR has been the presence of anti-retinal autoimmune antibodies (ARAs) in patients' sera, but patients can continue to have ARAs even when the disease has been quiescent for years. The accumulation of ARAs represents a breakdown of retinal immune tolerance with many different immunoreactive bands found at different reference weights in AIR patients. We began investigating cellular immunity using flow cytometry and found abnormal distributions (>2 StDev) of increased memory lymphocytes and NK cells and decreased regulatory B cell subsets in many AIR patients compared to normal controls. Culture of patient lymphocytes with small amounts (25 µg) of recoverin protein for 6 days led to significant elevations of interferon gamma (IFNγ) and in some cases tumor necrosis factor alpha (TNFα) production. We found the IFNγ/IL-10 ratio in response to recoverin was elevated in patients with more active disease (defined by visual field contraction between visits), but in some patients, there also appeared to be independent factors influencing severity, suggesting other autoimmune mechanisms were at play. These cellular immune parameters may provide improved markers for active AIR.

Urban particulate matter stimulation of human dendritic cells enhances priming of naive CD8 T lymphocytes.

Epidemiological studies have consistently shown associations between elevated concentrations of urban particulate matter (UPM) air pollution and exacerbations of asthma and chronic obstructive pulmonary disease, which are both associated with viral respiratory infections. The effects of UPM on dendritic cell (DC) -stimulated CD4 T lymphocytes have been investigated previously, but little work has focused on CD8 T-lymphocyte responses despite their importance in anti-viral immunity. To address this, we examined the effects of UPM on DC-stimulated naive CD8 T-cell responses. Expression of the maturation/activation markers CD83, CCR7, CD40 and MHC class I on human myeloid DCs (mDCs) was characterized by flow cytometry after stimulation with UPMin vitro in the presence/absence of granulocyte-macrophage colony-stimulating factor (GM-CSF). The capacity of these mDCs to stimulate naive CD8 T-lymphocyte responses in allogeneic co-culture was then assessed by measuring T-cell cytokine secretion using cytometric bead array, and proliferation and frequency of interferon-γ (IFN-γ)-producing T lymphocytes by flow cytometry. Treatment of mDCs with UPM increased expression of CD83 and CCR7, but not MHC class I. In allogeneic co-cultures, UPM treatment of mDCs enhanced CD8 T-cell proliferation and the frequency of IFN-γ+ cells. The secretion of tumour necrosis factor-α, interleukin-13, Granzyme A and Granzyme B were also increased. GM-CSF alone, and in concert with UPM, enhanced many of these T-cell functions. The PM-induced increase in Granzyme A was confirmed in a human experimental diesel exposure study. These data demonstrate that UPM treatment of mDCs enhances priming of naive CD8 T lymphocytes and increases production of pro-inflammatory cytokines. Such UPM-induced stimulation of CD8 cells may potentiate T-lymphocyte cytotoxic responses upon concurrent airway infection, increasing bystander damage to the airways.

Concurrent CCR7 Overexpression and RelB Knockdown in Immature Dendritic Cells Induces Immune Tolerance and Improves Skin-Graft Survival in a Murine Model.

BACKGROUND/AIMS: Skin transplantation aims to cover skin defects but often fails due to immune rejection of the transplantated tissue. Immature dendritic cells (imDCs) induce immune tolerance but have a low migration rate. After stimulation, imDCs transform into mature DCs, which activate immune rejection. Thus, inducing imDC to obtain a high migration counteracts development of immune tolerance. METHODS & RESULTS: We transfected imDCs with a recombinant adenovirus carrying the CCR7 gene (Ad-CCR7) and a small interfering RNA targeting RelB (RelB-siRNA) to concurrently overexpress CCR7 and downregulate RelB expression. Functionally, such cells showed a significantly enhanced migration rate in the chemotactic assay and decreased T-cell proliferation after lipopolysaccharide stimulation in mixed lymphocyte reactions. Cotransfected cells showed an increased ability to induce immune tolerance by upregulating T regulatory (Treg) cells and shifting the Th1/Th2 ratio. Cotransfection of Ad-CCR7 and RelB-siRNA endowed imDCs with resistance to apoptosis and cell death. CCR7 overexpression and RelB knockdown (KD) in imDCs improve skin-graft survival in a murine skin-transplantation model. CONCLUSION: Transfection with Ad-CCR7 and RelB KD in imDCs may be an effective approach inducing immune tolerance, thus being potentially valuable for inhibiting allograft rejection.

Adenovirus-Mediated CCR7 and BTLA Overexpression Enhances Immune Tolerance and Migration in Immature Dendritic Cells.

Our previous report revealed that immature dendritic cells (imDCs) with adenovirus-mediated CCR7 overexpression acquired an enhanced migratory ability but also exhibited the lower immune tolerance observed in more mature cells. In the present study, we aimed to investigate whether BTLA overexpression was sufficient to preserve immune tolerance in imDCs with exogenous CCR7 overexpression. Scanning electron microscopy and surface antigens analysis revealed that BTLA overexpression suppressed DC maturation, an effect further potentiated in CCR7 and BTLA cooverexpressing cells. Correspondingly, in vitro chemotaxis assays and mixed lymphocyte reactions demonstrated increased migratory potential and immune tolerance in CCR7 and BTLA coexpressing cells. Furthermore, CCR7 and BTLA cooverexpressed imDCs suppressed IFN-γ and IL-17 expression and promoted IL-4 and TGF-beta expression of lymphocyte, indicating an increase of T helper 2 (Th2) regulatory T cell (Treg). Thus, these data indicate that CCR7 and BTLA cooverexpression imparts an intermediate immune phenotype in imDCs when compared to that in CCR7- or BTLA-expressing counterparts that show a more immunocompetent or immunotolerant phenotype, respectively. All these results indicated that adenovirus-mediated CCR7 and BTLA overexpression could enhance immune tolerance and migration of imDCs. Our study provides a basis for further studies on imDCs in immune tolerance, with the goal of developing effective cellular immunotherapies for transplant recipients.

miR-199a-5p suppresses human bladder cancer cell metastasis by targeting CCR7.

BACKGROUND: C-C chemokine receptor type 7 (CCR7) overexpression correlated with lymphatic metastasis and poor prognosis is a major obstacle to bladder cancer treatment. Recent studies have revealed that miR-199a-5p was significantly abnormal expressed in several solid tumors and functioned as oncogene or tumor suppressor. This study was aimed to further investigate the effects of miR-199a-5p on the cell metastasis mediated by CCR7 in bladder cancer. METHODS: Quantitative Real Time PCR (qRT-PCR) was firstly performed to identified the expression of miR-199a-5p and CCR7 in human bladder cancer samples and cell lines. Following that, the effects of miR-199a-5p on cell migratory and invasive activities were assessed by wound healing and Matrigel invasion assays, respectively. Finally, luciferase reporter assay and western blot were employed to investigate whether CCR7 could directly interact with miR-199a-5p. RESULTS: miR-199a-5p downregulation and CCR7 upregulation were firstly observed in bladder cancer samples and cell lines. In addition, both miR-199a-5p downregulation and CCR7 upregulation were significantly involved in bladder cancer clinicopathological features. Moreover, overexpression of miR-199a-5p could inhibit baldder cancer cell migration and invasion. miR-199a-5p was confirmed to be able to target the 3' untranslated region (UTR) of CCR7 and regulate the expression of CCR7, Matrix metalloproteinases 9 (MMP-9) and Epithelial-Mesenchymal Transition (EMT)-related proteins. CONCLUSION: Our findings added newer insights into the multifaceted role played by miR-199a-5p/CCR7 in bladder cancer, prompting for the first time this miRNA/chemokine axis that regulates cell metastasis. The results strongly supported miR-199a-5p as a potential therapeutic agent and diagnostic marker of bladder cancer.

Tumor infiltrating T lymphocytes expressing FoxP3, CCR7 or PD-1 predict the outcome of prostate cancer patients subjected to salvage radiotherapy after biochemical relapse.

Tumor immunologic microenvironment is strongly involved in tumor progression and the presence of tumor infiltrating lymphocytes (TIL) with different phenotypes has been demonstrated to be of prognostic relevance in different malignancies. We investigated whether TIL infiltration of tumor tissues could also predict the outcome of prostate cancer patients. To this end, we carried out a retrospective analysis correlating the outcome of locally advanced prostate cancer patients undergone salvage radiotherapy upon relapse after radical surgery with the infiltration by different TIL populations. Twenty-two patients with resectable prostate cancer, with a mean age of 67 (+/-3.93) years, who received salvage radiotherapy with a mean of 69.66 (+/- 3.178) Gy in 8 weeks, between June 1999 and January 2009 and with a median follow up of 123 (+/- 55.82) months, were enrolled in this study. We evaluated, by immunohistochemistry, the intratumoral (t) and peripheral stroma (p) infiltration by CD45, CD3, CD4, CD8, CCR7, FoxP3 or PD-1-positive cells on tumor samples taken at the diagnosis (d) and relapse times (R). We correlated these variables with patients' biochemical progression free survival (bPFS), post-radiotherapy progression free survival (PFS), and overall survival (OS). Substantial changes in the rate of TIL subsets were found between the first and the second biopsy with progressive increase in CD4, CCR7, FoxP3, PD-1+ cells. Our analysis revealed that higher CD8p,R+ and lower PD-1R+ TIL scores correlated to a longer bPFS. Higher CD8p,R+ and CCR7t,R+ TIL scores and lower CD45p,R+ and FoxP3p,R+ TIL scores correlated to a prolonged PFS and OS. These results suggest that the immunological microenvironment of primary tumor is strictly correlated with patient outcome and provide the rationale for immunological treatment of prostate cancer.

Use of NRP1, a novel biomarker, along with VEGF-C, VEGFR-3, CCR7 and SEMA3E, to predict lymph node metastasis in squamous cell carcinoma of the tongue.

Lymph node (LN) metastasis has been suggested as a major prognostic factor for oral cancer. Knockdown of the growth factors and receptors involved in these metastatic mechanisms could significantly reduce LN metastasis and improve the survival of oral cancer patients after treatment. The present study, therefore, aimed to evaluate the expression levels of the following growth factors and receptors in squamous cell carcinoma (SCC) of the tongue: the vascular endothelial growth factor (VEGF)­C and VEGF­D, which bind to the cell surface tyrosine kinase receptor VEGF receptor­3 (VEGFR­3); C­C motif chemokine receptor 7 (CCR7); neuropilin (NRP)1 and NRP2; and semaphorin 3E (SEMA3E). Furthermore, we assessed microvessel density (MVD) and lymphatic vessel density (LVD) to demonstrate the correlation between these factors and regional LN metastasis, with respect to the clinicopathological features. Finally, we analyzed the correlation between these proteins and overall or disease­free survival, in order to demonstrate their prognostic value. Univariate analysis revealed a significant association between LN metastasis and the expression levels of VEGF­C, VEGFR­3, CCR7, NRP1, and SEMA3E, as well as LVD, in SCC cells. In contrast, multivariate analysis identified associations between LN metastasis and NRP1 expression, as well as between LN metastasis and LVD; however, no correlation was found between LN metastasis and the expression levels of the other proteins. The expression levels of VEGF­C, VEGFR­3, NRP1, and SEMA3E, as well as LVD, were correlated with disease­free survival time. These results indicate that LN metastasis is associated with poor survival in SCC. This study suggests that NRP1 expression and LVD are independent factors that are likely to predict the risk of LN metastasis in SCC of the tongue, whereas the expression of VEGF­C, VEGFR­3, CCR7, and SEMA3E are non­independent predictive factors.

CCL21-CCR7 promotes the lymph node metastasis of esophageal squamous cell carcinoma by up-regulating MUC1.

BACKGROUND: CCR7 and MUC1 are correlated with lymph node metastasis in ESCC, but the role of MUC1 in the CCR7-induced lymphatic metastasis and the underlying molecular mechanism is still unclear. METHODS: The expression of CCR7 and MUC1 was detected in the ESCC samples by IHC, and the clinical significance of CCR7 and MUC1 in ESCC was analyzed. The expression of CCR7 and MUC1 in ESCC cell lines was detected by qRT-PCR and western blot. The effect of CCL21 on the migration and invasion of ESCC cells was determined by transwell assay. The activity of MUC1 promoter was determined by luciferase reporter assay. The activation of Erk, Akt and Sp1 was detected by western blot and the binding of Sp1 to the MUC1 promoter was determined by ChIP. RESULTS: The co-expression of CCR7 and MUC1 was detected in 153 ESCC samples by IHC, and both were correlated with lymph node metastasis, regional lymphatic recurrence and poor prognosis. Correspondingly, increasing levels of MUC1 mRNA and protein were detected in the ESCC cell lines KYSE410 and Eca9706 after treatment with CCL21 in a time- and dose-dependent manner. Furthermore, silencing MUC1 could remarkably suppress the invasion and migration of ESCC cells induced by CCL21. Moreover, heterologous CCR7 promoted the invasion and migration of KYSE150 and up-regulated MUC1 expression. Increasing levels of activated ERK1/2 and Akt were detected in KYSE410 after treating the cells with CCL21, and inhibiting the activation of ERK1/2 but not Akt caused the increased transcription of MUC1. Finally, the phosphorylation of Sp1 induced by ERK1/2 and subsequent increases in the binding of Sp1 to the muc1 promoter at -99/-90 were confirmed to cause the up-regulation of MUC1 induced by CCL21-CCR7. CONCLUSIONS: Our findings suggested that MUC1 plays an important role in CCL21-CCR7-induced lymphatic metastasis and may serve as a therapeutic target in ESCC.

Inadequate activation of the HBsAg-specific Th cells by APCs leads to hyporesponsiveness to HBsAg vaccine in B10.S mice.

Hepatitis B can be effectively prevented by hepatitis B vaccination. However, hyporesponse to the hepatitis B vaccine has been found in both human and inbred mice with particular MHC alleles or haplotypes, but the mechanisms underlying this poor response remains elusive. In the present study, we investigated the mechanisms underlying the hyporesponse to hepatitis B vaccination using B10.S-H2s/SgMcdJ (B10.S, H-2(s), poor responder) and C57BL/10J (B10, H-2(b), good responder) mice. We observed that the B10.S mice displayed a hyporesponse to HBsAg vaccine but a normal response to 3 other foreign antigens (influenza A (H1N1) 2009 monovalent vaccine, tetanus toxoid and ovalbumin). In B10.S mice immunized with HBsAg, the levels of serum anti-HBs IgG, the number of HBsAg-specific IgG-secreting plasma cells and HBsAg-specific Th cells were considerably lower than that in B10 mice. Further, the findings of the insufficient maturation (CD86), co-stimulation (CD40) and migration (CCR7) activities of DCs together with the inadequate activation of the HBsAg-specific Th cells by APCs were identified as part of the reason for the HBsAg hyporesponse in B10.S mice, which supports the hypothesis that measures aimed at promoting the maturation, co-stimulation or migration of APCs to enhance Th cell activation may be a useful strategy for the development of new hepatitis B vaccines.

ß-Caryophyllene potently inhibits solid tumor growth and lymph node metastasis of B16F10 melanoma cells in high-fat diet-induced obese C57BL/6N mice.

We reported previously that high-fat diet (HFD) feeding stimulated solid tumor growth and lymph node (LN) metastasis in C57BL/6N mice injected with B16F10 melanoma cells. ß-caryophyllene (BCP) is a natural bicyclic sesquiterpene found in many essential oils and has been shown to exert anti-inflammatory activities. To examine whether BCP inhibits HFD-induced melanoma progression, 4-weeks old, male C57BL/6N mice were fed a control diet (CD, 10 kcal% fat) or HFD (60 kcal% fat + 0, 0.15 or 0.3% BCP) for the entire experimental period. After 16 weeks of feeding, B16F10s were subcutaneously injected into mice. Three weeks later, tumors were resected, and mice were killed 2 weeks post-resection. Although HFD feeding increased body weight gain, fasting blood glucose levels, solid tumor growth, LN metastasis, tumor cell proliferation, angiogenesis and lymphangiogenesis, it decreased apoptotic cells, all of which were suppressed by dietary BCP. HFD feeding increased the number of lipid vacuoles and F4/80+ macrophage (MΦ) and macrophage mannose receptor (MMR)+ M2-MΦs in tumor tissues and adipose tissues surrounding the LN, which was suppressed by BCP. HFD feeding increased the levels of CCL19 and CCL21 in the LN and the expression of CCR7 in the tumor; these changes were blocked by dietary BCP. In vitro culture results revealed that BCP inhibited lipid accumulation in 3T3-L1 preadipocytes; monocyte migration and monocyte chemoattractant protein-1 secretion by B16F10s, adipocytes and M2-MΦs; angiogenesis and lymphangiogenesis. The suppression of adipocyte and M2-cell accumulation and the inhibition of CCL19/21-CCR7 axis may be a part of mechanisms for the BCP suppression of HFD-stimulated melanoma progression.

Chlamydia pneumoniae and Chlamydia Trachomatis Infection Differentially Modulates Human Dendritic Cell Line (MUTZ) Differentiation and Activation.

Chlamydia trachomatis and Chlamydia pneumoniae are important human pathogens that infect the urogenital/anorectal and respiratory tracts, respectively. Whilst the ability of these bacteria to infect epithelia is well defined, there is also considerable evidence of infection of leucocytes, including dendritic cells (DCs). Using a human dendritic cell line (MUTZ), we demonstrate that the infection and replication of chlamydiae inside DCs is species and serovar specific and that live infection with C. pneumoniae is required to upregulate costimulatory markers CD80, CD83 and human leucocyte antigen (HLA)-DR on MUTZ cells, as well as induce secretion of interleukin (IL)-2, IL-6, IL-8, IL-12 (p70), interferon-gamma and tumour necrosis factor-alpha Conversely, C. trachomatis serovar D failed to upregulate DC costimulatory markers, but did induce secretion of high concentrations of IL-8. Interestingly, we also observed that infection of MUTZ cells with C. pneumoniae or C. trachomatis serovar L2, whilst not replicative, remained infectious and upregulated lymph node migratory marker CCR7 mRNA. Taken together, these data confirm the findings of other groups using primary DCs and demonstrate the utility of MUTZ cells for further studies of chlamydial infection.

Preclinical activity of anti-CCR7 immunotherapy in patients with high-risk chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) with deletions of the p53 locus on chromosome 17 and/or refractory to fludarabine chemoimmunotherapy remains a major clinical problem with few therapeutic options. Currently, these types of CLL are treated with approaches that do not target the p53 pathway, such as small molecules and monoclonal antibodies (mAb). We have previously postulated anti-CCR7 mAb therapy as a novel CLL treatment. In the present study, we evaluated the in vitro efficacy of anti-CCR7 mAb as a single agent in CLL patients with high-risk cytogenetics and/or refractory to fludarabine, by measuring CCR7 surface expression and complement-dependent cytotoxicity. Our results demonstrate that CCR7 is highly expressed in challenging and heavily treated CLL patients. In addition, the complement-mediated mechanism of action of this mAb effectively eradicates CLL cells while sparing subsets of T cells in these patients. Moreover, this mAb outperformed the activity of alemtuzumab, the mAb with the highest efficacy in these groups. Finally, in vitro activity was also demonstrated in patients with a disease refractory to both fludarabine and alemtuzumab, and patients harboring 11q22 deletion. Our results propose that anti-CCR7 mAb is an effective and promising future treatment in high-risk CLL.

CC-chemokine receptor 7 is overexpressed and correlates with growth and metastasis in prostate cancer.

Multiple studies have shown that CC-chemokine receptor 7 (CCR7) promotes cell proliferation in several human cancers. We investigated the expression and clinical significance of CCR7 in our large collection of prostate cancer (PCa) samples and explored its function on the proliferation and migration of PCa cells. In this study, the expression of CCR7 was examined by immunohistochemical staining and quantitative RT-PCR in primary PCa tissues from 60 patients who underwent radical prostatectomy. Then, we investigated the functional role of CCR7 in PCa cell proliferation and migration by small interfering RNA-mediated depletion. The positive rate of CCR7 staining was 88.33 % (53/60) in the PCa group and 16.67 % (10/60) in cases of benign prostate hyperplasia (BPH); the difference of CCR7 expression between PCa and BPH was statistically significant. The results were confirmed by quantitative real-time PCR. CCR7 was significantly elevated in all five PCa cell lines when compared to the RWPE-1 cells. Silencing of CCR7 inhibited the proliferation of PC3 cells which have a relatively high level of CCR7 in a time-dependent manner, and the invasion and migration of PC3 cells were distinctly suppressed. Our data suggest that the pathogenesis of human PCa maybe mediated by the CCR7, and thus CCR7 could represent selective targets for the molecularly targeted treatments of PCa.

Positive lymph-node breast cancer patients ­ activation of NF-κB in tumor-associated leukocytes stimulates cytokine secretion that promotes metastasis via C-C chemokine receptor CCR7.

Tumor metastasis to lymph nodes is most deadly complication among breast cancer patients. Herein, we investigated the molecular mechanism by which tumor-associated leukocytes (TALs) mediate lymph node metastasis. The density of different leukocyte subtypes infiltrating the tumor microenvironment of negative and positive lymph nodes (nLNs, pLNs) in breast cancer patients was measured using immunohistochemistry. In addition, we isolated TALs from blood drained from the axillary tributaries of nLN and pLN patients during breast surgery. Secretions of TALs were subjected to cytokine profiling using a cytokine antibody array. Our results showed an increase in the number of infiltrated CD45+ cells in the carcinoma tissues of pLN patients with the major proportion being myeloid subsets compared with nLN patients. Furthermore, TALs of pLN patients show a significant fivefold increase in the secretion of interleukin (IL)-1α, interferon-γ, IL-5, IL-3 and tumor necrosis factor-ß, and are characterized by enhanced constitutive NF-κB/p65 signaling compared with TALs isolated from nLN patients. Using an invasion assay, cytokines secreted by TALs of pLN patients were shown to augment the invasive phenotype of breast cancer MCF-7 and SKBR3 cells compared with nLN patients. Using flow cytometry, we found that C-C chemokine receptor 7 (CCR7) is significantly overexpressed in breast carcinoma of pLN patients compared with nLNs patients. Intriguingly, CCR7, a mechanistic clue for metastasis, is upregulated in MCF-7 cells upon stimulation with TAL-conditioned media of pLN patients. Our findings show that the molecular cues secreted by TALs alone or in combination with CCR7 may emerge as future therapeutic targets for lymph node metastasis in breast cancer patients.

Role of dendritic cells in the pathogenesis of Whipple's disease.

Accumulation of Tropheryma whipplei-stuffed macrophages in the duodenum, impaired T. whipplei-specific Th1 responses, and weak secretion of interleukin-12 (IL-12) are hallmarks of classical Whipple's disease (CWD). This study addresses dendritic cell (DC) functionality during CWD. We documented composition, distribution, and functionality of DC ex vivo or after in vitro maturation by fluorescence-activated cell sorting (FACS) and by immunohistochemistry in situ. A decrease in peripheral DC of untreated CWD patients compared to healthy donors was due to reduced CD11c(high) myeloid DC (M-DC). Decreased maturation markers CD83, CD86, and CCR7, as well as low IL-12 production in response to stimulation, disclosed an immature M-DC phenotype. In vitro-generated monocyte-derived DC from CWD patients showed normal maturation and T cell-stimulatory capacity under proinflammatory conditions but produced less IL-12 and failed to activate T. whipplei-specific Th1 cells. In duodenal and lymphoid tissues, T. whipplei was found within immature DC-SIGN(+) DC. DC and proliferating lymphocytes were reduced in lymph nodes of CWD patients compared to levels in controls. Our results indicate that dysfunctional IL-12 production by DC provides suboptimal conditions for priming of T. whipplei-specific T cells during CWD and that immature DC carrying T. whipplei contribute to the dissemination of the bacterium.

Correlation between CCR7 expression and lymph node metastatic potential of human tongue carcinoma.

Metastasis is an important cause of cancer-related mortality. In this study, we investigated the role of CCR7 in the lymph node metastasis of tongue carcinoma. Immunohistochemistry and Western blot revealed the expression of CCR7 in tongue SCC tissues and cell lines. In addition, we examined the expression of CCL21, a ligand of CCR7, in normal and diseased lymph nodes using immunohistochemistry and/or real-time PCR. The CCR7 expression was significantly correlated with cervical lymph node metastasis, tumor staging, and histological grade (P = 0.015, 0.040, and 0.015, respectively). The multivariate analysis showed that regional lymph node metastasis, the expression of CCR7, and LVD were the independent poor prognostic factors. Knockdown of CCR7 gene resulted in a significant inhibition of migration and invasion of SCC4 cells in vitro without affecting the proliferation and apoptosis of tumor cells. Also, CCR7 knockdown obviously inhibited cervical lymph node metastasis in an animal tumor model. Our study indicated that CCR7 may play an important role in progression of tongue SCC and could be a promising target for tongue SCC therapy.

C-type lectin-like receptor LOX-1 promotes dendritic cell-mediated class-switched B cell responses.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a pattern-recognition receptor for a variety of endogenous and exogenous ligands. However, LOX-1 function in the host immune response is not fully understood. Here, we report that LOX-1 expressed on dendritic cells (DCs) and B cells promotes humoral responses. On B cells LOX-1 signaling upregulated CCR7, promoting cellular migration toward lymphoid tissues. LOX-1 signaling on DCs licensed the cells to promote B cell differentiation into class-switched plasmablasts and led to downregulation of chemokine receptor CXCR5 and upregulation of chemokine receptor CCR10 on plasmablasts, enabling their exit from germinal centers and migration toward local mucosa and skin. Finally, we found that targeting influenza hemagglutinin 1 (HA1) subunit to LOX-1 elicited HA1-specific protective antibody responses in rhesus macaques. Thus, LOX-1 expressed on B cells and DC cells has complementary functions to promote humoral immune responses.