Impairment in two blood-brain barrier (BBB) efflux transporters, p-glycoprotein (Pgp) and low-density lipoprotein receptor-related protein-1 (LRP-1) are thought to contribute to the progression of Alzheimer's disease (AD) by resulting in the brain accumulation of their substrate amyloid beta peptide (Aβ). The initial cause of impaired efflux, however, is unknown. We have shown that induction of systemic inflammation by intraperitoneal administration of lipopolysaccharide impairs the efflux of Aβ from the brain, suggesting that systemic inflammation could be one such initiator. In this study, we determined whether pre-administration of the antioxidant N-aceytlcysteine (Nac) has a protective effect against LPS-induced Aβ transporter dysfunction. Our findings were that Nac protected against LPS-induced Aβ transport dysfunction at the BBB through an LRP-1-dependent and Pgp-independent mechanism. This was associated with Nac exerting antioxidant effects in the periphery but not the brain, despite an increased rate of entry of Nac into the brain following LPS. We also found that Nac pre-administration resulted in lower blood levels of the cytokines and chemokines interferon-γ, interleukin-10, CCL2, CCL4, and CCL5, but only lowered CCL4 in the cerebral cortex and hippocampus. Finally, we observed that hippocampal cytokine responses to LPS were decreased compared to cortex. These findings demonstrate a novel mechanism by which antioxidants prevent Aβ accumulation in the brain caused by inflammation, and therefore protect against AD.

OBJECTIVE

We recently described a monocyte pro-inflammatory state in patients with bipolar disorder (BD). We hypothesized that the CD4(+)T cell system is also activated and determined percentages of Th1, Th2, Th17 and CD4(+)CD25(high)FoxP3(+) regulatory T cells.

METHODS

We carried out a detailed FACS analysis to determine the various T cell subsets and used frozen stored peripheral blood mononuclear cells (PBMC) of 38 BD patients (of whom we previously had tested monocytes for pro-inflammatory gene expression (Drexhage et al., 2010b; Padmos et al., 2008)) and of 22 age/gender matched healthy controls (HC). In addition the cytokines CCL2, IL-1β, IL-6, TNF-α, PTX3, IL-10, IFN-γ, IL-17A, IL-4, IL-5 and IL-22 were measured in serum.

RESULTS

(a) Serum sCD25 levels and percentages of anti-inflammatory CD4(+)CD25(high)FoxP3+ regulatory T cells were higher, the latter in BD patients <40 years of age. Percentages of Th1, Th2 and Th17 cells were normal. (b) Of the pro-inflammatory monocyte cytokines CCL2 and PTX3 were raised in serum. (c) The monocyte pro-inflammatory state and the raised percentages of CD4(+)CD25(high)FoxP3(+) regulatory T cells occurred independently from each other. (d) In BD patients positive for thyroid autoimmune disease a significantly reduced percentage of CD4(+)CD25(high)FoxP3(+) regulatory T cells was found as compared to BD patients without AITD.

CONCLUSIONS

Our data show an enhancement of pro-inflammatory monocyte and anti-inflammatory T cell forces in BD patients. A lack of anti-inflammatory T cell forces co-occurred with AITD in BD patients.

UNASSIGNED

Little is known about how the immune microenvironment of breast cancer evolves during disease progression.

UNASSIGNED

We compared tumor infiltrating lymphocyte (TIL) count, PD-L1 protein expression by immunohistochemistry and mRNA levels of 730 immune-related genes using Nanostring technology in primary and metastatic cancer samples.

UNASSIGNED

TIL counts and PD-L1 positivity were significantly lower in metastases. Immune cell metagenes corresponding to CD8, T-helper, T-reg, Cytotoxic T, Dendritic and Mastoid cells, and expression of 13 of 29 immuno-oncology therapeutic targets in clinical development including PD1, PD-L1, and CTLA4 were significantly lower in metastases. There was also coordinated down regulation of chemoattractant ligand/receptor pairs (CCL19/CCR7, CXCL9/CXCR3, IL15/IL15R), interferon regulated genes (STAT1, IRF-1,-4,-7, IFI-27,-35), granzyme/granulysin, MHC class I and immune proteasome (PSMB-8,-9,-10) expression in metastases. Immunotherapy response predictive signatures were also lower. The expression of macrophage markers (CD163, CCL2/CCR2, CSF1/CSFR1, CXCR4/CXCL12), pro-tumorigenic toll like receptor pathway genes (CD14/TLR-1,-2,-4,-5,-6/MyD88), HLA-E, ecto-nuclease CD73/NT5E and inhibitory complement receptors (CD-59,-55,-46) remained high in metastases and represent potential therapeutic targets.

UNASSIGNED

Metastatic breast cancers are immunologically more inert than the corresponding primary tumors but some immune-oncology targets and macrophage and angiogenesis signatures show preserved expression and suggest therapeutic combinations for clinical testing.

Spiegelmers are synthetic target-binding oligonucleotides built from non-natural l-nucleotides. Like aptamers, Spiegelmers fold into distinct shapes that bind the targets with high affinity and selectivity. Furthermore, the mirror-image configuration confers plasma stability and immunological passivity. Various Spiegelmers against pharmacologically attractive targets were shown to be efficacious in animal models. Three Spiegelmer candidates: emapticap pegol (NOX-E36; anti-CCL2), olaptesed pegol (NOX-A12; anti-CXCL12) and lexaptepid pegol (NOX-H94; anti-hepcidin), underwent regulatory safety studies, demonstrated good safety profiles in healthy volunteers and were taken into Phase IIa studies in patients. Proof-of-concept for emapticap pegol has recently been demonstrated in diabetic nephropathy patients. Furthermore, promising interim Phase IIa data of olaptesed pegol and lexapteptid pegol also suggest efficacy in the respective patient populations.

The tumor microenvironment (TME) exerts critical pro-tumorigenic effects through cytokines and growth factors that support cancer cell proliferation, survival, motility and invasion. Insulin-like growth factor-1 (IGF-1) and signal transducer and activator of transcription 3 (STAT3) stimulate colorectal cancer development and progression via cell autonomous and microenvironmental effects. Using a unique inhibitor, NT157, which targets both IGF-1 receptor (IGF-1R) and STAT3, we show that these pathways regulate many TME functions associated with sporadic colonic tumorigenesis in CPC-APC mice, in which cancer development is driven by loss of the Apc tumor suppressor gene. NT157 causes a substantial reduction in tumor burden by affecting cancer cells, cancer-associated fibroblasts (CAF) and myeloid cells. Decreased cancer cell proliferation and increased apoptosis were accompanied by inhibition of CAF activation and decreased inflammation. Furthermore, NT157 inhibited expression of pro-tumorigenic cytokines, chemokines and growth factors, including IL-6, IL-11 and IL-23 as well as CCL2, CCL5, CXCL7, CXCL5, ICAM1 and TGFβ; decreased cancer cell migratory activity and reduced their proliferation in the liver. NT157 represents a new class of anti-cancer drugs that affect both the malignant cell and its supportive microenvironment.

The gene encoding F-box protein FBXW7 is frequently mutated in many human cancers. Although most previous studies have focused on the tumor-suppressive capacity of FBXW7 in tumor cells themselves, we determined that FBXW7 in the host microenvironment also suppresses cancer metastasis. Deletion of Fbxw7 in murine BM-derived stromal cells induced accumulation of NOTCH and consequent transcriptional activation of Ccl2. FBXW7-deficient mice exhibited increased serum levels of the chemokine CCL2, which resulted in the recruitment of both monocytic myeloid-derived suppressor cells and macrophages, thereby promoting metastatic tumor growth. Administration of a CCL2 receptor antagonist blocked the enhancement of metastasis in FBXW7-deficient mice. Furthermore, in human breast cancer patients, FBXW7 expression in peripheral blood was associated with serum CCL2 concentration and disease prognosis. Together, these results suggest that FBXW7 antagonizes cancer development in not only a cell-autonomous manner, but also a non-cell-autonomous manner, and that modulation of the FBXW7/NOTCH/CCL2 axis may provide a potential approach to suppression of cancer metastasis.

Spinal cord injury (SCI) often results in intractable chronic central pain syndromes. Recently chemokines such as CCL2 were identified as possible key integrators of neuropathic pain and inflammation after peripheral nerve lesion. The focus of the current study was the investigation of time-dependent CCL2 and CCR2 expression in relation to central neuropathic pain development after spinal cord impact lesions of 100, 150, or 200 kdyn force on spinal cord level T9 in adult rats. Below-level pain was monitored with weekly sensory testing for 42 days after SCI. In parallel expression of CCL2/CCR2 on cervical, thoracic, and lumbar levels was investigated by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry early (7 days [7d]), intermediate (15d), and late (42d) after lesion. Cellular source and anatomical pain related expression was determined by double-immunohistochemistry. Force-defined SCI led to acute mechanical hypersensitivity in all lesion groups, and to persistent below-level pain in severely injured animals. While in the early post-operative time course, CCL2 and CCR2 were expressed in astroglia and granulocytes only on level T9; there was additional astroglial CCL2 expression in dorsal columns and dorsal horns above and below T9 of severely injured animals 42d after lesion. In dorsal horns (level L3-L5) of animals exhibiting chronic below-level pain CCL2 was co-expressed with transmitters and receptors that are involved in nociceptive processing like calcitonin gene-related peptide (CGRP), Substance-P, vanilloid-receptor-1, and its activated phosphorylated form. These data demonstrate lesion grade dependence of below-level pain development and suggest chemokines as potential candidates for integrating inflammation and central neuropathic pain after SCI.

To glean biological differences and similarities of peripheral T-cell lymphoma-not otherwise specified [PTCL-NOS] to diffuse large B-cell lymphoma (DLBCL), a transcriptosome analysis was done on five PTCL-NOS and four DLBCL patients and validated by quantitative real-time reverse transcription-PCR on 10 selected genes. Normal peripheral blood T cells, peripheral blood B cells, and lymph node were used as controls. The resultant gene expression profile delineated distinct "tumor profile signatures" for PTCL-NOS and DLBCL. Several highly overexpressed genes in both PTCL-NOS and DLBCL involve the immune network, stroma, angiogenesis, and cell survival cascades that make important contributions to lymphomagenesis. Inflammatory chemokines and their receptors likely play a central role in these complex interrelated pathways: CCL2 and CXCR4 in PTCL-NOS and CCL5 and CCR1 in DLBCL. Highly overexpressed oncogenes unique to PTCL-NOS are SPI1, STK6, alpha-PDGFR, and SH2D1A, whereas in DLBCL they are PIM1, PIM2, LYN, BCL2A1, and RAB13. Oncogenes common to both lymphomas are MAFB, MET, NF-kappaB2, LCK, and LYN. Several tumor suppressors are also down-regulated (TPTE, MGC154, PTCH, ST5, and SUI1). This study illustrates the relevance of tumor-stroma immune trafficking and identified potential novel prognostic markers and targets for therapeutic intervention.

Morphine and related compounds are the first line of therapy in the treatment of moderate to severe pain. Over time, individuals taking opioids can develop an increasing sensitivity to noxious stimuli, even evolving into a painful response to previously non-noxious stimuli (opioid-induced hyperalgesia; OIH). The mechanism underlying OIH is not well understood although complex intracellular neural mechanisms, including opioid receptor desensitization and down-regulation, are believed to be major mechanisms underlying OIH. However, OIH may also be associated with changes in gene expression. A growing body of evidence suggests that cellular exposure to mu agonists upregulate chemokines/receptors and recent work from our laboratory implicates chemokine upregulation in a variety of neuropathic pain behaviors. Here we characterized the degree to which chemokines/receptors signaling is increased in primary afferent neurons of the dorsal root ganglion (DRG) following chronic morphine sulfate treatment and correlated these changes with tactile hyperalgesic behavior in rodents. We demonstrate that mRNA expression of the chemokine, stromal-derived factor-1 (SDF1/CXCL12) is upregulated following morphine treatment in sensory neurons of the rat. The release of SDF1 was found to be constitutive when compared with the activity dependent release of the C-C chemokine, monocyte chemoattractant protein-1 (MCP1/CCL2) in a line of F11 neuroblastoma-sensory neuron hybrid cells. We further determined that there is pronounced CXCR4 expression in satellite glial cells and following morphine treatment, increased functional CXCR4 expression in sensory neurons of the DRG. Moreover, intraperitoneal administration of the specific CXCR4 antagonist, AMD3100, completely reversed OIH in the rat. Taken together; the data suggest that opioid-induced SDF1/CXCR4 signaling is central to the development of long lasting OIH and that receptor antagonists represent a promising novel approach to the management of the side effects associated with the use of opioids for chronic pain management.

Clumping factor A (ClfA) is a fibrinogen-binding cell wall-attached protein and an important virulence factor of Staphylococcus aureus. Previous studies reported that an immunization with the fibrinogen-binding domain of ClfA (ClfA(40-559)) protected animals against S. aureus infection. It was reported that some cytokines are involved in the pathogenesis of staphylococcal diseases and in host defense against S. aureus infection. However, the role of cytokines in the protective effect of ClfA(40-559) as a vaccine has not been elucidated. In this study, we demonstrated that the spleen cells of ClfA(40-559)-immunized mice produced a large amount of interleukin-17A (IL-17A). The protective effect of immunization was exerted in wild-type mice but not in IL-17A-deficient mice. IL-17A mRNA expression was increased in the spleens and kidneys of immunized mice after infection. CXCL2 and CCL2 mRNA expression was increased in the spleens and kidneys, respectively. Consistent with upregulation of the mRNA expression, neutrophils infiltrated into the spleens extensively and macrophage infiltration was observed in the kidneys of immunized mice. These results suggest that immunization with ClfA(40-559) induces the IL-17A-producing cells and that IL-17-mediated cellular immunity is involved in the protective effect induced by immunization with ClfA(40-559) against S. aureus infection.

Macrophages and dendritic cells have been recognized as key players in the defense against mycobacterial infection. However, more recently, other cells in the lungs such as alveolar epithelial cells (AEC) have been found to play important roles in the defense and pathogenesis of infection. In the present study we first compared AEC with pulmonary macrophages (PuM) isolated from mice in their ability to internalize and control Bacillus Calmette-Guérin (BCG) growth and their capacity as APCs. AEC were able to internalize and control bacterial growth as well as present antigen to primed T cells. Secondly, we compared both cell types in their capacity to secrete cytokines and chemokines upon stimulation with various molecules including mycobacterial products. Activated PuM and AEC displayed different patterns of secretion. Finally, we analyzed the profile of response of AEC to diverse stimuli. AEC responded to both microbial and internal stimuli exemplified by TLR ligands and IFNs, respectively. The response included synthesis by AEC of several factors, known to have various effects in other cells. Interestingly, TNF could stimulate the production of CCL2/MCP-1. Since MCP-1 plays a role in the recruitment of monocytes and macrophages to sites of infection and macrophages are the main producers of TNF, we speculate that both cell types can stimulate each other. Also, another cell-cell interaction was suggested when IFNs (produced mainly by lymphocytes) were able to induce expression of chemokines (IP-10 and RANTES) by AEC involved in the recruitment of circulating lymphocytes to areas of injury, inflammation, or viral infection. In the current paper we confirm previous data on the capacity of AEC regarding internalization of mycobacteria and their role as APC, and extend the knowledge of AEC as a multifunctional cell type by assessing the secretion of a broad array of factors in response to several different types of stimuli.

The monocyte chemoattractant CCL2 is of major importance in inflammatory monocyte recruitment to the lungs in response to bacterial infection. Streptococcus pneumoniae is the most prevalent pathogen in community-acquired pneumonia causing significant morbidity and mortality worldwide. In the current study, we examined the role of CCL2 in lung-protective immunity against two strains of S. pneumoniae exhibiting different virulence profiles. Both wild-type mice and CCL2 knockout (KO) mice became septic within 24 h of infection with serotype 3 S. pneumoniae and died of infection by day 4 after challenge. In contrast, wild-type mice challenged with serotype 19 S. pneumoniae did not become septic or succumb to pneumococcal pneumonia, whereas CCL2 KO mice showed an early bacterial outgrowth in their lungs and sepsis starting by day 2 after infection, finally resulting in approximately 50% decreased survival compared with wild-type mice. This phenotype was not due to impaired lung neutrophil recruitment in the KO mice, but was characterized by a significantly reduced recruitment of lung exudate macrophages and conventional lung dendritic cells, suggesting that these two phagocyte subsets critically regulate protection against septic disease progression in mice. In conclusion, we show here a differential role for CCL2-dependent lung exudate macrophage and conventional dendritic cell recruitment that critically contributes to lung protective immunity against S. pneumoniae.

Age-related macular degeneration (AMD) is one of the leading cause of blindness among the elderly; however, current therapy options are limited. Epidemiological studies have shown that a diet that is high in omega-3 polyunsaturated (n-3) fatty acids can slow disease progression in patients with advanced AMD. In this study, we evaluated the effect of such a diet on the retinas of Ccl2(-/-)/Cx3cr1(-/-) mice, a model that develops AMD-like retinal lesions that include focal deep retinal lesions, abnormal retinal pigment epithelium, photoreceptor degeneration, and A2E accumulation. Ccl2(-/-)/Cx3cr1(-/-) mice that ingested a high n-3 fatty acid diet showed a slower progression of retinal lesions compared with the low n-3 fatty acids group. Some mice that were given high levels of n-3 fatty acids had lesion reversion. We found a shunted arachidonic acid metabolism that resulted in decreased pro-inflammatory derivatives (prostaglandin E(2) and leukotriene B(4)) and an increased anti-inflammatory derivative (prostaglandin D(2)). We also measured lower ocular TNF-alpha and IL-6 transcript levels in the mice fed a diet of high n-3 fatty acids. Our findings in these mice are in line with human studies of AMD risk reduction by long-chain n-3 fatty acids. This murine model provides a useful tool to evaluate therapies that might delay the development of AMD.

To assess the role of CC-chemokine ligand 5 (CCL5)/RANTES in opiate drug abuse and human immunodeficiency virus type 1 (HIV-1) comorbidity, the effects of systemic morphine and intrastriatal HIV-1 Tat on macrophage/microglial and astroglial activation were assessed in wild-type and CCL5 knockout mice. Mice were injected intrastriatally with vehicle or Tat and assessed after 7 days. Morphine was administered to some Tat-injected mice via time-release implant (5 mg/day, s.c. for 5 days) starting at 2 days post injection. Glial activation was significantly reduced in CCL5(-/-) compared to wild-type mice at 7 days following combined Tat and morphine exposure. Moreover, the percentage of 3-nitrotyrosine immunopositive macrophages/microglia was markedly reduced in CCL5(-/-) mice injected with Tat +/- morphine compared to wild-type counterparts, suggesting that CCL5 contributes to nitrosative stress in HIV-1 encephalitis. In CCL5(-/-) mice, the reductions in Tat +/- morphine-induced gliosis coincided with significant declines in the proportion of CCL2/MCP-1-immunoreactive astrocytes and macrophages/microglia compared to wild-type counterparts. In knockout mice, neither Tat alone nor in combination with morphine increased the proportion of CCL2-immunoreactive astrocytes above percentages seen in vehicle-injected controls. Macrophages/microglia differed showing modest, albeit significant, increases in the proportion of CCL2-positive cells with combined Tat and morphine exposure, suggesting that CCL5 preferentially affects CCL2 expression by astroglia. Thus, CCL5 mediates glial activation caused by Tat and morphine, thereby aggravating HIV-1 neuropathogenesis in opiate abusers and non-abusers. CCL5 is implicated as mediating the cytokine-driven amplification of CCL2 production by astrocytes and resultant macrophage/microglial recruitment and activation.

Acute lung injury (ALI) initiates protective responses involving genes downstream of the Nrf2 (Nfe2l2) transcription factor, including heme oxygenase-1 (HO-1), which stimulates mitochondrial biogenesis and related anti-inflammatory processes. We examined mitochondrial biogenesis during Staphylococcus aureus pneumonia in mice and the effect of Nrf2 deficiency on lung mitochondrial biogenesis and resolution of lung inflammation. S. aureus pneumonia established by nasal insufflation of live bacteria was studied in mitochondrial reporter (mt-COX8-GFP) mice, wild-type (WT) mice, and Nrf2⁻/⁻ mice. Bronchoalveolar lavage, wet/dry ratios, real-time RT-PCR and Western analysis, immunohistochemistry, and fluorescence microscopy were performed on the lung at 0, 6, 24, and 48 h. The mice survived S. aureus inoculations at 5×10⁸ CFU despite diffuse lung inflammation and edema, but the Nrf2⁻/⁻ lung showed increased ALI. In mt-COX8-GFP mice, mitochondrial fluorescence was enhanced in bronchial and alveolar type II (AT2) epithelial cells. WT mice displayed rapid HO-1 upregulation and lower proinflammatory TNF-α, IL-1β, and CCL2 and, especially in AT2 cells, higher anti-inflammatory IL-10 and suppressor of cytokine signaling-3 than Nrf2⁻/⁻ mice. In the alveolar region, WT but not Nrf2⁻/⁻ mice showed strongly induced nuclear respiratory factor-1, PGC-1α, mitochondrial transcription factor-A, SOD2, Bnip3, mtDNA copy number, and citrate synthase. These findings indicate that S. aureus pneumonia induces Nrf2-dependent mitochondrial biogenesis in the alveolar region, mainly in AT2 cells. Absence of Nrf2 suppresses the alveolar transcriptional network for mitochondrial biogenesis and anti-inflammation, which worsens ALI. The findings link redox activation of mitochondrial biogenesis to ALI resolution.

Because the potential of yerba maté (Ilex paraguariensis) has been suggested in the management of obesity, the aim of the present study was to evaluate the effects of yerba maté extract on weight loss, obesity-related biochemical parameters, and the regulation of adipose tissue gene expression in high-fat diet-induced obesity in mice. Thirty animals were randomly assigned to three groups. The mice were introduced to standard or high-fat diets. After 12 weeks on a high-fat diet, mice were randomly assigned according to the treatment (water or yerba maté extract 1.0 g/kg). After treatment intervention, plasma concentrations of total cholesterol, high-density lipoprotein cholesterol, triglyceride, low-density lipoprotein (LDL) cholesterol, and glucose were evaluated. Adipose tissue was examined to determine the mRNA levels of several genes such as tumor necrosis factor-alpha (TNF-alpha), leptin, interleukin-6 (IL-6), C-C motif chemokine ligand-2 (CCL2), CCL receptor-2 (CCR2), angiotensinogen, plasminogen activator inhibitor-1 (PAI-1), adiponectin, resistin, peroxisome proliferator-activated receptor-gamma(2) (PPAR-gamma(2)), uncoupling protein-1 (UCP1), and PPAR-gamma coactivator-1 alpha (PGC-1 alpha). The F4/80 levels were determined by immunoblotting. We found that obese mice treated with yerba maté exhibited marked attenuation of weight gain, adiposity, a decrease in epididymal fat-pad weight, and restoration of the serum levels of cholesterol, triglycerides, LDL cholesterol, and glucose. The gene and protein expression levels were directly regulated by the high-fat diet. After treatment with yerba maté extract, we observed a recovery of the expression levels. In conclusion, our data show that yerba maté extract has potent antiobesity activity in vivo. Additionally, we observed that the treatment had a modulatory effect on the expression of several genes related to obesity.

OBJECTIVE

Adoptive transfer of natural killer (NK) cells combined with tumor-specific monoclonal antibodies (mAb) has therapeutic potential for malignancies. We determined if large numbers of activated NK (aNK) cells can be grown ex vivo from peripheral blood mononuclear cells (PBMC) of children with high-risk neuroblastoma using artificial antigen-presenting cells (aAPC).

METHODS

Irradiated K562-derived Clone 9.mbIL21 aAPC were cocultured with PBMC, and propagated NK cells were characterized with flow cytometry, cytotoxicity assays, Luminex multicytokine assays, and a nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of disseminated neuroblastoma.

RESULTS

Coculturing patient PBMC with aAPC for 14 days induced 2,363- ± 443-fold expansion of CD56(+)CD3(-)CD14(-) NK cells with 83% ± 3% purity (n = 10). Results were similar to PBMC from normal donors (n = 5). Expression of DNAM-1, NKG2D, FcγRIII/CD16, and CD56 increased 6- ± 3-, 10- ± 2-, 21- ± 20-, and 18- ± 3-fold, respectively, on day 14 compared with day 0, showing activation of NK cells. In vitro, aNK cells were highly cytotoxic against neuroblastoma cell lines and killing was enhanced with GD2-specific mAb ch14.18. When mediating cytotoxicity with ch14.18, release of TNF-α, granulocyte macrophage colony-stimulating factor, IFN-γ, sCD40L, CCL2/MCP-1, CXCL9/MIG, and CXCL11/I-TAC by aNK cells increased 4-, 5-, 6-, 15-, 265-, 917-, and 363-fold (151-9,121 pg/mL), respectively, compared with aNK cells alone. Survival of NOD/SCID mice bearing disseminated neuroblastoma improved when treated with thawed and immediately intravenously infused cryopreserved aNK cells compared with untreated mice and was further improved when ch14.18 was added.

CONCLUSIONS

Propagation of large numbers of aNK cells that maintain potent antineuroblastoma activities when cryopreserved supports clinical testing of adoptive cell therapy with ch14.18.

BACKGROUND

Tissue fibrosis is an integral component of chronic inflammatory (liver and pancreas) diseases and pancreatic cancer. Activated pancreatic- (PSC) and hepatic- (HSC) stellate cells play a key role in fibrogenesis. To identify organ- and disease-specific stellate cell transcriptional fingerprints, we employed genome-wide transcriptional analysis of primary human PSC and HSC isolated from patients with chronic inflammation or cancer.

METHODS

Stellate cells were isolated from patients with pancreatic ductal adenocarcinoma (n = 5), chronic pancreatitis (n = 6), liver cirrhosis (n = 5) and liver metastasis of pancreatic ductal adenocarcinoma (n = 6). Genome-wide transcriptional profiles of stellate cells were generated using our 51K human cDNA microarray platform. The identified organ- and disease specific genes were validated by quantitative RT-PCR, immunoblot, ELISA, immunocytochemistry and immunohistochemistry.

RESULTS

Expression profiling identified 160 organ- and 89 disease- specific stellate cell transcripts. Collagen type 11a1 (COL11A1) was discovered as a novel PSC specific marker with up to 65-fold higher expression levels in PSC compared to HSC (p < 0.0001). Likewise, the expression of the cytokine CCL2 and the cell adhesion molecule VCAM1 were confined to HSC. PBX1 expression levels tend to be increased in inflammatory- vs. tumor- stellate cells. Intriguingly, tyrosine kinase JAK2 and a member of cell contact-mediated communication CELSR3 were found to be selectively up-regulated in tumor stellate cells.

CONCLUSIONS

We identified and validated HSC and PSC specific markers. Moreover, novel target genes of tumor- and inflammation associated stellate cells were discovered. Our data may be instrumental in developing new tailored organ- or disease-specific targeted therapies and stellate cell biomarkers.

OBJECTIVE

Local tumor growth is a major cause of morbidity and mortality in nearly 30% of patients with pancreatic ductal adenocarcinoma (PDAC). Radiotherapy is commonly used for local disease control in PDAC, but its efficacy is limited. We studied the impact of selectively intervening on radiotherapy-induced inflammation as an approach to overcome resistance to radiotherapy in PDAC.

METHODS

PDAC cell lines derived from primary pancreatic tumors arising spontaneously in KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx-1 Cre mice were implanted into syngeneic mice and tumors were focally irradiated using the Small Animal Radiation Research Platform (SARRP). We determined the impact of depleting T cells and Ly6C+ monocytes as well as inhibiting the chemokine CCL2 on radiotherapy efficacy. Tumors were analyzed by flow cytometry and IHC to detect changes in leukocyte infiltration, tumor viability, and vascularity. Assays were performed on tumor tissues to detect cytokines and gene expression.

RESULTS

Ablative radiotherapy alone had minimal impact on PDAC growth but led to a significant increase in CCL2 production by tumor cells and recruitment of Ly6C+CCR2+ monocytes. A neutralizing anti-CCL2 antibody selectively inhibited radiotherapy-dependent recruitment of monocytes/macrophages and delayed tumor growth but only in combination with radiotherapy (P < 0.001). This antitumor effect was associated with decreased tumor proliferation and vascularity. Genetic deletion of CCL2 in PDAC cells also improved radiotherapy efficacy.

CONCLUSIONS

PDAC responds to radiotherapy by producing CCL2, which recruits Ly6C+CCR2+ monocytes to support tumor proliferation and neovascularization after radiotherapy. Disrupting the CCL2-CCR2 axis in combination with radiotherapy holds promise for improving radiotherapy efficacy in PDAC. Clin Cancer Res; 23(1); 137-48. ©2016 AACR.

OBJECTIVE

Plasma microRNAs (miRNAs) are modulated during disease and are emerging biomarkers; they have not been characterized in HIV infection. Using our macaque/simian immunodeficiency virus (SIV) model of HIV, we sought to identify a plasma miRNA profile of acute lentiviral infection, evaluate its relationship with known cellular and viral determinants of lentivirus-associated central nervous system (CNS) disease, and explore the potential of miRNAs to predict CNS disease.

METHODS

Plasma samples were obtained before inoculation and 10 days after inoculation from SIV-infected macaques.

METHODS

Plasma miRNA expression profiles were determined by TaqMan low-density array for six individuals. miRNA expression was compared with levels of cytokines, virus, and plasma platelet count. miRNA results were confirmed by single miRNA-specific assays for 10 macaques. Nineteen individuals were used to validate a disease prediction test.

RESULTS

A 45-miRNA signature of acute infection (differential expression with P < 0.05 after multiple comparison correction) classified plasma as infected or not. Several differentially expressed miRNAs correlated with CNS disease-associated cytokines interleukin-6 and CCL2 and included predicted and/or validated regulators of the corresponding mRNAs. miRNAs tracked with viral load and platelet count were also predictors of CNS disease. At least six miRNAs were significantly differentially expressed in individuals with severe versus no CNS disease; in an unweighted expression test, they predicted CNS disease.

CONCLUSIONS

Acute-phase differential expression of plasma miRNAs predicts CNS disease and suggests that CNS damage or predisposition to disease progression begins in the earliest phase of infection. Plasma miRNAs should be investigated further as leading indicators of HIV diseases as early as acute infection.

Chemokines have been implicated in the promotion of leucocyte trafficking to diseased muscle. The purpose of this study was to determine whether a subset of inflammatory chemokines are able to directly drive myoblast proliferation, an essential early component of muscle regeneration, in a manner which is entirely independent of leucocytes. Cultured myoblasts (C2C12) were exposed to monocyte chemoattractant protein-1 (MCP-1; CCL2), macrophage inflammatory protein-1alpha (MIP-1alpha; CCL3) or MIP-1beta (CCL4). All chemokines induced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) and greatly increased myoblast proliferative responses. Chemokine-induced myoblast proliferation was abolished by pertussis toxin and the MEK1/2 inhibitor U0126, implicating both Galphai-coupled receptors and ERK1/2-dependent signalling. Myoblasts expressed receptors for all of the chemokines tested, and mitogenic responses were specifically inhibited by antibodies directed against CC family chemokine receptors 2 and 5 (CCR2 and CCR5). Within an in vitro myogenic wound healing assay devoid of leucocytes, all chemokines significantly accelerated the time course of myoblast wound closure after mechanical injury. Injections of MCP-1 into cardiotoxin-injured skeletal muscles in vivo also suppressed expression of the differentiation marker myogenin, consistent with a mitogenic effect. Taken together, our results indicate that CC chemokines have potent and direct effects on myoblast behaviour, thus indicating a novel role in muscle repair beyond leucocyte chemoattraction. Therefore, interventions aimed at modulating the balance between myoblast and leucocyte effects of CC chemokines in injured muscle could represent a novel strategy for the treatment of destructive muscle pathologies.

Many fibroblast-secreted proteins promote tumorigenicity, and several factors secreted by cancer cells have in turn been proposed to induce these proteins. It is not clear whether there are single dominant pathways underlying these interactions or whether they involve multiple pathways acting in parallel. Here, we identified 42 fibroblast-secreted factors induced by breast cancer cells using comparative genomic analysis. To determine what fraction was active in promoting tumorigenicity, we chose five representative fibroblast-secreted factors for in vivo analysis. We found that the majority (three out of five) played equally major roles in promoting tumorigenicity, and intriguingly, each one had distinct effects on the tumor microenvironment. Specifically, fibroblast-secreted amphiregulin promoted breast cancer cell survival, whereas the chemokine CCL7 stimulated tumor cell proliferation while CCL2 promoted innate immune cell infiltration and angiogenesis. The other two factors tested had minor (CCL8) or minimally (STC1) significant effects on the ability of fibroblasts to promote tumor growth. The importance of parallel interactions between fibroblasts and cancer cells was tested by simultaneously targeting fibroblast-secreted amphiregulin and the CCL7 receptor on cancer cells, and this was significantly more efficacious than blocking either pathway alone. We further explored the concept of parallel interactions by testing the extent to which induction of critical fibroblast-secreted proteins could be achieved by single, previously identified, factors produced by breast cancer cells. We found that although single factors could induce a subset of genes, even combinations of factors failed to induce the full repertoire of functionally important fibroblast-secreted proteins. Together, these results delineate a complex network of tumor-fibroblast interactions that act in parallel to promote tumorigenicity and suggest that effective anti-stromal therapeutic strategies will need to be multi-targeted.

CCL2 (MCP-1) has been shown to enhance HIV-1 replication. The expression of this chemokine by macrophages is up-modulated as a consequence of viral infection or gp120 exposure. In this study, we show for the first time that the phosphatidylcholine-specific phospholipase C (PC-PLC) is required for the production of CCL2 triggered by gp120 in human monocyte-derived macrophages (MDMs). Using a combination of pharmacologic inhibition, confocal laser-scanner microscopy, and enzymatic activity assay, we demonstrate that R5 gp120 interaction with CCR5 activates PC-PLC, as assessed by a time-dependent modification of its subcellular distribution and a concentration-dependent increase of its enzymatic activity. Furthermore, PC-PLC is required for NF-kB-mediated CCL2 production triggered by R5 gp120. Notably, PC-PLC activation through CCR5 is specifically induced by gp120, since triggering CCR5 through its natural ligand CCL4 (MIP-1beta) does not affect PC-PLC cellular distribution and enzymatic activity, as well as CCL2 secretion, thus suggesting that different signaling pathways can be activated through CCR5 interaction with HIV-1 or chemokine ligands. The identification of PC-PLC as a critical mediator of well-defined gp120-mediated effects in MDMs unravels a novel mechanism involved in bystander activation and may contribute to define potential therapeutic targets to block Env-triggered pathologic responses.

OBJECTIVE

These studies investigated cytokine and chemokine receptor profiles in nucleus pulposus (NP) cells, and the effects of receptor stimulation on mRNA levels of extracellular matrix (ECM) components, degrading enzymes and cytokine and chemokine expression.

METHODS

Immunohistochemistry (IHC) was performed to localise expression of CD4, CCR1, CXCR1 and CXCR2 in human NP tissue samples. Effects of cytokine and chemokine stimulation was performed to investigate effects related to ECM remodelling and modulation of cytokine and chemokine mRNA expression.

RESULTS

IHC identified CD4, CCR1, CXCR1 and CXCR2 expression by NP cells. Differential expression profiles were observed for CD4 and CXCR2 in tissue samples from degenerate and infiltrated IVDs. In vitro stimulations of primary human NP cultures with IL-16, CCL2, CCL3, CCL7 or CXCL8 did not identify any modulatory effects on parameters associated with ECM remodelling or expression of other cytokines and chemokines. Conversely, IL-1 was seen to modulate ECM remodelling and expression of all other cytokines and chemokines investigated.

CONCLUSIONS

This study demonstrates for the first time that NP cells express a number of cytokine and chemokine receptors and thus could respond in an autocrine or paracrine manner to cytokines and chemokines produced by NP cells, particularly during tissue degeneration. However, this study failed to demonstrate regulatory effects on ECM genes and degradative enzymes or other cytokines and chemokines for any target investigated, with the exception of IL-1. This suggests that IL-1 is a master regulator within the IVD and may exert regulatory potential over a plethora of other cytokines and chemokines.