Therapy With Carboplatin and Anti-PD-1 Antibodies Before Surgery Demonstrates Sustainable Anti-Tumor Effects for Secondary Cancers in Mice With Triple-Negative Breast Cancer.

Patients with triple-negative breast cancer (TNBC) suffer an unfavorable prognosis. Carboplatin (CBDCA) as a cytotoxic reagent has been widely administered to patients with cancer including TNBC. Programmed cell death protein 1 (PD-1) is an immune checkpoint, blockade of which unleashes T cell functions that kill cancer cells. However, the efficacy of CBDCA combined with anti-PD-1 antibodies in TNBC has not been determined. Patient-derived xenografts (PDX) were implanted to immune-deficient mice. Three mouse TNBC cell lines (4T1, EMT6, and E0771) were seeded to immune-competent mice. Tumor volumes and survival rates were monitored. CBDCA and anti-PD-1 antibodies were administered by intra-peritoneal injection at designated time points. Total CD8+ T cells, memory CD8+ T cells, and CD103+ dendritic cells (DC) in the tumor were measured by flow cytometry. Tumor-specific CD8+ T cells were quantified by the ELISpot assay. Administration of CBDCA to PDX-bearing mice induced increased levels of tumor cell necrosis and reduced tumor size. Treatment with CBDCA and anti-PD-1 antibodies reduced TNBC tumor volumes and slightly improved survival rates. More importantly, therapy with CBDCA and anti-PD-1 antibodies before surgery showed a remarkably improved, sustainable protection against a secondary tumor after surgery by a CD8+- T-cell-dependent manner, which required CCL4 expressed in the tumor and subsequently CD103+ DC recruited to the tumor microenvironment. Immunochemotherapy with CBDCA and anti-PD-1 antibodies before surgery improves the outcome of a secondary tumor after surgery via increasing the number of tumor-specific CD8+ T cells in the tumor microenvironment of murine TNBC. These results highlight the possibility to utilize this regimen in clinical practice.

Methylmercury induces the expression of chemokine CCL4 via SRF activation in C17.2 mouse neural stem cells.

Methylmercury is an environmental pollutant that causes specific and serious damage to the central nervous system. We have previously shown that C-C motif chemokine ligand 4 (CCL4) protects cultured neural cells from methylmercury toxicity and expression of CCL4 is specifically induced in mouse brain by methylmercury. In this study, we examined the transcriptional regulatory mechanism that induces CCL4 expression by methylmercury using C17.2 mouse neural stem cells. The promoter region of the CCL4 gene was analyzed by a reporter assay, revealing that the region up to 50 bp upstream from the transcription start site was necessary for inducing expression of CCL4 by methylmercury. Nine transcription factors that might bind to this upstream region and be involved in the induction of CCL4 expression by methylmercury were selected, and the induction of CCL4 expression by methylmercury was suppressed by the knockdown of serum response factor (SRF). In addition, the nuclear level of SRF was elevated by methylmercury, and an increase in the amount bound to the CCL4 gene promoter was also observed. Furthermore, we examined the upstream signaling pathway involved in the induction of CCL4 expression by SRF, and confirmed that activation of p38 and ERK, which are part of the MAPK pathway, are involved. These results suggest that methylmercury induces the expression of CCL4 by activating SRF via the p38 and ERK signaling pathway. Our findings are important for elucidating the mechanism involved in the brain-specific induction of CCL4 expression by methylmercury.

Hypoxia and macrophages promote glioblastoma invasion by the CCL4-CCR5 axis.

Glioblastoma (GBM) is a highly malignant brain tumor characterized by invasion tendency. Macrophage infiltration is associated with GBM invasion, but the mechanisms remain unclear. Hypoxia is an outstanding characteristic of GBM tissue. Hypoxia microenvironment modulates the biological behaviors of both tumor cells and infiltrated immune cells, including macrophages. In the present study, we analyzed the effects of hypoxia and macrophages on invasion of GBM cells and its potential mechanisms. We found that both hypoxia and macrophage supernatant promoted GBM cells invasion and matrix metalloproteinase (MMP)-9 expression, and hypoxia modulated the invasive activity of GBM cells by upregulating their CCR5 expression. The supernatant of hypoxic macrophages also showed greater pro-invasion effect than normoxic macrophages through the elevated secretion of CCL4. Moreover, we found that interferon regulatory factor-8 (IRF-8) was possibly involved in hypoxia-modulated CCL4 expression of macrophages. Taken together, the present study found that macrophages promoted GBM invasion by the CCL4-CCR5 axis, and hypoxia enhanced the interaction between these two types of cells by upregulating both CCL4 and CCR5 expression, respectively. The results of the present study suggested that hypoxia would be a potential target for the development of immune therapies of GBM.

Evaluation of chemokines in gingival crevicular fluid in children with dental caries and stainless steel crowns: A clinico-biochemical study.

AIMS AND OBJECTIVES: The study was conducted to detect the presence of macrophage inflammatory protein-1α (MIP-1α) and MIP-1ß and estimate their levels in gingival crevicular fluid (GCF) in children with dental caries and stainless steel crowns. MATERIALS AND METHODS: A total of 80 children with primary dentition were selected and categorized into four groups with twenty in each group; Group 1 - healthy subjects, Group 2 - dental caries, Group 3 - dental caries involving the pulp, and Group 4 - stainless steel crowns. GCF samples were collected by an extra-crevicular method with microcapillary pipettes. The GCF samples were quantified by ELISA and the levels of MIP-1α and MIP-1ß were determined. RESULTS: MIP-1α and MIP-1ß were detected in all the samples. Highest mean concentration in GCF was obtained for Group 3 followed by Groups 2 and 4 while the lowest concentration was seen in Group 1. This suggests that MIP-1α and MIP-1ß levels in GCF increased proportionately with the inflammation. CONCLUSIONS: GCF serves as a noninvasive diagnostic fluid to measure biomarkers released during dental caries initiation and progression. MIP-1α and MIP-1ß chemokines can be considered as novel biomarkers, in biological mechanism underlying the pathogenesis and inflammation in children with dental caries and stainless steel crowns.

Chemokines and their receptors in lung cancer progression and metastasis.

Lung cancer is the leading cause of cancer-related mortality around the world. Despite advancements in diagnosis, surgical techniques, and neoadjuvant chemoradiotherapy over the last decade, the mortality rate is still high and the 5-year survival is a dismal 15%. Fortunately, early detection by low-dose computed tomography (LDCT) scans has reduced mortality by 20%; yet, overall, 5-year-survival remains low at less than 20%. Therefore, in order to ameliorate this situation, a thorough understanding of the underlying molecular mechanisms is urgently needed. Chemokines and their receptors, crucial microenvironmental factors, play important roles in lung tumor genesis, progression, and metastasis, and exploring the mechanisms of this might bring new insights into early diagnosis and precisely targeted treatment. Consequently, this review will mainly focus on recent advancements on the axes of chemokines and their receptors of lung cancer.

Modulation of Hematopoietic Chemokine Effects In Vitro and In Vivo by DPP-4/CD26.

Dipeptidyl peptidase 4 (DPP4)/CD26 truncates certain proteins, and this posttranslational modification can influence their activity. Truncated (T) colony-stimulating factors (CSFs) are decreased in potency for stimulating proliferation of hematopoietic progenitor cells (HPCs). T-CXCL12, a modified chemokine, is inactive as an HPC chemotactic, survival, and enhancing factor for replating or ex-vivo expansion of HPCs. Moreover, T-CSFs and T-CXCL12 specifically downmodulates the positively acting effects of their own full-length molecule. Other chemokines have DPP4 truncation sites. In the present study, we evaluated effects of DPP4 inhibition (by Diprotin A) or gene deletion of HPC on chemokine inhibition of multicytokine-stimulated HPC, and on chemokine-enhancing effects on single CSF-stimulated HPC proliferation, as well as effects of DPP4 treatment of a number of chemokines. Myelosuppressive effects of chemokines with, but not without, a DPP4 truncation site were greatly enhanced in inhibitory potency by pretreating target bone marrow (BM) cells with Diprotin A, or by assaying their activity on dpp4/cd26(-/-) BM cells. DPP4 treatment of myelosuppressive chemokines containing a DPP4 truncation site produced a nonmyelosuppressive molecule, but one which had the capacity to block suppression by that unmodified chemokine both in vitro and in vivo. Additionally, DPP4 treatment ablated the single cytokine-stimulated HPC-enhancing activity of CCL3/MIP-1α and CCL4/MIP-1ß, and blocked the enhancing activity of each unmodified molecule, in vitro and in vivo. These results highlight the functional posttranslational modulating effects of DPP4 on chemokine activities, and information offering additional biological insight into chemokine regulation of hematopoiesis.

Inflammatory and antioxidant pattern unbalance in "clopidogrel-resistant" patients during acute coronary syndrome.

BACKGROUND: In acute coronary syndrome (ACS), inflammation and redox response are associated with increased residual platelet reactivity (RPR) on clopidogrel therapy. We investigated whether clopidogrel interaction affects platelet function and modulates factors related to inflammation and oxidation in ACS patients differently responding to clopidogrel. MATERIAL AND METHODS: Platelet aggregation was measured in 29 ACS patients on dual (aspirin/clopidogrel) antiplatelet therapy. Nonresponders (NR) were defined as RPR ≥70% by ADP. Several inflammatory and redox parameters were assayed and platelet proteome was determined. RESULTS: Eight (28%) out of 29 ACS patients resulted NR to clopidogrel. At 24 hours, the levels of Th2-type cytokines IL-4, IFNγ, and MCP-1 were higher in NR, while blood GSH (r-GSHbl) levels were lower in NR than responders (R). Proteomic analysis evidenced an upregulated level of platelet adhesion molecule, CD226, and a downregulation of the antioxidant peroxiredoxin-4. In R patients the proinflammatory cytokine IL-6 decreased, while the anti-inflammatory cytokine IL-1Ra increased. CONCLUSIONS: In patients with high RPR on clopidogrel therapy, an unbalance of inflammatory factors, platelet adhesion molecules, and circulatory and platelet antioxidant molecules was observed during the acute phase. Proinflammatory milieu persists in nonresponders for a long time after the acute event while antioxidant blood factors tend to conform to normal responsiveness.

[Cytokines and myeloma bone disease].

Multiple myeloma (MM) develops and expands almost exclusively in the bone marrow, and generates devastating bone destruction. MM cells produce a variety of cytokines to stimulate RANK ligand-mediated osteoclastogenesis and suppress osteoblastic differentiation from bone marrow stromal cells, leading to extensive bone destruction with rapid loss of bone. MM cells alter through bone destruction the microenvironment in bone where they colonize, which in turn favors tumor growth and survival, thereby forming a progressive vicious cycle between tumor expansion and bone destruction in MM.

Placental Hofbauer cells limit HIV-1 replication and potentially offset mother to child transmission (MTCT) by induction of immunoregulatory cytokines.

BACKGROUND: Despite readily detectable levels of the HIV-1 (co)-receptors CD4, CCR5 and DC-SIGN on placental macrophages (Hofbauer Cells [HCs]), the rate of HIV-1 infection in utero in the absence of interventions is only 7% of exposed infants. Here, we examine the replication kinetics of human HCs to the primary isolate HIV-1BaL. We also determined the infectivity of HIV-1-exposed HCs by co-culturing with isolated cord and peripheral blood mononuclear cells [CBMCs, PBMCs]. To understand the limiting nature of HCs to HIV-1 replication, we examined the effect of endogenously secreted cytokines on replication kinetics. RESULTS: HCs have reduced ability to replicate HIV-1 in vitro (p < 0.01) and to transmit virus to CBMCs and PBMCs (p < 0.001 for both) compared to standard infections of MDMs. HCs were shown to release HIV-1 particles at levels comparable to MDMs, however exhibit significant decreases in viral transcription (gag and env), which may account for lower levels of HIV-1 replication. Un-stimulated HCs constitutively express significantly higher levels of regulatory cytokines, IL-10 and TGF-ß, compared to MDMs (p < 0.01), which may contribute to immunoregulatory predominance at the placenta and possibly account for down-regulation of HIV-1 replication and infectivity by HCs. We further demonstrate that these regulatory cytokines inhibit HIV-1 replication within HCs in vitro. CONCLUSION: HCs have reduced ability to replicate and disseminate R5-tropic HIV-1BaLin vitro and potentially offset mother to child transmission (MTCT) of HIV-1 by the induction of immunoregulatory cytokines. Despite the potential for migration and infectivity, HCs are not present in the neighboring fetal circulation. These results implicate HCs as important mediators of protection at the feto-maternal interface during ongoing HIV-1 exposure.

Bone marrow-derived monocyte lineage cells recruited by MIP-1ß promote physiological revascularization in mouse model of oxygen-induced retinopathy.

Recent clinical observations have indicated that vascular endothelial growth factor (VEGF) is a key factor that stimulates the development of preretinal pathological neovascularization (NV). However, it has not been established how intraretinal physiological revascularization of hypoxic avascular areas is regulated. Our earlier study on the gene expression profile of hypoxic retinas in a mouse model of oxygen-induced retinopathy (OIR) showed that macrophage inflammatory protein-1ß (MIP-1ß) was the most upregulated protein. The purpose of this study was to investigate the role played by MIP-1ß in recruiting bone marrow-derived monocyte lineage cells (BM-MLCs) in a mouse model of OIR. Our results showed that MIP-1ß was upregulated, and its receptor, CCR5, was expressed in BM-MLCs in the hypoxic inner retina. Neutralizing Ab against MIP-1ß reduced the infiltration of BM-MLCs into the OIR retinas and increased the avascular area and preretinal neovascular tufts. A very strong significant correlation was found between the area of the preretinal neovascular tufts and the avascular area, regardless of the extent of BM-MLC infiltration into the OIR retinas. Additional treatment with VEGF-A-neutralizing Ab showed that the MIP-1ß-regulated pathological NV strongly depended on VEGF-A, which was probably secreted by the hypoxic avascular retinas. These results indicate that MIP-1ß is involved in the recruitment of BM-MLCs, which have a significant role in the physiological revascularization of hypoxic avascular retinas. Overall, these findings indicate that the MIP-1ß induction of BM-MLCs might possibly be used to promote intraretinal revascularization and thus prevent the abnormal NV in ischemic vision-threatening retinal diseases.

Cytokine responses and inducible nitrous oxide synthase expression patterns in neonatal chicken brain microglia infected with very virulent Marek's disease virus strain YL040920.

Purified and enriched brain microglia from neonatal chickens were infected with live Marek's disease virus (MDV)-both the very virulent (vv) YL040920 strain and the attenuated vaccine strain CVI988/Rispens in vitro. Although YL040920-infected microglia showed lower viral DNA loads compared with those infected with CVI988/Rispens at the same infectious dose (400 plaque-forming units for each), no significant differences in IFN-γ and IL-12p35 transcription were detected between the two MDV strains. Chicken microglia infected with live or fixed YL040920 expressed dramatically higher levels of IL-12p40, IL-8, and macrophage inflammatory protein-1ß (MIP-1ß) transcripts compared with those infected with CVI988/Rispens. On the other hand, CVI988/Rispens induced significantly higher levels of IFN-ß transcription than YL040920, especially the live virus. Inducible nitric oxide (NO) synthase (iNOS) transcription and NO production correlated with levels of both YL040920 and CVI988/Rispens live strain infection. Moreover, fixed MDVs induced higher levels of iNOS/NO than live viruses, especially with CVI988/Rispens. This study demonstrates that chicken microglial cells can become infected with live YL040920 and CVI988/Rispens and that microglia represent cellular sources of IL-12p40, IL-12p35, IFN-γ, IFN-ß, IL-8, MIP-1ß, iNOS mRNA, and NO expression after MDV infection in vitro. Transcription levels of IL-12p35 and IFN-γ were associated with MDV DNA replication, whereas transcription levels of IL-12p40, IFN-ß, IL-8, and MIP-1ß were associated with both MDV DNA replication and expression of viral specific genes. The transcription of iNOS was responsible for expression of viral specific genes, whereas it was suppressed by viral DNA replication during infection. Although YL040920, compared with CVI988/Rispens, induced similar levels of the typical Th1-type cytokine IFN-γ in microglia, vvMDV induced significant increases in other cytokines [IL-12 (p40 and 12p35), IL-8, and MIP-1ß]. More detailed investigation, as well as in vivo testing of the effects of vvMDV infection on Th1 responses, iNOS expression, and NO production in the brain of chickens should be undertaken.

Across-line SNP association study for direct and associative effects on feather damage in laying hens.

An association study between SNP markers and feather condition score on the back, rump and belly of laying hens was performed. Feather condition score is a measure of feather damage, which has been shown to be closely related to feather pecking behaviour in hens housed in groups. A population of 662 hens was genotyped for 1536 SNPs of which 1022 could be used for the association study. The analysis was conducted across 9 different lines of White Leghorn and Rhode Island Red origin. Across lines linkage disequilibrium is conserved at shorter distances than within lines; therefore, SNPs significantly associated with feather condition score across lines are expected to be closer to the functional mutations. The SNPs that had a significant across-line effect but did not show significant SNP-by-line interaction were identified, to test that the association was consistent across lines. Both the direct effect of the individual's genotype on its plumage condition, and the associative effect of the genotype of the cage mates on the individual's plumage condition were analysed. The direct genetic effect can be considered as the susceptibility to be pecked at, whereas the associative genetic effect can be interpreted as the propensity to perform feather pecking. Finally, 11 significant associations between SNPs and behavioural traits were detected in the direct model, and 81 in the associative model. A role of the gene for the serotonin receptor 2C (HTR2C) on chromosome 4 was found. This supports existing evidence of a prominent involvement of the serotonergic system in the modulation of this behavioural disorder in laying hens. The genes for IL9, IL4, CCL4 and NFKB were found to be associated to plumage condition, revealing relationships between the immune system and behaviour.

[Mechanisms for formation of myeloma bone disease].

Myeloma cells stimulate bone resorption by enhancing osteoclast formation and suppress bone formation by inhibiting osteoblast differentiation. Macrophage inflammatory protein (MIP)-1alpha and MIP-1beta as well as RANK ligand play a major role in the enhancement of bone resorption in myeloma. Myeloma cell-derived soluble Wnt inhibitors as well as TGF-beta released from the bone tissues through enhanced bone resorption are thought to suppress osteoblast differentiation. Such pathognomonically skewed cellular components in the bone marrow create a microenvironment suitable for myeloma cell growth and survival (a myeloma niche) , which should be targeted to suppress myeloma expansion along with amelioration of bone lesions.