Optimal conditions for adenoviral transduction of immature dendritic cells without affecting the tolerogenic activity of DC-based immunotherapy.

Dendritic cells (DCs) play a pivotal role in maintaining immune tolerance. Using recombinant adenovirus (rAd) to deliver vectors to immature dendritic cells (imDCs) is an important method for studying the tolerogenic function of DCs. We found that using RPMI medium and a higher MOI during transduction increased the expression of CD80, CD86, and MHC-II on the surface of imDCs. Our data reveal a significant increase in the secretion of the pro-inflammatory cytokine IL-6 in the group showing the most pronounced phenotypic changes. In the mouse heart transplant model, imDCs with unstable phenotype and function due to adenoviral transduction resulted in an increased proportion of Th1 and Th17 cells in recipients. However, these effects can be managed, and our proposed optimized transduction strategy significantly minimizes these adverse effects. Our study holds significant implications for the development and optimization of immunotherapy utilizing tolerogenic dendritic cells.

An integrated in silico-in vitro investigation to assess the skin sensitization potential of 4-Octylphenol.

One of the major challenges in chemical toxicity testing is the possibility to protect human health against adverse effects with non-animal methods. In this paper, 4-Octylphenol (OP) was tested for skin sensitization and immunomodulatory effects using an integrated in silico-in vitro test approach. In silico tools (QSAR TOOLBOX 4.5, ToxTree and VEGA) were used together with several in vitro tests including HaCaT cells (quantification of IL-6; IL-8; IL-1α and IL-18 by ELISA and expression of genes TNF, IL1A, IL6 and IL8 by RT- qPCR), RHE model (quantification of IL-6; IL-8; IL-1α and IL-18 by ELISA) and THP-1 activation assay (CD86/CD54 expression and IL-8 release). Additionally, the immunomodulatory effect of OP was investigated using lncRNAs MALAT1 and NEAT1 expression and LPS-induced THP-1 activation (CD86/CD54 expression and IL-8 release). The in silico tools predicted OP as a sensitizer. In vitro tests are also concordant with the in silico prediction. OP increased IL-6 expression (HaCaT cells); IL-18 and IL-8 expressions (RHE model). An irritant potential was also shown by a great expression of IL-1α (RHE model); and increased expression of CD54 marker and IL-8 in THP-1 cells. Immunomodulatory effects of OP were demonstrated by the downregulation of NEAT1, MALAT1 (epigenetic markers), IL6 and IL8; and an increase in LPS-induced CD54 and IL-8 expressions. Overall, results indicate that OP is a skin sensitizer, being positive in three key events of the AOP for skin sensitization, also showing immunomodulatory effects.

TRAM deletion attenuates monocyte exhaustion and alleviates sepsis severity.

Monocyte exhaustion characterized by immune-suppressive features can develop during sepsis and contribute to adverse patient outcomes. However, molecular mechanisms responsible for the establishment of immune-suppressive monocytes with reduced expression of immune-enhancing mediators such as CD86 during sepsis are not well understood. In this study, we identified that the TLR4 intracellular adaptor TRAM plays a key role in mediating the sustained reduction of CD86 expression on exhausted monocytes and generating an immune-suppressive monocyte state. TRAM contributes to the prolonged suppression of CD86 through inducing TAX1BP1 as well as SARM1, collectively inhibiting Akt and NFκB. TRAM deficient mice are protected from cecal slurry-induced experimental sepsis and retain immune-competent monocytes with CD86 expression. Our data reveal a key molecular circuitry responsible for monocyte exhaustion and provide a viable target for rejuvenating functional monocytes and treating sepsis.

Therapeutic gene editing of T cells to correct CTLA-4 insufficiency.

Heterozygous mutations in CTLA-4 result in an inborn error of immunity with an autoimmune and frequently severe clinical phenotype. Autologous T cell gene therapy may offer a cure without the immunological complications of allogeneic hematopoietic stem cell transplantation. Here, we designed a homology-directed repair (HDR) gene editing strategy that inserts the CTLA-4 cDNA into the first intron of the CTLA-4 genomic locus in primary human T cells. This resulted in regulated expression of CTLA-4 in CD4+ T cells, and functional studies demonstrated CD80 and CD86 transendocytosis. Gene editing of T cells isolated from three patients with CTLA-4 insufficiency also restored CTLA-4 protein expression and rescued transendocytosis of CD80 and CD86 in vitro. Last, gene-corrected T cells from CTLA-4-/- mice engrafted and prevented lymphoproliferation in an in vivo murine model of CTLA-4 insufficiency. These results demonstrate the feasibility of a therapeutic approach using T cell gene therapy for CTLA-4 insufficiency.

The Role of CD40, CD86, and Glutathione S-Transferase Omega 1 in the Pathogenesis of Chronic Obstructive Pulmonary Disease.

Objective: The aim of the study was to explore the relevance of CD40, CD86, and GSTO1 with the pathogenesis of COPD. Methods: Patients with acute exacerbation of COPD were contrasted with the healthy and nonsmoking ones and smoking but without COPD ones. The changes of CD40, CD86, and GSTO1 in the peripheral blood, collected from different groups, were detected by flow cytometry and western blotting, respectively. Results: Compared with the nonsmoking group and smoking but without the COPD group, the expression of CD40 and CD86 of the patients with COPD increased significantly, but the expression of GSTO1 decreased. CD40 and CD86 were negatively correlated with FEV1%, while GSTO1 was positively correlated with FEV1% and negatively correlated with CD40 and CD86. Conclusion: CD40, CD86, and GSTO1 may play a role in the pathogenesis of COPD, and they are related to the severity of COPD and the degree of changes in the lung function.

PDZ Proteins SCRIB and DLG1 Regulate Myeloma Cell Surface CD86 Expression, Growth, and Survival.

Despite advances in the treatment of multiple myeloma in the past decades, the disease remains incurable, and understanding signals and molecules that can control myeloma growth and survival are important for the development of novel therapeutic strategies. One such molecule, CD86, regulates multiple myeloma cell survival via its interaction with CD28 and signaling through its cytoplasmic tail. Although the CD86 cytoplasmic tail has been shown to be involved in drug resistance and can induce molecular changes in multiple myeloma cells, its function has been largely unexplored. Here, we show that CD86 cytoplasmic tail has a role in trafficking CD86 to the cell surface. This is due in part to a PDZ-binding motif at its C-terminus which is important for proper trafficking from the Golgi apparatus. BioID analysis revealed 10 PDZ domain-containing proteins proximal to CD86 cytoplasmic tail in myeloma cells. Among them, we found the planar cell polarity proteins, SCRIB and DLG1, are important for proper CD86 surface expression and the growth and survival of myeloma cells. These findings indicate a mechanism by which myeloma cells confer cellular survival and drug resistance and indicate a possible motif to target for therapeutic gain. IMPLICATIONS: These findings demonstrate the importance of proper trafficking of CD86 to the cell surface in myeloma cell survival and may provide a new therapeutic target in this disease.

Aqueous extract of Passiflora alata leaves modulates in vitro the indoleamine 2,3-dioxygenase (IDO) and CD86 expression in bone marrow-derived professional antigen-presenting cells polarizing NOD mice T cells to a Treg profile.

Dendritic cells (DCs) and macrophages are professional antigen-presenting cells (pAPCs), numerous in the pancreas of nonobese diabetic (NOD) mice and playing an essential role in the autoimmune response of type 1 diabetes. The expression of the enzyme indoleamine 2,3-dioxygenase (IDO) is a critical factor for the tolerogenic activity of pAPCs, acting in the catabolism of tryptophan, providing metabolites that suppress the T cell effectors and induce T regulatory cells differentiation. Here we investigated the in vitro mechanisms of lyophilized aqueous extract from Passiflora alata leaves (LAEPAL) that modulates bone marrow-derived professional antigen-presenting cells (BM-pAPCs), affecting their ability to polarize T cells. A cell culture model was defined using mixed cultures of BM-pAPCs and T lymphocytes NOD mice with stressed MIN-6 cells as a source of pancreatic ß cells antigens. We showed that the treatment with 300 µg/mL of LAEPAL induces a significant decrease in the CD4 and CD8 T effector lymphocytes proliferation from diabetic but not in non-diabetic mice, followed by a reduction of the IL-6 and IFN-γ cytokines release in the cell cultures supernatants. Moreover, we observed an increase of CD4+CD25+FoxP3+ Tregs in the cell cultures from diabetic mice. These results could be partially explained by the LAEPAL modulatory effects in BM-pAPCs, downregulating the CD86 co-stimulatory molecule expression, and increasing IDO-1 expression in F4/80+ BM-pAPCs. These results contribute to a better understanding of the polyphenols' immunomodulatory properties, meaning they could induce tolerogenic antigen-presenting cells, which could polarize T cells to a Treg profile and decrease the activity of CD4+ and CD8+ T effector cells.

ß-Glucans from Trametes versicolor (L.) Lloyd Is Effective for Prevention of Influenza Virus Infection.

Coriolus versicolor (C. versicolor) is a higher fungi or mushroom which is now known by its accepted scientific names as Trametes versicolor (L.) Lloyd. Many studies have shown that ß-glucans from C. versicolor have various physiological activities, including activating macrophages to protect against Salmonella infection. However, whether ß-glucans have antiviral effects has not been reported. Hence, the objective of this study was to confirm whether ß-glucans could boost the immune response to combat influenza virus in mouse and chick models. The results show that ß-glucans induced the expression of Dectin-1, costimulatory molecules (CD80/86) and cytokines IL-6, IL-1ß, IFN-ß and IL-10 in murine bone marrow dendritic cells (BMDCs). In addition, orally administered ß-glucans reduced weight loss, mortality and viral titers in the lungs of mice infected with influenza virus and attenuated pathological lung damage caused by the virus in the mice. Orally administered ß-glucans improved survival and reduced lung viral titers in chickens infected with H9N2 avian influenza virus. These results suggest that ß-glucans have a significant antiviral effect. Therefore, ß-glucans could become a potential immunomodulator against influenza virus.

Control of Memory Phenotype T Lymphocyte Homeostasis: Role of Costimulation.

Foxp3+ T regulatory cells (Tregs), CD4+Foxp3- T cells, and CD8+ T cells are composed of naive phenotype (NP) and memory phenotype (MP) subsets. Ten to 20% of each MP T cell population are cycling (Ki-67+) in vivo. We investigated the contribution of costimulatory (CD28) and coinhibitory (CTLA-4, PD-1) receptors on MP T cell homeostatic proliferation in vivo in the mouse. Blockade of CD28-CD80/CD86 signaling completely abolished MP Tregs and profoundly inhibited MP CD4+Foxp3- T cell proliferation, but it did not affect MP CD8+ T cell proliferation. Marked enhancement of homeostatic proliferation of MP Tregs and MP CD4+Foxp3- T cells was seen after blocking CTLA4-CD80/CD86 interactions and PD-1-PD-L1/2 interactions, and greater enhancement was seen with blockade of both pathways. The CD28 pathway also played an important role in the expansion of Tregs and MP T cells after treatment of mice with agonistic Abs to members of the TNF receptor superfamily, which can act directly (anti-GITR, anti-OX40, anti-4-1BB) or indirectly (anti-CD40) on T cells. Induction of a cytokine storm by blocking the interaction of NK inhibitory receptors with MHC class I had no effect on Treg homeostasis, enhanced MP CD4+ proliferation, and expansion in a CD28-dependent manner, but it enhanced MP CD8+ T cell proliferation in a CD28-independent manner. Because MP T cells exert potent biologic effects primarily before the induction of adaptive immune responses, these findings have important implications for the use of biologic agents designed to suppress autoimmune disease or enhance T effector function in cancer that may have negative effects on MP T cells.

Upregulation of CD86 and IL-12 by rhododendrol in THP-1 cells cocultured with melanocytes through ROS and ATP.

BACKGROUND: The tyrosinase inhibitor rhododendrol (RD), used as a skin whitening agent, reportedly has the potential to induce leukoderma. OBJECTIVE: Although an immune response toward melanocytes was demonstrated to be involved in leukoderma, the molecular mechanism is not fully understood. METHODS: We hypothesized that if RD is a pro-hapten and tyrosinase-oxidized RD metabolites are melanocyte-specific sensitizers, the sensitizing process could be reproduced by the human cell line activation test (h-CLAT) cocultured with melanocytes (h-CLATw/M) composed of human DC THP-1 cells and melanoma SK-MEL-37 cells. Cell surface expression, ROS generation and ATP release, mRNA expression, and the effects of several inhibitors were examined. RESULTS: When RD was added to the h-CLATw/M, the expression of cell-surface CD86 and IL-12 mRNA was greatly enhanced in THP-1 cells compared with those in the h-CLAT. The rapid death of melanoma cells was induced, with ROS generation and ATP release subsequently being greatly enhanced, resulting in the cooperative upregulation of CD86 and IL-12. Consistent with those observations, an ROS inhibitor, ATP receptor P2X7 antagonist, or PERK inhibitor antagonized the upregulation. CD86 upregulation was similarly observed with another leukoderma-inducible tyrosinase inhibitor, raspberry ketone, but not with the leukoderma noninducible skin-whitening agents ascorbic acid and tranexamic acid. CONCLUSION: RD is a pro-hapten sensitizer dependent on tyrosinase that induces ROS generation and ATP release from melanocytes for CD86 and IL-12 upregulation in DCs, possibly leading to the generation of tyrosinase-specific cytotoxic T lymphocytes. The coculture system h-CLATw/M may be useful for predicting the sensitizing potential to induce leukoderma.

CD40 and CD80/86 signaling in cDC1s mediate effective neoantigen vaccination and generation of antigen-specific CX3CR1+ CD8+ T cells.

The use of tumor mutation-derived neoantigen represents a promising approach for cancer vaccines. Preclinical and early phase human clinical studies have shown the successful induction of tumor neoepitope-directed responses; however, overall clinical efficacy of neoantigen vaccines has been limited. One major obstacle of this strategy is the prevailing lack of sufficient understanding of the mechanism underlying the generation of neoantigen-specific CD8+ T cells. Here, we report a correlation between antitumor efficacy of neoantigen/toll-like receptor 3 (TLR3)/CD40 agonists vaccination and an increased frequency of circulating antigen-specific CD8+ T cells expressing CX3C chemokine receptor 1 (CX3CR1) in a preclinical model. Mechanistic studies using mixed bone marrow chimeras identified that CD40 and CD80/86, but not CD70 signaling in Batf3-dependent conventional type 1 dendritic cells (cDC1s) is required for the antitumor efficacy of neoantigen vaccine and generation of neoantigen-specific CX3CR1+ CD8+ T cells. Although CX3CR1+ CD8+ T cells exhibited robust in vitro effector function, in vivo depletion of this subset did not alter the antitumor efficacy of neoantigen/TLR3/CD40 agonists vaccination. These findings indicate that the vaccine-primed CX3CR1+ subset is dispensable for antitumor CD8+ T cell responses, but can be used as a blood-based T-cell biomarker for effective priming of CD8+ T cells as post-differentiated T cells. Taken together, our results reveal a critical role of CD40 and CD80/86 signaling in cDC1s in antitumor efficacy of neoantigen-based therapeutic vaccines, and implicate the potential utility of CX3CR1 as a circulating predictive T-cell biomarker in vaccine therapy.

Characterisation of dendritic cell frequency and phenotype in bovine afferent lymph reveals kinetic changes in costimulatory molecule expression.

The bovine afferent lymphatic cannulation model allows collection of large volumes of afferent lymph and provides an opportunity to study lymphatic cells trafficking from the periphery directly ex-vivo. The technique requires surgical intervention, but influence of the procedure or time post-surgery on cells trafficking in the lymph has not been well documented. Here, we measured the volume of lymph and number of cells/mL collected daily over a two week time-course. Animal to animal variability was demonstrated but no consistent changes in lymph volume or cell density were observed in relation to time post-cannulation. Cell populations (dendritic cells, αß T-cells, γδ T-cells and NK cells) were analysed by flow cytometry at 1, 3 and 10 days post-cannulation (DPC) and a reduced percentage of γδ T-cells in afferent lymph was observed at 1 DPC. In addition, cell surface molecule expression by afferent lymphatic dendritic cells (ALDC) was assessed due to the key role of these cells in initiating an adaptive immune response. Co-stimulatory molecules CD80 and CD86 were upregulated by CD172a+ve ALDC early in the time-course, suggesting that the cannulation procedure and duration of experiment may impact the activation state of DCs in the naïve host. This should be considered when analysing the response of these cells to vaccines or pathogens.

Clinicopathological significance of CD28 overexpression in adult T-cell leukemia/lymphoma.

CD28, one of the costimulatory molecules, has a pivotal role in T-cell activation, and its expression is strictly regulated in normal T cells. Gain-of-function genetic alterations involving CD28 have been frequently observed in adult T-cell leukemia/lymphoma (ATLL). These abnormalities, such as CD28 fusions and copy number variations, may not only confer continuous, prolonged, and enhanced CD28 signaling to downstream pathways but also induce overexpression of the CD28 protein. In this study, 120 ATLL cases were examined by immunohistochemistry for CD28 and its ligands CD80 and CD86, and their expression on tumor cells was semiquantitatively evaluated. CD28 was overexpressed in 55 (46%) cases, and CD80 or CD86 (CD80/CD86) was infrequently overexpressed in 12 (11%). Compared with non-overexpressers, CD28 overexpressers showed a higher frequency of CD28 genetic alterations and had an increased number of CD80/CD86-positive non-neoplastic cells infiltrating tumor microenvironment. In the entire ATLL patient cohort, CD28 overexpressers showed a significantly poorer overall survival (OS) compared with non-overexpressers (P = .001). The same was true for a subgroup who were treated with multidrug regimens with or without mogamulizumab. CD28 overexpression had no prognostic impact in the group who received allogeneic hematopoietic stem cell transplantation. In the multivariate analysis for OS, CD28 overexpression was selected as an independent risk factor. These results suggest ATLL patients with CD28 overexpression have more aggressive clinical course and are more refractory to treatment with multidrug chemotherapy. CD28 overexpression appears to be a novel unfavorable prognostic marker in ATLL patients, and further prospective studies are warranted to establish its prognostic significance.

High Glucose Concentrations Impair the Processing and Presentation of Mycobacterium tuberculosis Antigens In Vitro.

Type 2 diabetes is an established risk factor for tuberculosis, but the underlying mechanisms are largely unknown. We established an in vitro model to analyze the effect of high glucose concentrations in antigen processing and presentation in antigen-presenting cells. Human monocyte-derived macrophages (MDMs) were exposed to high (11 mM and 30 mM) and low (5.5 mM) glucose concentrations and infected with Mycobacterium tuberculosis (Mtb). Flow cytometry was used to analyze the effect of high glucose concentrations in histocompatibility complex (MHC) class II molecules (HLA-DR) and co-stimulatory molecules (CD80 and CD86), indispensable for an adequate antigenic presentation and CD4+ T cell activation. HLA-DR and CD86 were significantly decreased by high glucose concentrations compared with low glucose concentrations. Confocal microscopy was used to detect Rab 5 and Lamp-1, proteins involved in the kinetics of antigen processing as early markers, and Rab 7 and cathepsin D as late markers. We observed a delay in the dynamics of the acquisition of Rab 7 and cathepsin D in high glucose concentrations. Moreover, the kinetics of the formation M. tuberculosis peptide-MHC II complexes in MDMs was decreased under high glucose concentrations, reducing their capacity for T cell activation. These findings suggest that high glucose concentrations directly affect antigenic processing, and therefore antigenic presentation.

Development of Exhausted Memory Monocytes and Underlying Mechanisms.

Pathogenic inflammation and immuno-suppression are cardinal features of exhausted monocytes increasingly recognized in septic patients and murine models of sepsis. However, underlying mechanisms responsible for the generation of exhausted monocytes have not been addressed. In this report, we examined the generation of exhausted primary murine monocytes through prolonged and repetitive challenges with high dose bacterial endotoxin lipopolysaccharide (LPS). We demonstrated that repetitive LPS challenges skew monocytes into the classically exhausted Ly6Chi population, and deplete the homeostatic non-classical Ly6Clo population, reminiscent of monocyte exhaustion in septic patients. scRNAseq analyses confirmed the expansion of Ly6Chi monocyte cluster, with elevation of pathogenic inflammatory genes previously observed in human septic patients. Furthermore, we identified CD38 as an inflammatory mediator of exhausted monocytes, associated with a drastic depletion of cellular NAD+; elevation of ROS; and compromise of mitochondria respiration, representative of septic monocytes. Mechanistically, we revealed that STAT1 is robustly elevated and sustained in LPS-exhausted monocytes, dependent upon the TRAM adaptor of the TLR4 pathway. TRAM deficient monocytes are largely resistant to LPS-mediated exhaustion, and retain the non-classical homeostatic features. Together, our current study addresses an important yet less-examined area of monocyte exhaustion, by providing phenotypic and mechanistic insights regarding the generation of exhausted monocytes.

Antioxidant support to ameliorate the oxaliplatin-dependent microglial alteration: morphological and molecular study.

Oxaliplatin is a third-generation chemotherapy drug mainly used for colorectal cancer treatment. However, it is also known to trigger neuropathy whose underlying neurobiological mechanisms are still under investigation and currently available treatments show limited efficacy. It is now established that neurons are not the only cell type involved in chronic pain and that glial cells, mainly astrocytes and microglia, are involved in the initiation and maintenance of neuropathy. Among all the pathogenetic factors involved in neuropathic pain, an oxaliplatin-dependent oxidative stress plays a predominant role. In our study, the antioxidant properties of magnesium (Mg), manganese (Mn) and zinc (Zn) salts were evaluated in order to counteract microglial activation induced by oxaliplatin. The antioxidant efficacy of these metals was evaluated by the means of molecular and morphological assays on the BV-2 microglial cell line. Our data clearly show that Mg, Mn and Zn are able to prevent oxaliplatin-dependent microglial alterations by reducing both oxidative and endoplasmic reticulum stress.

Micro/nano topography of selective laser melting titanium inhibits osteoclastogenesis via mediation of macrophage polarization.

Selective laser melting (SLM) titanium (Ti) implants have shown good prospects for personalized clinical application, but further research is necessary to develop stabilized long-term properties. Since surface modification has been proven bioactive for osseointegration, conventional Ti surface treatment technologies, including sandblasting/acid-etching (SLA) and sandblasting/alkali-heating (SAH), were applied to construct micro and micro/nano surfaces. The SAH group with netlike nano-structure topography exhibited appropriate surface roughness and high hydrophilicity, and as expected, the osseointegration capacities in vivo of the three groups were in order of SAH > SLA > SLM. Besides, both in vivo and in vitro studies revealed that the SLA- and SAH-treated SLM Ti implants significantly inhibited osteoclast activity of peri-implants. Considering the close associations between osteoclasts and macrophages, the effects of Ti surface topography on macrophage polarization were detected. The results showed that the SLA- and SAH-treated SLM Ti implants, especially the latter, had the capacity to promote macrophage polarization to the M2 phenotype. Moreover, the cell culture supernatants of M2 macrophages and RAW264.7 cells seeded on SLA- and SAH-treated SLM Ti surfaces had an adverse effect on osteoclastogenesis. Collectively, this study demonstrated that micro/nano topographies of SLM Ti implants were effective for osseointegration promotion, and their inhibition of osteoclastogenesis might be attributed to macrophage polarization. Our findings shed some light on clinical application of SLM Ti implants and also prove a specific association between macrophage polarization and osteoclastogenesis.

Pattern recognition receptor expression and maturation profile of dendritic cell subtypes in human tonsils and lymph nodes.

Dendritic cells (DCs) with capacity of antigen cross-presentation are of key interest for immunotherapy against cancer as they can induce antigen-specific cytotoxic T lymphocyte (CTL) responses. This study describes frequencies of DC subtypes in human tonsils and lymph nodes, and phenotypic aspects that may be targeted by adjuvant measures. From human tonsils and neck lymph nodes, DCs were identified through flow cytometry, and subsets of plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) were investigated. Maturity status was assessed and surface receptors with CTL-promoting potentials were studied. CD123+ pDCs as well as CD1c+, CD141+, and CD1c-CD141- mDCs were detected in tonsils and lymph nodes. Both sites featured a similar presence of DC subsets, with CD123+ pDC being dominant and CD141+ mDCs least frequent. Based on CD80/CD86 expression, all DC subtypes featured a low degree of maturation. Expression of pattern recognition receptors (PRRs) CD206, CD207, DC-SIGN, TLR2, and TLR4, as well as the chemokine receptor XCR1, indicated DC subset-specific receptor profiles. We conclude that tonsils and lymph nodes share common features in terms of DC subset frequency and maturation as well as PRR and XCR1 expression pattern. Our work suggests that both sites may be considered for vaccine deposition in DC-mediated immunotherapy.

The Ratio of CD86+/CD163+ Macrophages Predicts Postoperative Recurrence in Stage II-III Colorectal Cancer.

Tumor-associated macrophages (TAMs) are pivotal for tumor progression and metastasis. We investigated the stromal CD86+TAM/CD163+TAM (CD86/CD163) ratio as a novel prognostic biomarker for stage II-III colorectal cancer (CRC). Two independently clinical cohorts of stage II-III CRC were retrospectively enrolled in this study. TAMs were detected using immunohistochemical staining for CD86 and CD163. The stromal CD86/CD163 ratio was calculated as a prognostic biomarker for recurrence-free survival (RFS) and overall survival (OS). Patients with a low CD86/CD163 ratio had shorter RFS (HR=0.193, p<0.001) and OS (HR=0.180, p<0.001) than patients with a high CD86/CD163 ratio in the training cohort. CD86/CD163 ratio may be an independent predictor for RFS (HR=0.233, p<0.001) and OS (HR=0.224, p<0.001) in the training cohort. We obtained equivalent results in the validation cohort. The CD86/CD163 ratio tends to have better predictive values than tumor stage in the training (AUC: 0.682 vs 0.654, p=0.538) and validation (AUC: 0.697 vs 0.659, p=0.586) cohorts. CD86/CD163 ratio effectively predicts RFS for stage II (HR=0.203, p<0.001) and stage III CRC (HR=0.302, p<0.001). CD86/CD163 ratio also effectively predicts RFS in CRC patients with adjutant chemotherapy (HR=0.258, p<0.001) and without adjutant chemotherapy (HR=0.205, p<0.001). The stromal CD86/CD163 ratio could be used for individual risk assessment of recurrence and mortality for stage II-III CRC. Together with tumor stage, this ratio will aid in the personal treatment.

Characterization and immunomodulatory activity of sulfated galactan from the red seaweed Gracilaria fisheri.

Polysaccharides from the red seaweed Gracilaria fisheri possess many functions, which include antioxidant, antiviral, and antibacterial activities. However, detailed data on their immunomodulatory activities are scarce. Here, we isolated sulfated galactans (SG) from G. fisheri. We found that the predominant SG from G. fisheri, termed SG-1, had an estimated molecular mass of 100 kDa and activated murine J774A.1 macrophages via the dectin-1 signaling pathway. Furthermore, we observed enhancement of nitric oxide (NO) secretion, increased expression of inducible nitric oxide synthase (iNOS) mRNA, and increased mRNA levels of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukins IL-1ß and IL-6 by SG-1 in macrophages. Moreover, there was higher expression of intercellular adhesion molecule 1 (ICAM-1) and co-stimulatory molecules (B7-1 and B7-2) mRNA. Treatment with G. fisheri SG-1 at 50 µg/mL generally achieved or exceeded the pro-inflammatory activities of 100 ng/mL lipopolysaccharide. Our study demonstrates immune-stimulatory activities of G. fisheri SG that may be of value for immune-potentiating treatment in humans or livestock.