CEACAM1 regulates the IL-6 mediated fever response to LPS through the RP105 receptor in murine monocytes.

BACKGROUND: Systemic inflammation and the fever response to pathogens are coordinately regulated by IL-6 and IL-1ß. We previously showed that CEACAM1 regulates the LPS driven expression of IL-1ß in murine neutrophils through its ITIM receptor. RESULTS: We now show that the prompt secretion of IL-6 in response to LPS is regulated by CEACAM1 expression on bone marrow monocytes. Ceacam1-/- mice over-produce IL-6 in response to an i.p. LPS challenge, resulting in prolonged surface temperature depression and overt diarrhea compared to their wild type counterparts. Intraperitoneal injection of a 64Cu-labeled LPS, PET imaging agent shows confined localization to the peritoneal cavity, and fluorescent labeled LPS is taken up by myeloid splenocytes and muscle endothelial cells. While bone marrow monocytes and their progenitors (CD11b+Ly6G-) express IL-6 in the early response (< 2 h) to LPS in vitro, these cells are not detected in the bone marrow after in vivo LPS treatment perhaps due to their rapid and complete mobilization to the periphery. Notably, tissue macrophages are not involved in the early IL-6 response to LPS. In contrast to human monocytes, TLR4 is not expressed on murine bone marrow monocytes. Instead, the alternative LPS receptor RP105 is expressed and recruits MD1, CD14, Src, VAV1 and ß-actin in response to LPS. CEACAM1 negatively regulates RP105 signaling in monocytes by recruitment of SHP-1, resulting in the sequestration of pVAV1 and ß-actin from RP105. CONCLUSION: This novel pathway and regulation of IL-6 signaling by CEACAM1 defines a novel role for monocytes in the fever response of mice to LPS.

Generation of dual specific bivalent BiTEs (dbBIspecific T-cell engaging antibodies) for cellular immunotherapy.

BACKGROUND: Bispecific T-cell engaging antibodies (BiTES), comprising dual anti-CD3 and anti-tumor antigen scFv fragments, are important therapeutic agents for the treatment of cancer. The dual scFv construct for BiTES requires proper protein folding while their small molecular size leads to rapid kidney clearance. METHODS: An intact (150 kDa) anti-tumor antigen antibody to CEA was joined in high yield (ca. 30%) to intact (150 kDa) anti-murine and anti-human CD3 antibodies using hinge region specific Click chemistry to form dual-specific, bivalent BiTES (dbBiTES, 300 kDa). dbBiTEs were tested in vitro by EM, flow cytometry and cell cytoxicity and in vivo by PET tumor imaging and redirected T-cell therapy. RESULTS: The interlocked hinge regions are compatible with a structural model that fits the electron micrographs of 300 kDa particles. Compared to intact anti-CEA antibody, dbBiTES exhibit high in vitro cytotoxicity, high in vivo tumor targeting as demonstrated by PET imaging, and redirected dbBiTE coated T-cells (1 microgram/10 million cells) that kill CEA+ target cells in vivo in CEA transgenic mice. CONCLUSION: dbBiTE redirected T-cell therapy is a promising, efficient approach for targeting and killing cancer cells.

Regulation of adipose tissue T cell subsets by Stat3 is crucial for diet-induced obesity and insulin resistance.

Dysregulated inflammation in adipose tissue, marked by increased proinflammatory T-cell accumulation and reduced regulatory T cells (Tregs), contributes to obesity-associated insulin resistance. The molecular mechanisms underlying T-cell-mediated inflammation in adipose tissue remain largely unknown, however. Here we show a crucial role for signal transducer and activator of transcription 3 (Stat3) in T cells in skewing adaptive immunity in visceral adipose tissue (VAT), thereby contributing to diet-induced obesity (DIO) and insulin resistance. Stat3 activity is elevated in obese VAT and in VAT-resident T cells. Functional ablation of Stat3 in T cells reduces DIO, improves insulin sensitivity and glucose tolerance, and suppresses VAT inflammation. Importantly, Stat3 ablation reverses the high Th1/Treg ratio in VAT of DIO mice that is likely secondary to elevated IL-6 production, leading in turn to suppression of Tregs. In addition, Stat3 in T cells in DIO mice affects adipose tissue macrophage accumulation and M2 phenotype. Our study identifies Stat3 in VAT-resident T cells as an important mediator and direct target for regulating adipose tissue inflammation, DIO, and its associated metabolic dysfunctions.

IRF-1 regulates alternative mRNA splicing of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in breast epithelial cells generating an immunoreceptor tyrosine-based inhibition motif (ITIM) containing isoform.

BACKGROUND: Interferon regulatory factor-1 (IRF-1) is a master regulator of IFN-γ induced gene transcription. Previously we have shown that IRF-1 transcriptionally induces CEACAM1 via an ISRE (Interferon-Stimulated Response Element) in its promoter. CEACAM1 pre-mRNA undergoes extensive alternative splicing (AS) generating isoforms to produce either a short (S) cytoplasmic domain expressed primarily in epithelial cells or as an ITIM-containing long (L) isoform in immune cells. METHODS: The transcriptional and molecular mechanism of CEACAM1 minigenes AS containing promoter ISREs mutations in the breast epithelial, MDA-MB-468, cell line was detected using flow cytometry. In addition, transcriptome sequencing was utilized to determine whether IRF-1 could direct the AS of other genes as well. Tumor xenografts were used to evaluate CEACAM1 isoform expression on the leading edge of breast tumor cells. RESULTS: In the present study, we provide evidence that CEACAM1's promoter and variable exon 7 cross-talk allowing IRF-1 to direct AS events. Transcriptome sequencing shows that IRF-1 can also induce the global AS of genes involved in regulation of growth and differentiation as well as genes of the cytokine family. Furthermore, MDA-MB-468 cells grown as tumor xenografts exhibit an AS switch to the L-isoform of CEACAM1, demonstrating that an in vivo inflammatory milieu is also capable of generating the AS switch, similar to that found in human breast cancers Mol Cancer 7:46, 2008. CONCLUSIONS: The novel AS regulatory activities attributed to IRF-1 indicate that the IFN-γ response involves a global change in both gene transcription and AS in breast epithelial cells.

Development and characterization of DIA 12.3, a fully human intact anti-CEACAM1 monoclonal antibody.

Carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1), a homotypic cell adhesion molecule glycoprotein with apical expression on normal epithelial cells and activated lymphocytes, is overexpressed on many tumors and acts as an inhibitory receptor on NK cells, preventing their killing of CEACAM1 positive tumors. Production of humanized anti-CEACAM1 antibodies to block the inhibitory activity of CEACAM1 for immunotherapy and immunoimaging. Starting from a scFv, a fully human intact anti-CEACAM1 (DIA 12.3) that recognizes the N-terminal domain of CEACAM1 was developed and shown to bind CEACAM1 positive tumor cells and enhanced NK cell killing of CEACAM1 positive targets. DIA 12.3 bound to human neutrophils without activation, indicating they would be safe for human use. DIA 12.3 exhibited some cross-reactivity to CEACAM5, a tumor marker with high sequence homology to the N-terminal domain of CEACAM1. CEACAM1 PET imaging with 64Cu-COTA-DIA 12.3 showed excellent imaging of CEACAM1 positive tumors with reduced binding to CEACAM5 tumors. Based on its immunoinhibitory an immunoimaging activities, DIA 12.3 shows promise for therapeutic studies in man.

Comparison of IL-2-antibody to IL-2-Fc with or without stereotactic radiation therapy in CEA immunocompetent mice with CEA positive tumors.

BACKGROUND: The potent immune effects of interleukin-2 (IL-2) for cancer therapy can be increased by genetic fusion of IL-2 to the Fc domain of an antibody (IL-2-Fc) or tumor targeted by genetic fusion to a whole antibody known as an immunocytokine (ICK). METHODS: An anti-CEA ICK (M5A-IL-2) was compared to an IL-2-Fc fusion protein using tumor therapy and PET imaging in CEA transgenic immunocompetent mice bearing CEA positive colon or breast tumors. Combination with stereotactic radiation therapy (SRT) was performed with either ICK or IL-2-Fc. RESULTS: ICK and IL-2-Fc had comparable antitumor effects in both tumor models, although ICK had higher tumor uptake and slower blood clearance than an IL-2-Fc. Analysis of IFNγ+ /CD8+ and FoxP3+ /CD4+ T cells revealed higher levels of IFNγ-producing CD8+ T cells in ICK treated mice versus more efficient Treg elimination in IL-2-Fc treated mice. No significant or lasting toxicity was detected for either agent. Combination therapies with SRT revealed comparable efficacy and induction of immune memory for both ICK and IL-2-Fc when mice were rechallenged post-therapy. CONCLUSIONS: IL-2-Fc had comparable antitumor efficacy to CEA-targeted M5A-IL-2 ICK, while both fusion proteins induced immune memory when combined with SRT. Differences in the therapeutic mechanisms of both agents were observed.

DNA damage kinetics and apoptosis in ivermectin-treated Chinese hamster ovary cells.

A comet assay was used to analyze DNA damage kinetics in Chinese hamster ovary (CHO-K1) cells induced by antiparasitic ivermectin (IVM) and the IVM-containing technical formulation Ivomec® (IVO; 1% IVM). Cells were treated with 50 µg ml(-1) IVM and IVO for 80 min, washed and re-incubated in antiparasiticide-free medium for 0-24 h until assayed using the single-cell gel electrophoresis assay (SCGE). Cell viability remained unchanged up to 3 h of incubation. After 6 h of treatment, cell survival decreased up to 75% and 79% in IVM- and IVO-treated cultures, respectively, remaining unchanged within 12-24 h after treatment. For both anthelmintics, biphasic behavior in DNA damage occurred during the incubation time. A time-dependent increase of IVM- and IVO-induced DNA damage was observed within 0 to 3 h after pulse treatment, revealed by a progressive decrease of undamaged cells and an increase in slightly damaged and damaged cells. Finally, a time-dependent decrease in IVM- and IVO-induced DNA damage was revealed by a progressive decrease of slightly damaged cells and the absence of damaged cells simultaneously with an increase in the frequency of undamaged cells during the final 18 h of incubation. Flow cytometry analysis revealed that both compounds are able to induce a marked increase in early and late apoptosis. Based on our observations, we could conclude that the decrease in DNA lesions is mostly related to IVM-induced cytotoxicity rather than attributable to a repair process.

Generation of IL-2-Fc-antibody conjugates by click chemistry.

BACKGROUND: Immunocytokines (ICKs) are antibody directed cytokines produced by genetic fusion of an antibody to a cytokine. METHODS: We now show that antibodies conjugated by click chemistry to interleukin-2 (IL-2)-Fc form fully active conjugates, and in one example, equivalent activity to a genetically produced ICK. RESULTS: An IL-2-Fc fusion protein was optimized for click chemistry at hinge cysteines using protein stabilizing IL-2 mutations at Lys35 and Cys125 and Fc hinge mutations at Cys142 and Cys148. The IL-2-Fc fusion protein with K35E and C125S mutations with 3 intact hinge cysteines, designated as IL-2-Fc Par, was selected based on its minimal tendency to aggregate. IL-2-Fc-antibody clicked conjugates retained high IL-2 activity and bound target antigens comparable to parent antibodies. An IL-2-Fc-anti-CEA click conjugate showed comparable anti-tumor activity to an anti-CEA-IL-2 ICK in immunocompetent CEA transgenic mice bearing CEA positive orthotopic breast tumors. Significant increases in IFNγ+ /CD8+ and decreases in FoxP3+ /CD4+ T-cells were found for the clicked conjugate and ICK therapies, suggesting a common mechanism of tumor reduction. CONCLUSION: The production of antibody targeted IL-2 therapy via a click chemistry approach is feasible with comparable activity to genetically produced ICKs with the added advantage of multiplexing with other monoclonal antibodies.

Ex-vivo CS1-OKT3 dual specific bivalent antibody-armed effector T cells mediate cellular immunity against multiple myeloma.

Bispecific T cell engaging antibodies (bsAbs) have emerged as novel and powerful therapeutic agents for redirecting T cells towards antigen-specific tumor killing. The cell surface glycoprotein and SLAM family member, CS1, exhibits stable and high-level expression on malignant plasma cells including multiple myeloma, which is indicative of an ideal target for bsAb therapy. Here, we developed a CS1 bsAb (CS1-dbBiTE) using Click chemistry to conjugate intact anti-CS1 antibody (Elotuzumab) and anti-huOKT3 antibody at their respective hinge regions. Using a cellular therapy approach, human T cells were armed ex-vivo with CS1-dbBiTE prior to examining effector activity. Our data indicates that arming T cells with CS1-dbBiTE induced T cell activation and expansion and subsequent cytotoxic activity against CS1-bearing MM tumors, demonstrated by significant CD107a expression as well as inflammatory cytokine secretion. As expected, CS1-dbBiTE armed T cells showed significantly reduced effector activity in the absence of CS1 expression. Similarly, in MM mouse xenograft studies, armed T cells exhibited effective anti-tumor efficacy highlighted by reduced tumor burden in MM.1S tumor-bearing mice compared to controls. On the basis of these findings, the rationale for CS1 targeting by human T cells armed with CS1-dbBiTE presents a potentially effective therapeutic approach for targeting MM.

Inhibiting Stat3 signaling in the hematopoietic system elicits multicomponent antitumor immunity.

The immune system can act as an extrinsic suppressor of tumors. Therefore, tumor progression depends in part on mechanisms that downmodulate intrinsic immune surveillance. Identifying these inhibitory pathways may provide promising targets to enhance antitumor immunity. Here, we show that Stat3 is constitutively activated in diverse tumor-infiltrating immune cells, and ablating Stat3 in hematopoietic cells triggers an intrinsic immune-surveillance system that inhibits tumor growth and metastasis. We observed a markedly enhanced function of dendritic cells, T cells, natural killer (NK) cells and neutrophils in tumor-bearing mice with Stat3(-/-) hematopoietic cells, and showed that tumor regression requires immune cells. Targeting Stat3 with a small-molecule drug induces T cell- and NK cell-dependent growth inhibition of established tumors otherwise resistant to direct killing by the inhibitor. Our findings show that Stat3 signaling restrains natural tumor immune surveillance and that inhibiting hematopoietic Stat3 in tumor-bearing hosts elicits multicomponent therapeutic antitumor immunity.

Improved Tumor Responses with Sequential Targeted α-Particles Followed by Interleukin 2 Immunocytokine Therapies in Treatment of CEA-Positive Breast and Colon Tumors in CEA Transgenic Mice.

Targeted α-therapy (TAT) delivers high-linear-transfer-energy α-particles to tumors with the potential to generate tumor immune responses that may be augmented by antigen-targeted immunotherapy. Methods: This concept was evaluated in immunocompetent carcinoembryonic antigen (CEA) transgenic mice bearing CEA-positive mammary or colon tumors. Tumors were targeted with humanized anti-CEA antibody M5A labeled with 225Ac for its 10-d half-life and emission of 4 α-particles, as well as being targeted with the immunocytokine M5A-interleukin 2. Results: A dose response (3.7, 7.4, and 11.1 kBq) to TAT only, for orthotopic CEA-positive mammary tumors, was observed, with a tumor growth delay of 30 d and an increase in median survival from 20 to 36 d at the highest dose. Immunocytokine (4 times daily) monotherapy gave a tumor growth delay of 20 d that was not improved by addition of 7.4 kBq of TAT 5 d after the start of immunocytokine. However, TAT (7.4 kBq) followed by immunocytokine 10 d later led to a tumor growth delay of 38 d, with an increase in median survival to 45 d. Similar results were seen for TAT followed by immunocytokine at 5 versus 10 d. When a similar study was performed with subcutaneously implanted CEA-positive MC38 colon tumors, TAT (7.4 kBq) monotherapy gave an increase in median survival from 29 to 42 d. The addition of immunocytokine 10 d after 7.4 kBq of TAT increased median survival to 57 d. Immunophenotyping showed increased tumor-infiltrating interferon-γ-positive, CD8-positive T cells and an increased ratio of these cells to Foxp3-positive, CD4-positive regulatory T cells with sequential therapy. Immunohistochemistry confirmed there was an increase in tumor-infiltrating CD8-positive T cells in the sequential therapy group, strongly suggesting that immunocytokine augmented TAT can lead to an immune response that improves tumor therapy. Conclusion: Low-dose (7.4 kBq) TAT followed by a 4-dose immunocytokine regimen 5 or 10 d later gave superior tumor reductions and survival curves compared with either monotherapy in breast and colon cancer tumor models. Reversing the order of therapy to immunocytokine followed by TAT 5 d later was equivalent to either monotherapy in the breast cancer model.

T-cell surface generation of dual bivalent, bispecific T-cell engaging, RNA duplex cross-linked antibodies (dbBiTERs) for re-directed tumor cell lysis.

BACKGROUND: Genetic engineered Bispecific T-cell engagers (BiTEs) generate potent cytotoxic effects. METHODS: Alternately, click chemistry engineered, dual specific bivalent Bispecific T-cell engaging antibodies (dbBiTEs) on T-cell surfaces can be generated from parent monoclonal antibodies. RESULTS: We show the formation of dbBiTEs on the surface of T-cells along with the introduction of complementary 2'-OMe RNA 32-mer oligonucleotides allowing duplex formation between antibodies, designated as dbBiTERs. dbBiTERs generated in solution from anti-CEA and anti-CD3 OKT3 antibodies retained specific binding to CEA positive versus CEA negative cancer cells and to CD3 positive T-cells comparable to dbBiTEs. When T-cells were precoated with dbBiTEs or dbBiTERs and mixed with CEA positive versus CEA negative cancer cells, similar dose dependent and specific cytotoxicity were observed in redirected cell lysis assays. On-cell generated dbBiTERs exerted potent cytotoxic responses against CEA positive targets and were localized at the cell surface by immuno-gold EM. In addition, we demonstrate that target and T-cells, each coated separately with complementary 2'OMe-RNA-linked antibodies can be cross-linked by RNA duplex formation in vitro to generate redirected cell lysis. CONCLUSION: The facile generation of dbBiTERs with specific cytolytic activity from intact antibodies and their generation on-cell offers a new avenue for antigen specific T-cell therapy.

Isolation and expansion of murine γδ T cells from mouse splenocytes.

Elucidation of the function of gamma delta T cells (γδ T cells) requires robust models that show how γδ T cells are commonly involved in inflammation, since very little is known about the factors that promote and control their development and function. There are few studies of murine γδ T cells primarily because these cells have proven difficult to isolate, expand and characterize. Here, we describe a simple method that utilizes key expansion elements to isolate and expand murine CD4-CD8-CD3+ γδ T cells typically found in secondary lymphoid tissues. Expansion of γδ T cells reached 150-fold by day 8 of culture, depended on exogenous IL-2, αCD3, and αCD28, and supported efficient and reproducible in vitro differentiation. These studies showed high production of cytokines IFNγ and Granzyme B, with the novel finding of IL-24 upregulation as well. Expression analysis of expanded γδ T cells, after treatment with IL-2, revealed high levels of Granzyme B, Granzyme D, and IFNγ. Lactate dehydrogenase (LDH) cytotoxicity assays showed that expanded γδ T cells were effective at inducing >90% cytolysis of murine MC38 colon cancer, E0771 breast cancer, and B16 melanoma cells at 10:1 effector to target ratios. These findings indicated that murine γδ T cells can be successfully isolated, expanded, and used to perform preclinical therapy studies.

Stat3 mediates myeloid cell-dependent tumor angiogenesis in mice.

The underlying molecular mechanisms that cause immune cells, mediators of our defense system, to promote tumor invasion and angiogenesis remain incompletely understood. Constitutively activated Stat3 in tumor cells has been shown to promote tumor invasion and angiogenesis. Therefore, we sought to determine whether Stat3 activation in tumor-associated inflammatory cells has a similar function. We found that Stat3 signaling mediates multidirectional crosstalk among tumor cells, myeloid cells in the tumor stroma, and ECs that contributes to tumor angiogenesis in mice. Myeloid-derived suppressor cells and macrophages isolated from mouse tumors displayed activated Stat3 and induced angiogenesis in an in vitro tube formation assay via Stat3 induction of angiogenic factors, including VEGF and bFGF. Stat3-regulated factors produced by both tumor cells and tumor-derived myeloid cells also induced constitutive activation of Stat3 in tumor endothelium, and inhibiting Stat3 in ECs substantially reduced in vitro tumor factor-induced endothelial migration and tube formation. In vivo assays demonstrated the requirement for Stat3 signaling in tumor-associated myeloid cells for tumor angiogenesis. Our results indicate that, by virtue of the ability of Stat3 in tumor cells and tumor-derived myeloid cells to upregulate expression of factors that activate Stat3 in ECs, Stat3 mediates multidirectional crosstalk among tumor cells, tumor-associated myeloid cells, and ECs that contributes to tumor angiogenesis.

Tumor regression and immunity in combination therapy with anti-CEA chimeric antigen receptor T cells and anti-CEA-IL2 immunocytokine.

Targeted immunotherapy of solid cancers with chimeric antigen receptor (CAR) T cells and immunocytokines are attractive options in that they both rely on the specificity of tumor-targeted antibodies. Since carcinoembryonic antigen (CEA) expression in both colon and breast cancers is correlated with poor prognosis, it was chosen as a model tumor target in immunocompetent CEA transgenic (CEATg) mice. A second-generation anti-CEA CAR derived from CEA-specific antibody T84.66 was used to treat murine MC38 colon or E0771 breast carcinomas transfected with CEA. Anti-CEA CAR vs. mock transduced T cells exhibited a CEA-specific cytotoxic and IFN γ dose response to both CEA transfected cell lines vs. their CEA-negative controls. Anti-CEA CAR vs. mock transduced T cells delayed the median survival of CEA transfected s.c. MC38 or orthotopic E0771 tumor-bearing CEATg mice by 2 days. With the addition of one-day prior cyclophosphamide (CY) lymphodepletion, anti-CEA CAR T cell treatment delayed the median survival of MC38/CEA and E0771/CEA tumor-bearing CEATg mice by ten and 3 days, respectively. Since CAR T cells require IL2 for survival and expansion, anti-CEA-IL2 immunocytokine (ICK) treatment was performed post CAR T cell therapy. Single ICK treatment 1 day after CY plus anti-CEA CAR T cell therapy in the MC38/CEA model, and two ICK treatments every 3 days after CY plus anti-CEA CAR T cell therapy in the E0771/CEA model were ineffective, while four ICK treatments every 3 days after CY plus anti-CEA CAR T cell therapy completely eradicated MC38/CEA tumor growth and induced tumor immunity when the mice were re-challenged with tumor. These studies show the therapeutic potential of anti-CEA CAR T cells combined with ICK to treat CEA-positive tumors. Abbreviations: CAR: Chimeric antigen receptor, CEA: Carcinoembryonic antigen, CEACAM5, ICK: Immunocytokine, CY: Cyclophosphamide, CEATg mouse: transgenic CEA mouse, TDLN: Tumor-draining lymph node.

Potent immunomodulatory effects of an anti-CEA-IL-2 immunocytokine on tumor therapy and effects of stereotactic radiation.

While anti-CEA antibodies have no direct effect on CEA-positive tumors, they can be used to direct potent anti-tumor effects as an antibody-IL-2 fusion protein (immunocytokine, ICK), and at the same time reduce the toxicity of IL-2 as a single agent. Using a fusion protein of humanized anti-CEA with human IL-2 (M5A-IL-2) in a transgenic murine model expressing human CEA, we show high tumor uptake of the ICK to CEA-positive tumors with additional lymph node targeting. ICK treated CEA-positive tumors exhibit significant tumor eradication. Analysis of tumor-infiltrating lymphocytes shows a high frequency of both CD8+ and CD4+ T cells along with CD11b positive myeloid cells in ICK treated mice. The frequency of tumor-infiltrating FoxP3+ CD4+ T cells (Tregs) is significantly reduced vs anti-CEA antibody-treated controls, indicating that ICK did not preferentially stimulate migration or proliferation of Tregs to the tumor. Combination therapy with anti-PD-1 antibody did not improve tumor reduction over ICK therapy alone. Since stereotactic tumor irradiation (SRT), commonly used in cancer therapy has immunomodulatory effects, we tested combination SRT+ICK therapy in two tumor model systems. Use of fractionated vs single high dose SRT in combination with ICK resulted in greater tumor inhibition and immunity to tumor rechallenge. In particular, tumor microenvironment and myeloid cell composition appear to play a significant role in the response rate to ICK+SRT combination therapy.

Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury.

Although CEACAM1 (CC1) glycoprotein resides at the interface of immune liver injury and metabolic homeostasis, its role in orthotopic liver transplantation (OLT) remains elusive. We aimed to determine whether/how CEACAM1 signaling may affect hepatic ischemia-reperfusion injury (IRI) and OLT outcomes. In the mouse, donor liver CC1 null mutation augmented IRI-OLT (CC1-KO→WT) by enhancing ROS expression and HMGB1 translocation during cold storage, data supported by in vitro studies where hepatic flush from CC1-deficient livers enhanced macrophage activation in bone marrow-derived macrophage cultures. Although hepatic CC1 deficiency augmented cold stress-triggered ASK1/p-p38 upregulation, adjunctive ASK1 inhibition alleviated IRI and improved OLT survival by suppressing p-p38 upregulation, ROS induction, and HMGB1 translocation (CC1-KO→WT), whereas ASK1 silencing (siRNA) promoted cytoprotection in cold-stressed and damage-prone CC1-deficient hepatocyte cultures. Consistent with mouse data, CEACAM1 expression in 60 human donor liver biopsies correlated negatively with activation of the ASK1/p-p38 axis, whereas low CC1 levels associated with increased ROS and HMGB1 translocation, enhanced innate and adaptive immune responses, and inferior early OLT function. Notably, reduced donor liver CEACAM1 expression was identified as one of the independent predictors for early allograft dysfunction (EAD) in human OLT patients. Thus, as a checkpoint regulator of IR stress and sterile inflammation, CEACAM1 may be considered as a denominator of donor hepatic tissue quality, and a target for therapeutic modulation in OLT recipients.

Signal transducer and activator of transcription 3 is required for hypoxia-inducible factor-1alpha RNA expression in both tumor cells and tumor-associated myeloid cells.

Hypoxia-inducible factor 1 (HIF-1) is a potent tumorigenic factor. Its alpha subunit (HIF-1alpha), which is tightly regulated in normal tissues, is elevated in tumors due to hypoxia and overactive growth signaling pathways. Although much is known about HIF-1alpha regulation in cancer cells, crucial molecular targets that affect HIF-1alpha levels modulated by both hypoxia and oncogenic signaling pathways remain to be identified. Additionally, whether and how the tumor microenvironment contributes to HIF-1alpha accumulation is unclear. This study shows a novel mechanism by which HIF-1alpha availability is regulated in both cancer cells and in myeloid cells in the tumor microenvironment. We show a requirement of signal transducer and activator of transcription 3 (Stat3) for HIF-1alpha RNA expression under both hypoxia and growth signaling conditions. Furthermore, tumor-derived myeloid cells express elevated levels of HIF-1alpha mRNA relative to their counterparts from normal tissues in a Stat3-dependent manner. Additionally, Stat3 activity in the nontransformed cells in the tumor milieu affects HIF-1alpha RNA expression of the entire growing tumor. Consistent with a role of Stat3 in regulating HIF-1alpha RNA transcription, elevated Stat3 activity increases HIF-1alpha promoter activity, and Stat3 protein binds to the HIF-1alpha promoter in both transformed cells and in growing tumors. Taken together, these findings show a novel mode by which HIF-1alpha is regulated not only in cancer cells but also in the tumor-associated inflammatory cells, suggesting Stat3 as an important molecular target for inhibiting the oncogenic potential of HIF-1 induced by both hypoxia and overactive growth signaling pathways prevalent in cancer.

Bacteroides fragilis polysaccharide A induces IL-10 secreting B and T cells that prevent viral encephalitis.

The gut commensal Bacteroides fragilis or its capsular polysaccharide A (PSA) can prevent various peripheral and CNS sterile inflammatory disorders. Fatal herpes simplex encephalitis (HSE) results from immune pathology caused by uncontrolled invasion of the brainstem by inflammatory monocytes and neutrophils. Here we assess the immunomodulatory potential of PSA in HSE by infecting PSA or PBS treated 129S6 mice with HSV1, followed by delayed Acyclovir (ACV) treatment as often occurs in the clinical setting. Only PSA-treated mice survived, with dramatically reduced brainstem inflammation and altered cytokine and chemokine profiles. Importantly, PSA binding by B cells is essential for induction of regulatory CD4+ and CD8+ T cells secreting IL-10 to control innate inflammatory responses, consistent with the lack of PSA mediated protection in Rag-/-, B cell- and IL-10-deficient mice. Our data reveal the translational potential of PSA as an immunomodulatory symbiosis factor to orchestrate robust protective anti-inflammatory responses during viral infections.

Radiation-induced Vascular Damage and the Impact on the Treatment Outcome of Stereotactic Body Radiotherapy.

BACKGROUND/AIM: The aim of this study was to investigate radiation-induced tumour vascular damage and its impact thereof on the outcome of stereotactic body radiotherapy (SBRT). MATERIALS AND METHODS: Vessel densities in animal tumours before and after a single dose of 20 Gy were quantified and used as input for simulations of three-dimensional tumours with heterogeneous oxygenation. SBRT treatments of the modelled tumours in 1-8 fractions were simulated. The impact of vessel collapse on the outcome of SBRT was investigated by calculating tumour control probability (TCP) and the dose required to obtain a TCP of 50% (D50). RESULTS: A radiation-induced increase of acute hypoxia in tumours during SBRT treatment could be simulated based on the experimental data. The D50 values for these tumours were higher than for the simulated tumours without vessel collapse. CONCLUSION: The vascular changes after high doses of radiation could compromise the outcome of SBRT by increasing tumour hypoxia.