The portal venous circulation provides a conduit for pancreatic ductal adenocarcinoma (PDAC) tumor cells to the liver parenchyma sinusoids, a frequent site of metastasis. Turbulent flow in the portal circulation promotes retention of PDAC shed circulating tumor cells (CTC) and myeloid-derived immunosuppressor cells (MDSC). Excessive colony stimulating factor-1 receptor (CSF1R) signaling can induce myeloid differentiation to MDSC and transformation of MDSC to myeloid-derived fibroblasts (M-FB). Interactions between PDAC CTC and M-FB in the portal blood promotes the formation of immunoresistant clusters that enhance CTC proliferation, migration, and survival. Analysis of portal and peripheral blood samples collected intraoperatively from 30 PDAC patients undergoing pancreatico-duodenectomy showed that PDAC patient plasma contained high levels of macrophage colony stimulating factor (M-CSF/CSF1), granulocyte-macrophage colony stimulating factor (GM-CSF/CSF2), interleukin-8 (IL-8), and interleukin-34 (IL-34) compared to healthy control levels. Moreover, the level of M-CSF in portal blood was significantly higher than that detected in the peripheral blood of PDAC patients. PDAC CTC aseptically isolated by fluorescence activated cell sorting (FACS) out of freshly collected patient portal blood mononuclear cells (PortalBMC) had elevated RNA expression of IL34 (IL-34 gene) and CSF1 (M-CSF/CSF1 gene) which both signal through CSF1R. PDAC CTC also had high levels of RNA expression for CXCL8, the gene encoding chemokine interleukin-8 (IL-8) which can attract myeloid cells through their CXCR2 receptors. FACS-isolated portal PDAC CTC and M-FB co-cultured ex vivo had increased CTC proliferation, motility, and cluster formation compared to CTC cultured alone. CSF1R and CXCR2 cell surface expression were found on PDAC portal blood CTC and M-FB, suggesting that both cell types may respond to M-CSF, IL-34, and IL-8-mediated signaling. Portal PDAC CTC displayed enhanced RNA expression of CSF1 and IL34, while CTC+M-FB+ clusters formed in vivo had increased RNA expression of CSF2 and IL34. Portal M-FB were found to have high CSF1R RNA expression. CTC isolated from ex vivo 7-day cultures of PDAC patient portal blood mononuclear cells (PortalBMC) expressed elevated CSF1, IL34, and IL8 RNA, and CSF1 expression was elevated in M-FB. Treatment with rabbit anti-CSF1R antibodies decreased CTC proliferation. Treatment of PortalBMC cultures with humanized anti-CSF1R, humanized anti-IL-8, or anti-IL-34 antibodies disrupted CTC cluster formation and increased CTC apoptosis. U937 myeloid precursor cell line cultures treated with conditioned media from PortalBMC ex vivo cultures without treatment or treated with anti-IL-8 and/or anti-CSF1R did not prevent myeloid differentiation in the myeloid precursor cell line U937 to macrophage, dendritic cell, MDSC, and M-FB phenotypes; whereas, U937 cultures treated with conditioned media from PortalBMC ex vivo cultures exposed to anti-IL-34 were significantly inhibited in their myeloid differentiation to all but the M-FB phenotype. PDAC patient T cells that were found phenotypically anergic (CD3+CD25+CTLA4+PD1L1+) in PortalBMC could be re-activated (CD3+CD25+CTLA4-PD1L1-), and displayed increased interferon gamma (IFNγ) production when PortalBMC ex vivo cultures were treated with anti-CSF1R, anti-IL-8, and anti-IL-34 antibodies alone or in combination. These findings suggest that PDAC CTC have the potential to influence myeloid differentiation and/or antigen presenting cell activation in the PDAC portal blood microenvironment, and that disruption of CTC/M-FB interactions may be potential targets for reversing the immunosuppression supporting CTC survival in the portal blood.

Endothelial dysfunction and enhanced transforming growth factor-β (TGF-β)/Smad3 signalling are common features of progressive renal fibrosis. This study investigated a potential link between these mechanisms. In unilateral ureteric obstruction (UUO) we observed an acute (6 hr) down-regulation of nitric oxide synthase 3 (NOS3/eNOS) levels and increased phosphorylation of the linker region of Smad3 at T179 and S208 in Smad3/JNK complexes. These events preceded Smad3 C-terminal domain phosphorylation and the induction of myofibroblast proliferation at 48 hrs. Mice deficient in NOS3 showed enhanced myofibroblast proliferation and collagen accumulation compared to wild type mice in a 7 day UUO model. This was associated with enhanced phosphorylation of Smad3 T179 and S208 by 92% and 88%, respectively, whereas Smad3-C-terminal phosphorylation was not affected. Resolvin D1 (RvD1) can suppress renal fibrosis in the UUO model, and further analysis herein showed that RvD1 protected against endothelial dysfunction and suppressed Smad3/JNK complex formation with a consequent reduction in phosphorylation of Smad3 T179 and S208 by 78% and 65%, respectively, while Smad3 C-terminal phosphorylation was unaltered. In vitro, conditioned media from mouse microvascular endothelial cells (MMEC) treated with a general inhibitor of nitric oxide synthase (L-NAME) augmented the proliferation and collagen production of renal fibroblasts (NRK49F cells) compared to control MMEC media and this was associated with increased phosphorylation of JNK and Smad3 T179 and S208, whereas Smad3-C-terminal domain phosphorylation was unaffected. The addition of RvD1 to L-NAME treated MMEC abrogated these effects of the conditioned media on renal fibroblasts. Finally, Smad3 T179/V and S208/A mutations significantly inhibit TGF-β1 induced up-regulation collagen I promoter. In conclusion, these data suggest that endothelial dysfunction can exacerbate renal interstitial fibrosis through increased fibroblast proliferation and collagen production via enhanced Smad3 linker phosphorylation.

Modified Vaccinia virus has been shown to be a safe and immunogenic vector platform for delivery of HIV vaccines. Use of this vector is of particular importance to the military, with the implementation of a large scale smallpox vaccination campaign in 2002 in active duty and key civilian personnel in response to potential bioterrorist activities. Humoral immunity to smallpox vaccination was previously shown to be long lasting (up to 75 years) and protective. However, using vaccinia-vectored vaccine delivery for other diseases on a background of anti-vector antibodies (i.e. pre-existing immunity) may limit their use as a vaccine platform, especially in the military. In this pilot study, we examined the durability of vaccinia antibody responses in adult primary vaccinees in a healthy military population using a standard ELISA assay and a novel dendritic cell neutralization assay. We found binding and neutralizing antibody (NAb) responses to vaccinia waned after 5-10 years in a group of 475 active duty military, born after 1972, who were vaccinated as adults with Dryvax®. These responses decreased from a geometric mean titer (GMT) of 250 to baseline (<20) after 10-20 years post vaccination. This contrasted with a comparator group of adults, ages 35-49, who were vaccinated with Dryvax® as children. In the childhood vaccinees, titers persisted for >30 years with a GMT of 210 (range 112-3234). This data suggests limited durability of antibody responses in adult vaccinees compared to those vaccinated in childhood and further that adult vaccinia recipients may benefit similarly from receipt of a vaccinia based vaccine as those who are vaccinia naïve. Our findings may have implications for the smallpox vaccination schedule and support the ongoing development of this promising viral vector in a military vaccination program.

Secreted protein acidic and rich in cysteine (SPARC), a calcium-binding matricellular glycoprotein, is implicated in the progression of many cancers. In this study, we investigated the expression and function of SPARC in ovarian cancer.

The purpose of this study was to elucidate the role of gamma-tocotrienol (GT3)-mobilized progenitors in mitigating damage to mice exposed to a supralethal dose of cobalt-60 gamma-radiation. CD2F1 mice were transfused 24 h post-irradiation with whole blood or isolated peripheral blood mononuclear cells (PBMC) from donors that had received GT3 72 h prior to blood collection and recipient mice were monitored for 30 days. To understand the role of GT3-induced granulocyte colony-stimulating factor (G-CSF) in mobilizing progenitors, donor mice were administered a neutralizing antibody specific to G-CSF or its isotype before blood collection. Bacterial translocation from gut to heart, spleen and liver of irradiated recipient mice was evaluated by bacterial culture on enriched and selective agar media. Endotoxin in serum samples also was measured. We also analyzed the colony-forming units in the spleens of irradiated mice. Our results demonstrate that whole blood or PBMC from GT3-administered mice mitigated radiation injury when administered 24 h post-irradiation. Furthermore, administration of a G-CSF antibody to GT3-injected mice abrogated the efficacy of blood or PBMC obtained from such donors. Additionally, GT3-mobilized PBMC inhibited the translocation of intestinal bacteria to the heart, spleen, and liver, and increased colony forming unit-spleen (CFU-S) numbers in irradiated mice. Our data suggests that GT3 induces G-CSF, which mobilizes progenitors and these progenitors mitigate radiation injury in recipient mice. This approach using mobilized progenitor cells from GT3-injected donors could be a potential treatment for humans exposed to high doses of radiation.

Pulmonary arterial hypertension (PAH) is a disease of progressively increasing pulmonary vascular resistance, associated with mutations of the type 2 receptor for the BMP pathway, BMPR2. The canonical signaling pathway for BMPR2 is through the SMAD family of transcription factors. BMPR2 is expressed in every cell type, but the impact of BMPR2 mutations affecting SMAD signaling, such as Bmpr2delx4+, had only previously been investigated in smooth muscle and endothelium. In the present study, we created a mouse with universal doxycycline-inducible expression of Bmpr2delx4+ in order to determine if broader expression had an impact relevant to the development of PAH. We found that the most obvious phenotype was a dramatic, but patchy, increase in pulmonary inflammation. We crossed these double transgenic mice onto an NF-κB reporter strain, and by luciferase assays on live mice, individual organs and isolated macrophages, we narrowed down the origin of the inflammatory phenotype to constitutive activation of tissue macrophages. Study of bone marrow-derived macrophages from mutant and wild-type mice suggested a baseline difference in differentiation state in Bmpr2 mutants. When activated with LPS, both mutant and wild-type macrophages secrete BMP pathway inhibitors sufficient to suppress BMP pathway activity in smooth muscle cells (SMC) treated with conditioned media. Functionally, co-culture with macrophages results in a BMP signaling-dependent increase in scratch closure in cultured SMC. We conclude that SMAD signaling through BMP is responsible, in part, for preventing macrophage activation in both live animals and in cells in culture, and that activated macrophages secrete BMP inhibitors in sufficient quantity to cause paracrine effect on vascular smooth muscle.

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that is a common cause of acute and recurrent mucosal infections. One uncharacterized NTHi toxin-antitoxin (TA) module, NTHI1912-1913, is a host inhibition of growth (higBA) homologue. We hypothesized that this locus, which we designated toxAvapA, contributed to NTHi survival during infection. We deleted toxAvapA and determined that growth of the mutant in defined media was not different from the parent strain. We tested the mutant for persistence during long-term in vitro co-culture with primary human respiratory tissues, which revealed that the ΔtoxAvapA mutant was attenuated for survival. We then performed challenge studies using the chinchilla model of otitis media and determined that mutant survival was also reduced in vivo. Following purification, the toxin exhibited ribonuclease activity on RNA in vitro, while the antitoxin did not. A microarray comparison of the transcriptome revealed that the tryptophan biosynthetic regulon was significantly repressed in the mutant compared to the parent strain. HPLC studies of conditioned medium confirmed that there was no significant difference in the concentration of tryptophan remaining in the supernatant, indicating that the uptake of tryptophan by the mutant was not affected. We conclude that the role of the NTHi toxAvapA TA module in persistence following stress is multifactorial and includes effects on essential metabolic pathways.

Age-related Macular Degeneration (AMD) is one of the major vision-threatening diseases of the eye. Oxidative stress is one of the key factors in the onset and progression of AMD. In this study, metabolites associated with AMD pathology more so at the systemic level namely, oxidized LDL (oxLDL), homocysteine (Hcy), homocysteine thiolactone (HCTL), advanced glycation end product (AGE) were evaluated for their pro-oxidant nature in a localized ocular environment based on in vitro studies in human retinal pigment epithelial cells (ARPE-19 cells). Human ARPE-19 cells were treated with pro-oxidants 50 μg/mL oxLDL, 500 μM Hcy, 500 nM HCTL, 100 μg/mL AGE, 200 μM H2O2 and 200 μM H2O2 with and without pre-treatment of 5 mM N-acetyl cysteine (NAC). The cytokines IL-6, IL-8 and vascular endothelial growth factor (VEGF) secreted from ARPE-19 cells exposed to pro-oxidants were estimated by ELISA. In vitro angiogenesis assay was performed with conditioned media of the pro-oxidant treated ARPE-19 cells in Geltrex-Matrigel coated 96-well plate. The human acute monocytic leukemia cell line (THP-1) was differentiated into macrophages and its migration in response to conditioned media of ARPE-19 cells insulted with the pro-oxidants was studied by transwell migration assay. Western blot was performed to detect the protein expression of Bax, Bcl-2 and NF-κB to assess apoptotic changes. The compounds involved in the study showed a significant increase in reactive oxygen species (ROS) generation in ARPE-19 cells (oxLDL; Hcy; AGE: p < 0.001 and HCTL: p < 0.05). NAC pre-treatment significantly lowered the oxidative stress brought about by pro-oxidants as seen by lowered ROS and MDA levels in the cells. Treatment with pro-oxidants significantly increased the secretion of IL-6 (oxLDL: p < 0.05; Hcy, HCTL and AGE: p < 0.01) and IL-8 cytokines (oxLDL: p < 0.05; HCTL: p <. 001 and AGE: p < 0.01) in ARPE-19 cells. Serum samples of AMD patients (n = 23) revealed significantly higher IL-6 and IL-8 levels compared to control subjects (n = 23) (IL6: p < 0.01 and IL8: p < 0.05). The pro-oxidants also promoted VEGF secretion by ARPE-19 cells compared to untreated control (oxLDL: p < 0.001; Hcy: p < 0.01; HCTL and AGE: p < 0.05). In vitro angiogenesis assay showed that the conditioned media significantly increased the tube formation in RF/6A endothelial cells. Transwell migration assay revealed significant infiltration of macrophages in response to pro-oxidants. We further demonstrated that the pro-oxidants increased the Bax/Bcl-2 ratio and increased the NF-κB activation resulting in pro-apoptotic changes in ARPE-19 cells. Thus, oxLDL, Hcy, HCTL and AGE act as pro-oxidant metabolites in RPE that promote AMD through oxidative stress, inflammation, chemotaxis and neovascularization.

Brucella canis infection is an important cause of late-term abortion in pregnant bitches. The pathophysiological mechanisms leading to B. canis-induced abortion are unknown, but heavily infected trophoblasts are consistently observed. As trophoblasts responses to other pathogens contribute to placental inflammation leading to abortion, the aim of the present study was to characterize the cytokine response of canine trophoblasts to B. canis infection. To achieve this, trophoblasts isolated from term placenta of healthy female dogs were infected with B. canis, culture supernatants were harvested for cytokine determinations, and the load of intracellular viable B. canis was determined at different times post-infection. Additionally, cytokine responses were assessed in non-infected trophoblasts stimulated with conditioned media (CM) from B. canis-infected canine monocytes and neutrophils. Finally, cytokine response and bacteria replication were assessed in canine placental explants infected ex vivo. B. canis successfully infected and replicated in primary canine trophoblasts, eliciting an increase in IL-8 and RANTES (CCL5) secretion. Moreover, the stimulation of trophoblasts with CM from B. canis-infected monocytes and neutrophils induced a significant increase in IL-8, IL-6 and RANTES secretion. B. canis replication was confirmed in infected placental explants and the infection elicited an increased secretion of TNF-α, IL-8, IL-6 and RANTES. This study shows that canine trophoblasts produce proinflammatory cytokines in response to B. canis infection and/or to stimulation with factors produced by infected monocytes and neutrophils. These cytokines may contribute to placental inflammation leading to abortion in B. canis-infected pregnant bitches.

Glycosylation is an important and universal post-translational modification for many proteins, and regulates protein functions. However, simple and rapid methods to analyze glycans on individual proteins have not been available until recently.

To evaluate the effect of conditioned media (CM) and Extracellular Vesicles (EVs) derived from bovine oviduct epithelial cell (BOEC) lines on the developmental capacity of bovine zygotes and the quality of embryos produced in vitro, presumptive zygotes were cultured under specific conditions. In experiment 1, zygotes were cultured either on monolayers from BOEC extended culture (E), together with fresh BOEC suspension cells, or with BOEC-CM from fresh or E-monolayers. In experiment 2, EVs were isolated from BOEC-CM and characterized (150-200 nm) by Nanosight® and electron microscopy. Zygotes were cultured in the presence of 3x10(5) EVs/mL, 1.5x10(5) EVs/mL or 7.5x10(4) EVs/mL of fresh or frozen BOEC-EVs. In experiment 3, zygotes were cultured in absence of FCS but with EVs from BOEC-E that had been cultured in different culture media. In experiment 4, zygotes were cultured in SOF+5% normal-FCS, or EV-depleted-FCS. In all cases, cleavage rate (Day 2) and blastocyst development (Day 7-9) was assessed. Blastocysts on Days 7/8 were used for quality evaluation through differential cell count, cryotolerance and gene expression patterns. No differences were found among all FCS-containing groups in cleavage rate or blastocyst yield. However, embryos derived from BOEC-CM had more trophectoderm cells, while embryos derived from BOEC-EVs, both fresh and frozen, has more trophectoderm and total cells. More embryos survived vitrification in the BOEC-CM and BOEC-EV groups. In contrast, more embryos survived in the EV-depleted-FCS than in normal-FCS group. Gene expression patterns were modified for PAG1 for embryos cultured with EVs in the presence of FCS and for IFN-T, PLAC8, PAG1, CX43, and GAPDH in the absence of FCS. In conclusion, EVs from FCS have a deleterious effect on embryo quality. BOEC-CM and EVs during in vitro culture had a positive effect on the quality of in vitro produced bovine embryos, suggesting that EVs have functional communication between the oviduct and the embryo in the early stages of development.

Tumor progression and the immune response are intricately linked. While it is known that cancers alter macrophage inflammatory responses to promote tumor progression, little is known regarding how cancers affect macrophage-dependent innate host defense. In this study, murine bone-marrow-derived macrophages (BMDM) were exposed to murine carcinoma-conditioned media prior to assessment of the macrophage inflammatory response. BMDMs exposed to 4T1 mammary carcinoma-conditioned medium demonstrated enhanced production of pro-inflammatory cytokines tumor necrosis factor α, interleukin-6, and CCL2 in response to lipopolysaccharide (LPS) while production of interleukin-10 remained unchanged. The increased LPS-induced production of pro-inflammatory cytokines was transient and correlated with enhanced cytokine production in response to other Toll-like receptor agonists, including peptidoglycan and flagellin. In addition, 4T1-conditioned BMDMs exhibited strengthened LPS-induced nitric oxide production and enhanced phagocytosis of Escherichia coli. 4T1-mediated augmentation of macrophage responses to LPS was partially dependent on the NFκB pathway, macrophage-colony stimulating factor, and actin polymerization, as well as the presence of 4T1-secreted extracellular vesicles. Furthermore, peritoneal macrophages obtained from 4T1 tumor-bearing mice displayed enhanced pro-inflammatory cytokine production in response to LPS. These results suggest that uptake of 4T1-secreted factors and actin-mediated ingestion of 4T1-secreted exosomes by macrophages cause a transient enhancement of innate inflammatory responses. Mammary carcinoma-mediated regulation of innate immunity may have significant implications for our understanding of host defense and cancer progression.

Tumorigenic cells undergo cell aggregation and aggregate coalescence in a 3D Matrigel environment. Here, we expanded this 3D platform to assess the interactions of normal human dermal fibroblasts (NHDFs) and human primary mammary fibroblasts (HPMFs) with breast cancer-derived, tumorigenic cells (MDA-MB-231). Medium conditioned by MDA-MB-231 cells activates both types of fibroblasts, imbuing them with the capacity to accelerate the rate of aggregation and coalescence of MDA-MB-231 cells more than four fold. Acceleration is achieved 1) by direct physical interactions with MDA-MB-231 cells, in which activated fibroblasts penetrate the MDA-MB-231/Matrigel 3D environment and function as supporting scaffolds for MDA-MB-231 aggregation and coalescence, and 2) through the release of soluble accelerating factors, including matrix metalloproteinase (MMPs) and, in the case of activated NHDFs, SDF-1α/CXCL12. Fibroblast activation includes changes in morphology, motility, and gene expression. Podoplanin (PDPN) and fibroblast activation protein (FAP) are upregulated by more than nine-fold in activated NHDFs while activated HPMFs upregulate FAP, vimentin, desmin, platelet derived growth factor receptor A and S100A4. Overexpression of PDPN, but not FAP, in NHDF cells in the absence of MDA-MB-231-conditioned medium, activates NHDFs. These results reveal that complex reciprocal signaling between fibroblasts and cancer cells, coupled with their physical interactions, occurs in a highly coordinated fashion that orchestrates aggregation and coalescence, behaviors specific to cancer cells in a 3D environment. These in vitro interactions may reflect events involved in early tumorigenesis, particularly in cases of field cancerization, and may represent a new mechanism whereby cancer-associated fibroblasts (CAFs) promote tumor growth.

MiR-21 is an oncomir expressed by malignant cells and/or tumor microenvironment components. In this study we focused on understanding the effects of stromal miR-21 on esophageal malignant cells.

Endometrial cancer is the most commonly diagnosed gynecologic malignancy worldwide; yet the tumor microenvironment, especially the fibroblast cells surrounding the cancer cells, is poorly understood. We established four primary cultures of fibroblasts from human endometrial cancer tissues (cancer-associated fibroblasts, CAFs) using antibody-conjugated magnetic bead isolation. These relatively homogenous fibroblast cultures expressed fibroblast markers (CD90, vimentin and alpha-smooth muscle actin) and hormonal (estrogen and progesterone) receptors. Conditioned media collected from CAFs induced a dose-dependent proliferation of both primary cultures and cell lines of endometrial cancer in vitro (175%) when compared to non-treated cells, in contrast to those from normal endometrial fibroblast cell line (51%) (P<0.0001). These effects were not observed in fibroblast culture derived from benign endometrial hyperplasia tissues, indicating the specificity of CAFs in affecting endometrial cancer cell proliferation. To determine the mechanism underlying the differential fibroblast effects, we compared the activation of PI3K/Akt and MAPK/Erk pathways in endometrial cancer cells following treatment with normal fibroblasts- and CAFs-conditioned media. Western blot analysis showed that the expression of both phosphorylated forms of Akt and Erk were significantly down-regulated in normal fibroblasts-treated cells, but were up-regulated/maintained in CAFs-treated cells. Treatment with specific inhibitors LY294002 and U0126 reversed the CAFs-mediated cell proliferation (P<0.0001), suggesting for a role of these pathways in modulating endometrial cancer cell proliferation. Rapamycin, which targets a downstream molecule in PI3K pathway (mTOR), also suppressed CAFs-induced cell proliferation by inducing apoptosis. Cytokine profiling analysis revealed that CAFs secrete higher levels of macrophage chemoattractant protein (MCP)-1, interleukin (IL)-6, IL-8, RANTES and vascular endothelial growth factor (VEGF) than normal fibroblasts. Our data suggests that in contrast to normal fibroblasts, CAFs may exhibit a pro-tumorigenic effect in the progression of endometrial cancer, and PI3K/Akt and MAPK/Erk signaling may represent critical regulators in how endometrial cancer cells respond to their microenvironment.

We have previously demonstrated the role of gastrin-releasing peptide (GRP) as an autocrine growth factor for neuroblastoma. Here, we report that GRP silencing regulates cell signaling involved in the invasion-metastasis cascade. Using a doxycycline inducible system, we demonstrate that GRP silencing decreased anchorage-independent growth, inhibited migration and neuroblastoma cell-mediated angiogenesis in vitro, and suppressed metastasis in vivo. Targeted inhibition of GRP decreased the mRNA levels of oncogenes responsible for neuroblastoma progression. We also identified PTEN/AKT signaling as a key mediator of the tumorigenic properties of GRP in neuroblastoma cells. Interestingly, PTEN overexpression decreased GRP-mediated migration and angiogenesis; a novel role for this, otherwise, understated tumor suppressor in neuroblastoma. Furthermore, activation of AKT (pAKT) positively correlated with neuroblastoma progression in an in vivo tumor-metastasis model. PTEN expression was slightly decreased in metastatic lesions. A similar phenomenon was observed in human neuroblastoma sections, where, early-stage localized tumors had a higher PTEN expression relative to pAKT; however, an inverse expression pattern was observed in liver lesions. Taken together, our results argue for a dual purpose of targeting GRP in neuroblastoma--1) decreasing expression of critical oncogenes involved in tumor progression, and 2) enhancing activation of tumor suppressor genes to treat aggressive, advanced-stage disease.

Delayed wound healing and scar formation are among the most frequent complications after surgical interventions. Although biodegradable surgical sutures present an excellent drug delivery opportunity, their primary function is tissue fixation. Mesenchymal stem cells (MSC) act as trophic mediators and are successful in activating biomaterials. Here biodegradable sutures were filled with adipose-derived mesenchymal stem cells (ASC) to provide a pro-regenerative environment at the injured site. Results showed that after filling, ASCs attach to the suture material, distribute equally throughout the filaments, and remain viable in the suture. Among a broad panel of cytokines, cell-filled sutures constantly release vascular endothelial growth factor to supernatants. Such conditioned media was evaluated in an in vitro wound healing assay and showed a significant decrease in the open wound area compared to controls. After suturing in an ex vivo wound model, cells remained in the suture and maintained their metabolic activity. Furthermore, cell-filled sutures can be cryopreserved without losing their viability. This study presents an innovative approach to equip surgical sutures with pro-regenerative features and allows the treatment and fixation of wounds in one step, therefore representing a promising tool to promote wound healing after injury.

Herein, we aimed to identify the immunomodulatory role of tumor Syndecan-1 (CD138) in the polarization of CD4+ T helper (Th) subsets isolated from the tumor microenvironment of inflammatory breast cancer (IBC) and non-IBC patients. Lymphocytes and mononuclear cells isolated from the axillary tributaries of non-IBC and IBC patients during modified radical mastectomy were either stimulated with the secretome as indirect co-culture or directly co-cultured with control and Syndecan-1-silenced SUM-149 IBC cells. In addition, peripheral blood mononuclear cells (PBMCs) of normal subjects were used for the direct co-culture. Employing flow cytometry, we analyzed the expression of the intracellular IFN-γ, IL-4, IL-17, and Foxp3 markers as readout for basal and co-cultured Th1, Th2, Th17, and Treg CD4+ subsets, respectively. Our data revealed that IBC displayed a lower basal frequency of Th1 and Th2 subsets than non-IBC. Syndecan-1-silenced SUM-149 cells significantly upregulated only Treg subset polarization of normal subjects relative to controls. However, Syndecan-1 silencing significantly enhanced the polarization of Th17 and Treg subsets of non-IBC under both direct and indirect conditions and induced only Th1 subset polarization under indirect conditions compared to control. Interestingly, qPCR revealed that there was a negative correlation between Syndecan-1 and each of IL-4, IL-17, and Foxp3 mRNA expression in carcinoma tissues of IBC and that the correlation was reversed in non-IBC. Mechanistically, Syndecan-1 knockdown in SUM-149 cells promoted Th17 cell expansion via upregulation of IL-23 and the Notch ligand DLL4. Overall, this study indicates a low frequency of the circulating antitumor Th1 subset in IBC and suggests that tumor Syndecan-1 silencing enhances ex vivo polarization of CD4+ Th17 and Treg cells of non-IBC, whereby Th17 polarization is possibly mediated via upregulation of IL-23 and DLL4. These findings suggest the immunoregulatory role of tumor Syndecan-1 expression in Th cell polarization that may have therapeutic implications for breast cancer.

Fibroblast Growth Factor (FGF)-10 promotes the proliferation and survival of murine hepatoblasts during early stages of hepatogenesis through a Wnt-β-catenin dependent pathway. To determine the mechanism by which this occurs, we expanded primary culture of hepatoblasts enriched for progenitor markers CD133 and CD49f from embryonic day (E) 12.5 fetal liver and an established tumor initiating stem cell line from Mat1a(-/-) livers in media conditioned with recombinant (r) FGF10 or rFGF7. FGF Receptor (R) activation resulted in the downstream activation of MAPK, PI3K-AKT, and β-catenin pathways, as well as cellular proliferation. Additionally, increased levels of nuclear β-catenin phosphorylated at Serine-552 in cultured primary hepatoblasts, Mat1a(-/-) cells, and also in ex vivo embryonic liver explants indicate AKT-dependent activation of β-catenin downstream of FGFR activation; conversely, the addition of AKT inhibitor Ly294002 completely abrogated β-catenin activation. FGFR activation-induced cell proliferation and survival were also inhibited by the compound ICG-001, a small molecule inhibitor of β-catenin-CREB Binding Protein (CBP) in hepatoblasts, further indicating a CBP-dependent regulatory mechanism of β-catenin activity.

Due to their complex chemical and physical properties, the effects and mechanisms of action of natural sources of dietary fiber on the intestine are unclear. Pigs are commonly fed high-fiber diets to reduce production costs and non-starch polysaccharide (NSP)-degrading enzymes have been used to increase fiber digestibility. We evaluated the expression of mucin 2 (MUC2), presence of goblet cells, and ileal immune profile of pigs housed individually for 28 days and fed either a low fiber diet based on corn-soybean meal (CSB, n = 9), or two high fiber diets formulated adding 40% corn distillers' dried grains with solubles (DDGS, n = 9) or 30% wheat middlings (WM, n = 9) to CSB-based diet. Pigs were also fed those diets supplemented with a NSP enzymes mix (E) of xylanase, β-glucanase, mannanase, and galactosidase (n = 8, 10, and 9 for CSB+E, DDGS+E and WM+E, respectively). Feeding DDGS and WM diets increased ileal MUC2 expression compared with CSB diet, and this effect was reversed by the addition of enzymes. There were no differences in abundance of goblet cells among treatments. In general, enzyme supplementation increased gene expression and concentrations of IL-1β, and reduced the concentrations of IL-4, IL-17A and IL-11. The effects of diet-induced cytokines on modulating intestinal MUC2 were assessed in vitro by treating mouse and swine enteroids with 1 ng/ml of IL-4 and IL-1β. In accordance with previous studies, treatment with Il-4 induced Muc2 and expansion of goblet cells in mouse enteroids. However, swine enteroids did not change MUC2 expression or number of goblet cells when treated with IL-4 or IL-1β. Our results suggest that mucin and immune profile are regulated by diet in the swine intestine, but by mechanisms different to mouse, emphasizing the need for using appropriate models to study responses to dietary fiber in swine.