Liver Protein Expression in NASH Mice on a High-Fat Diet: Response to Multi-Mineral Intervention.

Male MS-NASH mice were maintained on a high-fat diet for 16 weeks with and without red algae-derived minerals. Obeticholic acid (OCA) was used as a comparator in the same strain and diet. C57BL/6 mice maintained on a standard (low-fat) rodent chow diet were used as a control. At the end of the in-life portion of the study, body weight, liver weight, liver enzyme levels and liver histology were assessed. Samples obtained from individual livers were subjected to Tandem Mass Tag labeling / mass spectroscopy for protein profile determination. As compared to mice maintained on the low-fat diet, all high-fat-fed mice had increased whole-body and liver weight, increased liver enzyme (aminotransferases) levels and widespread steatosis / ballooning hepatocyte degeneration. Histological evidence for liver inflammation and collagen deposition was also present, but changes were to a lesser extent. A moderate reduction in ballooning degeneration and collagen deposition was observed with mineral supplementation. Control mice on the high-fat diet alone demonstrated multiple protein changes associated with dysregulated fat and carbohydrate metabolism, lipotoxicity and oxidative stress. Cholesterol metabolism and bile acid formation were especially sensitive to diet. In mice receiving multi-mineral supplementation along with the high-fat diet, there was reduced liver toxicity as evidenced by a decrease in levels of several cytochrome P450 enzymes and other oxidant-generating moieties. Additionally, elevated expression of several keratins was also detected in mineral-supplemented mice. The protein changes observed with mineral supplementation were not seen with OCA. Our previous studies have shown that mice maintained on a high-fat diet for up to 18 months develop end-stage liver injury including hepatocellular carcinoma. Mineral-supplemented mice were substantially protected against tumor formation and other end-state consequences of high-fat feeding. The present study identifies early (16-week) protein changes occurring in the livers of the high-fat diet-fed mice, and how the expression of these proteins is influenced by mineral supplementation. These findings help elucidate early protein changes that contribute to end-stage liver injury and potential mechanisms by which dietary minerals may mitigate such damage.

Mechanisms involved in radiation enhancement of intratumoral dendritic cell therapy.

We have previously reported that local tumor irradiation, without inducing cell death, can augment the therapeutic efficacy of intratumoral (IT) dendritic cell (DC) vaccination. This study examined potential mechanisms underlying radiation enhancement of IT DC therapy in this setting. Even though ionizing radiation did not mediate tumor cell killing, bone marrow-derived DCs acquired in vitro tumor antigens from irradiated D5 murine melanoma cells more efficiently than from untreated cells. This radiation-enhanced loading of DCs did not induce DC maturation, but was associated with improved cross-priming of T cells both in vitro and in vivo. Furthermore, in vivo pulsing of DCs with irradiated versus untreated tumor cells resulted in superior presentation of tumor antigens to T cells. In addition, tumor irradiation facilitated homing of IT administered DCs to the draining lymph node, possibly by down-regulating CCL21 expression within the tumor mass. Studies of the tumor microenvironment in irradiated versus untreated tumors did not reveal significant inflammatory changes. Moreover, radiation did not promote accumulation of CD4 or CD8 effector T cells within solid tumors. Our results indicate that, without inducing cytotoxicity, tumor irradiation can enhance the ability of DCs to capture tumor antigens, migrate to the draining lymph node, and present processed antigens to T cells. These findings may prove useful in designing future strategies for human cancer immunotherapy.

Donor cell cycling, trafficking, and accumulation during adoptive immunotherapy for murine lung metastases.

Adoptive cellular immunotherapy treats metastatic cancer by infusing cultured T cells derived from resected tumors or primed lymph nodes. The infused cells must accumulate in metastatic lesions to suppress growth; however, this process and the resulting clinical response are dynamic and evolve during the days and weeks following cell infusion. This study used novel experimental techniques to determine the fate of infused, cultured tumor-draining lymph node (TDLN) cells during the treatment of murine pulmonary micrometastases. After infusion, the cultured TDLN cells accumulated in the pulmonary vasculature, systemic lymph nodes, and spleen. Donor cells were initially confined to alveolar capillaries with no movement into metastases. Within 4 h, TDLN cells began migrating across pulmonary postcapillary venules and first appeared within metastases. After 24 h, most donor cells in the lung were associated with tumor nodules. Donor cell proliferation within the lung and lymphoid organs was detected within 24 h of infusion and continued throughout the 5-day period of observation. Furthermore, those proliferating in lymphoid organs trafficked back to the tumor-bearing lungs, accounting for approximately 50% of the donor cells recovered from these sites after 5 days. Finally, donor T cells entering metastases both early (within 1-2 days) and late (after 2 days) suppressed tumor growth, but the early recruits accounted for most of the therapeutic response. Thus, cultured TDLN cells migrate directly into tumor-bearing organs and seed the recirculating pool of lymphocytes after infusion. Small fractions of the later differentiate in lymphoid organs and migrate into the lungs but appear less effective than effector cells in the initial bolus.

Sustained high-yield production of recombinant proteins in transiently transfected COS-7 cells grown on trimethylamine-coated (hillex) microcarrier beads.

The present study shows that COS-7 cells transiently transfected and maintained on positively charged (trimethylamine-coated) microcarrier beads synthesize recombinant protein at higher levels and for longer periods of time than cells transfected and maintained on polystyrene flasks in monolayer culture. Sustained, high-level synthesis was observed with secreted chimeric proteins (murine E-selectin- and P-selectin-human IgM chimeras) and a secreted hematopoietic growth factor (granulocyte-macrophage colony-stimulating factor). Studies with green fluorescent protein indicated that the transfected cells attached more firmly to the trimethylamine-coated microcarriers than to polystyrene flasks. After 10-14 days in culture, most of the transfected cells detached from the surface of the polystyrene flasks, whereas most transfected cells remained attached to the microcarriers. The transiently transfected microcarrier cultures produced higher levels of protein per transfected cell due to this prolonged attachment. The prolonged attachment and higher output of transfected cells on microcarriers resulted in a 5-fold increase in protein production from a single transfection over two weeks. Thus, microcarrier-based transient transfection yields quantities of recombinant proteins with a significant savings of time and reagents over monolayer culture.

CCR6 colocalizes with CD18 and enhances adhesion to activated endothelial cells in CCR6-transduced Jurkat T cells.

CCR6 is expressed by memory T cells (mTC) and is a requirement for efficient arrest of a subset of mTC to activated human dermal microvascular endothelial cells (HDMEC) under physiologic shear stress. We now address whether CCR6 alone is sufficient to induce arrest of a model T cell line (Jurkat) that shows low expression of all CCRs tested (CCR1-10). Herein, we transduced Jurkat (JK) T cells expressing fucosyltransferase VII with a chimeric chemokine receptor consisting of CCR6 fused to enhanced green fluorescent protein. In contrast to the starting JK lines, the resulting cell line (JK fucosyltransferase VII-CCR6) migrated 6-fold better to CCL20 in chemotaxis assays, arrested in response to CCL20 that was immobilized to plastic, and demonstrated a 2.5-fold increase in adhesion to activated HDMEC (p = 0.001). Adhesion was blocked by anti-CD18 mAb (p = 0.005) but not by anti-CD49d mAb (p = 0.3). After arrest on recombinant substrates, CCR6 clustered on the surface as detected by real-time observation of enhanced green fluorescent protein fluorescence. Dual-label confocal microscopy revealed that LFA-1 (CD18 and CD11a), but not CXCR4, colocalized with clustered CCR6 in the presence of immobilized CCL20. Thus, the functional expression of CCR6 is sufficient to provide the chemokine signaling necessary to induce arrest of a JK T cell line to activated HDMEC. Clustering of CCR6 and coassociation with critical integrins may serve to strengthen adhesion between T cells and activated endothelial cells.

Subset-specific reductions in lung lymphocyte accumulation following intratracheal antigen challenge in endothelial selectin-deficient mice.

We previously demonstrated induction and expression of CD62E and CD62P in the lungs of mice primed and then challenged with intratracheal (i.t.) SRBC. The current study examined accumulation of endogenous lymphocytes in the lungs of endothelial E- and P-selectin-deficient (E(-)P(-)) mice after i.t. SRBC challenge. Compared with syngeneic wild-type (wt) mice, E(-)P(-) mice showed an 85-95% decrease in CD8(+) T cells and B cells in the lungs at both early and late time points. In contrast, CD4(+) T cell accumulation was reduced by approximately 60% early, but equivalent to wt levels later. Surprisingly, many gammadelta T cells were found in lungs and blood of E(-)P(-) mice but were undetectable in the lungs and blood of wt mice. Absolute numbers of peripheral blood CD4, CD8, and B lymphocytes in E(-)P(-) mice equaled or exceeded the levels in wt mice, particularly after challenge. Trafficking studies using alphabeta T lymphoblasts confirmed that the recruitment of circulating cells after challenge was markedly reduced in E(-)P(-) mice. Furthermore, Ag priming occurred normally in both the selectin-deficient and wt mice, because primed lymphocytes from both groups transferred Ag sensitivity into naive wt mice. Lung production of mRNA for six CC and two CXC chemokines after challenge was equivalent by RT-PCR analysis in wt and E(-)P(-) mice. Therefore, reduced lung accumulation of alphabeta T cells and B cells in E(-)P(-) mice did not result from reduced delivery of circulating lymphocytes to the lungs, unsuccessful Ag priming, or defective pulmonary chemokine production. Selectin-dependent lymphocyte recruitment into the lungs following i.t.-SRBC challenge is subset specific and time dependent.

L-selectin serves as an E-selectin ligand on cultured human T lymphoblasts.

Previous studies reported that L-selectin (CD62L) on human peripheral blood neutrophils serves as an E-selectin ligand. This study shows that CD62L acquired E-selectin-binding activity following phorbol ester (PMA) treatment of the Jurkat T cell line and anti-CD3/IL-2-driven proliferation of human T lymphocytes in vitro. The recombinant porcine E-selectin/human Ig chimera P11.4 showed neuraminidase-sensitive and calcium-dependent attachment to PMA-stimulated human Jurkat T cells in a flow cytometry assay. The anti-CD62L mAb (DREG 56) blocked this binding interaction by approximately 60% and P11.4 precipitated CD62L from detergent lysates of PMA-activated Jurkat cells. In contrast, P11.4 precipitated minimal amounts of CD62L from detergent lysates of nonactivated human PBL. As reported previously, P-selectin glycoprotein ligand 1 and a distinct 130-kDa glycoprotein were the major species in these precipitates. However, T cell activation on plate-immobilized anti-CD3 and growth in low-dose IL-2 increased the percentage of CD62L molecules with E-selectin-binding activity. After two cycles of activation and culture, approximately 60-70% of the CD62L was precipitated with the P11.4 chimera. These cultured T lymphoblasts rolled avidly on both E-selectin and P-selectin at physiologic levels of linear shear stress. The DREG 56 Ab partially blocked rolling on the E-selectin substrate, whereas no effect was seen on P-selectin. Thus, CD62L on human cultured T lymphoblasts is one of several glycoproteins that interacts directly with E-selectin and contributes to rolling under flow.

The monoclonal antibody CHO-131 binds to a core 2 O-glycan terminated with sialyl-Lewis x, which is a functional glycan ligand for P-selectin.

Core 2 O-glycans terminated with sialyl-Lewis x (sLe(X)) are functionally important oligosaccharides that endow particular macromolecules with high-affinity glycan ligands for the selectin family. To date, antibodies that recognize these structures on leukocytes have not been described. We characterize such a monoclonal antibody (mAb) here (CHO-131). The binding specificity of CHO-131 was directly examined by means of synthetic glycopeptides containing precise O-glycan structures. CHO-131 bound to sLe(X) extended from a core 2 branch (C2-O-sLe(X)), but CHO-131 demonstrated no reactivity if this oligosaccharide lacked fucose or if sLe(X) was extended from a core 1 branch. Using transfected cell lines, we found that CHO-131 binding required the functional activity of the glycosyltransferases alpha2,3-sialyltransferase, alpha1,3-fucosyltransferase-VII, and core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT). The C2-O-sLe(X) motif occurs primarily on sialomucins and has been directly shown to contribute to high-affinity P-selectin glycoprotein ligand-1 binding by P-selectin. Indeed, CHO-131 staining of neutrophils was diminished following sialomucin removal by O-glycoprotease, and its reactivity with transfected hematopoietic cell lines correlated with the expression of P-selectin ligands. CHO-131 also stained a small population of lymphocytes that were primarily CD3(+), CD4(+), and CD45RO(+) and represented a subset (37.8% +/- 18.3%) of cutaneous lymphocyte-associated antigen (CLA) T cells, distinguished by the mAb HECA-452, which detects sLe(X)-related glycans. Unlike anti-sLe(X) mAbs, CHO-131 binding also indicates C2GnT activity and demonstrates that CLA T cells are heterogeneous based on the glycan structures they synthesize. These findings support evidence that differential C2GnT activity results in T-cell subsets that express ligands for E-selectin, P-selectin, or both.

Mice genetically lacking endothelial selectins are resistant to the lethality in septic peritonitis.

Leukocyte interactions with vascular endothelium are an initial step for leukocyte entry into infectious foci where endothelial selectins may play a key role. Infiltrating leukocyte is essential for bacterial clearance, suggesting that endothelial selectins would be important in host defense against microorganisms. To address this, E-, P-, and E/P-selectin-deficient mice (E(-/-), P(-/-), E/P(-/-)) and wild-type (WT) mice underwent cecal ligation and puncture (CLP). Neither leukocyte infiltration nor bacterial load in the peritoneum was altered in E(-/-), P(-/-), and E/P(-/-) mice compared to WT mice. However, E(-/-), P(-/-), and E/P(-/-) mice were resistant to the lethality induced by CLP. At the mechanistic level, E(-/-), P(-/-), and E/P(-/-) mice did not develop renal dysfunction, a possible cause of death during sepsis. The serum level of interleukin-13 in E(-/-), P(-/-), and E/P(-/-) mice that had undergone CLP was higher than that in WT mice, whereas levels of macrophage inflammatory protein-2, KC in serum, and KC in kidney were lower than those in WT mice. These experiments demonstrate that endothelial selectin-mediated leukocyte rolling is not required for leukocyte entry in septic peritonitis and that endothelial selectins may affect mice survival during sepsis by influencing the cytokine profiles.