We present the development of a new imaging technique for the early diagnosis of hepatocellular carcinoma that utilizes surface-modified gold nanoparticles in combination with x-ray imaging. Tissues labeled with these electron-dense particles show enhanced x-ray scattering over normal tissues, distinguishing cells containing gold nanoparticles from cells without gold in x-ray scatter images. Our results suggest that this novel approach could enable the in vivo detection of tumors as small as a few millimeters in size.
Dendritic cells (DCs) capture, process proteins and present peptides on the cell surface in the context of major histocompatibility complex (MHC1 and MHC11) molecules to induce antigen-specific T cell immune responses. The aims of this study were to (1) employ an expanded and purified DCs population and load them with aspartate-β-hydroxylase (ASPH), a highly expressed tumor associated cell surface protein, and (2) to determine if immunization induced anti-tumor effects in an orthotopic rat model of intrahepatic cholangiocarcinoma (ICC). Splenocytes were incubated with ASPH-coated beads and passed through a magnetic field to yield an 80% pure DC OX62+ population. This DC subset was stimulated with GM-CSF, IL-4, CD40L and IFN-γ, resulting in a 40-fold increase in IL12A mRNA expression to subsequently generate a Th1 type immune response. After incubation with the cytokine cocktail, DCs were found to have matured, as demonstrated by increased expression of CD40, CD80 and CD86 co-stimulatory molecules. Immunization with ASPH-loaded DCs induced antigen-specific immunity. A clone of the parental tumorigenic rat BDEneu cholangiocyte cell line, designated BDEneu-C24 found to have the highest number of cells expressing this surface protein (97%); it maintained the same phenotypic characteristics of the parental cell line and was used to produce intrahepatic tumors in immunocompetent syngeneic Fischer-344 rats. Immunization with ASPH-loaded DCs generated cytotoxicity against cholangiocarcinoma cells in vitro and significantly suppressed intrahepatic tumor growth and metastasis, and was associated with increased CD3+ lymphocyte infiltration into the tumors. Conclusions These findings suggest that immunization with ASPH-loaded DCs may constitute a novel therapeutic approach for ICC, since this protein also appears to be highly conserved and expressed on human hepatobiliary tumors.
Previous studies suggest that altered virus-specific T-cell responses observed during chronic ethanol exposure may be due to abnormal functioning of dendritic cells (DCs). Here we explored the effects of ethanol on exogenous antigen presentation by DCs. BALB/c, C57BL/6, and CBA/caj mice were fed ethanol or an isocaloric control diet for 8 weeks. The splenic DC population was expanded using an Flt3L expression plasmid via tail vein injection. DCs were purified and assessed for antigen presentation and processing and for peptide-major histocompatibility complex class I and II (MHCI and MHCII) formation on the cell surface. Interleukin-2 (IL-2) was measured as an indicator of antigen-specific T-cell activation by DCs in coculture. Antigen processing and peptide-MHCII complexes were evaluated by flow cytometry. We observed that ethanol not only suppressed allogeneic peptide presentation to T cells by DCs but also altered presentation of exogenous ovalbumin (OVA) peptide 323-339 to an OVA-specific DO11 T-cell line as well as to OVA-sensitized primary T cells. Smaller amounts of peptide-MHCII complexes were found on the DCs isolated from the spleens of ethanol-fed mice. In contrast to MHCII presentation, cross-presentation of exogenous OVA peptide via MHCI by DCs remained intact. More importantly, ethanol-exposed DCs had reduced B7-DC and enhanced ICOS-L (inhibitory) costimulatory molecule expression. Ethanol inhibits exogenous and allogeneic antigen presentation and affects the formation of peptide-MHCII complexes, as well as altering costimulatory molecule expression on the cell surface. Therefore, DC presentation of peptides in a favorable costimulatory protein environment is required to subsequently activate T cells and appears to be a critical target for the immunosuppressive effects of ethanol.
Objective Systemic sclerosis–associated interstitial lung disease (SSc‐ILD) is characterized by variable clinical outcomes, activation of innate immune pattern‐recognition receptors (PRRs), and accumulation of α‐smooth muscle actin (α‐SMA)–expressing myofibroblasts. The aim of this study was to identify an association between these entities and mitochondrial DNA (mtDNA), an endogenous ligand for the intracellular DNA–sensing PRRs Toll‐like receptor 9 (TLR‐9) and cyclic GMP‐AMP synthase/stimulator of interferon genes (cGAS/STING), which has yet to be determined. Methods Human lung fibroblasts (HLFs) from normal donors and SSc‐ILD explants were treated with synthetic CpG DNA and assayed for α‐SMA expression and extracellular mtDNA using quantitative polymerase chain reaction for the human MT‐ATP6 gene. Plasma MT‐ATP6 concentrations were evaluated in 2 independent SSc‐ILD cohorts and demographically matched controls. The ability of SSc‐ILD and control plasma to induce TLR‐9 and cGAS/STING activation was evaluated with commercially available HEK 293 reporter cells. Plasma concentrations of type I interferons (IFNs), interleukin‐6 (IL‐6), and oxidized DNA were measured using electrochemiluminescence and enzyme‐linked immunosorbent assay–based methods. Extracellular vesicles (EVs) precipitated from plasma were evaluated for MT‐ATP6 concentrations and proteomics via liquid chromatography mass spectrometry. Results Normal HLFs and SSc‐ILD fibroblasts developed increased α‐SMA expression and MT‐ATP6 release following CpG stimulation. Plasma mtDNA concentrations were increased in the 2 SSc‐ILD cohorts, reflective of ventilatory decline, and were positively associated with both TLR‐9 and cGAS/STING activation as well as type I IFN and IL‐6 expression. Plasma mtDNA was not oxidized and was conveyed by EVs displaying a proteomics profile consistent with a multicellular origin. Conclusion These findings demonstrate a previously unrecognized connection between EV‐encapsulated mtDNA, clinical outcomes, and intracellular DNA–sensing PRR activation in SSc‐ILD. Further study of these interactions could catalyze novel mechanistic and therapeutic insights into SSc‐ILD and related disorders.
Introduction Chronic ethanol consumption is associated with persistent hepatitis C viral (HCV) infection. This study explores the role of the host cellular immune response to HCV core protein in a murine model and how chronic ethanol consumption alters T-cell regulatory (Tregs) populations. Methods Balb/c mice were fed an isocaloric control or ethanol liquid diet. Dendritic cells (DCs) were isolated after expansion with a hFl3tL-expression plasmid and subsequently transfected with HCV core protein. Core-containing DCs (1 × 106) were subcutaneously injected (X3) in mice every 2 weeks. Splenocytes from immunized mice were isolated and stimulated with HCV core protein to measure generation of viral antigen specific Tregs, as well as secretion of IL-2, TNF-α, and IL-4. Cytotoxicity was measured by lactate dehydrogenase (LDH) release from HCV core-expressing syngeneic SP2/19 myeloma cells. Results Splenoctyes from mice immunized with ethanol-derived and HCV core-loaded DCs exhibited significantly lower in vitro cytotoxicity compared to mice immunized with HCV core-loaded DCs derived from isocaloric pair fed controls. Stimulation with HCV core protein triggered higher IL-2 TNF-α and IL-4 release in splenocytes following immunization with core-loaded DCs derived from controls as compared to chronic ethanol fed mice. Splenocytes derived from mice immunized with core-loaded DCs isolated from ethanol-fed mice exhibited a significantly higher CD25+FOXP3+ and CD4+FOXP3+ Treg population. Conclusions These results suggest that immunization with HCV core-containing DCs from ethanol-fed mice induces an increase in the CD25+FOXP3+ and CD4+FOXP3+ Treg population and may suppress HCV core-specific CD4+ and CD8+ T cell immune responses.
Background/Aims We have compared dendritic cell (DC) function derived from the alcoholic liver disease (ALD) sensitive Long–Evans (LE) and resistant Fischer rat strains to determine if the influence of ethanol on DCs was dependent on ALD. Methods The LE and Fischer rats were fed an ethanol-containing or isocaloric control liquid diet for 8 weeks and comparisons were made to LE rats injected with thioacetamide as a liver disease control. DCs were isolated from the spleen after expansion with human Fms-like tyrosine kinase receptor 3 ligand plasmid. Maturation markers CD86, CD80, CD40 and MHC-II were analysed by flow cytometry with or without lipopolysaccharide and poly I:C stimulation. Production of tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-12p40 and IL-10 cytokines and the antigen presentation ability of DCs was determined. Results Only LE rats developed ALD characterized by liver injury, elevated alanine aminotransferase levels and steatosis; CD86 and CD40 expression was decreased in LE but not Fischer rats. Reduced TNF-α, IFN-γ, IL-12, proinflammatory and enhanced IL-10 cytokine production was found in DCs isolated from ethanol-fed LE but not Fischer rats. Allostimulatory activity was reduced in LE compared with the Fischer strain. In contrast, DCs isolated from thioacetamide-induced liver damage displayed a reduction only in IL-12p40; TNF-α, IL-10 and IFN-α production as well as antigen presenting ability remained intact compared with controls. Conclusions ALD sensitive LE rats exhibited characteristics of a suppressed DC phenotype that was not observed following thioacetamide-induced liver disease, which suggests an important role for ALD in altering the host cellular and humoral immune responses.
Background Antimicrobial resistance is a global public health problem, particularly in low‐ and middle‐income countries (LMICs), where antibiotics are often obtained without a prescription. H. pylori antimicrobial resistance patterns are informative for patient care and gastric cancer prevention programs, have been shown to correlate with general antimicrobial consumption, and may guide antimicrobial stewardship programs in LMICs. We report H. pylori resistance and antimicrobial utilization patterns for western Honduras, representative of rural Central America. Methods In the context of the western Honduras gastric cancer epidemiology initiative, gastric biopsies from 189 patients were studied for culture and resistance patterns. Antimicrobial utilization was investigated for common H. pylori treatment regimens from regional public (7 antimicrobials) and national private (4 antimicrobials) data, analyzed in accordance with WHO anatomical therapeutic chemical defined daily doses (DDD) method and expressed as DDD/1000 inhabitants per day (DID) and per year (DIY). Results H. pylori was successfully cultured from 116 patients (56% males, mean age: 54), and nearly all strains were cagA+ and vacAs1m1+ positive (99% and 90.4%, respectively). Unexpectedly, high resistance was noted for levofloxacin (20.9%) and amoxicillin (10.7%), while metronidazole (67.9%) and clarithromycin (11.2%) were similar to data from Latin America. Significant associations with age, gender, or histology were not noted, with the exception of levofloxacin (28%, P = 0.01) in those with histology limited to non‐atrophic gastritis. Total antimicrobial usage in western Honduras of amoxicillin (17.3 DID) and the quinolones had the highest relative utilizations compared with other representative nations. Conclusions We observed significant H. pylori resistance to amoxicillin and levofloxacin in the context of high community antimicrobial utilization. This has implications in Central America for H. pylori treatment guidelines as well as antimicrobial stewardship programs.
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