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To enable rapid proliferation, colorectal tumor cells up-regulate epidermal growth factor receptor (EGFR) signaling and aerobic glycolysis, resulting in substantial lactate release into the tumor microenvironment and impaired anti-tumor immune responses. We hypothesized that a nutritional intervention designed to reduce aerobic glycolysis may boost the EGFR-directed antibody (Ab)-based therapy of pre-existing colitis-driven colorectal carcinoma (CRC). CRC development was induced by azoxymethane (AOM) and dextran sodium sulfate (DSS) administration to C57BL/6 mice. AOM/DSS-treated mice were fed a glucose-free, high-protein diet (GFHPD) or an isoenergetic control diet (CD) in the presence or absence of an i.p. injection of an anti-EGFR mIgG2a or respective controls. AOM/DSS-treated mice on a GFHPD displayed a reduced systemic glucose metabolism associated with reduced oxidative phosphorylation (OXPHOS) complex IV expression and diminished tumor loads. Comparable but not additive to an anti-EGFR-Ab therapy, the GFHPD was accompanied by enhanced tumoral goblet cell differentiation and decreased colonic PD-L1 and splenic CD3ε, as well as PD-1 immune checkpoint expression. In vitro, glucose-free, high-amino acid culture conditions reduced proliferation but improved goblet cell differentiation of murine and human CRC cell lines MC-38 and HT29-MTX in combination with down-regulation of PD-L1 expression. We here found GFHPD to systemically dampen glycolysis activity, thereby reducing CRC progression with a similar efficacy to EGFR-directed antibody therapy.

Rapid proliferation of cancer cells is enabled by favoring aerobic glycolysis over mitochondrial oxidative phosphorylation (OXPHOS). P32 ( C1QBP /gC1qR) is essential for mitochondrial protein translation and thus indispensable for OXPHOS activity. It is ubiquitously expressed and directed to the mitochondrial matrix in almost all cell types with an excessive up-regulation of p32 expression reported for tumor tissues. We recently demonstrated high levels of non-mitochondrial p32 to be associated with high-grade colorectal carcinoma. Mutations in human p32 are likely to disrupt proper mitochondrial function giving rise to various diseases including cancer. Hence, we aimed to investigate the impact of the most common single nucleotide polymorphism (SNP) rs56014026 in the coding sequence of p32 on tumor cell metabolism. In silico homology modeling of the resulting p.Thr130Met mutated p32 revealed that the single amino acid substitution potentially induces a strong conformational change in the protein, mainly affecting the mitochondrial targeting sequence (MTS). In vitro experiments confirmed an impaired mitochondrial import of mutated p32-T130M, resulting in reduced OXPHOS activity and a shift towards a low metabolic phenotype. Overexpression of p32-T130M maintained terminal differentiation of a goblet cell-like colorectal cancer cell line compared to p32-wt without affecting cell proliferation. Sanger sequencing of tumor samples from 128 CRC patients identified the heterozygous SNP rs56014026 in two well-differentiated, low proliferating adenocarcinomas, supporting our in vitro data. Together, the SNP rs56014026 reduces metabolic activity and proliferation while promoting differentiation in tumor cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest with the exception of BG and EP who receive royalties from the sale of the monoclonal anti-p32 antibody 60.11., (Copyright © 2021 Raschdorf, Sünderhauf, Skibbe, Ghebrehiwet, Peerschke, Sina and Derer.)

Background & Aims: Cell differentiation in the colonic crypt is driven by a metabolic switch from glycolysis to mitochondrial oxidation. Mitochondrial and goblet cell dysfunction have been attributed to the pathology of ulcerative colitis (UC). We hypothesized that p32/gC1qR/HABP1, which critically maintains oxidative phosphorylation, is involved in goblet cell differentiation and hence in the pathogenesis of UC., Methods: Ex vivo, goblet cell differentiation in relation to p32 expression and mitochondrial function was studied in tissue biopsies from UC patients versus controls. Functional studies were performed in goblet cell-like HT29-MTX cells in vitro. Mitochondrial respiratory chain complex V-deficient, ATP8 mutant mice were utilized as a confirmatory model. Nutritional intervention studies were performed in C57BL/6 mice., Results: In UC patients in remission, colonic goblet cell differentiation was significantly decreased compared to controls in a p32-dependent manner. Plasma/serum L-lactate and colonic pAMPK level were increased, pointing at high glycolytic activity and energy deficiency. Consistently, p32 silencing in mucus-secreting HT29-MTX cells abolished butyrate-induced differentiation and induced a shift towards glycolysis. In ATP8 mutant mice, colonic p32 expression correlated with loss of differentiated goblet cells, resulting in a thinner mucus layer. Conversely, feeding mice an isocaloric glucose-free, high-protein diet increased mucosal energy supply that promoted colonic p32 level, goblet cell differentiation and mucus production., Conclusion: We here describe a new molecular mechanism linking mucosal energy deficiency in UC to impaired, p32-dependent goblet cell differentiation that may be therapeutically prevented by nutritional intervention., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Adaptation of hepatitis C virus (HCV) to CD8 + T cell selection pressure is well described; however, it is unclear if HCV differentially adapts in different populations. Here, we studied HLA class I-associated viral sequence polymorphisms in HCV 1b isolates in a Chinese population and compared viral substitution patterns between Chinese and German populations. We identified three HLA class I-restricted epitopes in HCV NS3 with statistical support for selection pressure and found evidence for differential escape pathways between isolates from China and Germany depending on the HLA class I molecule. The substitution patterns particularly differed in the epitope VTLTHPITK 1635-1643 , which was presented by HLA-A*03 as well as HLA-A*11, two alleles with highly different frequencies in the two populations. In Germany, a substitution in position seven of the epitope was the most frequent substitution in the presence of HLA-A*03, functionally associated with immune escape and nearly absent in Chinese isolates. In contrast, the most frequent substitution in China was located at position two of the epitope and became the predominant consensus residue. Moreover, substitutions in position one of the epitope were significantly enriched in HLA-A*11-positive individuals in China and associated with different patterns of CD8 + T cell reactivity. Our study confirms the differential escape pathways selected by HCV that depended on different HLA class I alleles in Chinese and German populations, indicating that HCV differentially adapts to distinct HLA class I alleles in these populations. This result has important implications for vaccine design against highly variable and globally distributed pathogens, which may require matching antigen sequences to geographic regions for T cell-based vaccine strategies., (© 2018 The Authors. Journal of Viral Hepatitis Published by John Wiley & Sons Ltd.)

The complement system not only plays a critical role in efficient detection and clearance of bacteria, but also in intestinal immune homeostasis as mice deficient for key complement components display enhanced intestinal inflammation upon experimental colitis. Because underlying molecular mechanisms for this observation are unclear, we investigated the crosstalk between intestinal epithelial cells (IEC), bacteria and the complement system in the course of chronic colitis. Surprisingly, mouse intestinal epithelial cell lines constitutively express high mRNA levels of complement component 3 (C3), Toll-like receptor 2 (Tlr2) and Tlr4. Stimulation of these cells with lipopolysaccharide (LPS), but not with flagellin, LD-muramyldipeptide or peptidoglycan, triggered increased C3 expression, secretion and activation. Stimulation of the C3aR on these cell lines with C3a resulted in an increase of LPS-triggered pro-inflammatory response. Tissue biopsies from C57BL/6J mice revealed higher expression of C3, Tlr1, Tlr2 and Tlr4 in colonic primary IECs (pIECs) compared to ileal pIECs, while in germ-free mice no differences in C3 protein expression was observed. In DSS-induced chronic colitis mouse models, C3 mRNA expression was upregulated in colonic biopsies and ileal pIECs with elevated C3 protein in the lamina propria, IECs and the mucus. Notably, increased C3b opsonization of mucosa-attached bacteria and decreased fecal full-length C3 protein was observed in DSS-treated compared to untreated mice. Of significant interest, non-inflamed and inflamed colonic biopsy samples from CD but not UC patients displayed exacerbated C3 expression compared to controls. These findings suggest that a novel TLR4-C3 axis could control the intestinal immune response during chronic colitis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background/aims: Hypoxia occurs in many pathological conditions, including inflammation and cancer. Within this context, hypoxia was shown to inhibit but also to promote T cell responses. Due to this controversial function, we aimed to explore whether an insufficient anti-tumour response during colitis-associated colon cancer could be ascribed to a hypoxic microenvironment., Methods: Colitis-associated colon cancer was induced in wildtype mice, and hypoxia as well as T cell immunity were analysed in the colonic tumour tissues. In addition, CD4+ effector T cells and regulatory T cells were cultured under normoxic and hypoxic conditions and examined regarding their phenotype and function., Results: We observed severe hypoxia in the colon of mice suffering from colitis-associated colon cancer that was accompanied by a reduced differentiation of CD4+ effector T cells and an enhanced number and suppressive activity of regulatory T cells. Complementary ex vivo and in vitro studies revealed that T cell stimulation under hypoxic conditions inhibited the differentiation, proliferation and IFN-γ production of TH1 cells and enhanced the suppressive capacity of regulatory T cells. Moreover, we identified an active role for HIF-1α in the modulation of CD4+ T cell functions under hypoxic conditions., Conclusion: Our data indicate that oxygen availability can function as a local modulator of CD4+ T cell responses and thus influences tumour immune surveillance in inflammation-associated colon cancer., (© 2017 The Author(s)Published by S. Karger AG, Basel.)

Unlabelled: Antiviral CD8(+) T cells are a key component of the adaptive immune response against HCV, but their impact on viral control is influenced by preexisting viral variants in important target epitopes and the development of viral escape mutations. Immunodominant epitopes highly conserved across genotypes therefore are attractive for T cell based prophylactic vaccines. Here, we characterized the CD8(+) T cell response against the highly conserved HLA-B*51-restricted epitope IPFYGKAI1373-1380 located in the helicase domain of NS3 in people who inject drugs (PWID) exposed predominantly to HCV genotypes 1a and 3a. Despite this epitope being conserved in both genotypes, the corresponding CD8(+) T cell response was detected only in PWID infected with genotype 3a and HCV-RNA negative PWID, but not in PWID infected with genotype 1a. In genotype 3a, the detection of strong CD8(+) T cell responses was associated with epitope variants in the autologous virus consistent with immune escape. Analysis of viral sequences from multiple cohorts confirmed HLA-B*51-associated escape mutations inside the epitope in genotype 3a, but not in genotype 1a. Here, a distinct substitution in the N-terminal flanking region located 5 residues upstream of the epitope (S1368P; P = 0.00002) was selected in HLA-B*51-positive individuals. Functional assays revealed that the S1368P substitution impaired recognition of target cells presenting the endogenously processed epitope. The results highlight that, despite an epitope being highly conserved between two genotypes, there are major differences in the selected viral escape pathways and the corresponding T cell responses., Importance: HCV is able to evolutionary adapt to CD8(+) T cell immune pressure in multiple ways. Beyond selection of mutations inside targeted epitopes, this study demonstrates that HCV inhibits epitope processing by modification of the epitope flanking region under T cell immune pressure. Selection of a substitution five amino acids upstream of the epitope underlines that efficient antigen presentation strongly depends on its larger sequence context and that blocking of the multistep process of antigen processing by mutation is exploited also by HCV. The pathways to mutational escape of HCV are to some extent predictable but are distinct in different genotypes. Importantly, the selected escape pathway of HCV may have consequences for the destiny of antigen-specific CD8(+) T cells., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background & Aims: Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen., Methods: Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity., Results: Cell preparation yielded the following cell counts per gram of liver tissue: 2.0 ± 0.4 × 10(7) hepatocytes, 1.8 ± 0.5 × 10(6 )Kupffer cells, 4.3 ± 1.9 × 10(5) liver sinusoidal endothelial cells, and 3.2 ± 0.5 × 10(5) stellate cells. Hepatocytes were identified by albumin (95.5 ± 1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5 ± 1.2%) and exhibited phagocytic activity, as determined with 1 μm latex beads. Endothelial cells were CD146(+) (97.8 ± 1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of α-smooth muscle actin (97.1 ± 1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence., Conclusions: Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease.

Unlabelled: CD8+ T cells are an essential component of successful adaptive immune responses against hepatitis C virus (HCV). A major obstacle to vaccine design against HCV is its inherent viral sequence diversity. Here, we test the hypothesis that different sequence variants of an immunodominant CD8+ T cell epitope, all binding with high affinity to HLA class I, target different T cell receptor repertoires and thereby influence the quality of the CD8+ T cell response. The impacts of sequence differences in the HLA-A*02-restricted HCV NS31406-1415 epitope on in vitro priming of naive CD8+ T cells from seronegative donors and cross-reactivity of primed T cells with other epitope variants were characterized. Although the six epitope variants tested were all high-affinity binders to HLA-A*02:01, substantial differences in priming and cross-reactivity of CD8+ T cells were observed. The variant associated with the most reproducible priming and induction of T cells with broad cross-reactivity was a genotype 1b variant (KLSALGLNAV) that is more common in HCV isolates collected in Asia but is rare in sequences from Europe and North America. The superior immunogenicity and cross-reactivity of this relatively rare epitope variant were confirmed by using HCV-specific memory CD8+ T cells from people who inject drugs, who are frequently exposed to HCV. Collectively, the data suggest that sequence differences at the epitope level between HCV isolates substantially impact CD8+ T cell priming and the degree of cross-reactivity with other epitope variants., Importance: The results have important implications for vaccine design against highly variable pathogens and suggest that evidence-based selection of the vaccine antigen sequence may improve immunogenicity and T cell cross-reactivity. Cross-reactive CD8+ T cells are likely beneficial for immune control of transmitted viruses carrying epitope variants and for prevention of immune escape during acute infection. To this end, rare epitope variants and potentially even altered epitope sequences associated with priming of broadly cross-reactive T cell receptors should be considered for vaccine design and need further testing., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Antiviral CD8(+) T cells are a key component of the adaptive immune system against hepatitis C virus (HCV). For the development of immune therapies, it is essential to understand how CD8(+) T cells contribute to clearance of infection and why they fail so often. A mechanism for secondary failure is mutational escape of the virus. However, some substitutions in viral epitopes are associated with fitness costs and often require compensatory mutations. We hypothesized that compensatory mutations may point toward epitopes under particularly strong selection pressure that may be beneficial for vaccine design because of a higher genetic barrier to escape. We previously identified two HLA-B*15-restricted CD8(+) epitopes in NS5B (LLRHHNMVY(2450-2458) and SQRQKKVTF(2466-2474)), based on sequence analysis of a large HCV genotype 1b outbreak. Both epitopes are targeted in about 70% of HLA-B*15-positive individuals exposed to HCV. Reproducible selection of escape mutations was confirmed in an independent multicenter cohort in the present study. Interestingly, mutations were also selected in the epitope flanking region, suggesting that compensatory evolution may play a role. Covariation analysis of sequences from the database confirmed a significant association between escape mutations inside one of the epitopes (H2454R and M2456L) and substitutions in the epitope flanking region (S2439T and K2440Q). Functional analysis with the subgenomic replicon Con1 confirmed that the primary escape mutations impaired viral replication, while fitness was restored by the additional substitutions in the epitope flanking region. We concluded that selection of escape mutations inside an HLA-B*15 epitope requires secondary substitutions in the epitope flanking region that compensate for fitness costs.

Using crossed radioimmunoelectrophoresis ( CRIE ) aqueous extracts from pollen of Phleum pratense , Lolium perenne, Poa pratensis , Festuca pratensis and Alopecurus pratensis were investigated for allergen composition. We detected between 24 and 32 antigens. Employing sera from 11 patients with well established hay fever, IgE binding could be demonstrated in 15 out of 28 antigens in Phleum pratense , 13 out of 32 in Lolium perenne, 14 out of 26 in Poa pratensis , 12 out of 24 Festuca pratensis and 12 out of 24 antigens in Alopecurus pratensis . The 11 patients showed an individual pattern of sensitization against the various pollen allergens.

The seroprevalence and seasonal trend of antibody titers against equine monocytic ehrlichiosis (Potomac horse fever) were determined in apparently healthy horses in selected areas of Illinois in 1986. Sera from 1,367 horses (6 months to 29 years old) were evaluated for the presence of antibodies against Ehrlichia risticii with indirect immunofluorescence. The majority (88%) of the horses were Thoroughbred or Standardbred racehorses. The number of horses with antibodies against E risticii was 229/1,367 (16.75%). The titers in these horses ranged from 1:10 to 1:640. As the year progressed, the number of seropositive horses (titers greater than or equal to 1:10) and the magnitude of the titers increased significantly, both reaching a maximum in July and August, respectively (P less than 0.05). A relationship between seropositivity and gender was not detected. In the year prior to sampling, 56.8% of the seropositive horses had not been ill, whereas 0.8% had diarrhea, an episode of acute abdominal pain, or laminitis. It was concluded that a large number of horses in Illinois are exposed to E risticii, that maximal exposure occurs in July, and that the most common form of the disease in Illinois is not associated with clinical signs.