Pre-Operative SARS-CoV-2 Testing in Asymptomatic Heart Transplantation Recipients.

INTRODUCTION: From the start of the coronavirus disease 2019 (COVID-19) pandemic, international guidelines have recommended pre-operative screening for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) before heart transplantation (HTx). Due to the changing prevalence of COVID-19, the chances of false positive results have increased. Because of increased immunity in the population and evolution of SARS-CoV-2 to current Omicron variants, associated mortality and morbidity have decreased. We set out to investigate the yield and side effects of SARS-CoV-2 screening in our center. METHODS: We performed a retrospective cohort study in the University Medical Center Utrecht. The study period was from March 2019 to January 2023. All data from patients who underwent HTx were collected, including all pre-operative and post-operative SARS-CoV-2 tests. Furthermore, all clinical SARS-CoV-2 tests for the indication of potential HTx were screened. RESULTS: In the period under study, 51 patients underwent HTx. None of the recipients reported any symptoms of a viral infection. Fifty HTx recipients were screened for SARS-CoV-2. Forty-nine out of fifty patients tested negative. One patient had a false positive result, potentially delaying the HTx procedure. There were no cancelled HTx procedures due to a true positive SARS-CoV-2 test result. CONCLUSION: Pre-operative SARS-CoV-2 screening in asymptomatic HTx recipients did not lead to any true positive cases. In 2% of the cases, screening resulted in a false positive test result. With the current Omicron variants, in combination with a low-prevalence situation, we propose to abandon pre-operative SARS-CoV-2 screening and initiate a symptom-driven approach for the general viral testing of patients who are called in for a potential HTx.

Grp78 is required for intestinal Kras-dependent glycolysis proliferation and adenomagenesis.

In development of colorectal cancer, mutations in APC are often followed by mutations in oncogene KRAS The latter changes cellular metabolism and is associated with the Warburg phenomenon. Glucose-regulated protein 78 (Grp78) is an important regulator of the protein-folding machinery, involved in processing and localization of transmembrane proteins. We hypothesize that targeting Grp78 in Apc and Kras (AK)-mutant intestines interferes with the metabolic phenotype imposed by Kras mutations. In mice with intestinal epithelial mutations in Apc, Kras G12D and heterozygosity for Grp78 (AK-Grp78 HET ) adenoma number and size is decreased compared with AK-Grp78 WT mice. Organoids from AK-Grp78 WT mice exhibited a glycolysis metabolism which was completely rescued by Grp78 heterozygosity. Expression and correct localization of glucose transporter GLUT1 was diminished in AK-Grp78 HET cells. GLUT1 inhibition restrained the increased growth observed in AK-mutant organoids, whereas AK-Grp78 HET organoids were unaffected. We identify Grp78 as a critical factor in Kras-mutated adenomagenesis. This can be attributed to a critical role for Grp78 in GLUT1 expression and localization, targeting glycolysis and the Warburg effect.

Impaired SARS-CoV-2 specific T-cell response in patients with severe COVID-19.

Cellular immune responses are of pivotal importance to understand SARS-CoV-2 pathogenicity. Using an enzyme-linked immunosorbent spot (ELISpot) interferon-γ release assay with wild-type spike, membrane and nucleocapsid peptide pools, we longitudinally characterized functional SARS-CoV-2 specific T-cell responses in a cohort of patients with mild, moderate and severe COVID-19. All patients were included before emergence of the Omicron (B.1.1.529) variant. Our most important finding was an impaired development of early IFN-γ-secreting virus-specific T-cells in severe patients compared to patients with moderate disease, indicating that absence of virus-specific cellular responses in the acute phase may act as a prognostic factor for severe disease. Remarkably, in addition to reactivity against the spike protein, a substantial proportion of the SARS-CoV-2 specific T-cell response was directed against the conserved membrane protein. This may be relevant for diagnostics and vaccine design, especially considering new variants with heavily mutated spike proteins. Our data further strengthen the hypothesis that dysregulated adaptive immunity plays a central role in COVID-19 immunopathogenesis.

Differential abundance of IgG antibodies against the spike protein of SARS-CoV-2 and seasonal coronaviruses in patients with fatal COVID-19.

Infection with the novel pandemic SARS-CoV-2 virus has been shown to elicit a cross-reactive immune response that could lead to a back-boost of memory recall to previously encountered seasonal (endemic) coronaviruses (eCoVs). Whether this response is associated with a fatal clinical outcome in patients with severe COVID-19 remains unclear. In a cohort of hospitalized patients, we have previously shown that heterologous immune responses to eCoVs can be detected in severe COVID-19. Here, we report that COVID-19 patients with fatal disease have decreased SARS-CoV-2 neutralizing antibody titers at hospital admission, which correlated with lower SARS-CoV-2 spike-specific IgG and was paralleled by a relative abundance of IgG against spike protein of eCoVs of the genus Betacoronavirus. Additional research is needed to assess if eCoV-specific back-boosted IgG is a bystander phenomenon in severe COVID-19, or a factor that influences the development of an efficient anti-viral immune response.

Intestinal Apc-inactivation induces HSP25 dependency.

The majority of colorectal cancers (CRCs) present with early mutations in tumor suppressor gene APC. APC mutations result in oncogenic activation of the Wnt pathway, which is associated with hyperproliferation, cytoskeletal remodeling, and a global increase in mRNA translation. To compensate for the increased biosynthetic demand, cancer cells critically depend on protein chaperones to maintain proteostasis, although their function in CRC remains largely unexplored. In order to investigate the role of molecular chaperones in driving CRC initiation, we captured the transcriptomic profiles of murine wild type and Apc-mutant organoids during active transformation. We discovered a strong transcriptional upregulation of Hspb1, which encodes small heat shock protein 25 (HSP25). We reveal an indispensable role for HSP25 in facilitating Apc-driven transformation, using both in vitro organoid cultures and mouse models, and demonstrate that chemical inhibition of HSP25 using brivudine reduces the development of premalignant adenomas. These findings uncover a hitherto unknown vulnerability in intestinal transformation that could be exploited for the development of chemopreventive strategies in high-risk individuals.

Differential vaccine-induced kinetics of humoral and cellular immune responses in SARS-CoV-2 naive and convalescent health care workers.

Effective vaccination is a key element in the exit strategy from the current severe acute respiratory syndrome-CoV coronavirus-2 (SARS-CoV-2) pandemic, and may also offer protection against severe disease from future variants of concern. Here, we prospectively monitored T-cell responses over time, using ELISpot interferon-γ (INF-y) release assays, and B-cell responses, using serological tests, after vaccination and booster with BioNTech/Pfizer mRNA (Pfizer) and Janssen vector (Janssen/Johnson & Johnson) vaccines in hospital health care workers. Vaccine recipients were divided into seropositive and seronegative individuals at baseline, in order to determine the effect of natural immunity on vaccine-induced immune kinetics. We found that convalescent individuals mounted higher spike-specific INF-y-secreting T-cell responses and B-cell-mediated IgG responses, after receiving the Janssen vaccine or the first dose of the Pfizer vaccine. IgG levels corresponded to the virus neutralization capacity as measured by VNT assay. At 8 months postvaccination, spike-specific cellular immunity waned to low levels in individuals with or without prior natural immunity, whereas waning of humoral immunity occurred predominantly in naive individuals. The booster shot effectively reinduced both cellular and humoral immune responses. To conclude, our data supports the implemented single-dose mRNA booster strategy employed in the Netherlands. Furthermore, the level of pre-existing natural immunity may be factored into determining the optimal time window between future booster vaccines.

Heterologous Immune Responses of Serum IgG and Secretory IgA Against the Spike Protein of Endemic Coronaviruses During Severe COVID-19.

Defining immune correlates of disease severity is important to better understand the immunopathogenesis in COVID-19. Here we made use of a protein microarray platform to detect IgG- and IgA-reactive antibodies in sera and saliva respectively, and assess cross-reactivity between SARS-CoV-2 and endemic coronaviruses (eCoVs). IgG responses against the full protein of spike, but not the S1 subunit, were significantly higher in convalescent sera of patients with severe disease compared to mild disease and healthy controls. In addition, we detected reactivity of secretory IgA to eCoVs in saliva of patients with severe disease, not present in patients with moderate disease or seropositive healthy controls. These heterologous immune responses are in line with non-protective cross-reactivity, and support a potential role for immune imprinting in the pathogenesis of severe COVID-19.

The concerted action of oncogenic driver mutations directs global translation in intestinal epithelial cells.

Oncogenic transformation of colorectal cancer cells is driven by a set of mutations that cause aberrant signaling of growth factor-receptor pathways. Using organoids, we demonstrate that the most frequent driver mutations in APC, KRAS, SMAD4, and TP53 are enhancers of the global mRNA translational capacity, which is linked to intestinal cell growth in an mTOR-dependent manner.

Epithelial argininosuccinate synthetase is dispensable for intestinal regeneration and tumorigenesis.

The epithelial signaling pathways involved in damage and regeneration, and neoplastic transformation are known to be similar. We noted upregulation of argininosuccinate synthetase (ASS1) in hyperproliferative intestinal epithelium. Since ASS1 leads to de novo synthesis of arginine, an important amino acid for the growth of intestinal epithelial cells, its upregulation can contribute to epithelial proliferation necessary to be sustained during oncogenic transformation and regeneration. Here we investigated the function of ASS1 in the gut epithelium during tissue regeneration and tumorigenesis, using intestinal epithelial conditional Ass1 knockout mice and organoids, and tissue specimens from colorectal cancer patients. We demonstrate that ASS1 is strongly expressed in the regenerating and Apc-mutated intestinal epithelium. Furthermore, we observe an arrest in amino acid flux of the urea cycle, which leads to an accumulation of intracellular arginine. However, loss of epithelial Ass1 does not lead to a reduction in proliferation or increase in apoptosis in vivo, also in mice fed an arginine-free diet. Epithelial loss of Ass1 seems to be compensated by altered arginine metabolism in other cell types and the liver.

Early Life Antibiotics Influence In Vivo and In Vitro Mouse Intestinal Epithelium Maturation and Functioning.

BACKGROUND & AIMS: The use of antibiotics (ABs) is a common practice during the first months of life. ABs can perturb the intestinal microbiota, indirectly influencing the intestinal epithelial cells (IECs), but can also directly affect IECs independent of the microbiota. Previous studies have focused mostly on the impact of AB treatment during adulthood. However, the difference between the adult and neonatal intestine warrants careful investigation of AB effects in early life. METHODS: Neonatal mice were treated with a combination of amoxicillin, vancomycin, and metronidazole from postnatal day 10 to 20. Intestinal permeability and whole-intestine gene and protein expression were analyzed. IECs were sorted by a fluorescence-activated cell sorter and their genome-wide gene expression was analyzed. Mouse fetal intestinal organoids were treated with the same AB combination and their gene and protein expression and metabolic capacity were determined. RESULTS: We found that in vivo treatment of neonatal mice led to decreased intestinal permeability and a reduced number of specialized vacuolated cells, characteristic of the neonatal period and necessary for absorption of milk macromolecules. In addition, the expression of genes typically present in the neonatal intestinal epithelium was lower, whereas the adult gene expression signature was higher. Moreover, we found altered epithelial defense and transepithelial-sensing capacity. In vitro treatment of intestinal fetal organoids with AB showed that part of the consequences observed in vivo is a result of the direct action of the ABs on IECs. Lastly, ABs reduced the metabolic capacity of intestinal fetal organoids. CONCLUSIONS: Our results show that early life AB treatment induces direct and indirect effects on IECs, influencing their maturation and functioning.

Endoplasmic reticulum stress regulates the intestinal stem cell state through CtBP2.

Enforcing differentiation of cancer stem cells is considered as a potential strategy to sensitize colorectal cancer cells to irradiation and chemotherapy. Activation of the unfolded protein response, due to endoplasmic reticulum (ER) stress, causes rapid stem cell differentiation in normal intestinal and colon cancer cells. We previously found that stem cell differentiation was mediated by a Protein kinase R-like ER kinase (PERK) dependent arrest of mRNA translation, resulting in rapid protein depletion of WNT-dependent transcription factor c-MYC. We hypothesize that ER stress dependent stem cell differentiation may rely on the depletion of additional transcriptional regulators with a short protein half-life that are rapidly depleted due to a PERK-dependent translational pause. Using a novel screening method, we identify novel transcription factors that regulate the intestinal stem cell fate upon ER stress. ER stress was induced in LS174T cells with thapsigargin or subtilase cytotoxin (SubAB) and immediate alterations in nuclear transcription factor activity were assessed by the CatTFRE assay in which transcription factors present in nuclear lysate are bound to plasmid DNA, co-extracted and quantified using mass-spectrometry. The role of altered activity of transcription factor CtBP2 was further examined by modification of its expression levels using CAG-rtTA3-CtBP2 overexpression in small intestinal organoids, shCtBP2 knockdown in LS174T cells, and familial adenomatous polyposis patient-derived organoids. CtBP2 overexpression organoids were challenged by ER stress and ionizing irradiation. We identified a unique set of transcription factors with altered activation upon ER stress. Gene ontology analysis showed that transcription factors with diminished binding were involved in cellular differentiation processes. ER stress decreased CtBP2 protein expression in mouse small intestine. ER stress induced loss of CtBP2 expression which was rescued by inhibition of PERK signaling. CtBP2 was overexpressed in mouse and human colorectal adenomas. Inducible CtBP2 overexpression in organoids conferred higher clonogenic potential, resilience to irradiation-induced damage and a partial rescue of ER stress-induced loss of stemness. Using an unbiased proteomics approach, we identified a unique set of transcription factors for which DNA-binding activity is lost directly upon ER stress. We continued investigating the function of co-regulator CtBP2, and show that CtBP2 mediates ER stress-induced loss of stemness which supports the intestinal stem cell state in homeostatic stem cells and colorectal cancer cells.

Driver mutations of the adenoma-carcinoma sequence govern the intestinal epithelial global translational capacity.

Deregulated global mRNA translation is an emerging feature of cancer cells. Oncogenic transformation in colorectal cancer (CRC) is driven by mutations in APC, KRAS, SMAD4, and TP53, known as the adenoma-carcinoma sequence (ACS). Here we introduce each of these driver mutations into intestinal organoids to show that they are modulators of global translational capacity in intestinal epithelial cells. Increased global translation resulting from loss of Apc expression was potentiated by the presence of oncogenic KrasG12D Knockdown of Smad4 further enhanced global translation efficiency and was associated with a lower 4E-BP1-to-eIF4E ratio. Quadruple mutant cells with additional P53 loss displayed the highest global translational capacity, paralleled by high proliferation and growth rates, indicating that the proteome is heavily geared toward cell division. Transcriptional reprogramming facilitating global translation included elevated ribogenesis and activation of mTORC1 signaling. Accordingly, interfering with the mTORC1/4E-BP/eIF4E axis inhibited the growth potential endowed by accumulation of multiple drivers. In conclusion, the ACS is characterized by a strongly altered global translational landscape in epithelial cells, exposing a therapeutic potential for direct targeting of the translational apparatus.

Expression of UPR effector proteins ATF6 and XBP1 reduce colorectal cancer cell proliferation and stemness by activating PERK signaling.

The unfolded protein response (UPR) acts through its downstream branches, PERK-eIF2α signaling, IRE1α-XBP1 signaling and ATF6 signaling. In the intestine, activation of the UPR through the kinase PERK results in differentiation of intestinal epithelial stem cells and colon cancer stem cells, whereas deletion of XBP1 results in increased stemness and adenomagenesis. How downstream activation of XBP1 and ATF6 influences intestinal stemness and proliferation remains largely unknown. We generated colorectal cancer cells (LS174T) that harbor doxycycline inducible expression of the active forms of either XBP1(s) or ATF61-373. Activation of either XBP1 or ATF6 resulted in reduced cellular proliferation and reduced expression of markers of intestinal epithelial stemness. Moreover, XBP1 and ATF6 activation reduced global protein synthesis and lowered the threshold for UPR activation. XBP1-mediated loss of stemness and proliferation resulted from crossactivation of PERK-eIF2α signaling and could be rescued by constitutive expression of eIF2α phosphatase GADD34. We thus find that enforced activation of XBP1 and ATF6 results in reduction of stemness and proliferation. We expose a novel interaction between XBP1 and PERK-eIF2α signaling.

Heterozygosity of Chaperone Grp78 Reduces Intestinal Stem Cell Regeneration Potential and Protects against Adenoma Formation.

Deletion of endoplasmic reticulum resident chaperone Grp78 results in activation of the unfolded protein response and causes rapid depletion of the entire intestinal epithelium. Whether modest reduction of Grp78 may affect stem cell fate without compromising intestinal integrity remains unknown. Here, we employ a model of epithelial-specific, heterozygous Grp78 deletion by use of VillinCreERT2-Rosa26ZsGreen/LacZ-Grp78+/fl mice and organoids. We examine models of irradiation and tumorigenesis, both in vitro and in vivo Although we observed no phenotypic changes in Grp78 heterozygous mice, Grp78 heterozygous organoid growth was markedly reduced. Irradiation of Grp78 heterozygous mice resulted in less frequent regeneration of crypts compared with nonrecombined (wild-type) mice, exposing reduced capacity for self-renewal upon genotoxic insult. We crossed mice to Apc-mutant animals for adenoma studies and found that adenomagenesis in Apc heterozygous-Grp78 heterozygous mice was reduced compared with Apc heterozygous controls (1.43 vs. 3.33; P < 0.01). In conclusion, epithelium-specific Grp78 heterozygosity compromises epithelial fitness under conditions requiring expansive growth such as adenomagenesis or regeneration after γ-irradiation. These results suggest that Grp78 may be a therapeutic target in prevention of intestinal neoplasms without affecting normal tissue.Significance: Heterozygous disruption of chaperone protein Grp78 reduces tissue regeneration and expansive growth and protects from tumor formation without affecting intestinal homeostasis. Cancer Res; 78(21); 6098-106. ©2018 AACR.

No evidence to support a role for Helicobacter pylori infection and plasminogen binding protein in autoimmune pancreatitis and IgG4-related disease in a UK cohort.

BACKGROUND AND OBJECTIVES: Helicobacter pylori (H.pylori) plasminogen binding protein (PBP) has been proposed as an antigen triggering autoimmune pancreatitis (AIP), the pancreatic manifestation of IgG4-related disease (IgG4-RD). We investigated exposure to H. pylori infection, cytokine response and immunological memory to H. pylori PBP in a prospective IgG4-RD cohort in the UK. METHODS: Clinical and endoscopic evidence of peptic ulceration, serological H. pylori exposure and serum IgG4 levels were obtained in 55 IgG4-RD patients and 52 disease controls (DC) with autoimmune or inflammatory conditions with an elevated serum IgG4. Gastric and duodenal tissues were assessed for H. pylori and immunostained for IgG4. B and T cell ELISpot and cytokine luminex assays were used to detect immune responses to H. pylori PBP. RESULTS: 85% of IgG4-RD patients had pancreatic and/or biliary disease, 89% had extra-pancreatic manifestations, and 84% had an increased serum IgG4. Clinical dyspepsia (35.2%), gastritis (58%), peptic ulceration (7.4%) and H. pylori colonisation (24%) in IgG4-RD was similar to DC. In IgG4-RD, gastric tissue contained a chronic inflammatory infiltrate with a low IgG4+ plasma-cell count (<10/HPF; range 1-4/HPF), and duodenal specimens had an increased IgG4 count (>10/HPF; range 7-54) compared with DC (p < 0.01). Th1 and Th2 cytokine response and immunological B-cell memory to H. pylori PBP did not differ between IgG4-RD and DC. CONCLUSIONS: In a prospective UK cohort, the prevalence of gastric ulceration, exposure to H. pylori, cytokine response and immunological memory to H. pylori PBP did not differ in IgG4-RD patients compared with DC. This study does not support a role for H. pylori PBP as a microbial antigen in IgG4-RD. KEYWORDS FOR ABSTRACT: Peptic ulceration, Antigens, B cells, T cells, Interleukins, Helicobacter pylori.

New Thoughts on Immunoglobulin G4-Related Sclerosing Cholangitis.

Immunoglobulin G4 (IgG4)-related sclerosing cholangitis (IgG4-SC) is the biliary manifestation of the multisystem IgG4-related disease. IgG4-SC presents with biliary strictures and/or masses that can bear a striking similarity to other malignant and inflammatory diseases. Diagnosis is based on a combination of clinical, biochemical, radiological, and histologic findings with careful exclusion of malignant disease. Corticosteroids are the mainstay of treatment with good clinical, biochemical, and radiological responses. This review provides a comprehensive overview of the current knowledge of the prevalence, clinical features, radiology and histology findings, diagnosis, treatment, natural history, and pathophysiology of IgG4-SC.

Characterization and treatment of persistent hepatocellular secretory failure.

BACKGROUND & AIMS: Hepatocellular secretory failure induced by drugs, toxins or transient biliary obstruction may sometimes persist for months after removal of the initiating factor and may then be fatal without liver transplantation. We characterized patients with severe persistent hepatocellular secretory failure (PHSF) and treated them with the pregnane X receptor (PXR) agonist, rifampicin. We also studied the effect of rifampicin on PXR-dependent expression of genes involved in biotransformation and secretion in vitro. METHODS: Thirteen patients (age 18-81 years, 6 male) with hepatocellular secretory failure that persisted after removal of the inducing factor (drugs/toxin: 9) or biliary obstruction (4) were identified over 6 years. Six of these patients were screened for ATP8B1 or ABCB11 mutations. All were treated with rifampicin (300 mg daily) for 1-10 weeks. Expression of genes involved in biotransformation and secretion was determined by rtPCR in human hepatocytes and intestinal cells incubated with rifampicin (10 µmol/L). RESULTS: Serum bilirubin of patients with PHSF ranged from 264 to 755 µmol/L. Normal γGT was found in 10/13 patients of whom 3/6 tested positive for ATP8B1/ABCB11 mutations. Serum bilirubin declined to <33 µmol/L after 1-10 weeks of rifampicin treatment. In vitro, rifampicin PXR-dependently upregulated biotransformation phase 1 (CYP3A4), phase 2 (UGT1A1) and phase 3 (MRP2) enzymes/carriers as well as the basolateral bile salt exporter OSTß. CONCLUSION: Persistent hepatocellular secretory failure may develop in carriers of transporter gene mutations. In severe cases, rifampicin may represent an effective therapeutic option of PHSF. PXR-dependent induction of CYP3A4, UGT1A1, MRP2 and OSTß could contribute to the anticholestatic effect of rifampicin in PHSF.

The emerging mysteries of IgG4-related disease.

IgG4-related disease (IgG4-RD) is increasingly recognised in Western societies as a multi-system, inflammatory, fibrosing disease of unknown aetiology that typically, though not exclusively, presents in older men. The clinical manifestations are diverse and almost any organ may be affected. The cardinal histological features are a lymphoplasmacytic infiltrate, storiform fibrosis, obliterative phlebitis and an abundance of IgG4+ plasma cells in affected organs. Serum IgG4 levels are elevated in approximately 70% of patients and are a useful biomarker when present. IgG4-RD is frequently misdiagnosed as malignancy. Making the correct diagnosis is important as the disease is usually steroid responsive, although relapse rates are high. Second-line immunosuppressive agents and B-cell depletion therapy have also been used in retreatment strategies. Recent data suggests that the disease is associated with both progressive organ failure and malignancy. The biological mechanisms driving IgG4-RD remain unclear but this is currently an area of intense scientific investigation. Broadly, IgG4+ B cells are thought to exhibit a regulatory phenotype, but it is not known if these are pathogenic or simply represent a bystander effect. Extending our understanding of the role of IgG4 immunoglobulins in health and disease, the assessment of B and T cell immune phenotype, and large genetic studies of IgG4-RD may enhance our understanding of disease pathogenesis. Ultimately it may be that there is not a single, simple unifying aetiology and so careful stratification of disease by clinical phenotype will be required in multi-centre prospective clinical cohorts. These cohorts will also be essential for the study of treatment outcomes with novel therapies.

Tritrophic interactions between aphids (Aphis jacobaeae Schrank), ant species, Tyria jacobaeae L., and Senecio jacobaea L. lead to maintenance of genetic variation in pyrrolizidine alkaloid concentration.

We hypothesize that the tritrophic interaction between ants, the aphid Aphis jacobaeae, the moth Tyria jacobaeae, and the plant Senecio jacobaea can explain the genetic variation observed in pyrrolizidine alkaloid concentration in natural populations of S. jacobaea. The ant Lasius niger effectively defends S. jacobaea plants infested with A. jacobaeae against larvae of T. jacobaeae. S. jacobaea plants with A. jacobaeae which are defended by ants escape regular defoliation by T. jacobaeae. Plants with aphids and ants have a lower pyrrolizidine alkaloid concentration than plants without aphids and ants. When these data are fitted to an existing theoretical model for temporal variation in fitness it is shown that varying herbivore pressure by T. jacobaeae in interaction with ants defending aphid-infested plants with a low pyrrolizidine alkaloid concentration can lead to a stable polymorphism in pyrrolizidine alkaloid concentration. Costs of the production and maintenance of pyrrolizidine alkaloids are not accounted for in the model.