Proton pump inhibitor effect on esophageal protein signature of eosinophilic esophagitis, prediction, and evaluation of treatment response.

BACKGROUND: Recently, we have identified a dysregulated protein signature in the esophageal epithelium of eosinophilic esophagitis (EoE) patients including proteins associated with inflammation and epithelial barrier function; however, the effect of proton pump inhibitor (PPI) treatment on this signature is unknown. Herein, we used a proteomic approach to investigate: (1) whether PPI treatment alters the esophageal epithelium protein profile observed in EoE patients and (2) whether the protein signature at baseline predicts PPI response. METHODS: We evaluated the protein signature of esophageal biopsies using a cohort of adult EoE (n = 25) patients and healthy controls (C) (n = 10). In EoE patients, esophageal biopsies were taken before (pre) and after (post) an 8-week PPI treatment, determining the histologic response. Eosinophil count PostPPI was used to classify the patients: ≥15 eosinophils/hpf as non-responders (non-responder) and < 15 eosinophils/hpf as responders (R). Protein signature was determined and differentially accumulated proteins were characterized to identify altered biological processes and signaling pathways. RESULTS: Comparative analysis of differentially accumulated proteins between groups revealed common signatures between three groups of patients with inflammation (responder-PrePPI, non-responder-PrePPI, and non-responder-PostPPI) and without inflammation (controls and responder-PostPPI). PPI therapy almost reversed the EoE specific esophageal protein signature, which is enriched in pathways associated with inflammation and epithelial barrier function, in responder-PostPPI. Furthermore, we identified a set of candidate proteins to differentiate responder-PrePPI and non-responder-PrePPI EoE patients before treatment. CONCLUSION: These findings provide evidence that PPI therapy reverses the alterations in esophageal inflammatory and epithelial proteins characterizing EoE, thereby providing new insights into the mechanism of PPI clinical response. Interestingly, our results also suggest that PPI response could be predicted at baseline in EoE.

Fibrous Remodeling in Eosinophilic Esophagitis: Clinical Facts and Pathophysiological Uncertainties.

Eosinophilic esophagitis (EoE) is a chronic, progressive, type 2 inflammatory disease with increasing global prevalence. An eosinophil-predominant inflammation that permeates the epithelium and deeper esophageal layers characterizes the disease. Several cytokines, mainly derived from inflammatory T-helper 2 (Th2) cells and epithelial cells, are involved in perpetuating inflammatory responses by increasing surface permeability and promoting tissue remodeling characterized by epithelial-mesenchymal transition (EMT) and collagen deposition. This leads to esophageal strictures and narrow caliber esophagi, which are proportional a patient's age and untreated disease length. Pathophysiological mechanisms leading to EoE have been described in recent years, and transforming growth factor beta (TGF)-beta have been involved in fibrotic phenomena in EoE. However, evidence on the dependence of these phenomena on TGF-beta is scarce and contradictory. This review provides state-of-the art knowledge on intimate mechanisms of esophageal fibrosis in EoE and its clinical consequences.

Impact of STAT6 Variants on the Response to Proton Pump Inhibitors and Comorbidities in Patients with Eosinophilic Esophagitis.

Proton pump inhibitors (PPIs) are the first-line drug for eosinophilic esophagitis (EoE), although it is estimated that there is a lack of histological remission in 50% of patients. This research aimed to identify pharmacogenetic biomarkers predictive of PPI effectiveness and to study their association with disease features. Peak eosinophil count (PEC) and the endoscopic reference score (EREFS) were determined before and after an eight-week PPI course in 28 EoE patients. The impact of the signal transducer and activator of transcription 6 (STAT6), CYP2C19, CYP3A4, CYP3A5, and ABCB1 genetic variations on baseline PEC and EREFS, their reduction and histological response, and on EoE symptoms and comorbidities was analyzed. PEC reduction was higher in omeprazole-treated patients (92.5%) compared to other PPIs (57.9%, p = 0.003). STAT6 rs12368672 (g.18453G>C) G/G genotype showed higher baseline PEC values compared to G/C and C/C genotypes (83.2 vs. 52.9, p = 0.027). EREFS reduction in STAT6 rs12368672 G/G and G/C genotypes was higher than in the C/C genotype (36.7% vs. -75.0% p = 0.011). However, significance was lost after Bonferroni correction. Heartburn incidence was higher in STAT6 rs167769 (g.27148G>A) G/G patients compared to G/A (54.55% vs. 11.77%, p = 0.030). STAT6 rs12368672G>C and rs167769G>A variants might have a relevant impact on EoE status and PPI response. Further research is warranted to clarify the clinical relevance of these variants.

Proteomic analysis of the esophageal epithelium reveals key features of eosinophilic esophagitis pathophysiology.

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic non-IgE-mediated allergic disease of the esophagus. An unbiased proteomics approach was performed to investigate pathophysiological changes in esophageal epithelium. Additionally, an RNAseq-based transcriptomic analysis in paired samples was also carried out. METHODS: Total proteins were purified from esophageal endoscopic biopsies in a cohort of adult EoE patients (n = 25) and healthy esophagus controls (n = 10). Differentially accumulated (DA) proteins in EoE patients compared to control tissues were characterized to identify altered biological processes and signaling pathways. Results were also compared with a quantitative proteome dataset of the human esophageal mucosa. Next, results were contrasted with those obtained after RNAseq analysis in paired samples. Finally, we matched up protein expression with two EoE-specific mRNA panels (EDP and Eso-EoE panel). RESULTS: A total of 1667 proteins were identified, of which 363 were DA in EoE. RNA sequencing in paired samples identified 1993 differentially expressed (DE) genes. Total RNA and protein levels positively correlated, especially in DE mRNA-proteins pairs. Pathway analysis of these proteins in EoE showed alterations in immune and inflammatory responses for the upregulated proteins, and in epithelial differentiation, cornification and keratinization in those downregulated. Interestingly, a set of DA proteins, including eosinophil-related and secreted proteins, were not detected at the mRNA level. Protein expression positively correlated with EDP and Eso-EoE, and corresponded with the most abundant proteins of the human esophageal proteome. CONCLUSIONS: We unraveled for the first time key proteomic features involved in EoE pathogenesis. An integrative analysis of transcriptomic and proteomic datasets provides a deeper insight than transcriptomic alone into understanding complex disease mechanisms.

Blood-Based Biomarkers for Eosinophilic Esophagitis and Concomitant Atopic Diseases: A Look into the Potential of Extracellular Vesicles.

Eosinophilic esophagitis (EoE) is a chronic, Th2-inflammatory disease of the esophagus that can severely affect food intake. Currently, diagnosis and assessing response to treatment of EoE is highly invasive and requires endoscopy with esophageal biopsies. Finding non-invasive and accurate biomarkers is important for improving patient well-being. Unfortunately, EoE is usually accompanied by other atopies, which make it difficult to identify specific biomarkers. Providing an update of circulating EoE biomarkers and concomitant atopies is therefore timely. This review summarizes the current knowledge in EoE blood biomarkers and two of its most common comorbidities, bronchial asthma (BA) and atopic dermatitis (AD), focusing on dysregulated proteins, metabolites, and RNAs. It also revises the current knowledge on extracellular vesicles (EVs) as non-invasive biomarkers for BA and AD, and concludes with the potential use of EVs as biomarkers in EoE.

Comparison of a new rapid method for the determination of adalimumab serum levels with two established ELISA kits.

Background Therapeutic drug monitoring (TDM) of adalimumab (ADA) in inflammatory bowel diseases (IBDs) has gained increased attention since several studies showed a correlation between drug levels and mucosal healing. The limitations of routine usage of enzyme-linked immunoabsorbent assay (ELISA) kits for measuring serum ADA concentrations have prompted the development of rapid methods, such as Quantum Blue (QB). We evaluated the interchangeability and agreement between the QB method and two established ELISA kits, Promonitor (PM) and Lisa-Tracker (LT). Methods Fifty samples from patients with IBD were included. Quantitative analysis was performed using the ANOVA test for repeated measures, Deming regression and the Bland-Altman plot. Clinical implications were evaluated by concordance in classifying patients into therapeutic windows according to the proposed cut-off levels for subtherapeutic (either <5 or <7.5 µg/mL) and supratherapeutic (>12 µg/mL) ranges. Results Statistical differences were detected between the QB method and the two ELISA kits, with QB overestimating ADA serum values compared to them. A lack of interchangeability was observed between methods, with greater differences as ADA levels increased. An analysis of a sub-set of samples with ADA values below 9 µg/mL (n = 25) showed that QB fulfilled the criteria to be interchangeable with the LT assay. Concordance for patient classification into ADA therapeutic windows was better for QB vs. LT than for QB vs. PM, with high agreement (>75%) for subtherapeutic levels among the three methods. Conclusions Although quantitative differences existed between the rapid method and ELISA kits that hampered their interchangeability, the agreement for identifying patients with subtherapeutic values of ADA was high.

Proton-Pump Inhibitors in Eosinophilic Esophagitis: A Review Focused on the Role of Pharmacogenetics.

Proton-pump inhibitors (PPIs) are the most administered first-line treatment for eosinophilic esophagitis (EoE). However, only around half of EoE patients respond histologically to a double dosage of PPI. In addition, 70% of responders maintain EoE in remission after tapering the PPI dose. In order to avoid endoscopy with biopsies-the only accurate method of assessing PPI response-efforts have been made to identify PPI responder patients. The clinical or endoscopic features and biomarkers evaluated so far, however, have not proven to be sufficient in predicting PPI response. Although new approaches based on omics technologies have uncovered promising biomarkers, the specialized and complex procedures required are difficult to implement in clinical settings. Alternatively, PPI pharmacogenetics based on identifying variations in CYP2C19 and STAT6 genes have shown promising results in EoE, and could easily be performed in most laboratories. Other genetic variations have also been associated with PPI response and may explain those cases not related to CYP2C19 or STAT6. Here, we provide an overview of PPI treatment in EoE and evidence of how genetic variations in CYP2C19 and other genes could affect PPI effectiveness, and also discuss studies evaluating the role of pharmacogenetics in predicting PPI response in patients with EoE.

Circulating immunome fingerprint in eosinophilic esophagitis is associated with clinical response to proton pump inhibitor treatment.

Objectives: The aim of the study was to characterize the circulating immunome of patients with EoE before and after proton pump inhibitor (PPI) treatment in order to identify potential non-invasive biomarkers of treatment response. Methods: PBMCs from 19 healthy controls and 24 EoE patients were studied using a 39-plex spectral cytometry panel. The plasmacytoid dendritic cell (pDC) population was differentially characterized by spectral cytometry analysis and immunofluorescence assays in esophageal biopsies from 7 healthy controls and 13 EoE patients. Results: Interestingly, EoE patients at baseline had lower levels of circulating pDC compared with controls. Before treatment, patients with EoE who responded to PPI therapy had higher levels of circulating pDC and classical monocytes, compared with non-responders. Moreover, following PPI therapy pDC levels were increased in all EoE patients, while normal levels were only restored in PPI-responding patients. Finally, circulating pDC levels inversely correlated with peak eosinophil count and pDC count in esophageal biopsies. The number of tissue pDCs significantly increased during active EoE, being even higher in non-responder patients when compared to responder patients pre-PPI. pDC levels decreased after PPI intake, being further restored almost to control levels in responder patients post-PPI. Conclusions: We hereby describe a unique immune fingerprint of EoE patients at diagnosis. Moreover, circulating pDC may be also used as a novel non-invasive biomarker to predict subsequent response to PPI treatment.

Prognostic Factors at Admission for In-Hospital Mortality from COVID-19 Infection in an Older Rural Population in Central Spain.

BACKGROUND: Risk factors for in-hospital mortality from severe coronavirus disease 2019 (COVID-19) infection have been identified in studies mainly carried out in urban-based teaching hospitals. However, there is little data for rural populations attending community hospitals during the first wave of the pandemic. METHODS: A retrospective, single-center cohort study was undertaken among inpatients at a rural community hospital in Spain. Electronic medical records of the 444 patients (56.5% males) admitted due to severe SARS-CoV-2 infection during 26 February 2020-31 May 2020 were reviewed. RESULTS: Mean age was 71.2 ± 14.6 years (rank 22-98), with 69.8% over 65. At least one comorbidity was present in 410 patients (92.3%), with chronic obstructive pulmonary disease (COPD) present in 21.7%. Overall in-hospital mortality was 32%. Multivariate analysis of factors associated with death identified patients' age (with a cumulative effect per decade), COPD as a comorbidity, and respiratory insufficiency at the point of admission. No additional comorbid conditions proved significant. Among analytical values, increased serum creatinine, LDH > 500 mg/dL, thrombocytopenia (<150 × 109/per L), and lymphopenia (<1000 cells/µL) were all independently associated with mortality during admission. CONCLUSIONS: Age remained the major determinant for COVID-19-caused mortality; COPD was the only comorbidity independently associated with in-hospital death, together with respiratory insufficiency and analytical markers at admission.

Esophageal perforation in eosinophilic esophagitis: A systematic review on clinical presentation, management and outcomes.

There is evidence of an increased fragility in the inflamed esophagus of patients with eosinophilic esophagitis (EoE). We performed a systematic review on presentation, management and outcomes of and surgical interventions for esophageal perforation in these patients, by searching in the MEDLINE, Embase and Scopus databases. Of the 599 references identified, 41 full-papers and 9 abstract met the inclusion criteria. Overall, 76 esophageal perforation episodes in 70 individual patients aged between 9 and 65 years were reported. 51 patients had not been diagnosed with EoE at the time of perforation; 14 patients had an untreated disease and the remaining were non responsive to therapy. Acute or progressive pain after long-lasting dysphagia and food impaction was the most common symptom leading to diagnosis in 42 patients who presented with Boerhaave syndrome. Pushing impacted food into the stomach led to perforation in 5 cases. Eight episodes appeared after dilation. CT scans demonstrated perforation in 82.4% of patients. Conservative management (including esophageal stenting) was used in 67.1% patients. The 25 remaining patients underwent surgery. Recovery was uneventful in the vast majority of patients. No death was reported. Active inflammation due to undiagnosed or untreated EoE was present in most cases of esophageal perforation. Conservative treatment of perforation should always be considered in EoE.

Poor Sensitivity of Fecal Gluten Immunogenic Peptides and Serum Antibodies to Detect Duodenal Mucosal Damage in Celiac Disease Monitoring.

A lifelong gluten-free diet (GFD) is the only current treatment for celiac disease (CD), but strict compliance is complicated. Duodenal biopsies are the "gold standard" method for diagnosing CD, but they are not generally recommended for disease monitoring. We evaluated the sensitivity and specificity of fecal gluten immunogenic peptides (GIPs) to detect duodenal lesions in CD patients on a GFD and compared them with serum anti-tissue transglutaminase (tTG) IgA antibodies. A prospective study was conducted at two tertiary centers in Spain on a consecutive series of adolescents and adults with CD who maintained a long-lasting GFD. Adherence to a GFD and health-related quality of life were scored with validated questionnaires. Mucosal damage graded according to the Marsh-Oberhüber classification (Marsh 1/2/3) was used as the reference standard. Of the 97 patients included, 27 presented duodenal mucosal damage and 70 had normal biopsies (Marsh 0). The sensitivity (33%) and specificity (81%) of GIPs were similar to those provided by the two assays used to measure anti-tTG antibodies. Scores in questionnaires showed no association with GIP, but an association between GIPs and patients' self-reported gluten consumption was found (p = 0.003). GIP displayed low sensitivity but acceptable specificity for the detection of mucosal damage in CD.

Systematic review with meta-analysis: the growing incidence and prevalence of eosinophilic oesophagitis in children and adults in population-based studies.

BACKGROUND: The frequency of eosinophilic oesophagitis (EoE) occurrence is escalating. Current diagnostic criteria recently proposed for the disease, determine that previous estimates of incidence and prevalence are outdated. AIM: To gauge the current incidence and prevalence of EoE by performing a systematic review of population-based studies. METHODS: Three electronic databases were searched from their inception dates to September 2018. A total of 2386 documents were screened; 29 studies reported on the prevalence and incidence of EoE in the general population. RESULTS: The pooled prevalence of EoE was 34.4 cases per 100 000 inhabitants (95% CI, 23.1-47.5), and was higher for adults (42.2; 95% CI, 31.1-55) than for children (34; 95% CI, 22.3-49.2). The pooled EoE incidence rates were 6.6/100 000 person-years (95% CI, 3-11.7) in children and 7.7/100 000 (95% CI, 1.8-17.8) in adults. No differences were found between North American and European studies using varied sources of data (insurance and administrative databases compared to hospital-bases case series). Subgroup analysis according to risk of bias did not change results significantly. A steady rise in EoE incidence and prevalence rates was observed over time, comparing studies conducted under subsequent definitions for EoE. No significant publication bias was found. CONCLUSIONS: In a systematic review and meta-analysis, we found a sharp increase, higher than previous estimates, in the incidence and prevalence of EoE in population based studies. Results from studies carried out in developed countries show broad consistency and provide evidence of increasing pooled prevalence and incidence of EoE rates over time.

Determinant factors of quality of life in adult patients with eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) affects health-related quality of life (HRQoL). Data on determinant factors and the influence of dietary interventions are scarce. OBJECTIVE: The objective of this article is to evaluate factors influencing HRQoL in adult EoE patients. METHODS: We conducted a multicenter observational, cross-sectional study. A validated Spanish version of the self-administered Adult Eosinophilic Esophagitis Quality of Life (EoE-QoL-A) questionnaire and specific surveys were used. Multiple linear regression was used to identify and quantify determinant factors of HRQoL. RESULTS: Responses provided by 170 patients were assessed (73.5% male; mean age 33.5 ± 11.4 years). Overall mean score for the EoE-QoL-A index was 1.4 ± 0.8, with no differences between patients on dietary or pharmacological therapy (1.82 ± 0.8 vs. 1.62 ± 0.8; p = 0.132). Disease anxiety showed the highest mean score (2.13 ± 0.9 points), followed by choking anxiety (1.97 ± 1.1); social impact (1.77 ± 1.1), and diet/eating impact (1.68 ± 0.9). Emotional impact had the lowest rating (1.15 ± 0.9), and only with a significantly worse score in patients under dietary restrictions. Recurrent food impaction, a higher educational level, dietary interventions and symptom duration were all independent determinant factors significantly impairing HRQoL. Female gender and empiric elimination diets negatively influenced on diet/eating impact. CONCLUSION: Recurrent food impaction, dietary interventions and symptom duration are the most important factors influencing the perception of HRQoL in adults with EoE.

Toll-like receptors-mediated pathways activate inflammatory responses in the esophageal mucosa of adult eosinophilic esophagitis.

OBJECTIVES: Esophageal microbiota and regulation of adaptive immunity are increasingly being investigated in eosinophilic esophagitis (EoE). Toll-like receptors (TLRs) play a central role in the initiation and maintenance of innate immune activity. Our objective was to characterize the esophageal and duodenal innate immune response in EoE and its modulation by dietary therapy. METHODS: Esophageal and duodenal biopsy samples were collected from 10 adults with untreated EoE, before and after effective treatment with a six-food elimination diet (SFED), and 10 controls with normal esophagus. In all cases, bacterial load (by mRNA expression of 16S), TLRs, mucins, transcription factors, interleukins, components of the NKG2D system, and innate immunity effectors were assessed by qPCR. Protein expression of TLRs were also determined by immunofluorescence. RESULTS: Bacterial load and TLR1, TLR2, TLR4, and TLR9 were overexpressed on biopsies with active EoE compared with controls. Muc1 and Muc5B genes were downregulated while Muc4 was overexpressed. Upregulation of MyD88 and NFκB was found together with IL-1ß, IL-6, IL-8, and IL-10 mediators and PER-1, iNOS, and GRZA effectors. NG-K2D components (KLRK1, IL-15, MICB) were also upregulated. In all cases, changes in active EoE were normalized following SFED and mucosal healing. Duodenal samples also showed increased expressions of TLR-1, TLR-2, and TLR-4, but not 16S or any other mediators nor effectors of inflammation. CONCLUSIONS: Esophageal TLR-dependent signaling pathways in EoE support the potential implication of microbiota and the innate immune system in the pathogenesis of this disease.

The role of endoscopy in eosinophilic esophagitis: from diagnosis to therapy.

INTRODUCTION: Eosinophilic esophagitis (EoE) has arisen as a common disorder in current clinical and endoscopic gastroenterology practice. Areas covered: A comprehensive review of the literature to summarize and update different aspects related with the use of endoscopy in the diagnostic workout and treatment of pediatric and adult EoE patients is conducted. Expert commentary: Endoscopic features in EoE are frequently subtle, so were inadverted in some initial reports of the disease. Literature has described a wide number of EoE-associated features, systematized in the EREFS classification, which standardized the grade and severity of exudates, rings, edema, furrows, and strictures. The insufficient reliability of these features to predict eosinophilic inflammation still makes biopsies essential in diagnosing or monitoring EoE. EoE causes half of the food impactions requiring endoscopy; food impaction leads to EoE diagnosis in up to half of cases. Long term consequences of EoE include esophageal remodeling leading to strictures and narrowing, thus impairing symptoms and needs dilation. Recognizing the risks from dilation in EoE required carrying out a safe technique to avoid the high complication rate reported in the early literature. Endoscopic dilation should be considered in patients with esophageal narrowing and dysphagia/food impaction unresponsive to diet or drugs-based anti-inflammatory treatment.

Abrogation of the p38 MAPK α signaling pathway does not promote radioresistance but its activity is required for 5-Fluorouracil-associated radiosensitivity.

The p38 Mitogen Activated Protein Kinase (MAPK) signaling pathway has become a major player in the response to DNA-damage. A growing body of evidences has been relating this signaling pathway to the cellular response to ionizing radiation (IR), suggesting a role in radioresistance. Here, we study the implication of this signaling pathway in the response to IR in terms of radioresistance. To this end we used 10 different cell lines derived from several types of tumors (colorectal, non-small cell lung cancer -NSCLC-, renal and glioblastoma). Although p38 MAPK is transiently activated by IR, our data, obtained by genetic and chemical approaches, showed that this signaling pathway is not implicated in cellular viability after IR exposure. Indeed, down-modulation of this signaling pathway promotes a mild radiosensitivity depending on the cell line. However, it is remarkable that lack of p38 MAPK α abrogates the radiosensitizing effect of 5-Fluorouracil (5-FU) in HCT116 cell line, supporting the role of this MAPK in the radiosensitizing action of 5-FU.

ERK5/BMK1 is a novel target of the tumor suppressor VHL: implication in clear cell renal carcinoma.

Extracellular signal-regulated kinase 5 (ERK5), also known as big mitogen-activated protein kinase (MAPK) 1, is implicated in a wide range of biologic processes, which include proliferation or vascularization. Here, we show that ERK5 is degraded through the ubiquitin-proteasome system, in a process mediated by the tumor suppressor von Hippel-Lindau (VHL) gene, through a prolyl hydroxylation-dependent mechanism. Our conclusions derive from transient transfection assays in Cos7 cells, as well as the study of endogenous ERK5 in different experimental systems such as MCF7, HMEC, or Caki-2 cell lines. In fact, the specific knockdown of ERK5 in pVHL-negative cell lines promotes a decrease in proliferation and migration, supporting the role of this MAPK in cellular transformation. Furthermore, in a short series of fresh samples from human clear cell renal cell carcinoma, high levels of ERK5 correlate with more aggressive and metastatic stages of the disease. Therefore, our results provide new biochemical data suggesting that ERK5 is a novel target of the tumor suppressor VHL, opening a new field of research on the role of ERK5 in renal carcinomas.

ERK2, but not ERK1, mediates acquired and "de novo" resistance to imatinib mesylate: implication for CML therapy.

Resistance to Imatinib Mesylate (IM) is a major problem in Chronic Myelogenous Leukaemia management. Most of the studies about resistance have focused on point mutations on BCR/ABL. However, other types of resistance that do not imply mutations in BCR/ABL have been also described. In the present report we aim to study the role of several MAPK in IM resistance not associate to BCR/ABL mutations. Therefore we used an experimental system of resistant cell lines generated by co-culturing with IM (K562, Lama 84) as well as primary material from resistant and responder patient without BCR/ABL mutations. Here we demonstrate that Erk5 and p38MAPK signaling pathways are not implicated in the acquired resistance phenotype. However, Erk2, but not Erk1, is critical for the acquired resistance to IM. In fact, Bcr/Abl activates preferentially Erk2 in transient transfection in a dose dependent fashion through the c-Abl part of the chimeric protein. Finally, we present evidences demonstrating how constitutive activation of Erk2 is a de novo mechanism of resistance to IM. In summary our data support the use of therapeutic approaches based on Erk2 inhibition, which could be added to the therapeutic armamentarium to fight CML, especially when IM resistance develops secondary to Erk2 activation.