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BACKGROUND AND AIMS: During early phases of inflammation, activated neutrophils extrude neutrophil extracellular traps (NETs) in a PAD4-dependent manner, aggravating tissue injury and remodelling. In this study, we investigated the potential pro-fibrotic properties and signalling of NETs in Crohn's disease (CD). METHODS: NETs and activated fibroblasts were labelled on resected ileum from CD patients by multiplex immunofluorescence staining. NETs-treated human primary intestinal fibroblasts were analysed by bulk RNA-sequencing to uncover cell signalling pathways, and by high-throughput imaging to assess collagen production and migratory activity. Consequentially, TLR2/NF-kB pathway was evaluated by transfection of CCD-18Co fibroblasts with NF-kB-luciferase reporter plasmid, incorporating C29 to block TLR2 signalling. A chronic DSS mouse model was used to define the specific role of PAD4 deletion in neutrophils (MRP8-Cre, Pad4fl/fl). RESULTS: Immunofluorescence showed spatial co-localisation of NETs and activated fibroblasts in ileal ulcerations of CD patients. Transcriptomic analysis revealed upregulation of pro-fibrotic genes and activation of TLR-signalling pathways in NETs-treated fibroblasts. NETs treatment induced fibroblast proliferation, diminished migratory capability, and increased collagen release. Transfection experiments indicated a substantial increase in NF-kB expression with NETs, whereas C29 led to decreased expression and release of collagen. In line, a significantly reduction in collagen content was observed in the colon of MRP8-Cre, Pad4fl/fl mice subjected to chronic DSS colitis. CONCLUSIONS: NETs potentially serve as an initial stimulus for pathological activation of fibroblasts within the intestine via the TLR2/NF-kB pathway. Given their early involvement in inflammation, inhibition of PAD4 might offer a strategy to modulate both inflammation and fibrogenesis in CD.
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BACKGROUND: A role for eosinophils in intestinal inflammation and fibrosis in the context of inflammatory bowel disease has been suggested, yet the precise nature, whether causal or secondary remains debated. Hence, it remains unclear whether targeting eosinophils should be further explored as a treatment option in inflammatory bowel disease. METHODS: Acute and chronic dextran sulfate sodium colitis was induced in wild-type C57BL/6 mice. Eosinophils were depleted by anti-CCR3 injections before colitis induction in a chronic model and after colitis onset in an acute model in order to investigate the impact of eosinophil depletion on pre-existing colitis. Inflammation was assessed using the disease activity index, macroscopic damage, and histological disease activity score. In the chronic model, fibrosis was assessed by examining colon weight/length ratio, collagen deposition through Martius Scarlet Blue staining, hydroxyproline assay, and COL1A1 expression. Protein and gene expression were assessed using the Meso Scale Discovery platform and real-time quantitative polymerase chain reaction. RESULTS: In the acute and chronic colitis model, eosinophil depletion resulted in reduced disease activity and faster recovery, as observed via the total area under the curve of the disease activity index (P = .004 and P = .02, respectively), macroscopic damage score (P = .009 and P = .08, respectively), and histological disease activity score (P = .09 and P = .002, respectively). In the acute model, the accelerated recovery was accompanied by an increase in interleukin (IL)-10 (P = .03) and a decrease in IL-4 (P = .03) and IL-6 (P = .009). Colon weight/length ratio and collagen deposition were not affected by eosinophil depletion. CONCLUSIONS: Eosinophil depletion prevents and decreases intestinal inflammation in a preclinical dextran sulfate sodium model without affecting fibrosis. These results pave the way for exploring eosinophil depletion as a novel treatment modality in addressing intestinal inflammation.
This study investigated the effect of eosinophil depletion on intestinal inflammation and fibrosis in a dextran sulfate sodium colitis model. Our findings suggest a proinflammatory role for eosinophils and therefore warrant further investigation into eosinophil targeting as a treatment option for inflammatory bowel disease patients.
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Crohn's disease (CD) is marked by recurring intestinal inflammation and tissue injury, often resulting in fibrostenosis and bowel obstruction, necessitating surgical intervention with high recurrence rates. To elucidate the mechanisms underlying fibrostenosis in CD, we analyzed the transcriptome of cells isolated from the transmural ileum of patients with CD, including a trio of lesions from each patient: non-affected, inflamed, and stenotic ileum samples, and compared them with samples from patients without CD. Our computational analysis revealed that profibrotic signals from a subset of monocyte-derived cells expressing CD150 induced a disease-specific fibroblast population, resulting in chronic inflammation and tissue fibrosis. The transcription factor TWIST1 was identified as a key modulator of fibroblast activation and extracellular matrix (ECM) deposition. Genetic and pharmacological inhibition of TWIST1 prevents fibroblast activation, reducing ECM production and collagen deposition. Our findings suggest that the myeloid-stromal axis may offer a promising therapeutic target to prevent fibrostenosis in CD.
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Doença de Crohn , Fibroblastos , Fibrose , Monócitos , Proteína 1 Relacionada a Twist , Doença de Crohn/metabolismo , Doença de Crohn/patologia , Doença de Crohn/imunologia , Humanos , Fibroblastos/metabolismo , Fibroblastos/patologia , Proteína 1 Relacionada a Twist/metabolismo , Proteína 1 Relacionada a Twist/genética , Monócitos/metabolismo , Monócitos/patologia , Monócitos/imunologia , Masculino , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Feminino , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/genética , Íleo/patologia , Íleo/metabolismo , Íleo/imunologia , Comunicação Celular , Adulto , Endopeptidases/metabolismo , Endopeptidases/genética , Animais , CamundongosRESUMO
INTRODUCTION: The diagnosis of eosinophilic gastrointestinal diseases is largely based on mucosal eosinophil counts, but thresholds and normal ranges beyond the esophagus are debated, calling for much-needed methodological standardization. We aimed to develop a standardized workflow for duodenal cell quantification and estimate duodenal eosinophil and mast cell numbers in healthy controls. METHODS: Software-based histological cell quantification using free-sized or fixed-sized regions was developed and applied to digitized hematoxylin and eosin (H&E)-stained slides from 58 individuals (healthy controls [HCs] and patients with functional dyspepsia). Intraclass correlation coefficients (ICCs) compared inter-rater reliability between software-based and microscopic quantification. Reproducibility of the software-based method was validated in an independent cohort of 37 control and functional dyspepsia subjects. Eosinophil identification on H&E staining was compared to immunohistochemistry (IHC). Normal eosinophil (H&E) and mast cell (cKit) ranges were determined in 70 adult HCs. RESULTS: Eosinophil quantification on digitized slides demonstrated excellent (ICC = 0.909) and significantly improved reproducibility over microscopic evaluation (ICC = 0.796, P = 0.0014), validated in an independent cohort (ICC = 0.910). Duodenal eosinophils were more abundant around crypts than in villi ( P < 0.0001), while counts were similar on matched H&E- and IHC-stained slides ( P = 0.55). Mean ± SD (95th percentile) duodenal eosinophils and mast cells in HC were 228.8/mm 2 ± 94.7 (402.8/mm 2 ) and 419.5/mm 2 ± 132.2 (707.6/mm 2 ), respectively. DISCUSSION: We developed and validated a standardized approach to duodenal histological cell quantification, generalizable to various mucosal cell types. Implementation of software-based quantification identified 400 eosinophils/mm 2 and 700 mast cells/mm 2 as thresholds for abnormal duodenal infiltration.
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Duodeno , Eosinófilos , Mastócitos , Software , Humanos , Eosinófilos/patologia , Eosinófilos/citologia , Duodeno/patologia , Duodeno/citologia , Mastócitos/patologia , Reprodutibilidade dos Testes , Adulto , Masculino , Feminino , Pessoa de Meia-Idade , Eosinofilia/patologia , Eosinofilia/diagnóstico , Contagem de Células , Contagem de Leucócitos/métodos , Imuno-Histoquímica , Dispepsia/patologia , Dispepsia/diagnóstico , Mucosa Intestinal/patologia , Mucosa Intestinal/citologia , Idoso , Estudos de Casos e Controles , Adulto Jovem , Variações Dependentes do ObservadorRESUMO
Background: Failure to close the abdominal wall after intestinal transplantation (ITx) or multivisceral Tx remains a surgical challenge. An attractive method is the use of nonvascularized rectus fascia (NVRF) in which both layers of the donor abdominal rectus fascia are used as an inlay patch without vascular anastomosis. How this graft integrates over time remains unknown. The study aims to provide a multilevel analysis of the neovascularization and integration process of the NVRF. Methods: Three NVRF-Tx were performed after ITx. Clinical, radiological, histological, and immunological data were analyzed to get insights into the neovascularization and integration process of the NVRF. Moreover, cryogenic contrast-enhanced microfocus computed tomography (microCT) analysis was used for detailed reconstruction of the vasculature in and around the NVRF (3-dimensional histology). Results: Two men (31- and 51-y-old) and 1 woman (49-y-old) underwent 2 multivisceral Tx and 1 combined liver-ITx, respectively. A CT scan showed contrast enhancement around the fascia graft at 5 days post-Tx. At 6 weeks, newly formed blood vessels were visualized around the graft with Doppler ultrasound. Biopsies at 2 weeks post-Tx revealed inflammation around the NVRF and early fibrosis. At 6 months, classical 2-dimensional histological analysis of a biopsy confirmed integration of the fascia graft with strong fibrotic reaction without signs of rejection. A cryogenic contrast-enhanced microCT scan of the same biopsy revealed the presence of microvasculature, enveloping and penetrating the donor fascia. Conclusions: We showed clinical, histological, and microCT evidence of the neovascularization and integration process of the NVRF after Tx.
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Lynch syndrome (LS) and constitutional mismatch repair deficiency (CMMRD) are distinct cancer syndromes caused, respectively, by mono- and bi-allelic germline mismatch repair (MMR) variants. LS predisposes to mainly gastrointestinal and genitourinary cancers in adulthood. CMMRD predisposes to brain, haematological, and LS-spectrum cancers from childhood. Two suspected LS patients with first cancer diagnosis aged 27 or 38 years were found to be homozygous for an MMR (likely) pathogenic variant, MSH6 c.3226C>T (p.(Arg1076Cys)), or variant of uncertain significance (VUS), MLH1 c.306G>A (p.(Glu102=)). MLH1 c.306G>A was shown to cause leaky exon 3 skipping. The apparent genotype-phenotype conflict was resolved by detection of constitutional microsatellite instability in both patients, a hallmark feature of CMMRD. A hypomorphic effect of these and other variants found in additional late onset CMMRD cases, identified by literature review, likely explains a LS-like phenotype. CMMRD testing in carriers of compound heterozygous or homozygous MMR VUS may find similar cases and novel hypomorphic variants. Individualised management of mono- and bi-allelic carriers of hypomorphic MMR variants is needed until we better characterise the associated phenotypes.
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BACKGROUND & AIMS: Seventeen percent of patients with ulcerative colitis that undergo proctocolectomy with pouch surgery will develop chronic pouchitis. We evaluated the efficacy of ustekinumab for these patients. METHODS: We performed a prospective study of patients with chronic pouchitis receiving ustekinumab intravenously at baseline (â¼6 mg/kg) and 90 mg ustekinumab subcutaneously every 8 weeks thereafter. The Modified Pouchitis Disease Activity Index (mPDAI) was assessed at baseline and weeks 16 and 48. The primary endpoint was the proportion of patients achieving steroid-free remission (mPDAI <5 and reduction by ≥2 points) at week 16. Secondary endpoints included the proportion of patients achieving remission at week 48, the proportion of patients achieving response (reduction of mPDAI by ≥2 points) at weeks 16 and 48, and change in mPDAI. RESULTS: We enrolled 22 patients (59% male; median age, 42.2 years). Remission was achieved in 27.3% at week 16 and 36.4% at week 48. Response was achieved in 54.5% both at weeks 16 and 48. The median mPDAI decreased from 8 (interquartile range [IQR], 7-10) to 7 (IQR, 4-9) at week 16 (P = .007) and 4 (IQR, 1.75-7.25) at week 48 (P < .001). The clinical mPDAI subscore decreased from 3.5 (IQR, 2-4) to 2 (IQR, 1-3) at week 16 (P = .009) and 1 (IQR, 0-2.25) at week 48 (P = .001). The endoscopic mPDAI subscore decreased from 5.5 (IQR, 4-6) to 4 (IQR, 3-6) at week 16 (P = .032) and 3 (IQR, 1.75-4.25) at week 48 (P = .001). CONCLUSION: Ustekinumab was efficacious in one-half of the patients suffering from chronic pouchitis. Ustekinumab should therefore be positioned in the treatment algorithm of chronic pouchitis. (ClinicalTrials.gov Number NCT04089345).
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INTRODUCTION: Approximately 50% of patients with Crohn's disease (CD) develop intestinal strictures necessitating surgery. The immune cell distribution in these strictures remains uncharacterized. We aimed to identify the immune cells in intestinal strictures of patients with CD. METHODS: During ileocolonic resections, transmural sections of terminal ileum were sampled from 25 patients with CD and 10 non-inflammatory bowel disease controls. Macroscopically unaffected, fibrostenotic, and inflamed ileum was collected and analyzed for immune cell distribution (flow cytometry) and protein expression. Collagen deposition was assessed through a Masson Trichrome staining. Eosinophil and fibroblast colocalization was assessed through immunohistochemistry. RESULTS: The Masson Trichrome staining confirmed augmented collagen deposition in both the fibrotic and the inflamed regions, though with a significant increased collagen deposition in the fibrotic compared with inflamed tissue. Distinct Th1, Th2, regulatory T cells, dendritic cells, and monocytes were identified in fibrotic and inflamed CD ileum compared with unaffected ileum of patients with CD as non-inflammatory bowel disease controls. Only minor differences were observed between fibrotic and inflamed tissue, with more active eosinophils in fibrotic deeper layers and increased eosinophil cationic protein expression in inflamed deeper layers. Last, no differences in eosinophil and fibroblast colocalization were observed between the different regions. DISCUSSION: This study characterized immune cell distribution and protein expression in fibrotic and inflamed ileal tissue of patients with CD. Immunologic, proteomic, and histological data suggest inflammation and fibrosis are intertwined, with a large overlap between both tissue types. However strikingly, we did identify an increased presence of active eosinophils only in the fibrotic deeper layers, suggesting their potential role in fibrosis development.
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Colágeno , Doença de Crohn , Eosinófilos , Fibrose , Íleo , Humanos , Doença de Crohn/patologia , Doença de Crohn/imunologia , Doença de Crohn/metabolismo , Eosinófilos/patologia , Eosinófilos/imunologia , Masculino , Feminino , Adulto , Íleo/patologia , Íleo/imunologia , Pessoa de Meia-Idade , Colágeno/metabolismo , Colágeno/análise , Fibroblastos/patologia , Fibroblastos/metabolismo , Estudos de Casos e Controles , Adulto Jovem , Constrição Patológica/patologia , Citometria de Fluxo , Células Dendríticas/imunologia , Células Dendríticas/patologia , Células Dendríticas/metabolismo , Imuno-HistoquímicaRESUMO
INTRODUCTION: Treatment goals for ulcerative colitis (UC) are evolving from the achievement of clinical remission to more rigorous goals defined by endoscopic and histologic healing. Achievement of deeper remission targets aims to reduce the risk of colectomy, hospitalizations, and colorectal cancer. AREAS COVERED: This review covers histologic assessments, histologic remission as a clinical trial endpoint, and the association between histologic disease activity and clinical outcomes. Future directions are also discussed, including the use of advanced imaging and artificial intelligence technologies, as well as potential future treatment targets beyond histologic remission. EXPERT OPINION: Histologic assessments are used for their sensitivity in measuring mucosal inflammatory changes in UC. Due to correlation with disease activity, histologic assessments may support clinical decision-making regarding treatment decisions as such assessments can be associated with rates of clinical relapse, hospitalization, colectomy, and neoplasia. While histologic remission is limited by varying definitions and multiple histologic indices, work is ongoing to create a consensus on the use of histologic assessments in clinical trials. As research advances, aspirational targets beyond histologic remission, such as molecular healing and disease clearance, are being explored.
Ulcerative colitis (UC) is the most common inflammatory bowel disease and often results in bloody diarrhea, frequent bowel movements, and bowel urgency. Patients with UC are at greater risk for hospitalization, surgery, and colorectal cancer. To reduce these risks, the goals of UC treatment are changing from mainly addressing symptoms to reducing inflammation at a deeper histologic, or microscopic, level. The inflammation in UC causes distinct microscopic changes in the colon, which can be assessed after collecting biopsies or tissue samples. This review provides an overview of histologic remission (when no signs of inflammation are seen in tissue samples viewed under a microscope) as a treatment goal in UC.Histologic remission has been shown to be associated with lower rates of relapse, hospitalization, surgical removal of the colon, and colorectal cancer. However, using histologic remission as a treatment target can be difficult due to varying definitions and the many different scoring assessments available to healthcare providers. Updated guidance from regulatory agencies and academic organizations has helped align definitions of histologic remission and how to assess histologic healing in clinical trials.The introduction of targeted advanced therapies has allowed for deeper healing with the potential for histologic resolution. This enables clinicians and researchers to aim for treatment targets that are harder to achieve but have a greater impact for patients in the course of their disease. New technologies such as artificial intelligence, high-resolution endoscopy, and digital pathology have also led to targets beyond histologic healing, aiming to restore the function of the colon's mucosal barrier and disease clearance.
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Colite Ulcerativa , Humanos , Colite Ulcerativa/terapia , Colite Ulcerativa/tratamento farmacológico , Inteligência Artificial , Endoscopia , Colectomia/efeitos adversos , Indução de Remissão , Índice de Gravidade de DoençaRESUMO
AIMS: Patients with Crohn's disease (CrD) have an elevated risk for the development of small bowel adenocarcinomas (SBAs). Actionable isocitrate dehydrogenase 1 (IDH1) mutations have been reported to be more frequent in CrD-SBAs than in sporadic SBAs. The present study aimed to investigate the clinicopathological and immunophenotypical features, as well as methylation profiles, of IDH1-mutated CrD-SBAs. METHODS AND RESULTS: An international multicentre series of surgically resected CrD-SBAs was tested for IDH1 mutation. Clinicopathological features, immunophenotypical marker expression and O6-methylguanine-DNA methyltransferase (MGMT) and long interspersed nuclear element-1 (LINE-1) methylation were compared between IDH1-mutated and IDH1 wild-type CrD-SBAs. Ten (20%) of the 49 CrD-SBAs examined harboured an IDH1 mutation and all the mutated cancers harboured the R132C variant. Compared to IDH1 wild-type cases, IDH1-mutated CrD-SBAs showed significantly lower rates of cytokeratin 7 expression (P = 0.005) and higher rates of p53 overexpression (P = 0.012) and MGMT methylation (P = 0.012). All three dysplastic growths associated with IDH1-mutated SBAs harboured the same IDH1 variant (R132C) of the corresponding invasive cancer, and all were of non-conventional subtype (two serrated dysplastic lesions and one goblet cell-deficient dysplasia). In particular, non-conventional serrated dysplasia was significantly associated with IDH1-mutated CrD-SBAs (P = 0.029). No significant cancer-specific survival difference between IDH1-mutated CrD-SBA patients and IDH1 wild-type CrD-SBA patients was found (hazard ratio = 0.55, 95% confidence interval = 0.16-1.89; P = 0.313). CONCLUSIONS: IDH1-mutated CrD-SBAs, which represent approximately one-fifth of total cases, are characterised by distinctive immunophenotypical features and methylation profiles, with potential therapeutic implications. Moreover, IDH1-mutated non-conventional, serrated dysplasia is likely to represent a precursor lesion to such CrD-SBAs.
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Adenocarcinoma , Neoplasias Encefálicas , Doença de Crohn , Neoplasias Duodenais , Humanos , Doença de Crohn/genética , Metilação de DNA , Adenocarcinoma/genética , Adenocarcinoma/patologia , Neoplasias Duodenais/genética , Metilases de Modificação do DNA/genética , Hiperplasia , Isocitrato Desidrogenase/genética , Mutação , Neoplasias Encefálicas/patologia , Prognóstico , Proteínas Supressoras de Tumor/genética , Enzimas Reparadoras do DNA/genéticaRESUMO
BACKGROUND AND AIMS: We explored the potential for differential efficacy of vedolizumab between "early" and "late" ulcerative colitis (UC) with evaluation of clinical, endoscopic, and histological endpoints. METHODS: This was a multicentre, multinational open-label study in patients with moderately-to-severely active UC, defining "early" UC by a disease duration <4 years and bio-naïve and "late" UC by a disease duration >4 years and additional exposure to tumour necrosis factor antagonists. Patients received standard treatment with intravenous vedolizumab for 52 weeks (300 mg weeks 0-2-6, every 8 weeks thereafter without escalation). The primary endpoint was corticosteroid-free clinical remission with endoscopic improvement (total Mayo score ≤2 with no subscore >1) at both week 26 and 52. RESULTS: A total of 121 patients were included: in the "early" group 25/59 (42.4%) achieved the primary endpoint versus 19/62 (30.6%) in the "late" group (P = 0.18). There were no significant differences between the two groups in endoscopic improvement (week 26: "early" 32/59 [54.2%] vs. "late" 29/62 [46.8%]; P = 0.412; week 52: 27/59 [45.8%] vs. 25/62 [40.3%]; P = 0.546) or histological remission (Robarts Histopathology Index <3 without neutrophils in the epithelium and lamina propria) (week 26: 24/59 [40.7%] vs. 21/62 [33.9%]; P = 0.439; week 52: 22/59 [37.3%] vs. 22/62 [35.5%]; P = 0.837). CONCLUSIONS: No significant differences in clinical, endoscopic, and histological outcomes were observed between "early" and "late" disease.
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We aimed to investigate the role of the CD40-CD40 ligand (CD40L) pathway in inflammation-mediated angiogenesis in proliferative diabetic retinopathy (PDR). We analyzed vitreous fluids and epiretinal fibrovascular membranes from PDR and nondiabetic patients, cultures of human retinal microvascular endothelial cells (HRMECs) and Müller glial cells and rat retinas with ELISA, immunohistochemistry, flow cytometry and Western blot analysis. Functional tests included measurement of blood-retinal barrier breakdown, in vitro angiogenesis and assessment of monocyte-HRMEC adherence. CD40L and CD40 levels were significantly increased in PDR vitreous samples. We demonstrated CD40L and CD40 expression in vascular endothelial cells, leukocytes and myofibroblasts in epiretinal membranes. Intravitreal administration of soluble (s)CD40L in normal rats significantly increased retinal vascular permeability and induced significant upregulation of phospho-ERK1/2, VEGF, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). sCD40L induced upregulation of VEGF, MMP-9, MCP-1 and HMGB1 in cultured Müller cells and phospo-ERK1/2, p65 subunit of NF-ĸB, VCAM-1 and VEGF in cultured HRMECS. TNF-α induced significant upregulation of CD40 in HRMECs and Müller cells and VEGF induced significant upregulation of CD40 in HRMECs. sCD40L induced proliferation and migration of HRMECs. We provide experimental evidence supporting the involvement of the CD40L-CD40 pathway and how it regulates inflammatory angiogenesis in PDR.
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Diabetes Mellitus , Retinopatia Diabética , Humanos , Ratos , Animais , Retinopatia Diabética/metabolismo , Ligante de CD40/metabolismo , Células Endoteliais/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ratos Sprague-Dawley , Inflamação/metabolismo , Diabetes Mellitus/metabolismoRESUMO
BACKGROUND & AIMS: Histologic evaluation of mucosal healing in Crohn's disease is an evolving treatment target. We evaluated histologic outcomes for mirikizumab efficacy and associations with endoscopic and 1-year outcomes. METHODS: Biopsy specimens from 1 ileal and 4 colonic segments were evaluated at weeks 0, 12, and 52 from each of the 170 SERENITY participants. Criteria for the weeks 12 and 52 histologic response were no epithelial neutrophils or epithelial damage, or >50% decrease in either the Robarts Histopathology Index or the active Global Histologic Disease Activity Score, and remission (no mucosal neutrophils and no epithelial damage) had to be met in all biopsy specimens. Agreement was evaluated between histologic and endoscopic end points. Associations between 1-year outcomes and week 12 histologic and endoscopic response were evaluated. RESULTS: At week 12, 1000 mg mirikizumab resulted in greater rates of histologic response (66% vs 27%; P < .001) and remission (26% vs 6%; P < .01) than placebo. Rates were numerically similar at 1 year (mirikizumab pooled response, 46%-69%; remission, 13%-31%). Agreement between week 12 histologic and endoscopic response was 69% (Cohen's kappa coefficient [κ] = 0.40) and remission was 83% (κ = 0.38) in all pooled arms, including placebo. At 1 year, the percentage of participants who received any dose of mirikizumab and achieved endoscopic remission differed by their week 12 response: histologic (20%), endoscopic (25%), combined histology-endoscopy (45%), or neither (4%) (P = .003). CONCLUSIONS: In a post hoc analysis of phase 2 data, mirikizumab induced and sustained histologic response and remission in Crohn's disease over 52 weeks. Early combined histologic-endoscopic response was associated with endoscopic remission after 1 year of treatment with mirikizumab (ClinicalTrials.gov NCT02891226).
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CD8+ T cell activation via immune checkpoint blockade (ICB) is successful in microsatellite instable (MSI) colorectal cancer (CRC) patients. By comparison, the success of immunotherapy against microsatellite stable (MSS) CRC is limited. Little is known about the most critical features of CRC CD8+ T cells that together determine the diverse immune landscapes and contrasting ICB responses. Hence, we pursued a deep single cell mapping of CRC CD8+ T cells on transcriptomic and T cell receptor (TCR) repertoire levels in a diverse patient cohort, with additional surface proteome validation. This revealed that CRC CD8+ T cell dynamics are underscored by complex interactions between interferon-γ signaling, tumor reactivity, TCR repertoire, (predicted) TCR antigen-specificities, and environmental cues like gut microbiome or colon tissue-specific 'self-like' features. MSI CRC CD8+ T cells showed tumor-specific activation reminiscent of canonical 'T cell hot' tumors, whereas the MSS CRC CD8+ T cells exhibited tumor unspecific or bystander-like features. This was accompanied by inflammation reminiscent of 'pseudo-T cell hot' tumors. Consequently, MSI and MSS CRC CD8+ T cells showed overlapping phenotypic features that differed dramatically in their TCR antigen-specificities. Given their high discriminating potential for CD8+ T cell features/specificities, we used the single cell tumor-reactive signaling modules in CD8+ T cells to build a bulk tumor transcriptome classification for CRC patients. This "Immune Subtype Classification" (ISC) successfully distinguished various tumoral immune landscapes that showed prognostic value and predicted immunotherapy responses in CRC patients. Thus, we deliver a unique map of CRC CD8+ T cells that drives a novel tumor immune landscape classification, with relevance for immunotherapy decision-making.
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BACKGROUND: Very few risk factors for postoperative recurrence (POR) of Crohn's Disease (CD) after ileocecal resection have been identified. The aim of the present study was to verify the association between an a priori defined list of intraoperative macroscopic findings and POR. METHODS: This was a prospective observational study including patients undergoing primary ileocecal resection for CD. Four intraoperative factors were independently evaluated by 2 surgeons: length of resected ileum, mesentery thickness, presence of areas of serosal fat infiltration, or abnormal serosal vasodilation on normal bowel proximal to the resected bowel. The primary end point was early endoscopic POR at month 6 and defined as modified Rutgeerts score ≥i2b. Secondary end points were clinical and surgical recurrence. RESULTS: Between September 2020 and November 2022, 83 consecutive patients were included. Early endoscopic recurrence occurred in 45 of 76 patients (59.2%). Clinical and biochemical recurrence occurred in 17.3% (95% confidence interval, [CI], 10.4%-28.0%) and 14.6% of the patients after 12 months. The risk of developing endoscopic and clinical recurrence was 1.127 (95% CI, 0.448;2.834, P = .799) and 0.896 (95% CI, 0.324-2.478, P = .832) when serosal fat infiltration was observed, and 1.388 (95% CI, 0.554-3.476, P = .484), and 1.153 (95% CI, 0.417;3.187, P = .783) when abnormal serosal vasodilation was observed. Similarly, length of the resected bowel and mesentery thickness showed no association with POR. A subgroup analysis on patients who received no postoperative medical prophylaxis did not identify any risk factor for endoscopic POR. CONCLUSIONS: The macroscopic appearance of the bowel and associated mesentery during surgery does not seem to be predictive of POR after ileocecal resection for CD.
Prospective studies investigating risk factors for Crohn's disease recurrence after surgery are scarce. In a prospective cohort of 83 patients undergoing primary ileocecal resection, no association between few intraoperative macroscopic findings and postoperative endoscopic/clinical recurrence was observed.
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Intestinal ischemia is a potentially catastrophic emergency, with a high rate of morbidity and mortality. Currently, no specific pharmacological treatments are available. Previous work demonstrated that pre-treatment with obeticholic acid (OCA) protected against ischemia reperfusion injury (IRI). Recently, a more potent and water-soluble version has been synthesized: Intercept 767 (INT-767). The aim of this study was to investigate if intravenous treatment with INT-767 can improve outcomes after IRI. In a validated rat model of IRI (60 min ischemia + 60 min reperfusion), three groups were investigated (n = 6/group): (i) sham: surgery without ischemia; (ii) IRI + vehicle; and (iii) IRI + INT-767. The vehicle (0.9% NaCl) or INT-767 (10 mg/kg) were administered intravenously 15 min after start of ischemia. Endpoints were 7-day survival, serum injury markers (L-lactate and I-FABP), histology (Park-Chiu and villus length), permeability (transepithelial electrical resistance and endotoxin translocation), and cytokine expression. Untreated, IRI was uniformly lethal by provoking severe inflammation and structural damage, leading to translocation and sepsis. INT-767 treatment significantly improved survival by reducing inflammation and preserving intestinal structural integrity. This study demonstrates that treatment with INT-767 15 min after onset of intestinal ischemia significantly decreases IRI and improves survival. The ability to administer INT-767 intravenously greatly enhances its clinical potential.
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Ácidos e Sais Biliares , Intestinos , Receptores Citoplasmáticos e Nucleares , Receptores Acoplados a Proteínas G , Traumatismo por Reperfusão , Animais , Ratos , Inflamação/tratamento farmacológico , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Traumatismo por Reperfusão/tratamento farmacológico , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Ácidos e Sais Biliares/uso terapêutico , Intestinos/irrigação sanguíneaRESUMO
BACKGROUND AND AIMS: Histologic disease activity in Inflammatory Bowel Disease (IBD) is associated with clinical outcomes and is an important endpoint in drug development. We developed deep learning models for automating histological assessments in IBD. METHODS: Histology images of intestinal mucosa from phase 2 and phase 3 clinical trials in Crohn's disease (CD) and Ulcerative Colitis (UC) were used to train artificial intelligence (AI) models to predict the Global Histology Activity Score (GHAS) for CD and Geboes histopathology score for UC. Three AI methods were compared. AI models were evaluated on held-back testing sets and model predictions were compared against an expert central reader and five independent pathologists. RESULTS: The model based on multiple instance learning and the attention mechanism (SA-AbMILP) demonstrated the best performance among competing models. AI modeled GHAS and Geboes sub-grades matched central readings with moderate to substantial agreement, with accuracies ranging from 65% to 89%. Furthermore, the model was able to distinguish the presence and absence of pathology across four selected histological features with accuracies for colon, in both CD and UC, ranging from 87% to 94% and, for CD ileum, ranging from 76% to 83%. For both CD and UC, and across anatomical compartments (ileum and colon) in CD, comparable accuracies against central readings were found between the model assigned scores and scores by an independent set of pathologists. CONCLUSIONS: Deep learning models based upon GHAS and Geboes scoring systems were effective at distinguishing between the presence and absence of IBD microscopic disease activity.
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Inflammation and fibrosis are key features of proliferative vitreoretinal disorders. We aimed to define the macrophage phenotype and investigate the role of macrophage-myofibroblast transition (MMT) in the contribution to myofibroblast populations present in epiretinal membranes. Vitreous samples from proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR) and nondiabetic control patients, epiretinal fibrovascular membranes from PDR patients and fibrocellular membranes from PVR patients, human retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied by ELISA, immunohistochemistry and flow cytometry analysis. Myofibroblasts expressing α-SMA, fibroblast activation protein-α (FAP-α) and fibroblast-specific protein-1 (FSP-1) were present in all membranes. The majority of CD68+ monocytes/macrophages co-expressed the M2 macrophage marker CD206. In epiretinal membranes, cells undergoing MMT were identified by co-expression of the macrophage marker CD68 and myofibroblast markers α-SMA and FSP-1. Further analysis revealed that CD206+ M2 macrophages co-expressed α-SMA, FSP-1, FAP-α and ß-catenin. Soluble (s) CD206 and sFAP-α levels were significantly higher in vitreous samples from PDR and PVR patients than in nondiabetic control patients. The proinflammatory cytokine TNF-α and the hypoxia mimetic agent cobalt chloride induced upregulation of sFAP-α in culture media of Müller cells but not of HRMECs. The NF-Ä¸ß inhibitor BAY11-7085 significantly attenuated TNF-α-induced upregulation of sFAP-α in Müller cells. Our findings suggest that the process of MMT might contribute to myofibroblast formation in epiretinal membranes, and this transition involved macrophages with a predominant M2 phenotype. In addition, sFAP-α as a vitreous biomarker may be derived from M2 macrophages transitioned to myofibroblasts and from Müller cells.
Assuntos
Retinopatia Diabética , Membrana Epirretiniana , Oftalmopatias , Vitreorretinopatia Proliferativa , Humanos , Células Endoteliais , Miofibroblastos , Fator de Necrose Tumoral alfaRESUMO
Intestinal ischemia-reperfusion injury (IRI) is a common clinical entity, and its outcome is unpredictable due to the triad of inflammation, increased permeability and bacterial translocation. Polyethylene glycol (PEG) is a polyether compound that is extensively used in pharmacology as an excipient in various products. More recently, this class of products have shown to have potent anti-inflammatory, anti-apoptotic, immunosuppressive and cell-membrane-stabilizing properties. However, its effects on the outcome after intestinal IRI have not yet been investigated. We hypothesized that PEG administration would reduce the effects of intestinal IRI in rodents. In a previously described rat model of severe IRI (45 min of ischemia followed by 60 min of reperfusion), we evaluated the effect of IV PEG administration at different doses (50 and 100 mg/kg) before and after the onset of ischemia. In comparison to control animals, PEG administration stabilized the endothelial glycocalyx, leading to reduced reperfusion edema, bacterial translocation and inflammatory reaction as well as improved 7-day survival. These effects were seen both in a pretreatment and in a treatment setting. The fact that this product is readily available and safe should encourage further clinical investigations in settings of intestinal IRI, organ preservation and transplantation.
Assuntos
Traumatismo por Reperfusão , Roedores , Ratos , Animais , Polietilenoglicóis/farmacologia , Polietilenoglicóis/uso terapêutico , Traumatismo por Reperfusão/tratamento farmacológico , Intestinos , Preservação de ÓrgãosRESUMO
Rationale: Invasive pulmonary aspergillosis has emerged as a frequent coinfection in severe coronavirus disease (COVID-19), similarly to influenza, yet the clinical invasiveness is more debated. Objectives: We investigated the invasive nature of pulmonary aspergillosis in histology specimens of influenza and COVID-19 ICU fatalities in a tertiary care center. Methods: In this monocentric, descriptive, retrospective case series, we included adult ICU patients with PCR-proven influenza/COVID-19 respiratory failure who underwent postmortem examination and/or tracheobronchial biopsy during ICU admission from September 2009 until June 2021. Diagnosis of probable/proven viral-associated pulmonary aspergillosis (VAPA) was made based on the Intensive Care Medicine influenza-associated pulmonary aspergillosis and the European Confederation of Medical Mycology (ECMM) and the International Society for Human and Animal Mycology (ISHAM) COVID-19-associated pulmonary aspergillosis consensus criteria. All respiratory tissues were independently reviewed by two experienced pathologists. Measurements and Main Results: In the 44 patients of the autopsy-verified cohort, 6 proven influenza-associated and 6 proven COVID-19-associated pulmonary aspergillosis diagnoses were identified. Fungal disease was identified as a missed diagnosis upon autopsy in 8% of proven cases (n = 1/12), yet it was most frequently found as confirmation of a probable antemortem diagnosis (n = 11/21, 52%) despite receiving antifungal treatment. Bronchoalveolar lavage galactomannan testing showed the highest sensitivity for VAPA diagnosis. Among both viral entities, an impeded fungal growth was the predominant histologic pattern of pulmonary aspergillosis. Fungal tracheobronchitis was histologically indistinguishable in influenza (n = 3) and COVID-19 (n = 3) cases, yet macroscopically more extensive at bronchoscopy in influenza setting. Conclusions: A proven invasive pulmonary aspergillosis diagnosis was found regularly and with a similar histological pattern in influenza and in COVID-19 ICU case fatalities. Our findings highlight an important need for VAPA awareness, with an emphasis on mycological bronchoscopic work-up.