Background & Aims There is evidence from clinical studies that compromised intestinal epithelial permeability contributes to the development of nonalcoholic steatohepatitis (NASH), but the exact ...mechanisms are not clear. Mice with disruption of the gene ( F11r ) encoding junctional adhesion molecule A (JAM-A) have defects in intestinal epithelial permeability. We used these mice to study how disruption of the intestinal epithelial barrier contributes to NASH. Methods Male C57BL/6 (control) or F11r-/- mice were fed a normal diet or a diet high in saturated fat, fructose, and cholesterol (HFCD) for 8 weeks. Liver and intestinal tissues were collected and analyzed by histology, quantitative reverse-transcription polymerase chain reaction, and flow cytometry. Intestinal epithelial permeability was assessed in mice by measuring permeability to fluorescently labeled dextran. The intestinal microbiota were analyzed using 16S ribosomal RNA sequencing. We also analyzed biopsy specimens from proximal colons of 30 patients with nonalcoholic fatty liver disease (NAFLD) and 19 subjects without NAFLD (controls) undergoing surveillance colonoscopy. Results F11r-/- mice fed a HFCD, but not a normal diet, developed histologic and pathologic features of severe NASH including steatosis, lobular inflammation, hepatocellular ballooning, and fibrosis, whereas control mice fed a HFCD developed only modest steatosis. Interestingly, there were no differences in body weight, ratio of liver weight:body weight, or glucose homeostasis between control and F11r-/- mice fed a HFCD. In these mice, liver injury was associated with significant increases in mucosal inflammation, tight junction disruption, and intestinal epithelial permeability to bacterial endotoxins, compared with control mice or F11r-/- mice fed a normal diet. The HFCD led to a significant increase in inflammatory microbial taxa in F11r-/- mice, compared with control mice. Administration of oral antibiotics or sequestration of bacterial endotoxins with sevelamer hydrochloride reduced mucosal inflammation and restored normal liver histology in F11r-/- mice fed a HFCD. Protein and transcript levels of JAM-A were significantly lower in the intestinal mucosa of patients with NAFLD than without NAFLD; decreased expression of JAM-A correlated with increased mucosal inflammation. Conclusions Mice with defects in intestinal epithelial permeability develop more severe steatohepatitis after a HFCD than control mice, and colon tissues from patients with NAFLD have lower levels of JAM-A and higher levels of inflammation than subjects without NAFLD. These findings indicate that intestinal epithelial barrier function and microbial dysbiosis contribute to the development of NASH. Restoration of intestinal barrier integrity and manipulation of gut microbiota might be developed as therapeutic strategies for patients with NASH.
Tubulointerstitial injury in the kidney is complex, involving a number of independent and overlapping cellular and molecular pathways, with renal interstitial fibrosis and tubular atrophy (IFTA) as ...the final common pathway. Furthermore, there are multiple ways to assess IFTA.
Cells involved include tubular epithelial cells, fibroblasts, fibrocytes, myofibroblasts, monocyte/macrophages, and mast cells with complex and still incompletely characterized cell-molecular interactions. Molecular mediators involved are numerous and involve pathways such as transforming growth factor (TGF)-β, bone morphogenic protein (BMP), platelet-derived growth factor (PDGF), and hepatocyte growth factor (HGF). Recent genomic approaches have shed insight into some of these cellular and molecular pathways. Pathologic evaluation of IFTA is central in assessing the severity of chronic disease; however, there are a variety of methods used to assess IFTA. Most assessment of IFTA relies on pathologist assessment of special stains such as trichrome, Sirius Red, and collagen III immunohistochemistry. Visual pathologist assessment can be prone to intra and interobserver variability, but some methods employ computerized morphometery, without a clear consensus as to the best method.
IFTA results from on orchestration of cell types and molecular pathways. Opinions vary on the optimal qualitative and quantitative assessment of IFTA.
Hepatic steatosis droplet quantification with histology biopsies has high clinical significance for risk stratification and management of patients with fatty liver diseases and in the decision to use ...donor livers for transplantation. However, pathology reviewing processes, when conducted manually, are subject to a high inter- and intra-reader variability, due to the overwhelmingly large number and significantly varying appearance of steatosis instances. This process is challenging as there is a large number of overlapped steatosis droplets with either missing or weak boundaries. In this study, we propose a deep-learning-based region-boundary integrated network for precise steatosis quantification with whole slide liver histopathology images. The proposed model consists of two sequential steps: a region extraction and a boundary prediction module for foreground regions and steatosis boundary prediction, followed by an integrated prediction map generation. Missing steatosis boundaries are next recovered from the predicted map and assembled from adjacent image patches to generate results for the whole slide histopathology image. The resulting steatosis measures both at the pixel level and steatosis object-level present strong correlation with pathologist annotations, radiology readouts and clinical data. In addition, the segregated steatosis object count is shown as a promising alternative measure to the traditional metrics at the pixel level. These results suggest a high potential of artificial intelligence-assisted technology to enhance liver disease decision support using whole slide images.
Accurate quantification of steatosis in liver biopsies is a key step in the treatment of patients with fatty liver diseases. To assist pathologists for such analysis tasks, we develop a novel deep learning-based framework to segment overlapped steatosis droplets in whole slide liver biopsy images. Quantitative measurements of steatosis at both pixel and object-level present strong correlation with clinical data, suggesting its potential for clinical decision support.
The XV. Banff conference for allograft pathology was held in conjunction with the annual meeting of the American Society for Histocompatibility and Immunogenetics in Pittsburgh, PA (USA) and focused ...on refining recent updates to the classification, advances from the Banff working groups, and standardization of molecular diagnostics. This report on kidney transplant pathology details clarifications and refinements to the criteria for chronic active (CA) T cell–mediated rejection (TCMR), borderline, and antibody‐mediated rejection (ABMR). The main focus of kidney sessions was on how to address biopsies meeting criteria for CA TCMR plus borderline or acute TCMR. Recent studies on the clinical impact of borderline infiltrates were also presented to clarify whether the threshold for interstitial inflammation in diagnosis of borderline should be i0 or i1. Sessions on ABMR focused on biopsies showing microvascular inflammation in the absence of C4d staining or detectable donor‐specific antibodies; the potential value of molecular diagnostics in such cases and recommendations for use of the latter in the setting of solid organ transplantation are presented in the accompanying meeting report. Finally, several speakers discussed the capabilities of artificial intelligence and the potential for use of machine learning algorithms in diagnosis and personalized therapeutics in solid organ transplantation.
This report focuses on the clarification of the criteria for chronic active T cell–mediated rejection and antibody‐mediated rejection and the optimization of the inflammation threshold for the diagnosis of borderline for acute T cell–mediated rejection, and discusses the potential to use machine learning in diagnostics and personalized therapeutics in solid organ transplantation.
Background & Aims A high-fat diet (HFD) can cause serious health problems, including alteration of gastrointestinal transit, the exact mechanism of which is not clear. Several microRNAs (miRNAs) are ...involved in energy homeostasis, lipid metabolism, and HFD-induced weight gain. We investigated the role of miRNAs in HFD-induced damage to the enteric nervous system. Methods Male mice were fed a HFD (60% calories from fat) or regular diets (18% calories from fat) for 11 weeks. Mice on regular diets and HFDs were given intraperitoneal injections of Mir375 inhibitor or a negative control. Body weights, food intake, stool indices, and gastrointestinal transit (following Evans blue gavage) were measured. An enteric neuronal cell line (immorto-fetal enteric neuronal) and primary enteric neurons were used for in vitro studies. Results HFD delayed intestinal transit, which was associated with increased apoptosis and loss of colonic myenteric neurons. Mice fed a low-palmitate HFD did not develop a similar phenotype. Palmitate caused apoptosis of enteric neuronal cells associated with mitochondrial dysfunction and endoplasmic reticulum stress. Palmitate significantly increased the expression of Mir375 in vitro; transfection of cells with a Mir375 inhibitor prevented the palmitate-induced enteric neuronal cell apoptosis. Mir375 expression was increased in myenteric ganglia of mice fed HFD and associated with decreased levels of Mir375 target messenger RNAs, including Pdk1. Systemic injection of a Mir375 inhibitor for 5 weeks prevented HFD-induced delay in intestinal transit and morphologic changes. Conclusions HFDs delay colonic transit, partly by inducing apoptosis in enteric neuronal cells. This effect is mediated by Mir375 and is associated with reduced levels of Pdk1. Mir375 might be targeted to increase survival of enteric neurons and gastrointestinal motility.
Donor-specific antibodies are associated with increased risk of antibody-mediated rejection and decreased allograft survival. Therefore, reducing the risk of these antibodies remains a clinical need ...in transplantation. Plasma cells are a logical target of therapy given their critical role in antibody production.
To target plasma cells, we treated sensitized rhesus macaques with daratumumab (anti-CD38 mAb). Before transplant, we sensitized eight macaques with two sequential skin grafts from MHC-mismatched donors; four of them were also desensitized with daratumumab and plerixafor (anti-CXCR4). We also treated two patients with daratumumab in the context of transplant.
The animals treated with daratumumab had significantly reduced donor-specific antibody levels compared with untreated controls (57.9% versus 13% reduction;
<0.05) and prolonged renal graft survival (28.0 days versus 5.2 days;
<0.01). However, the reduction in donor-specific antibodies was not maintained because all recipients demonstrated rapid rebound of antibodies, with profound T cell-mediated rejection. In the two clinical patients, a combined heart and kidney transplant recipient with refractory antibody-mediated rejection and a highly sensitized heart transplant candidate, we also observed a significant decrease in class 1 and 2 donor-specific antibodies that led to clinical improvement of antibody-mediated rejection and to heart graft access.
Targeting CD38 with daratumumab significantly reduced anti-HLA antibodies and anti-HLA donor-specific antibodies in a nonhuman primate model and in two transplant clinical cases before and after transplant. This supports investigation of daratumumab as a potential therapeutic strategy; however, further research is needed regarding its use for both antibody-mediated rejection and desensitization.
Renal allograft rejection diagnosis depends on assessment of parameters such as interstitial inflammation; however, studies have shown interobserver variability regarding interstitial inflammation ...assessment. Since automated image analysis quantitation can be reproducible, we devised customized analysis methods for CD3+ T-cell staining density as a measure of rejection severity and compared them with established commercial methods along with visual assessment. Renal biopsy CD3 immunohistochemistry slides (
n
= 45), including renal allografts with various degrees of acute cellular rejection (ACR) were scanned for whole slide images (WSIs). Inflammation was quantitated in the WSIs using pathologist visual assessment, commercial algorithms (Aperio nuclear algorithm for CD3+ cells/mm
2
and Aperio positive pixel count algorithm), and customized open source algorithms developed in ImageJ with thresholding/positive pixel counting (custom CD3+%) and identification of pixels fulfilling “maxima” criteria for CD3 expression (custom CD3+ cells/mm
2
). Based on visual inspections of “markup” images, CD3 quantitation algorithms produced adequate accuracy. Additionally, CD3 quantitation algorithms correlated between each other and also with visual assessment in a statistically significant manner (
r
= 0.44 to 0.94,
p
= 0.003 to < 0.0001). Methods for assessing inflammation suggested a progression through the tubulointerstitial ACR grades, with statistically different results in borderline versus other ACR types, in all but the custom methods. Assessment of CD3-stained slides using various open source image analysis algorithms presents salient correlations with established methods of CD3 quantitation. These analysis techniques are promising and highly customizable, providing a form of on-slide “flow cytometry” that can facilitate additional diagnostic accuracy in tissue-based assessments.
Glomerular number and size in autopsy kidneys: The relationship to birth weight.
In the Southeast United States, African Americans have an estimated incidence of hypertension and end-stage renal ...disease (ESRD) that is five times greater than Caucasians. Higher rates of low birth weight (LBW) among African Americans is suggested to predispose African Americans to the higher risk, possibly by reducing the number of glomeruli that develop in the kidney. This study investigates the relationships between age, race, gender, total glomerular number (Nglom), mean glomerular volume (Vglom), body surface area (BSA), and birth weight.
Stereologic estimates of Nglom and Vglom were obtained using the physical disector/fractionator combination for autopsy kidneys from 37 African Americans and 19 Caucasians.
Nglom was normally distributed and ranged from 227,327 to 1,825,380, an 8.0-fold difference. A direct linear relationship was observed between Nglom and birth weight (r = 0.423, P = 0.0012) with a regression coefficient that predicted an increase of 257,426 glomeruli per kilogram increase in birth weight (α = 0.050:0.908). Among adults there was a 4.9-fold range in Vglom, and in adults, Vglom was strongly and inversely correlated with Nglom (r =-0.640, P = 0.000002). Adult Vglom showed no significant correlation with BSA for males (r = -0.0150, P = 0.936), although it did for females (r = 0.606, P = 0.022). No racial differences in average Nglom or Vglom were observed.
Birth weight is a strong determinant of Nglom and thereby of glomerular size in the postnatal kidney. The findings support the hypothesis that LBW by impairing nephron development is a risk factor for hypertension and ESRD in adulthood.
The Banff Working Group on Liver Allograft Pathology met in September 2022. Participants included hepatologists, surgeons, pathologists, immunologists, and histocompatibility specialists. ...Presentations and discussions focused on the evaluation of long-term allograft health, including noninvasive and tissue monitoring, immunosuppression optimization, and long-term structural changes. Potential revision of the rejection classification scheme to better accommodate and communicate late T cell-mediated rejection patterns and related structural changes, such as nodular regenerative hyperplasia, were discussed. Improved stratification of long-term maintenance immunosuppression to match the heterogeneity of patient settings will be central to improving long-term patient survival. Such personalized therapeutics are in turn contingent on a better understanding and monitoring of allograft status within a rational decision-making approach, likely to be facilitated in implementation with emerging decision-support tools. Proposed revisions to rejection classification emerging from the meeting include the incorporation of interface hepatitis and fibrosis staging. These will be opened to online testing, modified accordingly, and subject to consensus discussion leading up to the next Banff conference.
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