Publications by authors named "Alton B Farris"

96 Publications

Artificial Intelligence and Algorithmic Computational Pathology: Introduction with Renal Allograft Examples.

Histopathology 2020 Nov 19. Epub 2020 Nov 19.

Department of Surgery, Emory University, Atlanta, GA, U.S.A.

Whole slide imaging (WSI), an important technique in the field of digital pathology, has recently been the subject of increased interest and avenues for utilization; and with more widespread WSI utilization, there will also be increased interest in and implementation of image analysis techniques. Image analysis includes artificial intelligence (AI) and targeted or hypothesis-driven algorithms. In the overall pathology field, citations related to these topics have increased in recent years. Renal pathology is one anatomic pathology subspecialty that has utilized WSIs and image analysis algorithms; and it can be argued that renal transplant pathology could be particularly suited for WSI and image analysis, since renal transplant pathology is frequently classified using the semiquantitative Banff Classification of Renal Allograft Pathology. Hypothesis-driven/targeted algorithms have been used in the past for the assessment of a variety of features in the kidney (e.g., interstitial fibrosis and tubular atrophy and inflammation); and in recent years, research has particularly increased in the area of AI/machine learning for the identification of glomeruli, for histologic segmentation, and other applications. Deep learning is the form of machine learning most often used for such AI approaches to the "big data" of pathology WSIs, and deep learning methods such as artificial neural networks (ANNs)/convolutional neural networks (CNNs) are utilized. Unsupervised and supervised AI algorithms can be employed to accomplish image or semantic classification. In this review, AI and other image analysis algorithms applied to WSIs are discussed; and examples from renal pathology are covered, with an emphasis on renal transplant pathology.
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http://dx.doi.org/10.1111/his.14304DOI Listing
November 2020

Assessment of a computerized quantitative quality control tool for whole slide images of kidney biopsies.

J Pathol 2021 Mar 5;253(3):268-278. Epub 2021 Jan 5.

Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH, USA.

Inconsistencies in the preparation of histology slides and whole-slide images (WSIs) may lead to challenges with subsequent image analysis and machine learning approaches for interrogating the WSI. These variabilities are especially pronounced in multicenter cohorts, where batch effects (i.e. systematic technical artifacts unrelated to biological variability) may introduce biases to machine learning algorithms. To date, manual quality control (QC) has been the de facto standard for dataset curation, but remains highly subjective and is too laborious in light of the increasing scale of tissue slide digitization efforts. This study aimed to evaluate a computer-aided QC pipeline for facilitating a reproducible QC process of WSI datasets. An open source tool, HistoQC, was employed to identify image artifacts and compute quantitative metrics describing visual attributes of WSIs to the Nephrotic Syndrome Study Network (NEPTUNE) digital pathology repository. A comparison in inter-reader concordance between HistoQC aided and unaided curation was performed to quantify improvements in curation reproducibility. HistoQC metrics were additionally employed to quantify the presence of batch effects within NEPTUNE WSIs. Of the 1814 WSIs (458 H&E, 470 PAS, 438 silver, 448 trichrome) from n = 512 cases considered in this study, approximately 9% (163) were identified as unsuitable for subsequent computational analysis. The concordance in the identification of these WSIs among computational pathologists rose from moderate (Gwet's AC1 range 0.43 to 0.59 across stains) to excellent (Gwet's AC1 range 0.79 to 0.93 across stains) agreement when aided by HistoQC. Furthermore, statistically significant batch effects (p < 0.001) in the NEPTUNE WSI dataset were discovered. Taken together, our findings strongly suggest that quantitative QC is a necessary step in the curation of digital pathology cohorts. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5590DOI Listing
March 2021

Correlation Patterns Among B7 Family Ligands and Tryptophan Degrading Enzymes in Hepatocellular Carcinoma.

Front Oncol 2020 3;10:1632. Epub 2020 Sep 3.

School of Biology, Georgia Institute of Technology, Atlanta, GA, United States.

Mechanisms of dysfunctional T cell immunity in Hepatocellular Carcinoma (HCC) need to be well defined. B7 family molecules provide both co-stimulatory and co-inhibitory signals to T cells while tryptophan degrading enzymes like Indoleamine 2,3 dioxygenase (IDO) and Tryptophan 2,3 Dioxygenase (TDO) mediate tumor immune tolerance. It is necessary to identify their correlative expression, which informs targets for combined immunotherapy approaches. We investigated B7 family molecules, IDO, TDO and immune responsive effectors in the tumor tissues of patients with HCC ( = 28) using a pathway-focused quantitative nanoscale chip real-time PCR. Four best correlative expressions, namely (1) B7-1 & PD-L2, (2) B7-H2 & B7-H3, (3) B7-2 & PD-L1, (4) PD-L1 & PD-L2, were identified among B7 family ligands, albeit they express at different levels. Although TDO expression is higher than IDO, PD-L1 correlates only with IDO but not TDO. Immune effector (Granzyme B) and suppressive (PD-1 and TGF-β) genes correlate with IDO and B7-1, B7-H5, PD-L2. Identification of the correlation of PD-L1, PD-L2 and IDO suggest their cumulative immuno suppressive role in HCC. The distinct correlations among B7-1, B7-2, B7-H2, and B7-H3, correlation of PD-1 with non-cognate ligands such as B7-1 and B7-H5, and correlation of tumor lytic enzyme Granzyme B with IDO and PD-L2 suggest that HCC microenvironment is complexly orchestrated with both stimulatory and inhibitory molecules which together neutralize and blunt anti-HCC immunity. Functional assays demonstrate that both PDL-1 and IDO synergistically inhibit T cell responses. Altogether, the present data suggest the usage of combined immune checkpoint blocking strategies targeting co-inhibitory B7 molecules and IDO for HCC management.
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http://dx.doi.org/10.3389/fonc.2020.01632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494748PMC
September 2020

Quantitative assessment of liver fibrosis by digital image analysis reveals correlation with qualitative clinical fibrosis staging in liver transplant patients.

PLoS One 2020 28;15(9):e0239624. Epub 2020 Sep 28.

Department of Pathology, Emory University, Atlanta, Georgia, United States of America.

Technologies for digitizing tissues provide important quantitative data for liver histopathology investigation. We aimed to assess liver fibrosis degree with quantitative morphometric measurements of histopathological sections utilizing digital image analysis (DIA) and to further investigate if a correlation with histopathologic scoring (Scheuer staging) exists. A retrospective study of patients with at least two post-liver transplant biopsies having a Scheuer stage of ≤ 2 at baseline were gathered. Portal tract fibrotic percentage (%) and size (μm2) were measured by DIA, while clinical fibrosis score was measured by the Scheuer system. Correlations between DIA measurements and Scheuer scores were computed by Spearman correlation analysis. Differences between mean levels of fibrosis (score, size, and percentage) at baseline versus second visit were computed by Student's t-test. P values < 0.05 were considered significant. Of 22 patients who met the study criteria, 54 biopsies were included for analysis. Average levels ±standard error [S.E.] of portal tract fibrotic percentage (%) and size (μm2) progressed from 46.5 ± 3.6% at baseline to 61.8 ± 3.8% at the second visit (P = 0.005 by Student's t-test), and from 28,075 ± 3,232 μm2 at base line to 67,146 ± 10,639 μm2 at the second visit (P = 0.002 by Student's t-test), respectively. Average levels of Scheuer fibrosis scores progressed from 0.55±0.19 at baseline to 1.14±0.26 at the second visit (P = 0.02 by Student's t-test). Portal tract fibrotic percentage (%) and portal tract fibrotic size were directly correlated with clinical Scheuer fibrosis stage, with Spearman correlation coefficient and P value computed as r = 0.70, P < 0.0001 and r = 0.41, P = 0.002, respectively. Digital quantitative assessment of portal triad size and fibrosis percentage demonstrates a strong correlation with visually assessed histologic stage of liver fibrosis and complements the standard assessment for allograft monitoring, suggesting the utility of future WSI analysis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0239624PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7521727PMC
November 2020

Preoperative carfilzomib and lulizumab based desensitization prolongs graft survival in a sensitized non-human primate model.

Kidney Int 2021 01 6;99(1):161-172. Epub 2020 Sep 6.

Duke Transplant Center, Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA. Electronic address:

Sensitized patients are difficult to transplant due to pre-formed anti-donor immunity. We have previously reported successful desensitization using carfilzomib and belatacept in a non-human primate (NHP) model. Here we evaluated selective blockade of the co-stimulatory signal (CD28-B7) with Lulizumab, which preserves the co-inhibitory signal (CTLA4-B7). Five maximally MHC-mismatched pairs of NHPs were sensitized to each other with two sequential skin transplants. Individuals from each pair were randomized to either desensitization with once-weekly Carfilzomib (27mg/m IV) and Lulizumab (12.5mg/kg SC) over four weeks, or no desensitization (Control). NHPs then underwent life-sustaining kidney transplantation from their previous skin donor. Rhesus-specific anti-thymocyte globulin was used as induction therapy and immunosuppression maintained with tacrolimus, mycophenolate, and methylprednisolone. Desensitized subjects demonstrated a significant reduction in donor-specific antibody, follicular helper T cells (CD4PD-1ICOS), and proliferating B cells (CD20Ki67) in the lymph nodes. Interestingly, regulatory T cell (CD4CD25CD127) frequency was maintained after desensitization in addition to increased frequency of naïve CD4 T cells (CCR7CD45RA) and naïve B cells (IgDCD27CD20) in circulation. This was associated with significant prolongation in graft survival (MST = 5.8 ± 4.0 vs. 64.8 ± 36.3; p<0.05) and lower antibody-mediated rejection scores compared to control animals. However, all desensitized animals eventually developed AMR and graft failure. Desensitization with CFZ and Lulizumab improves allograft survival in allosensitized NHPs, by transient control of the germinal center and shifting of the immune system to a more naive phenotype. This regimen may translate into clinical practice to improve outcomes of highly sensitized transplant patients.
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http://dx.doi.org/10.1016/j.kint.2020.08.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7785689PMC
January 2021

Deep-learning-based accurate hepatic steatosis quantification for histological assessment of liver biopsies.

Lab Invest 2020 10 13;100(10):1367-1383. Epub 2020 Jul 13.

Department of Mathematics and Statistics, Georgia State University, Atlanta, GA, 30303, USA.

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.
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http://dx.doi.org/10.1038/s41374-020-0463-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502534PMC
October 2020

Avoidance of CNI and steroids using belatacept-Results of the Clinical Trials in Organ Transplantation 16 trial.

Am J Transplant 2020 12 13;20(12):3599-3608. Epub 2020 Jul 13.

Emory Transplant Center, Emory University School of Medicine, Atlanta, Georgia, USA.

Immunosuppression devoid of corticosteroids has been investigated to avoid long-term comorbidities. Likewise, alternatives to calcineurin inhibitors have been investigated as a strategy to improve long-term kidney function following transplanion. Costimulatory blockade strategies that include corticosteroids have recently shown promise, despite their higher rates of early acute rejection. We designed a randomized clinical trial utilizing depletional induction therapy to mitigate early rejection risk while limiting calcineurin inhibitors and corticosteroids. This trial, Clinical Trials in Organ Transplantation 16 (CTOT-16), sought to evaluate novel belatacept-based strategies employing tacrolimus and corticosteroid avoidance. Sixty-nine kidney transplant recipients were randomized from 4 US transplant centers comparing a control group of with rabbit antithymocyte globulin (rATG) induction, rapid steroid taper, and maintenance mycophenolate and tacrolimus, to 2 arms using maintenance belatacept. There were no graft losses but there were 2 deaths in the control group. However, the trial was halted early because of rejection in the belatacept treatment groups. Serious adverse events were similar across groups. Although rejection was not uniform in the belatacept maintenance therapy groups, the frequency of rejection limits the practical implementation of this strategy to avoid both calcineurin inhibitors and corticosteroids at this time.
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http://dx.doi.org/10.1111/ajt.16152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710570PMC
December 2020

The Banff 2019 Kidney Meeting Report (I): Updates on and clarification of criteria for T cell- and antibody-mediated rejection.

Am J Transplant 2020 09 28;20(9):2318-2331. Epub 2020 May 28.

Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Canada.

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.
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http://dx.doi.org/10.1111/ajt.15898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496245PMC
September 2020

Low-grade fibromyxoid sarcoma: a potentially useful histologic finding.

Histopathology 2020 08 2;77(2):329-331. Epub 2020 Jul 2.

Mayo Clinic, Jacksonville, FL, USA.

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http://dx.doi.org/10.1111/his.14142DOI Listing
August 2020

Cultured thymus tissue implantation promotes donor-specific tolerance to allogeneic heart transplants.

JCI Insight 2020 06 4;5(11). Epub 2020 Jun 4.

Department of Immunology, and.

Eighty-six infants born without a thymus have been treated with allogeneic cultured thymus tissue implantation (CTTI). These infants, who lack T cells and are profoundly immunodeficient at birth, after CTTI from an unmatched donor develop T cells similar to those of recipient that are tolerant to both their own major histocompatibility antigens and those of the donor. We tested use of CTTI with the goal of inducing tolerance to unmatched heart transplants in immunocompetent rats. We thymectomized and T cell-depleted Lewis rats. The rats were then given cultured thymus tissue from F1 (Lewis × Dark Agouti ) under the kidney capsule and vascularized Dark Agouti (DA) heart transplants in the abdomen. Cyclosporine was administered for 4 months. The control group did not receive CTTI. Recipients with CTTI showed repopulation of naive and recent thymic emigrant CD4 T cells; controls had none. Recipients of CTTI did not reject DA cardiac allografts. Control animals did not reject DA grafts, due to lack of functional T cells. To confirm donor-specific unresponsiveness, MHC-mismatched Brown Norway (BN) hearts were transplanted 6 months after the initial DA heart transplant. LW rats with LWxDA CTTI rejected the third-party BN hearts (mean survival time 10 days); controls did not. CTTI recipients produced antibody against third-party BN donor but not against the DA thymus donor, demonstrating humoral donor-specific tolerance. Taken together, F1(LWxDA) CTTI given to Lewis rats resulted in specific tolerance to the allogeneic DA MHC expressed in the donor thymus, with resulting long-term survival of DA heart transplants after withdrawal of all immunosuppression.
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http://dx.doi.org/10.1172/jci.insight.129983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7308047PMC
June 2020

Targeting Calcium Release-activated Calcium Channel Is Not Sufficient to Prevent Rejection in Nonhuman Primate Kidney Transplantation.

Transplantation 2020 05;104(5):970-980

Duke Transplant Center, Department of Surgery, Duke University, Durham, NC.

Background: Calcineurin inhibitors successfully control rejection of transplanted organs but also cause nephrotoxicity. This study, using a rhesus monkey renal transplantation model, sought to determine the applicability of a new immunomodulatory drug inhibiting the store-operated calcium release-activated calcium channel of lymphocytes to control transplant rejection without nephrotoxicity.

Methods: Animals underwent kidney transplantation and were treated with tacrolimus alone (n = 3), a CRACM1 inhibitor (PRCL-02) (n = 6) alone, or with initial tacrolimus monotherapy followed by gradual conversion at 3 weeks to PRCL-02 alone (n = 3). PRCL-02 was administered via a surgically inserted gastrostomy tube BID.

Results: Dose-related drug exposure in monkeys was established and renal transplants were then performed using PRCL-02 monotherapy. Oral dosing of PRCL-02 was well tolerated and resulted in suppressed T-cell proliferation in in vitro MLR comparable to animals in the tacrolimus control arm. Animals receiving tacrolimus monotherapy were e on day 100 without rejection. PRCL-02 monotherapy only marginally prolonged graft survival (MST = 13.16 d; group 2) compared with untreated controls. Animals treated initially with tacrolimus and converted to PRCL-02 monotherapy had a mean graft survival of 35.3 days which was prolonged compared with PRCL-02 monotherapy but not compared with the tacrolimus-treated group. Pharmacokinetic studies showed inconsistent drug exposures despite attempts to adjust dose and exposure which may have contributed to the rejections.

Conclusions: We conclude that, in this nonhuman primate model of kidney transplantation, PRCL-02 demonstrated evidence of in vivo immunosuppressive activity but was inferior to tacrolimus treatment with respect to suppressing immune transplant rejection.
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http://dx.doi.org/10.1097/TP.0000000000003078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7182068PMC
May 2020

Western diet-induced increase in colonic bile acids compromises epithelial barrier in nonalcoholic steatohepatitis.

FASEB J 2020 05 10;34(5):7089-7102. Epub 2020 Apr 10.

Division of Experimental Pathology, Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA.

There is compelling evidence implicating intestinal permeability in the pathogenesis of nonalcoholic steatohepatitis (NASH), but the underlying mechanisms remain poorly understood. Here we examined the role of bile acids (BA) in western diet (WD)-induced loss of colonic epithelial barrier (CEB) function in mice with a genetic impairment in intestinal epithelial barrier function, junctional adhesion molecule A knockout mice, F11r . WD-fed knockout mice developed severe NASH, which was associated with increased BA concentration in the cecum and loss of CEB function. Analysis of cecal BA composition revealed selective increases in primary unconjugated BAs in the WD-fed mice, which correlated with increased abundance of microbial taxa linked to BA metabolism. In vitro permeability assays revealed that chenodeoxycholic acid (CDCA), which was elevated in the cecum of WD-fed mice, increased paracellular permeability, while the BA-binding resin sevelamer hydrochloride protected against CDCA-induced loss of barrier function. Sequestration of intestinal BAs by in vivo delivery of sevelamer to WD-fed knockout mice attenuated colonic mucosal inflammation and improved CEB. Sevelamer also reduced hepatic inflammation and fibrosis, and improved metabolic derangements associated with NASH. Collectively, these findings highlight a hitherto unappreciated role for BAs in WD-induced impairment of the intestinal epithelial barrier in NASH.
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http://dx.doi.org/10.1096/fj.201902687RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7831197PMC
May 2020

Banff Digital Pathology Working Group: Going digital in transplant pathology.

Am J Transplant 2020 09 19;20(9):2392-2399. Epub 2020 Apr 19.

University of Alberta, Edmonton, Canada.

The Banff Digital Pathology Working Group (DPWG) was formed in the time leading up to and during the joint American Society for Histocompatibility and Immunogenetics/Banff Meeting, September 23-27, 2019, held in Pittsburgh, Pennsylvania. At the meeting, the 14th Banff Conference, presentations directly and peripherally related to the topic of "digital pathology" were presented; and discussions before, during, and after the meeting have resulted in a list of issues to address for the DPWG. Included are practice standardization, integrative approaches for study classification, scoring of histologic parameters (eg, interstitial fibrosis and tubular atrophy and inflammation), algorithm classification, and precision diagnosis (eg, molecular pathways and therapeutics). Since the meeting, a survey with international participation of mostly pathologists (81%) was conducted, showing that whole slide imaging is available at the majority of centers (71%) but that artificial intelligence (AI)/machine learning was only used in ≈12% of centers, with a wide variety of programs/algorithms employed. Digitalization is not just an end in itself. It also is a necessary precondition for AI and other approaches. Discussions at the meeting and the survey highlight the unmet need for a Banff DPWG and point the way toward future contributions that can be made.
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http://dx.doi.org/10.1111/ajt.15850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496838PMC
September 2020

Decreased Hepatocyte Autophagy Leads to Synergistic IL-1β and TNF Mouse Liver Injury and Inflammation.

Hepatology 2020 Aug 23;72(2):595-608. Epub 2020 Jun 23.

Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, GA.

Background And Aims: The proinflammatory cytokine IL-1β has been implicated in the pathophysiology of nonalcoholic and alcoholic steatohepatitis. How IL-1β promotes liver injury in these diseases is unclear, as no IL-1β receptor-linked death pathway has been identified. Autophagy functions in hepatocyte resistance to injury and death, and findings of decreased hepatic autophagy in many liver diseases suggest a role for impaired autophagy in disease pathogenesis. Recent findings that autophagy blocks mouse liver injury from lipopolysaccharide led to an examination of autophagy's function in hepatotoxicity from proinflammatory cytokines.

Approach And Results: AML12 cells with decreased autophagy from a lentiviral autophagy-related 5 (Atg5) knockdown were resistant to toxicity from TNF, but sensitized to death from IL-1β, which was markedly amplified by TNF co-treatment. IL-1β/TNF death was necrosis by trypan blue and propidium iodide positivity, absence of mitochondrial death pathway and caspase activation, and failure of a caspase inhibitor or necrostatin-1s to prevent death. IL-1β/TNF depleted autophagy-deficient cells of ATP, and ATP depletion and cell death were prevented by supplementation with the energy substrate pyruvate or oleate. Pharmacological inhibitors and genetic knockdown studies demonstrated that IL-1β/TNF-induced necrosis resulted from lysosomal permeabilization and release of cathepsins B and L in autophagy-deficient cells. Mice with a tamoxifen-inducible, hepatocyte-specific Atg5 knockout were similarly sensitized to cathepsin-dependent hepatocellular injury and death from IL-1β/TNF in combination, but neither IL-1β nor TNF alone. Knockout mice had increased hepatic inflammation, and IL-1β/TNF-treated, autophagy-deficient AML12 cells secreted exosomes with proinflammatory damage-associated molecular patterns.

Conclusions: The findings delineate mechanisms by which decreased hepatocyte autophagy promotes IL-1β/TNF-induced necrosis from impaired energy homeostasis and lysosomal permeabilization and inflammation through the secretion of exosomal damage-associated molecular patterns.
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http://dx.doi.org/10.1002/hep.31209DOI Listing
August 2020

Association Between Molecular Subtypes of Colorectal Tumors and Patient Survival, Based on Pooled Analysis of 7 International Studies.

Gastroenterology 2020 06 20;158(8):2158-2168.e4. Epub 2020 Feb 20.

Epidemiology Department, University of Washington, Seattle, Washington; Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington.

Background And Aims: The heterogeneity among colorectal tumors is probably due to differences in developmental pathways and might associate with patient survival times. We studied the relationship among markers of different subtypes of colorectal tumors and patient survival.

Methods: We pooled data from 7 observational studies, comprising 5010 patients with colorectal cancer. All the studies collected information on microsatellite instability (MSI), CpG island methylator phenotype (CIMP), and mutations in KRAS and BRAF in tumors. Tumors with complete marker data were classified as type 1 (MSI-high, CIMP-positive, with pathogenic mutations in BRAF but not KRAS), type 2 (not MSI-high, CIMP-positive, with pathogenic mutations in BRAF but not KRAS), type 3 (not MSI-high or CIMP, with pathogenic mutations in KRAS but not BRAF), type 4 (not MSI-high or CIMP, no pathogenic mutations in BRAF or KRAS), or type 5 (MSI-high, no CIMP, no pathogenic mutations in BRAF or KRAS). We used Cox regression to estimate hazard ratios (HR) and 95% confidence intervals (CIs) for associations of these subtypes and tumor markers with disease-specific survival (DSS) and overall survival times, adjusting for age, sex, stage at diagnosis, and study population.

Results: Patients with type 2 colorectal tumors had significantly shorter time of DSS than patients with type 4 tumors (HR 1.66; 95% CI 1.33-2.07), regardless of sex, age, or stage at diagnosis. Patients without MSI-high tumors had significantly shorter time of DSS compared with patients with MSI-high tumors (HR 0.42; 95% CI 0.27-0.64), regardless of other tumor markers or stage, or patient sex or age.

Conclusions: In a pooled analysis of data from 7 observational studies of patients with colorectal cancer, we found that tumor subtypes, defined by combinations of 4 common tumor markers, were associated with differences in survival time. Colorectal tumor subtypes might therefore be used in determining patients' prognoses.
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http://dx.doi.org/10.1053/j.gastro.2020.02.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7282955PMC
June 2020

Stathmin 1 Induces Murine Hepatocyte Proliferation and Increased Liver Mass.

Hepatol Commun 2020 Jan 7;4(1):38-49. Epub 2019 Nov 7.

Division of Digestive Diseases Department of Medicine Emory University School of Medicine Atlanta GA.

The endogenous cellular signals that initiate the transition of hepatocytes from quiescence to proliferation remain unclear. The protein stathmin 1 (STMN1) is highly expressed in dividing cells, including hepatocytes, and functions to promote cell mitosis through physical interactions with tubulin and microtubules that regulate mitotic spindle formation. The recent finding that STMN1 mediates the resistance of cultured hepatocytes to oxidant stress led to an examination of the expression and function of this protein in the liver . STMN1 messenger RNA (mRNA) and protein were essentially undetectable in normal mouse liver but increased markedly in response to oxidant injury from carbon tetrachloride. Similarly, levels of STMN1 mRNA and protein were increased in human livers from patients with acute fulminant hepatic failure. To determine STMN1 function in the liver , mice were infected with a control or -expressing adenovirus. expression induced spontaneous liver enlargement with a doubling of the liver to body weight ratio. The increase in liver mass resulted, in part, from hepatocellular hypertrophy but mainly from an induction of hepatocyte proliferation. STMN1 expression led to marked increases in the numbers of 5-bromo-2'-deoxyuridine-positive and mitotic hepatocytes and hepatic nuclear levels of cyclins and cyclin-dependent kinases. STMN1-induced hepatocyte proliferation was followed by an apoptotic response and a return of the liver to its normal mass. STMN1 promotes entry of quiescent hepatocytes into the cell cycle. STMN1 expression by itself in the absence of any reduction in liver mass is sufficient to stimulate a hepatic proliferative response that significantly increases liver mass.
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http://dx.doi.org/10.1002/hep4.1447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6939544PMC
January 2020

Pretransplant Desensitization with Costimulation Blockade and Proteasome Inhibitor Reduces DSA and Delays Antibody-Mediated Rejection in Highly Sensitized Nonhuman Primate Kidney Transplant Recipients.

J Am Soc Nephrol 2019 12 28;30(12):2399-2411. Epub 2019 Oct 28.

Department of Surgery, Duke Transplant Center,

Background: Patients with broad HLA sensitization have poor access to donor organs, high mortality while waiting for kidney transplant, and inferior graft survival. Although desensitization strategies permit transplantation lowering of donor-specific antibodies, the B cell-response axis from germinal center activation to plasma cell differentiation remains intact.

Methods: To investigate targeting the germinal center response and plasma cells as a desensitization strategy, we sensitized maximally MHC-mismatched rhesus pairs with two sequential skin transplants. We administered a proteasome inhibitor (carfilzomib) and costimulation blockade agent (belatacept) to six animals weekly for 1 month; four controls received no treatment. We analyzed blood, lymph node, bone marrow cells, and serum before desensitization, after desensitization, and after kidney transplantation.

Results: The group receiving carfilzomib and belatacept exhibited significantly reduced levels of donor-specific antibodies (=0.05) and bone marrow plasma cells (=0.02) compared with controls, with a trend toward reduced lymph node T follicular helper cells (=0.06). Compared with controls, carfilzomib- and belatacept-treated animals had significantly prolonged graft survival (=0.02), and renal biopsy at 1 month showed significantly reduced antibody-mediated rejection scores (=0.02). However, four of five animals with long-term graft survival showed gradual rebound of donor-specific antibodies and antibody-mediated rejection.

Conclusions: Desensitization using proteasome inhibition and costimulation blockade reduces bone marrow plasma cells, disorganizes germinal center responses, reduces donor-specific antibody levels, and prolongs allograft survival in highly sensitized nonhuman primates. Most animals experienced antibody-mediated rejection with humoral-response rebound, suggesting desensitization must be maintained after transplantation using ongoing suppression of the B cell response.
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http://dx.doi.org/10.1681/ASN.2019030304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900797PMC
December 2019

Daratumumab in Sensitized Kidney Transplantation: Potentials and Limitations of Experimental and Clinical Use.

J Am Soc Nephrol 2019 07 21;30(7):1206-1219. Epub 2019 Jun 21.

Nephrology and Transplantation Department, Cancerology-Immunity-Transplantation-Infectiology, Clinical Investigation Center-Biotherapies, Henri Mondor Hospital, Assistance Publique-Hôpitaux de Paris, INSERM U955, Paris-Est-Créteil University, Paris, France;

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.
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http://dx.doi.org/10.1681/ASN.2018121254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6622431PMC
July 2019

LIVER STEATOSIS SEGMENTATION WITH DEEP LEARNING METHODS.

Proc IEEE Int Symp Biomed Imaging 2019 Apr 11;2019:24-27. Epub 2019 Jul 11.

Department of Mathematics and Statistics, Georgia State University, Atlanta, GA, 30303, USA.

Liver steatosis is known as the abnormal accumulation of lipids within cells. An accurate quantification of steatosis area within the liver histopathological microscopy images plays an important role in liver disease diagnosis and transplantation assessment. Such a quantification analysis often requires a precise steatosis segmentation that is challenging due to abundant presence of highly overlapped steatosis droplets. In this paper, a deep learning model Mask-RCNN is used to segment the steatosis droplets in clumps. Extended from Faster R-CNN, Mask-RCNN can predict object masks in addition to bounding box detection. With transfer learning, the resulting model is able to segment overlapped steatosis regions at 75.87% by Average Precision, 60.66% by Recall,65.88% by F1-score, and 76.97% by Jaccard index, promising to support liver disease diagnosis and allograft rejection prediction in future clinical practice.
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http://dx.doi.org/10.1109/isbi.2019.8759600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363395PMC
April 2019

Long-term survival of pig-to-rhesus macaque renal xenografts is dependent on CD4 T cell depletion.

Am J Transplant 2019 08 5;19(8):2174-2185. Epub 2019 Apr 5.

Emory Transplant Center, Department of Surgery, School of Medicine, Emory University, Atlanta, Georgia.

The shortage of available organs remains the greatest barrier to expanding access to transplant. Despite advances in genetic editing and immunosuppression, survival in experimental models of kidney xenotransplant has generally been limited to <100 days. We found that pretransplant selection of recipients with low titers of anti-pig antibodies significantly improved survival in a pig-to-rhesus macaque kidney transplant model (6 days vs median survival time 235 days). Immunosuppression included transient pan-T cell depletion and an anti-CD154-based maintenance regimen. Selective depletion of CD4 T cells but not CD8 T cells resulted in long-term survival (median survival time >400 days vs 6 days). These studies suggested that CD4 T cells may have a more prominent role in xenograft rejection compared with CD8 T cells. Although animals that received selective depletion of CD8 T cells showed signs of early cellular rejection (marked CD4 infiltrates), animals receiving selective CD4 depletion exhibited normal biopsy results until late, when signs of chronic antibody rejection were present. In vitro study results suggested that rhesus CD4 T cells required the presence of SLA class II to mount an effective proliferative response. The combination of low pretransplant anti-pig antibody and CD4 depletion resulted in consistent, long-term xenograft survival.
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http://dx.doi.org/10.1111/ajt.15329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658347PMC
August 2019

IL-21 Biased Alemtuzumab Induced Chronic Antibody-Mediated Rejection Is Reversed by LFA-1 Costimulation Blockade.

Front Immunol 2018 15;9:2323. Epub 2018 Oct 15.

Department of Surgery, Duke Transplant Center, Duke University Medical Center, Durham, NC, United States.

Despite its excellent efficacy in controlling T cell mediated acute rejection, lymphocyte depletion may promote a humoral response. While T cell repopulation after depletion has been evaluated in many aspects, the B cell response has not been fully elucidated. We tested the hypothesis that the mechanisms also involve skewed T helper phenotype after lymphocytic depletion. Post-transplant immune response was measured from alemtuzumab treated hCD52Tg cardiac allograft recipients with or without anti-LFA-1 mAb. Alemtuzumab induction promoted serum DSA, allo-B cells, and CAV in humanized CD52 transgenic (hCD52Tg) mice after heterotopic heart transplantation. Additional anti-LFA-1 mAb treatment resulted in reduced DSA (Fold increase 4.75 ± 6.9 vs. 0.7 ± 0.5; < 0.01), allo-specific B cells (0.07 ± 0.06 vs. 0.006 ± 0.002 %; < 0.01), neo-intimal hyperplasia (56 ± 14% vs. 23 ± 13%; < 0.05), arterial disease (77.8 ± 14.2 vs. 25.8 ± 20.1%; < 0.05), and fibrosis (15 ± 23.3 vs. 4.3 ± 1.65%; < 0.05) in this alemtuzumab-induced chronic antibody-mediated rejection (CAMR) model. Surprisingly, elevated serum IL-21 levels in alemtuzumab-treated mice was reduced with LFA-1 blockade. In accordance with the increased serum IL-21 level, alemtuzumab treated mice showed hyperplastic germinal center (GC) development, while the supplemental anti-LFA-1 mAb significantly reduced the GC frequency and size. We report that the incomplete T cell depletion inside of the GC leads to a systemic IL-21 dominant milieu with hyperplastic GC formation and CAMR. Conventional immunosuppression, such as tacrolimus and rapamycin, failed to reverse AMR, while co-stimulation blockade with LFA-1 corrected the GC hyperplastic response. The identification of IL-21 driven chronic AMR elucidates a novel mechanism that suggests a therapeutic approach with cytolytic induction.
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http://dx.doi.org/10.3389/fimmu.2018.02323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6196291PMC
September 2019

Dual targeting: Combining costimulation blockade and bortezomib to permit kidney transplantation in sensitized recipients.

Am J Transplant 2019 03 17;19(3):724-736. Epub 2018 Sep 17.

Emory Transplant Center, Department of Surgery, Emory School of Medicine, Atlanta, GA, USA.

Previous evidence suggests that a homeostatic germinal center (GC) response may limit bortezomib desensitization therapy. We evaluated the combination of costimulation blockade with bortezomib in a sensitized non-human primate kidney transplant model. Sensitized animals were treated with bortezomib, belatacept, and anti-CD40 mAb twice weekly for a month (n = 6) and compared to control animals (n = 7). Desensitization therapy-mediated DSA reductions approached statistical significance (P = .07) and significantly diminished bone marrow PCs, lymph node follicular helper T cells, and memory B cell proliferation. Graft survival was prolonged in the desensitization group (P = .073). All control animals (n = 6) experienced graft loss due to antibody-mediated rejection (AMR) after kidney transplantation, compared to one desensitized animal (1/5). Overall, histological AMR scores were significantly lower in the treatment group (n = 5) compared to control (P = .020). However, CMV disease was common in the desensitized group (3/5). Desensitized animals were sacrificed after long-term follow-up with functioning grafts. Dual targeting of both plasma cells and upstream GC responses successfully prolongs graft survival in a sensitized NHP model despite significant infectious complications and drug toxicity. Further work is planned to dissect underlying mechanisms, and explore safety concerns.
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http://dx.doi.org/10.1111/ajt.15067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7185755PMC
March 2019

Murine Pancreatic Cancer Alters T Cell Activation and Apoptosis and Worsens Survival After Cecal Ligation and Puncture.

Shock 2019 06;51(6):731-739

Department of Surgery and Emory Critical Care Center, Emory University School of Medicine, Atlanta, Georgia.

Patients with cancer who develop sepsis have a markedly higher mortality than patients who were healthy prior to the onset of sepsis. Potential mechanisms underlying this difference have previously been examined in two preclinical models of cancer followed by sepsis. Both pancreatic cancer/pneumonia and lung cancer/cecal ligation and puncture (CLP) increase murine mortality, associated with alterations in lymphocyte apoptosis and intestinal integrity. However, pancreatic cancer/pneumonia decreases lymphocyte apoptosis and increases gut apoptosis while lung cancer/CLP increases lymphocyte apoptosis and decreases intestinal proliferation. These results cannot distinguish the individual roles of cancer versus sepsis since different models of each were used. We therefore created a new cancer/sepsis model to standardize each variable. Mice were injected with a pancreatic cancer cell line and 3 weeks later cancer mice and healthy mice were subjected to CLP. Cancer septic mice had a significantly higher 10-day mortality than previously healthy septic mice. Cancer septic mice had increased CD4 T cells and CD8 T cells, associated with decreased CD4 T cell apoptosis 24 h after CLP. Further, splenic CD8+ T cell activation was decreased in cancer septic mice. In contrast, no differences were noted in intestinal apoptosis, proliferation, or permeability, nor were changes noted in local bacterial burden, renal, liver, or pulmonary injury. Cancer septic mice thus have consistently reduced survival compared with previously healthy septic mice, independent of the cancer or sepsis model utilized. Changes in lymphocyte apoptosis are common to cancer model and independent of sepsis model, whereas gut apoptosis is common to sepsis model and independent of cancer model. The host response to the combination of cancer and sepsis is dependent, at least in part, on both chronic comorbidity and acute illness.
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http://dx.doi.org/10.1097/SHK.0000000000001203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309343PMC
June 2019

Successful desensitization with proteasome inhibition and costimulation blockade in sensitized nonhuman primates.

Blood Adv 2017 Nov 26;1(24):2115-2119. Epub 2017 Oct 26.

Duke Transplant Center, Department of Surgery, Duke University Medical Center, Durham, NC.

The detrimental effects of donor-directed antibodies in sensitized transplant patients remain a difficult immunologic barrier to successful organ transplantation. Antibody removal is often followed by rebound. Proteasome inhibitors (PIs) deplete antibody-producing plasma cells (PCs) but have shown marginal benefit for desensitization. In an allosensitized nonhuman primate (NHP) model, we observed increased germinal center (GC) formation after PI monotherapy, suggesting a compensatory PC repopulation mediated via GC activation. Here we show that costimulation blockade (CoB) targets GC follicular helper T (Tfh) cells in allosensitized NHPs. Combined PI and CoB significantly reduces bone marrow PCs (CD19CD20CD38), Tfh cells (CD4ICOSPD-1), and GC B cells (BCL-6CD20); controls the homeostatic GC response to PC depletion; and sustains alloantibody decline. Importantly, dual PC and CoB therapy prolongs rejection-free graft survival in major histocompatibility complex incompatible kidney transplantation without alloantibody rebound. Our study illustrates a translatable desensitization method and provides mechanistic insight into maintenance of alloantibody sensitization.
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http://dx.doi.org/10.1182/bloodadvances.2017010991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737135PMC
November 2017

Development of CD3 cell quantitation algorithms for renal allograft biopsy rejection assessment utilizing open source image analysis software.

Virchows Arch 2018 Feb 8;472(2):259-269. Epub 2017 Nov 8.

Department of Pathology, Emory University, Atlanta, GA, USA.

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 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). 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.
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http://dx.doi.org/10.1007/s00428-017-2260-6DOI Listing
February 2018

Honokiol Increases CD4+ T Cell Activation and Decreases TNF but Fails to Improve Survival Following Sepsis.

Shock 2018 08;50(2):178-186

Department of Surgery and Emory Critical Care Center, Emory University School of Medicine, Atlanta, Georgia.

Honokiol is a biphenolic isolate extracted from the bark of the magnolia tree that has been used in traditional Chinese and Japanese medicine, and has more recently been investigated for its anti-inflammatory and antibacterial properties. Honokiol has previously been demonstrated to improve survival in sepsis models that have rapid 100% lethality. The purpose of this study was to determine the impact of Honokiol on the host response in a model of sepsis that more closely approximates human disease. Male and female C57BL/6 mice underwent cecal ligation and puncture to induce polymicrobial intra-abdominal sepsis. Mice were then randomized to receive an injection of either Honokiol (120 mg/kg/day) or vehicle and were sacrificed after 24 h for functional studies or followed 7 days for survival. Honokiol treatment after sepsis increased the frequency of CD4 T cells and increased activation of CD4 T cells as measured by the activation marker CD69. Honokiol also increased splenic dendritic cells. Honokiol simultaneously decreased frequency and number of CD8 T cells. Honokiol decreased systemic tumor necrosis factor without impacting other systemic cytokines. Honokiol did not have a detectable effect on kidney function, lung physiology, liver function, or intestinal integrity. In contrast to prior studies of Honokiol in a lethal model of sepsis, Honokiol did not alter survival at 7 days (70% mortality for Honokiol vs. 60% mortality for vehicle). Honokiol is thus effective in modulating the host immune response and inflammation following a clinically relevant model of sepsis but is not sufficient to alter survival.
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http://dx.doi.org/10.1097/SHK.0000000000001021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5895534PMC
August 2018

Alloimmunity But Not Viral Immunity Promotes Allograft Loss in a Mouse Model of Polyomavirus-Associated Allograft Injury.

Transplant Direct 2017 Jun 12;3(6):e161. Epub 2017 May 12.

Emory University School of Medicine, Emory Transplant Center, Atlanta, GA.

Background: The interplay between viral infection and alloimmunity is known to influence the fate of transplanted organs. Clarifying how local virus-associated inflammation/injury and antiviral immunity can alter host alloimmune responses in transplantation remains a critical question.

Methods: We used a mouse model of polyomavirus (PyV) infection and kidney transplantation to investigate the roles of direct viral pathology, the antiviral immune response, and alloimmunity in the pathogenesis of PyV-associated allograft injury. We have previously shown that an effective primary T cell response is required in PyV-associated graft injury.

Results: Here we show that the transfer of primed antidonor, but not antiviral, T cells results in PyV-associated allograft injury. In further studies, we use a surrogate minor antigen model (ovalbumin) and show that only antidonor specific T cells and not antiviral specific T cells are sufficient to mediate injury. Lastly, we demonstrate that local but not systemic virus-mediated inflammation and injury within the graft itself are required.

Conclusions: These data suggest that in this mouse model, the predominant mechanism of allograft injury in PyV-associated injury is due to an augmented alloimmune T cell response driven by virus-induced inflammation/injury within the graft. These studies highlight the important interplay between viral infection and alloimmunity in a model system.
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http://dx.doi.org/10.1097/TXD.0000000000000677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5464780PMC
June 2017

Myosin light chain kinase knockout improves gut barrier function and confers a survival advantage in polymicrobial sepsis.

Mol Med 2017 08 7;23:155-165. Epub 2017 Jun 7.

Department of Surgery and Emory Critical Care Center, Emory University School of Medicine, Atlanta, GA.

Sepsis-induced intestinal hyperpermeability is mediated by disruption of the epithelial tight junction, which is closely associated with the peri-junctional actin-myosin ring. Myosin light chain kinase (MLCK) phosphorylates the myosin regulatory light chain, resulting in increased permeability. The purpose of this study was to determine whether genetic deletion of MLCK would alter gut barrier function and survival from sepsis. MLCK and wild type (WT) mice were subjected to cecal ligation and puncture and assayed for both survival and mechanistic studies. Survival was significantly increased in MLCK mice (95% vs. 24%, p<0.0001). Intestinal permeability increased in septic WT mice compared to unmanipulated mice. In contrast, permeability in septic MLCK mice was similar to that seen in unmanipulated animals. Improved gut barrier function in MLCK mice was associated with increases in the tight junction mediators ZO-1 and claudin 15 without alterations in claudin 1, 2, 3, 4, 5, 7, 8, 13, occludin or JAM-A. Other components of intestinal integrity (apoptosis, proliferation and villus length) were unaffected by MLCK deletion as were local peritoneal inflammation and distant lung injury. Systemic IL-10 was decreased greater than 10-fold in MLCK mice; however, survival was similar between septic MLCK mice given exogenous IL-10 or vehicle. These data demonstrate that deletion of MLCK improves survival following sepsis, associated with normalization of intestinal permeability and selected tight junction proteins.
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http://dx.doi.org/10.2119/molmed.2016.00256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568914PMC
August 2017

Establishment of human metastatic colorectal cancer model in rabbit liver: A pilot study.

PLoS One 2017 5;12(5):e0177212. Epub 2017 May 5.

Division of Interventional Radiology, Department of Diagnostic Radiology, Yale University School of Medicine, New Haven, CT, United States of America.

Rationale And Objectives: To develop a human metastatic colorectal cancer (mCRC) model in a rabbit liver.

Materials And Methods: Immunosuppression in 4 adult New Zealand White rabbits weighing 3.5 to 4.5 kg was induced with daily subcutaneous injection of 15 mg/kg Cyclosporine A (CsA). On day 3 open mini-laparotomy was performed and 0.2 ml (1.8x105 cells) suspension of HCT-116 and HT-29 human CRC cells were injected into the left and right medial lobe respectively. On day 10 the CsA dose was reduced to 10 mg/kg daily maintenance dose. Rabbits were weighed weekly, closely monitored for CsA side effects (weight loss, gingival hyperplasia and gut modification). Rabbits were sacrificed 5, 6, 7, and 8 weeks after cells injection. Liver tumors were collected for histopathology and immunohistochemical analysis.

Results: HT-29 Tumor growth was observed in 3 rabbits (75%). Tumors measured 3, 4 and 6 mm after 5, 6 and 8 weeks respectively. Microscopically, tumors contained hyperchromatic, pleomorphic cells that stained for monoclonal carcinoembryonic antigen (CEA), polyclonal CEA, cytokeratin 20, vascular markers (CD31, CD34), and vascular endothelial growth factor (VEGF) by immunohistochemistry, supporting involvement by the poorly differentiated HT-29 colorectal cancer cell line. No gross tumor growth or microscopic viability was observed from HCT-116 cell injection. CsA extra-hepatic manifestations included minimal gum hyperplasia and decrease in gut motility in 3 rabbits (75%), which was treated with Azithromycin 15 mg/kg and Cisapride 0.5 mg/kg every 12 hours, respectively.

Conclusion: We successfully developed a human metastatic colon cancer model in immunosuppressed rabbit liver using HT-29 cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0177212PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5419650PMC
September 2017