Involvement of eNAMPT/TLR4 inflammatory signaling in progression of non-alcoholic fatty liver disease, steatohepatitis, and fibrosis.

Although the progression of non-alcoholic fatty liver disease (NAFLD) from steatosis to steatohepatitis (NASH) and cirrhosis remains poorly understood, a critical role for dysregulated innate immunity has emerged. We examined the utility of ALT-100, a monoclonal antibody (mAb), in reducing NAFLD severity and progression to NASH/hepatic fibrosis. ALT-100 neutralizes eNAMPT (extracellular nicotinamide phosphoribosyltransferase), a novel damage-associated molecular pattern protein (DAMP) and Toll-like receptor 4 (TLR4) ligand. Histologic and biochemical markers were measured in liver tissues and plasma from human NAFLD subjects and NAFLD mice (streptozotocin/high-fat diet-STZ/HFD, 12 weeks). Human NAFLD subjects (n = 5) exhibited significantly increased NAMPT hepatic expression and significantly elevated plasma levels of eNAMPT, IL-6, Ang-2, and IL-1RA compared to healthy controls, with IL-6 and Ang-2 levels significantly increased in NASH non-survivors. Untreated STZ/HFD-exposed mice displayed significant increases in NAFLD activity scores, liver triglycerides, NAMPT hepatic expression, plasma cytokine levels (eNAMPT, IL-6, and TNFα), and histologic evidence of hepatocyte ballooning and hepatic fibrosis. Mice receiving the eNAMPT-neutralizing ALT-100 mAb (0.4 mg/kg/week, IP, weeks 9 to 12) exhibited marked attenuation of each index of NASH progression/severity. Thus, activation of the eNAMPT/TLR4 inflammatory pathway contributes to NAFLD severity and NASH/hepatic fibrosis. ALT-100 is potentially an effective therapeutic approach to address this unmet NAFLD need.

Immunotherapy in treatment of metastatic prostate cancer: An approach to circumvent immunosuppressive tumor microenvironment.

Prostate cancer is the second most common cause of cancer-related death in men in the United States and the fifth worldwide. Most prostate cancer arises as an androgen-dependent tumor but eventually progresses into castration-resistance prostate cancer, incurable by the current androgen deprivation therapy and chemotherapy. The development of immunotherapy in cancer treatment has brought an exciting era of antiprostate cancer therapy through antitumor immune responses. Prostate cancer is recognized as a poorly immunogenic tissue with immunological ignorance showing low levels of antigen-presenting process and cytotoxic T-cell activation, high levels of immune checkpoint molecules and immunosuppressive cytokines/chemokines, and recruitment of immunosuppressive cells. Immunotherapies for prostate cancer have been developed to activate the innate and adaptive immune responses, such as vaccines and adoptive CAR-T cells, or to inhibit immunosuppressive molecules, such as immune checkpoint inhibitors or antibodies. The U.S Food and Drug Administration has approved Sipuleucel-T for the treatment of asymptomatic or minimally symptomatic metastatic castrate-resistant prostate cancer (mCRPC) and immune checkpoint inhibitor pembrolizumab for the treatment of all solid tumors, including prostate cancer, with impaired mismatch repair genes/microsatellite instability; however, the current clinical outcomes still need to be improved. As various immunosuppressive mechanisms coexist and cross-interact within the tumor microenvironment, different immunotherapy approaches may have to be combined and selected in a highly personalized way. It is hoped that this rapidly evolving field of immunotherapy will achieve successful treatment for mCRPC and will be applied to a wider range of prostate cancer patients.

Genetic and epigenetic regulation of the non-muscle myosin light chain kinase isoform by lung inflammatory factors and mechanical stress.

RATIONALE: The myosin light chain kinase gene, MYLK, encodes three proteins via unique promoters, including the non-muscle isoform of myosin light chain kinase (nmMLCK), a cytoskeletal protein centrally involved in regulation of vascular integrity. As MYLK coding SNPs are associated with severe inflammatory disorders (asthma, acute respiratory distress syndrome (ARDS)), we explored clinically relevant inflammatory stimuli and promoter SNPs in nmMLCK promoter regulation. METHODS: Full-length or serially deleted MYLK luciferase reporter promoter activities were measured in human lung endothelial cells (ECs). SNP-containing non-muscle MYLK (nmMYLK) DNA fragments were generated and nmMYLK promoter binding by transcription factors (TFs) detected by protein-DNA electrophoretic mobility shift assay (EMSA). Promoter demethylation was evaluated by 5-aza-2'-deoxycytidine (5-Aza). A preclinical mouse model of lipopolysaccharide (LPS)-induced acute lung injury (ALI) was utilized for nmMLCK validation. RESULTS: Lung EC levels of nmMLCK were significantly increased in LPS-challenged mice and LPS, tumor necrosis factor-α (TNF-α), 18% cyclic stretch (CS) and 5-Aza each significantly up-regulated EC nmMYLK promoter activities. EC exposure to FG-4592, a prolyl hydroxylase inhibitor that increases hypoxia-inducible factor (HIF) expression, increased nmMYLK promoter activity, confirmed by HIF1α/HIF2α silencing. nmMYLK promoter deletion studies identified distal inhibitory and proximal enhancing promoter regions as well as mechanical stretch-, LPS- and TNFα-inducible regions. Insertion of ARDS-associated SNPs (rs2700408, rs11714297) significantly increased nmMYLK promoter activity via increased transcription binding (glial cells missing homolog 1 (GCM1) and intestine-specific homeobox (ISX), respectively). Finally, the MYLK rs78755744 SNP (-261G/A), residing within a nmMYLK CpG island, significantly attenuated 5-Aza-induced promoter activity. CONCLUSION: These findings indicate nmMYLK transcriptional regulation by clinically relevant inflammatory factors and ARDS-associated nmMYLK promoter variants are consistent with nmMLCK as a therapeutic target in severe inflammatory disorders.

Submucosal lifting agent ORISE gel causes extensive foreign body granuloma post endoscopic resection.

BACKGROUND: Submucosal injection of lifting solution is often performed to facilitate endoscopic mucosal resection or endoscopic submucosal dissection. ORISETM gel is a synthetic solution recently approved by the US Food and Drug Administration (FDA) in 2018 for use as submucosal lifting solution for endoscopic resection procedures and has gained popularity for its convenient pre-filled syringe. However, here the present two cases show that ORISETM gel induces marked foreign body giant cell granulomatous reaction in the submucosa and muscularis propria following endoscopic resection. METHODS AND RESULTS: A 73-year-old female underwent laparoscopic right colectomy after her initial endoscopic polypectomy of a hyperplastic polyp, and a 78-year-old male had partial gastrectomy following endoscopic mucosal resection of an invasive adenocarcinoma. Both patients had submucosal injection of ORISETM gel during endoscopic procedures, and the surgical resection specimens showed ORISETM gel deposition and extensive foreign body giant cell granulomatous reaction in the submucosa and muscularis propria. CONCLUSION: These cases raise the awareness that ORISETM gel stimulates extensive foreign body reaction at the injection site, and the further consequence is still unclear.

Direct Extracellular NAMPT Involvement in Pulmonary Hypertension and Vascular Remodeling. Transcriptional Regulation by SOX and HIF-2α.

We previously demonstrated involvement of NAMPT (nicotinamide phosphoribosyltransferase) in pulmonary arterial hypertension (PAH) and now examine NAMPT regulation and extracellular NAMPT's (eNAMPT's) role in PAH vascular remodeling. NAMPT transcription and protein expression in human lung endothelial cells were assessed in response to PAH-relevant stimuli (PDGF [platelet-derived growth factor], VEGF [vascular endothelial growth factor], TGF-ß1 [transforming growth factor-ß1], and hypoxia). Endothelial-to-mesenchymal transition was detected by SNAI1 (snail family transcriptional repressor 1) and PECAM1 (platelet endothelial cell adhesion molecule 1) immunofluorescence. An eNAMPT-neutralizing polyclonal antibody was tested in a PAH model of monocrotaline challenge in rats. Plasma eNAMPT concentrations, significantly increased in patients with idiopathic pulmonary arterial hypertension, were highly correlated with indices of PAH severity. eNAMPT increased endothelial-to-mesenchymal transition, and each PAH stimulus significantly increased endothelial cell NAMPT promoter activity involving transcription factors STAT5 (signal transducer and activator of transcription 5), SOX18 (SRY-box transcription factor 18), and SOX17 (SRY-box transcription factor 17), a PAH candidate gene newly defined by genome-wide association study. The hypoxia-induced transcription factor HIF-2α (hypoxia-inducible factor-2α) also potently regulated NAMPT promoter activity, and HIF-2α binding sites were identified between -628 bp and -328 bp. The PHD2 (prolyl hydroxylase domain-containing protein 2) inhibitor FG-4592 significantly increased NAMPT promoter activity and protein expression in an HIF-2α-dependent manner. Finally, the eNAMPT-neutralizing polyclonal antibody significantly reduced monocrotaline-induced vascular remodeling, PAH hemodynamic alterations, and NF-κB activation. eNAMPT is a novel and attractive therapeutic target essential to PAH vascular remodeling.

Immune checkpoint inhibitor-related colitis in patients on immunotherapy for cancer.

OBJECTIVES: Immune checkpoint inhibitors, a revolutionary class of cancer immunotherapy drugs, have transformed cancer treatment by bolstering antitumor immunity for various advanced-stage solid cancers. The US Food and Drug Administration has approved 7 immune checkpoint inhibitors that target 3 major immune checkpoint proteins: cytotoxic T-lymphocyte-associated protein 4, programmed cell death 1 protein, and programmed cell death 1 ligand 1. In addition to their remarkable efficacy, however, these inhibitors have been observed causing immune-related adverse events, particularly immune checkpoint inhibitor-related colitis, which often results in severe or life-threatening clinical issues. METHODS: The diagnosis of immune checkpoint inhibitor-related colitis relies on incorporation of clinical evaluation as well as endoscopic and histopathologic examination, with exclusion of other potential etiologies. RESULTS: The common histopathologic manifestations of immune checkpoint inhibitor-related colitis are acute active colitis, chronic active colitis, microscopic colitis (collagenous or lymphocytic), and ischemic colitis, with patterns overlapping. Notably, enterocyte apoptosis is a unique feature of immune checkpoint inhibitor toxicity. The proposed mechanisms for the pathogenesis of immune checkpoint inhibitor-related colitis are primarily associated with autoimmune-type dysregulation and gut microbiome alteration. This review summarizes the clinical and pathologic characteristics of immune checkpoint inhibitor-related colitis and elucidates its underlying pathogenic mechanisms. CONCLUSIONS: Future successful management of this form of colitis relies on our comprehension of the intricate interplay between tumoral and systemic immune responses to immune checkpoint inhibitors and innovative approaches to modify these responses, along with specific immune cell populations, to preclude immune-related adverse events while achieving antitumor therapeutic outcomes.

Motionless volumetric structured light sheet microscopy.

To meet the increasing need for low-cost, compact imaging technology with cellular resolution, we have developed a microLED-based structured light sheet microscope for three-dimensional ex vivo and in vivo imaging of biological tissue in multiple modalities. All the illumination structure is generated directly at the microLED panel-which serves as the source-so light sheet scanning and modulation is completely digital, yielding a system that is simpler and less prone to error than previously reported methods. Volumetric images with optical sectioning are thus achieved in an inexpensive, compact form factor without any moving parts. We demonstrate the unique properties and general applicability of our technique by ex vivo imaging of porcine and murine tissue from the gastrointestinal tract, kidney, and brain.

Targeting SELPLG/P-selectin glycoprotein ligand 1 in preclinical ARDS: Genetic and epigenetic regulation of the SELPLG promoter.

We previously identified a missense single nucleotide polymorphism rs2228315 (G>A, Met62Ile) in the selectin-P-ligand gene (SELPLG), encoding P-selectin glycoprotein ligand 1 (PSGL-1), to be associated with increased susceptibility to acute respiratory distress syndrome (ARDS). These earlier studies demonstrated that SELPLG lung tissue expression was increased in mice exposed to lipopolysaccharide (LPS)- and ventilator-induced lung injury (VILI) suggesting that inflammatory and epigenetic factors regulate SELPLG promoter activity and transcription. In this report, we used a novel recombinant tandem PSGL1 immunoglobulin fusion molecule (TSGL-Ig), a competitive inhibitor of PSGL1/P-selectin interactions, to demonstrate significant TSGL-Ig-mediated decreases in SELPLG lung tissue expression as well as highly significant protection from LPS- and VILI-induced lung injury. In vitro studies examined the effects of key ARDS stimuli (LPS, 18% cyclic stretch to simulate VILI) on SELPLG promoter activity and showed LPS-mediated increases in SELPLG promoter activity and identified putative promoter regions associated with increased SELPLG expression. SELPLG promoter activity was strongly regulated by the key hypoxia-inducible transcription factors, HIF-1α, and HIF-2α as well as NRF2. Finally, the transcriptional regulation of SELPLG promoter by ARDS stimuli and the effect of DNA methylation on SELPLG expression in endothelial cell was confirmed. These findings indicate SELPLG transcriptional regulation by clinically-relevant inflammatory factors with the significant TSGL-Ig-mediated attenuation of LPS and VILI highly consistent with PSGL1/P-selectin as therapeutic targets in ARDS.

Current Microsatellite Instability Testing in Management of Colorectal Cancer.

Colorectal cancer (CRC) is the third most common cancer worldwide. In the past decade, mismatch repair deficiency (dMMR), manifested as microsatellite instability-high (MSI-H), has been recognized as a distinct mechanism promoting tumorigenesis in 15% of CRCs including 3% Lynch syndrome and 12% sporadic CRCs. As the molecular classifications of CRCs are continuously evolving, MSI-H CRCs appear to be the most homogeneous CRCs with distinct molecular, morphologic, and clinical features. MSI-H CRCs have dMMR causing MSI-H and genetic hypermutation but with diploid chromosomes. Morphologically, MSI-H CRCs appear as poorly differentiated or mucinous adenocarcinoma with characteristic lymphocytic infiltration. Most importantly, MSI-H CRCs have better stage-adjusted survival, do not respond well to standard 5-fluorouracil-based adjuvant chemotherapy, but do respond to immunotherapy. The United States Food and Drug Administration granted accelerated approval to immune checkpoint inhibitors, anti-programmed cell death protein-1 antibodies pembrolizumab and nivolumab, and the combination of nivolumab with anti-CTLA4 antibody ipilimumab for the second-line treatment of patients with stage IV MSI-H CRCs in 2017. There are still ongoing phase III clinical trials evaluating pembrolizumab and anti-programmed death-ligand 1 antibody atezolizumab as the first-line treatment in stage IV MSI-H CRCs and a phase I study on the combination of nivolumab and ipilimumab in patients with early stage CRC. These ongoing clinical studies on immunotherapy may lead to practice-changing results in the management of MSI-H CRCs. The National Comprehensive Cancer Network 2018 guidelines recommended MSI to be tested in all newly diagnosed CRCs. The MSI test will become increasingly vital in guiding adjuvant chemotherapy and immunotherapy in the management of CRCs.

Submucosal gastric heterotopia presenting as an upper esophageal nodule.

Esophageal gastric heterotopia (GH), the presence of differentiated gastric tissue in the esophagus, is estimated in up to 14% of populations worldwide and has always been reported on the surface of the esophagus, where it is also known as inlet patch. However, submucosal GH, in any tissue, is a rare finding. We report the case of a 50 year-old male presenting with chronic cough, heartburn and raspy vocalizations. Endoscopic examination showed a single 7 mm esophageal nodule, 20 cm from the incisors, interpreted as a submucosal mass. Pathologic examination of the endoscopically excised nodule showed well-differentiated gastric mucosa within the submucosa underneath the overlying squamous mucosa, consistent with submucosal GH. This case raises the awareness of an atypical presentation and location of GH seen as a submucosal mass on endoscopy.

A Humanized Monoclonal Antibody Targeting Extracellular Nicotinamide Phosphoribosyltransferase Prevents Aggressive Prostate Cancer Progression.

Prostate cancer (PCa) is the major cause of cancer-related death in males; however, effective treatments to prevent aggressive progression remain an unmet need. We have previously demonstrated that secreted extracellular nicotinamide phosphoribosyltransferase (eNAMPT) is a multifunctional innate immunity regulator that promotes PCa invasion. In the current study, we further investigate the therapeutic effects of an eNAMPT-neutralizing humanized monoclonal antibody (ALT-100 mAb) in preclinical PCa orthotopic xenograft models. We utilized human aggressive PCa cells (DU145 or PC3) for prostate implantation in SCID mice receiving weekly intraperitoneal injections of either ALT-100 mAb or IgG/PBS (control) for 12 weeks. Prostatic tumors and solid organs were examined for tumor growth, invasion, and metastasis and for biochemical and immunohistochemistry evidence of NFκB activation. ALT-100 mAb treatment significantly improved overall survival of SCID mice implanted with human PCa orthotopic prostate xenografts while inducing tumor necrosis, decreasing PCa proliferation and reducing local invasion and distal metastases. The ALT-100 mAb inhibits NFκB phosphorylation and signaling in PCa cells both in vitro and in vivo. This study demonstrates that eNAMPT neutralization effectively prevents human PCa aggressive progression in preclinical models, indicating its high potential to directly address the unmet need for an effective targeted therapy for patients with aggressive PCa.

Role of secreted extracellular nicotinamide phosphoribosyltransferase (eNAMPT) in prostate cancer progression: Novel biomarker and therapeutic target.

BACKGROUND: There remains a serious need to prevent the progression of invasive prostate cancer (PCa). We previously showed that secreted extracellular nicotinamide phosphoribosyltransferase (eNAMPT) is a multifunctional innate immunity regulator via TLR4 ligation which has been implicated in PCa progression. Here we investigate the role of eNAMPT as a diagnostic biomarker and therapeutic target in the progression of PCa. METHODS: Tumor NAMPT expression and plasma eNAMPT level were evaluated in human subjects with various PCa tumor stages and high risk subjects followed-up clinically for PCa. The genetic regulation of NAMPT expression in PCa cells and the role of eNAMPT in PCa invasion were investigated utilizing in vitro and in vivo models. FINDINGS: Marked NAMPT expression was detected in human extraprostatic-invasive PCa tissues compared to minimal expression of organ-confined PCa. Plasma eNAMPT levels were significantly elevated in PCa subjects compared to male controls, and significantly greater in subjects with extraprostatic-invasive PCa compared to subjects with organ-confined PCa. Plasma eNAMPT levels showed significant predictive value for diagnosing PCa. NAMPT expression and eNAMPT secretion were highly upregulated in human PCa cells in response to hypoxia-inducible factors and EGF. In vitro cell culture and in vivo preclinical mouse model studies confirmed eNAMPT-mediated enhancement of PCa invasiveness into muscle tissues and dramatic attenuation of PCa invasion by weekly treatment with an eNAMPT-neutralizing polyclonal antibody. INTERPRETATION: This study suggests that eNAMPT is a potential biomarker for PCa, especially invasive PCa. Neutralization of eNAMPT may be an effective therapeutic approach to prevent PCa invasion and progression.

Graft-Versus-Host Disease With Early Cytomegalovirus Infection in Gastrointestinal Tract Biopsies.

Gastrointestinal (GI) graft-versus-host disease (GVHD) and cytomegalovirus (CMV) infection often simulate each other. However, distinction between GVHD and CMV infection is critical in the management of immunosuppression for transplant recipients. This study retrospectively reviewed 16 patients diagnosed with GVHD from 2010 to 2016 and found 4 cases (25%) coinfected with CMV. Two cases were initially diagnosed as GVHD only but found to have CMV infection by serological testing within 3 days after immunosuppression treatment for GVHD. The remarkable histological feature of CMV infection appeared to be significant acute inflammation in addition to apoptotic epithelial injuries, and particularly in an early stage of CMV replication, acute inflammation is possibly the only detectable feature of CMV infection.

Metachronous bilateral testicular seminoma developing after an interval of 31 years: case report and review of the literature.

Men diagnosed with testicular germ cell tumors are at higher risk for development of a second germ cell tumor in the contralateral testis. Metachronous bilateral testicular germ cell tumors usually occur within 5 years. Here, we report a case of a 63-year-old man previously diagnosed with testicular seminoma and treated with a left orchiectomy followed by radiation, developing contralateral testicular seminoma after an interval of 31 years. The patient was asymptomatic and found to have an enlarged, nontender right testis on routine urological examination. Further workup did not reveal evidence of metastatic disease or lymphadenopathy. The surgery specimen revealed a 4.2 × 3.1 × 1.8 cm distinct mass without tumor involvement of tunica albuginea or the tunica vaginalis. Microscopy showed classic seminoma with venous/lymphatic tumor invasion. The current case underscores the importance of recommending lifelong follow-up for patients with testicular germ cell tumors.

Cystic nephroma/mixed epithelial stromal tumor: a benign neoplasm with potential for recurrence.

Cystic nephroma (CN) is a rare, benign, renal neoplasm composed of epithelial and stromal elements. Only about 200 cases have been reported since 1892 and recurrence has rarely been observed. We report a 32-year-old Hispanic woman, with a history of a right, complex cystic, renal mass treated by robotic decortication 2 years ago, who presented with flank pain, hematuria, and recurrent urinary tract infection. A magnetic resonance imaging study showed a 3.4-cm multicystic lesion with thickened septa and enhancement at the right kidney. The partial nephrectomy specimen revealed a well-circumscribed, multicystic tumor abutting the renal pelvis, with thick septa and smooth walls, filled with clear fluid. Microscopic examination showed variably sized cysts lined by cuboidal epithelium with focal hobnailing, without significant cytologic atypia and mitosis. The epithelial lining was positive for CK19, high molecular weight cytokeratin, and α-methylacyl-CoA racemase suggesting a primitive tubular epithelial phenotype. Primitive glomeruli-like structures were also present. The ovarian-like stroma was condensed around the cysts and was variably cellular with areas of muscle differentiation and thick-walled vessels. The stroma was positive for desmin, estrogen receptor, progesterone receptor, and CD10. We suggest that CN represents a variable mixture of epithelial and stromal elements, immature glomerular, tubular, muscle, and vascular elements, which may be present in variable proportions creating a spectrum of lesions previously described as CN and mixed epithelial and stromal tumors (MEST). This case emphasizes that CN/MEST clinically/radiologically mimics other cystic renal neoplasms, especially cystic renal cell carcinoma and tubulocystic carcinoma, necessitating histopathological examination and immunohistochemial studies for definitive diagnosis. Additionally, CN has the tendency to recur when not completely excised initially.