C-Type Lectin Receptor DCAR Recognizes Mycobacterial Phosphatidyl-Inositol Mannosides to Promote a Th1 Response during Infection.

Phosphatidyl-inositol mannosides (PIM) are glycolipids unique to mycobacteria and other related bacteria that stimulate host immune responses and are implicated in mycobacteria pathogenicity. Here, we found that the FcRγ-coupled C-type lectin receptor DCAR (dendritic cell immunoactivating receptor; gene symbol Clec4b1) is a direct receptor for PIM. Mycobacteria activated reporter cells expressing DCAR, and delipidation of mycobacteria abolished this activity. Acylated PIMs purified from mycobacteria were identified as ligands for DCAR. DCAR was predominantly expressed in small peritoneal macrophages and monocyte-derived inflammatory cells in lungs and spleen. These cells produced monocyte chemoattractant protein-1 (MCP-1) upon PIM treatment, and absence of DCAR or FcRγ abrogated MCP-1 production. Upon mycobacterial infection, Clec4b1-deficient mice showed reduced numbers of monocyte-derived inflammatory cells at the infection site, impaired IFNγ production by T cells, and an increased bacterial load. Thus, DCAR is a critical receptor for PIM that functions to promote T cell responses against mycobacteria.

Beclin1 is essential for the pancreas development.

Beclin1 (Becn1) is a multifunctional protein involved in autophagy regulation, membrane trafficking, and tumor suppression. In this study, we examined the roles of Becn1 in the pancreas development by generating mice with conditional deletion of Becn1 in the pancreas using pancreatic transcriptional factor 1a (Ptf1a)-Cre mice (Becn1f/f; Ptf1aCre/+). Surprisingly, loss of Becn1 in the pancreas resulted in severe pancreatic developmental defects, leading to insufficient exocrine and endocrine pancreatic function. Approximately half of Becn1f/f; Ptf1aCre/+ mice died immediately after birth. However, duodenum and neural tissue development were almost normal, indicating that pancreatic insufficiency was the cause of death. These findings demonstrated a novel role for Becn1 in pancreas morphogenesis, differentiation, and growth, and suggested that loss of this factor leaded to pancreatic agenesis at birth.

BAP1 depletion in human B-lymphoblast cells affects the production of innate immune cytokines and chemokines.

BRCA1 associated protein 1 (BAP1) is a ubiquitin C-terminal hydrolase that deubiquitinates histone H2AK119ub and other proteins and regulates the expression of multiple genes. The knockout of this tumor suppressor gene results in severe thymic atrophy, complete loss of the T cell lineage, and abnormal B cell development in mice. In the current study, we investigated in vitro effects of BAP1 knockout on cytokine and chemokine production using the human B-lymphoblast cell line TSCE5. We confirmed that knockout changed the production of innate immune-associated genes and their receptors. The CCL19, CCR7, CCL2, and CXCR5 genes associated with T and B cell migration were upregulated. Knockout cells producing high levels of CCL19 showed acceleration of actin polymerization, which is essential for cell migration. CD69, PTPRC, and TLR3 genes that activate inflammation were downregulated. The tumor necrosis factor ligand genes TNF, LTA, and TNFSF10 were downregulated by knockout. In knockout cells, TNFα production was strongly downregulated upon the addition of H2 O2 , but NF-κB in the basal condition and when TNFα was added was augmented, suggesting that these cells could respond to TNFα. These results indicated that BAP1 affects the expression of chemokines and cytokines, T and B cell migration, and activated inflammation associating with innate immunity.

Risk prediction for metastasis of clear cell renal cell carcinoma using digital multiplex ligation-dependent probe amplification.

Precise quantification of copy-number alterations (CNAs) in a tumor genome is difficult. We have applied a comprehensive copy-number analysis method, digital multiplex ligation-dependent probe amplification (digitalMLPA), for targeted gene copy-number analysis in clear cell renal cell carcinoma (ccRCC). Copy-number status of all chromosomal arms and 11 genes was determined in 60 ccRCC samples. Chromosome 3p loss and 5q gain, known as early changes in ccRCC development, as well as losses at 9p and 14q were detected in 56/60 (93.3%), 31/60 (51.7%), 11/60 (18.3%), and 33/60 (55%), respectively. Through gene expression analysis, a significant positive correlation was detected in terms of 14q loss determined using digitalMLPA and downregulation of mRNA expression ratios with HIF1A and L2HGDH (P = .0253 and .0117, respectively). Patients with early metastasis (<1 y) (n = 18) showed CNAs in 6 arms (in median), whereas metastasis-free patients (n = 34) showed those in significantly less arms (3 arms in median) (P = .0289). In particular, biallelic deletion of CDKN2A/2B was associated with multiple CNAs (≥7 arms) in 3 tumors. Together with sequence-level mutations in genes VHL, PBRM1, SETD2, and BAP1, we performed multiple correspondence analysis, which identified the association of 9p loss and 4q loss with early metastasis (both P < .05). This analysis indicated the association of 4p loss and 1p loss with poor survival (both, P < .05). These findings suggest that CNAs have essential roles in aggressiveness of ccRCC. We showed that our approach of measuring CNA through digitalMLPA will facilitate the selection of patients who may develop metastasis.

Double deficiency of cathepsin B and L in the mouse pancreas alters trypsin activity without affecting acute pancreatitis severity.

BACKGROUND: Premature intracellular trypsinogen activation has long been considered a key initiator of acute pancreatitis (AP). Cathepsin B (CTSB) activates trypsinogen, while cathepsin L (CTSL) inactivates trypsin(ogen), and both proteins play a role in the onset of AP. METHODS: AP was induced by 7 hourly intraperitoneal injections of cerulein (50 µg/kg) in wild-type and pancreas-specific conditional Ctsb knockout (CtsbΔpan), Ctsl knockout (CtslΔpan), and Ctsb;Ctsl double-knockout (CtsbΔpan;CtslΔpan) mice. Pancreatic samples were collected and analyzed by histology, immunohistochemistry, real-time PCR, and immunoblots. Trypsin activity was measured in pancreatic homogenates. Peripheral blood was collected, and serum amylase activity was measured. RESULTS: Double deletion of Ctsb and Cstl did not affect pancreatic development or mouse growth. After 7 times cerulein injections, double Ctsb and Ctsl deficiency in mouse pancreases increased trypsin activity to the same extent as that in Ctsl-deficient mice, while Ctsb deficiency decreased trypsin activity but did not affect the severity of AP. CtsbΔpan;CtslΔpan mice had comparable serum amylase activity and histopathological changes and displayed similar levels of proinflammatory cytokines, apoptosis, and autophagy activity compared with wild-type, CtsbΔpan, and CtslΔpan mice. CONCLUSION: Double deletion of Ctsb and Ctsl in the mouse pancreas altered intrapancreatic trypsin activity but did not affect disease severity and inflammatory response after cerulein-induced AP.

Fusobacterium nucleatum confers chemoresistance by modulating autophagy in oesophageal squamous cell carcinoma.

BACKGROUND: Fusobacterium nucleatum (F. nucleatum) is a gut microbe implicated in gastrointestinal tumorigenesis. Predicting the chemotherapeutic response is critical to developing personalised therapeutic strategies for oesophageal cancer patients. The present study investigated the relationship between F. nucleatum and chemotherapeutic resistance in oesophageal squamous cell carcinoma (ESCC). METHODS: We examined the relationship between F. nucleatum and chemotherapy response in 120 ESCC resected specimens and 30 pre-treatment biopsy specimens. In vitro studies using ESCC cell lines and co-culture assays further uncovered the mechanism underlying chemotherapeutic resistance. RESULTS: ESCC patients with F. nucleatum infection displayed lesser chemotherapeutic response. The infiltration and subsistence of F. nucleatum in the ESCC cells were observed by transmission electron microscopy and laser scanning confocal microscopy. We also observed that F. nucleatum modulates the endogenous LC3 and ATG7 expression, as well as autophagosome formation to induce chemoresistance against 5-FU, CDDP, and Docetaxel. ATG7 knockdown resulted in reversal of F. nucleatum-induced chemoresistance. In addition, immunohistochemical studies confirmed the correlation between F. nucleatum infection and ATG7 expression in 284 ESCC specimens. CONCLUSIONS: F. nucleatum confers chemoresistance to ESCC cells by modulating autophagy. These findings suggest that targeting F. nucleatum, during chemotherapy, could result in variable therapeutic outcomes for ESCC patients.

Selective expression of a C-type lectin receptor, Clec12b, on skin mast cells.

Mast cells (MCs) are present in various organs including the skin, peritoneal cavity, lung, and intestine and involved in the development of allergic diseases and host defense against infection. However, the regulatory mechanism of mast cell activation remains incompletely understood. We found in a database that Clec12b encoding a C-type lectin receptor Clec12b is preferentially expressed in skin MCs in mice. However, neither MCs in other tissues such as trachea, tongue, esophagus, or peritoneal cavity nor most lymphocytes and myeloid cells express Clec12b. To analyze the protein expression of Clec12b, we newly generated a monoclonal antibody (named TX109), which recognizes both mouse and human Clec12b. Consistent with the gene expression profile, flow cytometry analysis demonstrated that Clec12b is expressed only on MCs in the skin, but not on any other immune cell types in various tissues, in mice. Similarly, Clec12b is also expressed on skin MCs, but not on circulating lymphocytes and myeloid cells, in humans. Our results suggest that Clec12b plays an important role in the regulation of MCs activation in the skin.

Lung fibroblasts produce IL-33 in response to stimulation with retinoblastoma-binding protein 9 via production of prostaglandin E2.

Intestinal nematode infection induces pulmonary eosinophilia via IL-33, although the mechanism of pulmonary IL-33 induction remains unclear. Because nematode migration damages lungs, we speculated that lung-derived damage-associated molecular patterns (DAMPs) possess an IL-33-inducing activity (IL33ia). Indeed, intra-nasal administration of a lung extract induced IL-33 production in lungs. Additionally, lung extracts increased Il33 mRNA expression in primary lung fibroblasts. Proteomic analysis identified retinoblastoma-binding protein 9 (RBBP9) as a major DAMP with IL33ia. RBBP9 was originally discovered as a protein that provides cells with resistance to the growth inhibitory effect of transforming growth factor (TGF)-ß1. Here, we found that stimulation by RBBP9 induced primary fibroblasts to produce prostaglandin E2 (PGE2) that, in turn, induced fibroblasts to produce IL-33. RBBP9-activated fibroblasts expressed mRNAs of cyclooxygenase-2 (COX-2) and PGE2 synthase-1 that convert arachidonic acid to PGE2. Furthermore, they expressed PGE2 receptors E-prostanoid (EP) 2 and EP4. Thus, treatment with a COX-2 inhibitor or EP2 and/or EP4 receptor antagonists inhibited RBBP9-induced IL-33 production. Nematode infection induced pulmonary Il33 mRNA expression, which was inhibited by the COX-2 inhibitor or EP2 and EP4 antagonists, suggesting that nematode infection induced pulmonary Il33 mRNA via PGE2. RBBP9 was expressed constitutively in the lung in the steady state, which did not increase after nematode infection. Finally, we found that Rbbp9-deficient mice had a significantly diminished capacity to increase pulmonary Il33 mRNA expression following nematode infection. Thus, the PGE2-EP2/EP4 pathway activated by RBBP9 released from damaged lungs is important for pulmonary IL-33 production in nematode-infected animals.

Deletion in the Cobalamin Synthetase W Domain-Containing Protein 1 Gene Is associated with Congenital Anomalies of the Kidney and Urinary Tract.

BACKGROUND: Researchers have identified about 40 genes with mutations that result in the most common cause of CKD in children, congenital anomalies of the kidney and urinary tract (CAKUT), but approximately 85% of patients with CAKUT lack mutations in these genes. The anomalies that comprise CAKUT are clinically heterogenous, and thought to be caused by disturbances at different points in kidney development. However, identification of novel CAKUT-causing genes remains difficult because of their variable expressivity, incomplete penetrance, and heterogeneity. METHODS: We investigated two generations of a family that included two siblings with CAKUT. Although the parents and another child were healthy, the two affected siblings presented the same manifestations, unilateral renal agenesis and contralateral renal hypoplasia. To search for a novel causative gene of CAKUT, we performed whole-exome and whole-genome sequencing of DNA from the family members. We also generated two lines of genetically modified mice with a gene deletion present only in the affected siblings, and performed immunohistochemical and phenotypic analyses of these mice. RESULTS: We found that the affected siblings, but not healthy family members, had a homozygous deletion in the Cobalamin Synthetase W Domain-Containing Protein 1 (CBWD1) gene. Whole-genome sequencing uncovered genomic breakpoints, which involved exon 1 of CBWD1, harboring the initiating codon. Immunohistochemical analysis revealed high expression of Cbwd1 in the nuclei of the ureteric bud cells in the developing kidneys. Cbwd1-deficient mice showed CAKUT phenotypes, including hydronephrosis, hydroureters, and duplicated ureters. CONCLUSIONS: The identification of a deletion in CBWD1 gene in two siblings with CAKUT implies a role for CBWD1 in the etiology of some cases of CAKUT.

Six1 is required for signaling center formation and labial-lingual asymmetry in developing lower incisors.

BACKGROUND: The structure of the mouse incisor is characterized by its asymmetric accumulation of enamel matrix proteins on the labial side. The asymmetric structure originates from the patterning of the epithelial incisor placode through the interaction with dental mesenchymal cells. However, the molecular basis for the asymmetric patterning of the incisor germ is largely unknown. RESULTS: A homeobox transcription factor SIX1 was shown to be produced in the mandibular mesenchyme, and its localization patterns changed dynamically during lower incisor development. Six1-/- mice exhibited smaller lower incisor primordia than wild-type mice. Furthermore, Six1-/- mice showed enamel matrix production on both the lingual and labial sides and disturbed odontoblast maturation. In the earlier stages of development, the formation of signaling centers, the initiation knot and the enamel knot, which are essential for the morphogenesis of tooth germs, were impaired in Six1-/- embryos. Notably, Wnt signaling activity, which shows an anterior-posterior gradient, and the expression patterns of genes involved in incisor formation were altered in the mesenchyme in Six1-/- embryos. CONCLUSION: Our results indicate that Six1 is required for signaling center formation in lower incisor germs and the labial-lingual asymmetry of the lower incisors by regulating the anterior-posterior patterning of the mandibular mesenchyme.

Impact of loss-of-function mutations at the RNF43 locus on colorectal cancer development and progression.

RNF43 mutations are frequently detected in colorectal cancer cells and lead to a loss of function of the ubiquitin E3 ligase. Here, we investigated the clinical significance of RNF43 mutations in a large Japanese cohort and the role of RNF43 at various stages of colorectal cancer development and progression. Mutation analysis of the RNF43 gene locus with pyrosequencing technology detected RNF43 hotspot mutations in one (0.88%) of 113 colorectal polyp cases and in 30 (6.45%) of 465 colorectal cancer cases. Moreover, patients with colorectal cancer harbouring mutated RNF43 experienced a higher recurrence rate than those harbouring non-mutated RNF43. In addition, the growth of RNF43 wild-type colorectal cancer cell lines was significantly increased by RNF43 silencing. We generated Rnf43 knockout mice in a C57BL/6 N background by using the CRISPR-Cas9 system. Although intestinal organoids from Rnf43 knockout mice did not show continuous growth in the absence of R-spondin, an azoxymethane/dextran sodium sulphate mouse model demonstrated that tumours were markedly larger in Rnf43 knockout mice than in wild-type mice. These findings provide evidence that Wnt signalling activation by RNF43 mutations during the tumourigenic stage enhances tumour growth and promotes a high recurrence rate in colorectal cancer patients. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

High-density array-CGH with targeted NGS unmask multiple noncontiguous minute deletions on chromosome 3p21 in mesothelioma.

We used a custom-made comparative genomic hybridization array (aCGH; average probe interval 254 bp) to screen 33 malignant mesothelioma (MM) biopsies for somatic copy number loss throughout the 3p21 region (10.7 Mb) that harbors 251 genes, including BRCA1 (breast cancer 1)-associated protein 1 (BAP1), the most commonly mutated gene in MM. We identified frequent minute biallelic deletions (<3 kb) in 46 of 251 genes: four were cancer-associated genes: SETD2 (SET domain-containing protein 2) (7 of 33), BAP1 (8 of 33), PBRM1 (polybromo 1) (3 of 33), and SMARCC1 (switch/sucrose nonfermentable- SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily c, member 1) (2 of 33). These four genes were further investigated by targeted next-generation sequencing (tNGS), which revealed sequence-level mutations causing biallelic inactivation. Combined high-density aCGH and tNGS revealed biallelic gene inactivation in SETD2 (9 of 33, 27%), BAP1 (16 of 33, 48%), PBRM1 (5 of 33, 15%), and SMARCC1 (2 of 33, 6%). The incidence of genetic alterations detected is much higher than reported in the literature because minute deletions are not detected by NGS or commercial aCGH. Many of these minute deletions were not contiguous, but rather alternated with segments showing oscillating copy number changes along the 3p21 region. In summary, we found that in MM: (i) multiple minute simultaneous biallelic deletions are frequent in chromosome 3p21, where they occur as distinct events involving multiple genes; (ii) in addition to BAP1, mutations of SETD2, PBRM1, and SMARCC1 are frequent in MM; and (iii) our results suggest that high-density aCGH combined with tNGS provides a more precise estimate of the frequency and types of genes inactivated in human cancer than approaches based exclusively on NGS strategy.

Lack of Cathepsin D in the Renal Proximal Tubular Cells Resulted in Increased Sensitivity against Renal Ischemia/Reperfusion Injury.

Cathepsin D is one of the major lysosomal aspartic proteases that is essential for the normal functioning of the autophagy-lysosomal system. In the kidney, cathepsin D is enriched in renal proximal tubular epithelial cells, and its levels increase during acute kidney injury. To investigate how cathepsin D-deficiency impacts renal proximal tubular cells, we employed a conditional knockout CtsDflox/-; Spink3Cre mouse. Immunohistochemical analyses using anti-cathepsin D antibody revealed that cathepsin D was significantly decreased in tubular epithelial cells of the cortico-medullary region, mainly in renal proximal tubular cells of this mouse. Cathepsin D-deficient renal proximal tubular cells showed an increase of microtubule-associated protein light chain 3 (LC3; a marker for autophagosome/autolysosome)-signals and an accumulation of abnormal autophagic structures. Renal ischemia/reperfusion injury resulted in an increase of early kidney injury marker, Kidney injury molecule 1 (Kim-1), in the cathepsin D-deficient renal tubular epithelial cells of the CtsDflox/-; Spink3Cre mouse. Inflammation marker was also increased in the cortico-medullary region of the CtsDflox/-; Spink3Cre mouse. Our results indicated that lack of cathepsin D in the renal tubular epithelial cells led to an increase of sensitivity against ischemia/reperfusion injury.

Generation of and characterization of anti-IL-11 antibodies using newly established Il11-deficient mice.

Interleukin (IL)-11 belongs to the members of the IL-6 family of cytokines and is involved in a variety of biological responses, including hematopoiesis, bone development, and carcinogenesis. However, the cellular sources of IL-11 and regulation of IL-11 expression under physiological and pathological conditions are not fully understood. One of the causes to prevent characterization of IL-11 in vivo is due to the lack of reliable antibodies that detect IL-11 by immunohistochemistry. Moreover, although mice lacking Il11ra have been generated and extensively characterized, Il11-deficient mice have not been characterized yet. Here we generated two anti-IL-11 antibodies that blocked biological activities of IL-11 and detected IL-11 by immunohistochemistry, respectively. One clone of anti-IL-11 antibodies blocked IL-11-, but not IL-6-induced cell proliferation and IL-11-induced phosphorylation of STAT3 of an IL-11-dependent cell line. Moreover, we used recently established Il11-deficient mice to test the specificity of anti-IL-11 antibodies for immunohistochemistry. Another clone of anti-IL-11 antibodies stained stromal cells surrounding tumors of the colon of wild-type, but not Il11-deficient mice following treatment with Azoxymethane plus dextran sulfate sodium. Together, these newly developed anti-IL-11 antibodies provide a better understanding of the functions of IL-11 in vivo under various physiological and pathological conditions.

Depletion of myeloid cells exacerbates hepatitis and induces an aberrant increase in histone H3 in mouse serum.

Tissue-resident macrophages and bone marrow (BM)-derived monocytes play a crucial role in the maintenance of tissue homeostasis; however, their contribution to recovery from acute tissue injury is not fully understood. To address this issue, we generated an acute murine liver injury model using hepatocyte-specific Cflar-deficient (CflarHep-low ) mice. Cellular FLICE-inhibitory protein expression was down-regulated in Cflar-deficient hepatocytes, which thereby increased susceptibility of hepatocytes to death receptor-induced apoptosis. CflarHep-low mice developed acute hepatitis and recovered with clearance of apoptotic hepatocytes at 24 hours after injection of low doses of tumor necrosis factor α (TNFα), which could not induce hepatitis in wild-type (WT) mice. Depletion of Kupffer cells (KCs) by clodronate liposomes did not impair clearance of dying hepatocytes or exacerbate hepatitis in CflarHep-low mice. To elucidate the roles of BM-derived monocytes and neutrophils in clearance of apoptotic hepatocytes, we examined the effect of depletion of these cells on TNFα-induced hepatitis in CflarHep-low mice. We reconstituted CflarHep-low mice with BM cells from transgenic mice in which human diphtheria toxin receptor (DTR) was expressed under control of the lysozyme M (LysM) promoter. TNFα-induced infiltration of myeloid cells, including monocytes and neutrophils, was completely ablated in LysM-DTR BM-reconstituted CflarHep-low mice pretreated with diphtheria toxin, whereas KCs remained present in the livers. Under these experimental conditions, LysM-DTR BM-reconstituted CflarHep-low mice rapidly developed severe hepatitis and succumbed within several hours of TNFα injection. We found that serum interleukin-6 (IL-6), TNFα, and histone H3 were aberrantly increased in LysM-DTR BM-reconstituted, but not in WT BM-reconstituted, CflarHep-low mice following TNFα injection. CONCLUSION: These findings indicate an unexpected role of myeloid cells in decreasing serum IL-6, TNFα, and histone H3 levels via the suppression of TNFα-induced hepatocyte apoptosis. (Hepatology 2017;65:237-252).

Significance of lymph node metastasis in pancreatic neuroendocrine tumor.

PURPOSE: Pancreatic neuroendocrine tumor (PNET) is relatively rare and has a generally better prognosis than does pancreatic cancer. However, as its prognosis in patients with lymph node metastasis (LNM) is unclear, lymph node dissection for PNET is controversial. Our study aimed to clarify the significance of LNM in PNET. METHODS: We retrospectively examined 83 PNET patients who underwent pancreatic resections with lymph node dissection at Kumamoto University Hospital, Saiseikai Kumamoto Hospital, and Kumamoto Regional Medical Center from April 2001 to December 2014. Their clinicopathological parameters were analyzed by the absence or presence of LNM, and with regard to the disease-free survival (DFS) and overall survival (OS). A predictive score of LNM was also made using the age, tumor size, primary tumor location, and tumor function. RESULTS: Although the 5-year OS was 74.8% for LNM+ and 94.6% for LNM- (P = 0.002), LNM was not an independent risk factor for the OS in a multivariate analysis. However, tumors larger than 1.8 cm were found to be an independent prognostic factor, and the cut-off value for the predictive score was 1.69. CONCLUSIONS: Although LNM was not an independent prognostic factor, lymph node dissection is recommended for patients whose predictive score is larger than 1.69.

Short form FLICE-inhibitory protein promotes TNFα-induced necroptosis in fibroblasts derived from CFLARs transgenic mice.

Cellular FLICE-inhibitory protein (cFLIP) is a catalytically inactive homolog of the initiator caspase, caspase 8 and blocks apoptosis through binding to caspase 8. Human CFLAR gene encodes two proteins, a long form cFLIP (cFLIPL) and a short form cFLIP (cFLIPs) due to an alternative splicing. Recent studies have shown that expression of cFLIPs, but not cFLIPL promotes programmed necrosis (also referred to as necroptosis) in an immortalized human keratinocyte cell line, HaCaT. Here, we found that expression of cFLIPs similarly promoted necroptosis in immortalized fibroblasts. To further expand this observation and exclude the possibility that immortalization process of keratinocytes or fibroblasts might affect the phenotype induced by cFLIPs expression, we generated human CFLARs transgenic (Tg) mice. Primary fibroblasts derived from CFLARs Tg mice were increased in susceptibility to TNFα-induced necroptosis, but not apoptosis compared to wild-type (WT) fibroblasts. Moreover, hallmarks of necroptosis, such as phosphorylation of receptor-interacting protein kinase (RIPK)1 and RIPK3, and oligomer formation of mixed lineage kinase domain-like (MLKL) were robustly induced in CFLARs Tg fibroblasts compared to wild-type fibroblasts following TNFα stimulation. Thus, cFLIPs-dependent promotion of necroptosis is not unique to immortalized keratinocytes or fibroblasts, but also to generalized to primary fibroblasts.

Cathepsin D in pancreatic acinar cells is implicated in cathepsin B and L degradation, but not in autophagic activity.

Cathepsin D (CD) is the major lysosomal aspartic protease and is widely distributed in the cells of various mammalian tissues. CD participates in various physiological events such as regulation of programmed cell death, activation of enzymatic precursors, and metabolic degradation of intracellular proteins through macroautophagy. To investigate the role of CD in pancreatic acinar cells, which constitute the exocrine pancreas, we generated and examined mice specifically deficient for CD in pancreatic acinar cells. CD deficient mice showed normal pancreatic development and autophagic activity, although LC3-II, which is a marker of the autophagosome, accumulates in both physiological and pancreatitis conditions. Moreover, CD deficiency leads to accumulation of matured cathepsin B (CB) and cathepsin L (CL) which are members of the cysteine protease family. We therefore conclude that CD in pancreatic acinar cells is implicated in CB and CL degradation but not in autophagic activity.

Residual total pancreatectomy: Short- and long-term outcomes.

BACKGROUND/OBJECTIVES: Because of limited numbers of patients, there are limited data available regarding outcomes after residual total pancreatectomy (R-TP). This study aimed to assess outcomes after the R-TP vs the one-stage total pancreatectomy (O-TP), especially focused on the pancreatic adenocarcinoma cases. METHODS: From 2005 to 2014, all patients who underwent the R-TP (n = 8) and the O-TP (n = 12) for pancreatic primary malignancy were prospectively enrolled. RESULTS: The median time from the initial operation to the R-TP was 30 months. Ten patients in the O-TP group and 8 in the R-TP had pancreatic adenocarcinoma. Postoperative complications occurred in two O-TP patients and one R-TP patient. There was no in-hospital mortality. At 12 months after surgery, the median insulin dose was 27 U/day after the O-TP and 24 U/day after the R-TP, the median hemoglobin A1c was 7.2% after the O-TP and 6.9% after the R-TP. There was a significantly larger reduction in body weight after the O-TP than after the R-TP. Postoperative fatty liver disease occurred in about half of the patients in each group. In patients with pancreatic adenocarcinoma, the 2-year overall survival rate was not significantly different (68.6% after the O-TP vs 71.4% after the R-TP). CONCLUSIONS: Although the postoperative morbidity and nutritional statuses should be improved, these favorable short- and long-term outcomes demonstrate that the R-TP is a feasible procedure for patients with malignant tumor in the remnant pancreas.