Endoscopic main duct stenting in refractory postoperative pancreatic fistula after distal pancreatectomy - a friend or a foe?

BACKGROUND: Clinically relevant (CR) postoperative pancreatic fistula (POPF) after distal pancreatectomy (DP) are common. Endoscopic treatment (ET) has only scarcely been explored. The aim of this study was to evaluate risk factors for CR POPF after DP and the efficacy of ET in adjunct to standard therapy. METHODS: Consecutive patients without previous pancreatic surgery who underwent DP between 2011 and 2020 were evaluated, analyzing risk factors for CR POPF. The choice and performance of ET, main pancreatic duct (MPD) stenting, was not standardized. Healing time and complications after ET were registered. RESULTS: 406 patients underwent DP, CR POPF occurred in 29.6%. ET was performed in 17 patients 27 days (median) after index surgery. Risk for CR POPF was increased in ASA-PS 1-2 patients, MPD ≤ 3 mm, procedure time ≥ 3 h, and CRP ≥ 180 on postoperative day 3. POPF resolved with standard treatment after 32 days and 59 days in the ET group (p < 0.001). There was one mortality in the ET-group (not procedure related). Mild post-ERCP pancreatitis occurred in three patients. CONCLUSIONS: CR POPF is common after DP. Long operating time, a narrow MPD, low ASA score, and high postoperative CRP were risk factors for CR POPF. ET was not beneficial but proper evaluation was not possible due to few patients and non-standardized treatment. Complications after ET appeared mild.

Matrix metalloproteinases at a glance.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases that belong to the group of endopeptidases or matrixins. They are able to cleave a plethora of substrates, including components of the extracellular matrix and cell-surface-associated proteins, as well as intracellular targets. Accordingly, MMPs play key roles in a variety of physiological and pathological processes, such as tissue homeostasis and cancer cell invasion. MMP activity is exquisitely regulated at several levels, including pro-domain removal, association with inhibitors, intracellular trafficking and transport via extracellular vesicles. Moreover, the regulation of MMP activity is currently being rediscovered for the development of respective therapies for the treatment of cancer, as well as infectious, inflammatory and neurological diseases. In this Cell Science at a Glance article and the accompanying poster, we present an overview of the current knowledge regarding the regulation of MMP activity, the intra- and extra-cellular trafficking pathways of these enzymes and their diverse groups of target proteins, as well as their impact on health and disease.

KIF16B drives MT1-MMP recycling in macrophages and promotes co-invasion of cancer cells.

The matrix metalloproteinase MT1-MMP is a central effector of cellular proteolysis. Accordingly, regulation of the surface-localized pool of MT1-MMP is crucial for cell migration and invasion. Here, we identify the superprocessive kinesin KIF16B as a major driver of fast recycling of MT1-MMP to the surface of primary human macrophages. KIF16B associates with MT1-MMP on Rab14-positive vesicles, and its depletion results in strongly reduced MT1-MMP surface levels, as shown by microscopical, biochemical, and cell-sorting approaches. As a consequence, KIF16B-depleted macrophages exhibit strongly reduced matrix degradation and invasion. We further identify the cargo-binding C-terminus of KIF16B as a critical element of MT1-MMP transport, as its overexpression uncouples MT1-MMP vesicles from the endogenous motor, thus leading to a reduction of surface-associated MT1-MMP and to reduced matrix degradation and invasion. Importantly, depletion of KIF16B in primary macrophages also reduces the co-invasion of cancer cells from tumor spheroids, pointing to the KIF16B-driven recycling pathway in macrophages as an important regulatory element of the tumor microenvironment.

Get a grip: Podosomes as potential players in phagocytosis.

Podosomes have been known for several decades as micron-sized, F-actin-rich structures that play a pivotal role in cell migration and invasion, as they are able to mediate both cell-matrix attachment as well as extracellular matrix degradation. Particularly in monocytic cells, podosomes have been shown to fulfill a variety of additional functions such as sensing of substrate rigidity and topography, or cell-cell fusion. Increasing evidence now points to the involvement of podosome-like structures also during phagocytosis by immune cells such as macrophages, dendritic cells, and neutrophils. Here, we compare the different cell models and experimental set ups where "phagocytic podosomes" have been described. We also discuss the composition and architecture of these structures, their potential involvement in mechanosensing and particle disruption, as well as the pros and cons for addressing them as bona fide podosomes.

Proteolysis of CD44 at the cell surface controls a downstream protease network.

The cell surface receptor cluster of differentiation 44 (CD44) is the main hyaluronan receptor of the human body. At the cell surface, it can be proteolytically processed by different proteases and was shown to interact with different matrix metalloproteinases. Upon proteolytic processing of CD44 and generation of a C-terminal fragment (CTF), an intracellular domain (ICD) is released after intramembranous cleavage by the γ-secretase complex. This intracellular domain then translocates to the nucleus and induces transcriptional activation of target genes. In the past CD44 was identified as a risk gene for different tumor entities and a switch in CD44 isoform expression towards isoform CD44s associates with epithelial to mesenchymal transition (EMT) and cancer cell invasion. Here, we introduce meprin ß as a new sheddase of CD44 and use a CRISPR/Cas9 approach to deplete CD44 and its sheddases ADAM10 and MMP14 in HeLa cells. We here identify a regulatory loop at the transcriptional level between ADAM10, CD44, MMP14 and MMP2. We show that this interplay is not only present in our cell model, but also across different human tissues as deduced from GTEx (Gene Tissue Expression) data. Furthermore, we identify a close relation between CD44 and MMP14 that is also reflected in functional assays for cell proliferation, spheroid formation, migration and adhesion.

Tumor cell integrin ß4 and tumor stroma E-/P-selectin cooperatively regulate tumor growth in vivo.

BACKGROUND: The immunological composition of the tumor microenvironment has a decisive influence on the biological course of cancer and is therefore of profound clinical relevance. In this study, we analyzed the cooperative effects of integrin ß4 (ITGB4) on tumor cells and E-/P-selectin on endothelial cells within the tumor stroma for regulating tumor growth by shaping the local and systemic immune environment. METHODS: We used several preclinical mouse models for different solid human cancer types (xenograft and syngeneic) to explore the role of ITGB4 (shRNA-mediated knockdown in tumor cells) and E-/P-selectins (knockout in mice) for tumor growth; effects on apoptosis, proliferation and intratumoral signaling pathways were determined by histological and biochemical methods and 3D in vitro experiments; changes in the intratumoral and systemic immune cell composition were determined by flow cytometry and immunohistochemistry; chemokine levels and their attracting potential were measured by ELISA and 3D invasion assays. RESULTS: We observed a very robust synergism between ITGB4 and E-/P-selectin for the regulation of tumor growth, accompanied by an increased recruitment of CD11b+ Gr-1Hi cells with low granularity (i.e., myeloid-derived suppressor cells, MDSCs) specifically into ITGB4-depleted tumors. ITGB4-depleted tumors undergo apoptosis and actively attract MDSCs, well-known to promote tumor growth in several cancers, via increased secretion of different chemokines. MDSC trafficking into tumors crucially depends on E-/P-selectin expression. Analyses of clinical samples confirmed an inverse relationship between ITGB4 expression in tumors and number of tumor-infiltrating leukocytes. CONCLUSIONS: These findings suggest a distinct vulnerability of ITGB4Lo tumors for MDSC-directed immunotherapies.

MT1-MMP and ADAM10/17 exhibit a remarkable overlap of shedding properties.

Membrane-type-I matrix metalloproteinase (MT1-MMP) is one of six human membrane-bound MMPs and is responsible for extracellular matrix remodelling by degrading several substrates like fibrillar collagens, including types I-III, or fibronectin. Moreover, MT1-MMP was described as a key player in cancer progression and it is involved in various inflammatory processes, as well as in the pathogenesis of Alzheimer's disease (AD). The membrane-tethered metalloprotease meprin ß as well as a disintegrin and metalloproteinase 10 (ADAM10) and ADAM17 are also associated with these diseases. Interestingly, meprin ß, ADAM10/17 and MT1-MMP also have a shared substrate pool including the interleukin-6 receptor and the amyloid precursor protein. We investigated the interaction of these proteases, focusing on a possible connection between MT1-MMP and meprin ß, to elucidate the potential mutual regulations of both enzymes. Herein, we show that besides ADAM10/17, MT1-MMP is also able to shed meprin ß from the plasma membrane, leading to the release of soluble meprin ß. Mass spectrometry-based cleavage site analysis revealed that the cleavage of meprin ß by all three proteases occurs between Pro602 and Ser603 , N-terminal of the EGF-like domain. Furthermore, only inactive human pro-meprin ß is shed by MT1-MMP, which is again in accordance with the shedding capability observed for ADAM10/17. Vice versa, meprin ß also appears to shed MT1-MMP, indicating a complex regulatory network. Further studies will elucidate this well-orchestrated proteolytic web under distinct conditions in health and disease and will possibly show whether the loss of one of the above-mentioned sheddases can be compensated by the other enzymes.

Mechanisms and roles of podosomes and invadopodia.

Cell invasion into the surrounding extracellular matrix or across tissue boundaries and endothelial barriers occurs in both physiological and pathological scenarios such as immune surveillance or cancer metastasis. Podosomes and invadopodia, collectively called 'invadosomes', are actin-based structures that drive the proteolytic invasion of cells, by forming highly regulated platforms for the localized release of lytic enzymes that degrade the matrix. Recent advances in high-resolution microscopy techniques, in vivo imaging and high-throughput analyses have led to considerable progress in understanding mechanisms of invadosomes, revealing the intricate inner architecture of these structures, as well as their growing repertoire of functions that extends well beyond matrix degradation. In this Review, we discuss the known functions, architecture and regulatory mechanisms of podosomes and invadopodia. In particular, we describe the molecular mechanisms of localized actin turnover and microtubule-based cargo delivery, with a special focus on matrix-lytic enzymes that enable proteolytic invasion. Finally, we point out topics that should become important in the invadosome field in the future.

Development of an artificial synovial fluid useful for studying Staphylococcus epidermidis joint infections.

Staphylococcus epidermidis is a major causative agent of prosthetic joint infections (PJI). The ability to form biofilms supports this highly selective pathogenic potential. In vitro studies essentially relying on phenotypic assays and genetic approaches have provided a detailed picture of the molecular events contributing to biofilm assembly. A major limitation in these studies is the use of synthetic growth media, which significantly differs from the environmental conditions S. epidermidis encounters during host invasion. Building on evidence showing that growth in serum substantially affects S. epidermidis gene expression profiles and phenotypes, the major aim of this study was to develop and characterize a growth medium mimicking synovial fluid, thereby facilitating research addressing specific aspects related to PJI. Using fresh human plasma, a protocol was established allowing for the large-scale production of a medium that by biochemical analysis matches key characteristics of synovial fluid and therefore is referred to as artificial synovial fluid (ASF). By analysis of biofilm-positive, polysaccharide intercellular adhesion (PIA)-producing S. epidermidis 1457 and its isogenic, PIA- and biofilm-negative mutant 1457-M10, evidence is provided that the presence of ASF induces cluster formation in S. epidermidis 1457 and mutant 1457-M10. Consistent with the aggregative properties, both strains formed multilayered biofilms when analyzed by confocal laser scanning microscopy. In parallel to the phenotypic findings, expression analysis after growth in ASF found upregulation of genes encoding for intercellular adhesins (icaA, aap, and embp) as well as atlE, encoding for the major cell wall autolysin being responsible for eDNA release. In contrast, growth in ASF was associated with reduced expression of the master regulator agr. Collectively, these results indicate that ASF induces expression profiles that are able to support intercellular adhesion in both PIA-positive and PIA-negative S. epidermidis. Given the observation that ASF overall induced biofilm formation in a collection of S. epidermidis isolates from PJI, the results strongly support the idea of using growth media mimicking host environments. ASF may play an important role in future studies related to the pathogenesis of S. epidermidis PJI.

Impact of spatio-temporal recurrence pattern on overall survival for invasive intraductal papillary mucinous neoplasia - A comparison with pancreatic ductal adenocarcinoma.

BACKGROUND: Resections for intraductal papillary mucinous neoplasia (IPMN) have increased dramatically during the last decade. Recurrence pattern and impact of adjuvant chemotherapy for solid pancreatic ductal adenocarcinoma (PDAC) is well known, but not for invasive IPMN (inv-IPMN). OBJECTIVES: To elucidate the impact of spatio-temporal recurrence pattern and adjuvant chemotherapy on overall survival for inv-IPMN compared with PDAC. METHODS: We conducted a retrospective single-center observational study of consecutive patients ≥18 years of age who underwent resection for inv-IPMN or PDAC at Karolinska University Hospital, between 2009 and 2018. Different initial recurrence sites and time frames as well as predictors for death were assessed with multivariable Cox and logistic regressions. Survival analyses were performed using the Kaplan-Meier model and log rank test. RESULTS: Of 396 resected patients, 92 were inv-IPMN and 304 PDAC. Both recurrence rate and death rate within three-years were lower for inv-IPMN compared to PDAC (p = 0.006 and p = 0.007 respectively). Across the whole cohort, the most common recurrence patterns were multi-site (25%), single-site liver (21%) and single-site locoregional (10%) recurrence. The most prominent predictors for death in multivariable Cox regression, especially if occurred within the first year, were multi-site (HR 17.0), single-site peritoneal (HR 13.6) and single-site liver (HR 13.1) recurrence. These predictors were less common in inv-IPMN compared to PDAC (p = 0.007). The effect of adjuvant chemotherapy was similar in the two groups. CONCLUSION: Resected inv-IPMN exhibits a less aggressive recurrence pattern than PDAC that translates into a more favorable overall survival.

Prognostic impact of para-aortic lymph node status in resected pancreatic ductal adenocarcinoma and invasive intraductal papillary mucinous neoplasm - Time to consider a reclassification?

BACKGROUND: Para-aortic lymph node (PALN) metastases in pancreatic ductal adenocarcinoma (PDAC) correlates with poor prognosis. The role of PALN in invasive intraductal papillary mucinous neoplasms (inv-IPMN) has not been well explored. The present study investigated the rate of metastatic PALN, lymph node ratio (LNR) and the overall nodal (N) status as prognostic factors in PDAC and inv-IPMN. METHODS: This consecutive single-center series included patients with PDAC or inv-IPMN in the pancreatic head who underwent pancreatoduodenectomy or total pancreatectomy, including PALN resection between 2009 and 2018. Median overall survival (mOS) and impact of clinicopathological factors, including PALN status on survival, were evaluated. RESULTS: 403 patients were included, 314 had PDAC and 89 inv-IPMN. PALN were metastatic in 16% of PDAC and 17% of inv-IPMN. N0 status was present in 6% of the patients with PDAC and 16% of inv-IPMN patients (p = 0.007). LNR >15% was more common in PDAC (52%) than in inv-IPMN (34%) (p = 0.004). mOS was 12.7 months in the presence of PALN metastases and 22.7 months without (p < 0.0001). Age >70 years, CA19-9 >200 U/mL, PDAC and N2 status were significantly associated with worse survival in a multivariable analysis. PALN status and LNR were not independent prognostic factors. In N2 status mOS was similar regardless the presence of PALN metastases. CONCLUSION: The frequency of PALN metastases was similar in PDAC and inv-IPMN. Although PALN positive status entailed a shorter mOS, it was not an independent risk factor for death, and did not influence survival in N2-staged disease. The M1-status for PALN positivity may need reconsideration.

The circle of life: Phases of podosome formation, turnover and reemergence.

Podosomes are highly dynamic actin-rich structures in a variety of cell types, especially monocytic cells. They fulfill multiple functions such as adhesion, mechanosensing, or extracellular matrix degradation, thus allowing cells to detect and respond to a changing environment. These abilities are based on an intricate architecture that enables podosomes to sense mechanical properties of their substratum and to transduce them intracellularly in order to generate an appropriate cellular response. These processes are enabled through the tightly orchestrated interplay of more than 300 different components that are dynamically recruited during podosome formation and turnover. In this review, we discuss the different phases of the podosome life cycle and the current knowledge on regulatory factors that impact on the genesis, activity, dissolution and reemergence of podosomes. We also highlight mechanoregulatory processes that become important during these different stages, on the level of individual podosomes, and also at podosome sub- and superstructures.

Does moral commitment predict resistance to corruption? experimental evidence from a bribery game.

Corruption is ubiquitous in practice and has severe negative consequences for organizations and societies at large. Drawing on a laboratory experiment, we propose that individuals high in moral commitment are less likely to engage in corrupt behaviors and prefer foregoing financial benefits. Specifically, we posit that individuals refrain from corruption (i) the more they endorse integrity (incorruptibility) as a protected value and (ii) the higher their level of Honesty-Humility. The results of a two-step experiment largely support our expectations: people who treat compromises to integrity as unacceptable were less willing to accept bribes, and Honesty-Humility decreased bribe-giving. The findings are robust to demographic variables (e.g., age, gender, cultural background) and additional personal characteristics (e.g., risk tolerance, dispositional greed) and have important implications for ongoing theory-building efforts and business practice.

There and back again: Intracellular trafficking, release and recycling of matrix metalloproteinases.

Matrix metalloproteinases are a family of zinc-dependent endopeptidases that are involved in a large variety of proteolytic processes in physiological and pathological scenarios, including immune cell surveillance, tissue homeostasis, or tumor cell metastasis. This is based on their ability to cleave a plethora of substrates that include components of the extracellular matrix, but also cell surface-associated and intracellular proteins. Accordingly, a tight regulatory web has evolved that closely regulates spatiotemporal activity of specific MMPs. An often underappreciated mechanism of MMP regulation involves their trafficking to and from specific subcellular sites that require MMP activity only for a certain period. In this review, we focus on the current knowledge of MMP intracellular trafficking, their secretion or surface exposure, as well as their recycling back from the cell surface. We discuss molecular mechanisms that enable these steps, in particular microtubule-dependent motility of vesicles that is driven by molecular motors and directed by vesicle regulatory proteins. Finally, we also point out open questions in the field of MMP motility that may become important in the future.

Nectin stabilization at adherens junctions is counteracted by Rab5a-dependent endocytosis.

Cell-cell junctions undergo constant remodeling, which is crucial for the control of vascular integrity. Indeed, transport of junctional components such as cadherins is understood in increasing depth. However, little is known about the respective pathways regulating localization of nectin at cell-cell junctions. Here, we performed an siRNA-based screen of vesicle regulators of the RabGTPase family, leading to the identification of a novel role for Rab5a in the endocytosis nectin-2 at adherens junctions of primary human endothelial cells (HUVEC). Confocal microscopy experiments revealed disordered nectin-2 localization at adherens junctions upon Rab5a depletion. In addition, internalized nectin-2 was shown to prominently localize to Rab5a-positive vesicles in both fixed and living cells. As shown previously, nectin-2 stabilization at junctions is achieved via drebrin-dependent coupling to the subcortical actin cytoskeleton. Consistently, depletion of drebrin in this study leads to enhanced internalization of nectin-2 from junctions. Strikingly, simultaneous silencing of Rab5a and drebrin restored the junctional localization of nectin-2, pointing to Rab5a as counteracting the drebrin-dependent stabilization of nectin-2 at adherens junctions. This mechanism could be further validated by transendothelial resistance measurements. Collectively, our results identify Rab5a as a key player in the endocytosis of nectin-2 and thus in the regulation of adherens junction integrity in primary human endothelial cells.

Cargo-specific recruitment in clathrin- and dynamin-independent endocytosis.

Spatially controlled, cargo-specific endocytosis is essential for development, tissue homeostasis and cancer invasion. Unlike cargo-specific clathrin-mediated endocytosis, the clathrin- and dynamin-independent endocytic pathway (CLIC-GEEC, CG pathway) is considered a bulk internalization route for the fluid phase, glycosylated membrane proteins and lipids. While the core molecular players of CG-endocytosis have been recently defined, evidence of cargo-specific adaptors or selective uptake of proteins for the pathway are lacking. Here we identify the actin-binding protein Swiprosin-1 (Swip1, EFHD2) as a cargo-specific adaptor for CG-endocytosis. Swip1 couples active Rab21-associated integrins with key components of the CG-endocytic machinery-Arf1, IRSp53 and actin-and is critical for integrin endocytosis. Through this function, Swip1 supports integrin-dependent cancer-cell migration and invasion, and is a negative prognostic marker in breast cancer. Our results demonstrate a previously unknown cargo selectivity for the CG pathway and a role for specific adaptors in recruitment into this endocytic route.

Molecular Mechanisms of Borrelia burgdorferi Phagocytosis and Intracellular Processing by Human Macrophages.

Lyme disease is the most common vector-borne illness in North America and Europe. Its causative agents are spirochetes of the Borrelia burgdorferi sensu latu complex. Infection with borreliae can manifest in different tissues, most commonly in the skin and joints, but in severe cases also in the nervous systems and the heart. The immune response of the host is a crucial factor for preventing the development or progression of Lyme disease. Macrophages are part of the innate immune system and thus one of the first cells to encounter infecting borreliae. As professional phagocytes, they are capable of recognition, uptake, intracellular processing and final elimination of borreliae. This sequence of events involves the initial capture and internalization by actin-rich cellular protrusions, filopodia and coiling pseudopods. Uptake into phagosomes is followed by compaction of the elongated spirochetes and degradation in mature phagolysosomes. In this review, we discuss the current knowledge about the processes and molecular mechanisms involved in recognition, capturing, uptake and intracellular processing of Borrelia by human macrophages. Moreover, we highlight interactions between macrophages and other cells of the immune system during these processes and point out open questions in the intracellular processing of borreliae, which include potential escape strategies of Borrelia.

FIB-SEM-based analysis of Borrelia intracellular processing by human macrophages.

Borrelia burgdorferi is the causative agent of Lyme disease, a multisystemic disorder affecting primarily skin, joints and nervous system. Successful internalization and intracellular processing of borreliae by immune cells, like macrophages, is decisive for the outcome of a respective infection. Here, we use, for the first time, focused ion beam scanning electron microscopy tomography (FIB-SEM tomography) to visualize the interaction of borreliae with primary human macrophages with high resolution. We report that interaction between macrophages and the elongated and highly motile borreliae can lead to formation of membrane tunnels that extend deeper into the host cytoplasm than the actual phagosome, most probably as a result of partial extrication of captured borreliae. We also show that membrane tubulation at borreliae-containing phagosomes, a process suggested earlier as a mechanism leading to phagosome compaction but hard to visualize in live-cell imaging, is apparently a frequent phenomenon. Finally, we demonstrate that the endoplasmic reticulum (ER) forms multiple STIM1-positive contact sites with both membrane tunnels and phagosome tubulations, confirming the important role of the ER during uptake and intracellular processing of borreliae.

The podosome cap: past, present, perspective.

Podosomes are prominent actin-based adhesion structures in a variety of cell types. They feature an extensive repertoire of functions, which requires exquisite spatiotemporal fine-tuning. Accordingly, podosomes consist of hundreds of different components, which fulfill specific structural and regulatory functions. Moreover, it has become apparent that podosome architecture is more intricate than previously believed. The classical model of an actin-rich core surrounded by a ring structure containing adhesion plaque proteins thus had to be expanded, and several additional substructures have been described, most notably the podosome cap on top of the actin-rich core. Here, we discuss the known components of the podosome cap, the history of their detection and their potential regulatory roles in podosome turnover and function. We also point out strategies for identifying further cap components and present a new model for the podosome cap as a multilayered module that fine-tunes actomyosin contractility, a central requirement for podosome architecture, dynamics and function.