Release Of Proteases Research Articles

Cathepsin G Activity as a New Marker for Detecting Airway Inflammation by Microscopy and Flow Cytometry.

Muco-obstructive lung diseases feature extensive bronchiectasis due to the uncontrolled release of neutrophil serine proteases into the airways. To assess if cathepsin G (CG) is a novel key player in chronic lung inflammation, we developed membrane-bound (mSAM) and soluble (sSAM) FRET reporters. The probes quantitatively revealed elevated CG activity in samples from 46 patients. For future basic science and personalized clinical applications, we developed a rapid, highly informative, and easily translatable small-molecule FRET flow cytometry assay for monitoring protease activity including cathepsin G. We demonstrated that mSAM distinguished healthy from patient cells by FRET-based flow cytometry with excellent correlation to confocal microscopy data.

Japanese encephalitis virus neuropenetrance is driven by mast cell chymase

Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis. However, the mechanisms of JEV penetration of the blood-brain-barrier (BBB) remain poorly understood. Mast cells (MCs) are granulated innate immune sentinels located perivascularly, including at the BBB. Here we show that JEV activates MCs, leading to the release of granule-associated proteases in vivo. MC-deficient mice display reduced BBB permeability during JEV infection compared to congenic wild-type (WT) mice, indicating that enhanced vascular leakage in the brain during JEV infection is MC-dependent. Moreover, MCs promoted increased JEV infection in the central nervous system (CNS), enhanced neurological deficits, and reduced survival in vivo. Mechanistically, chymase, a MC-specific protease, enhances JEV-induced breakdown of the BBB and cleavage of tight-junction proteins. Chymase inhibition reversed BBB leakage, reduced brain infection and neurological deficits during JEV infection, and prolonged survival, suggesting chymase is a novel therapeutic target to prevent JEV encephalitis.

Tumour cell blebbing and extracellular vesicle shedding: key role of matrikines and ribosomal protein SA

BackgroundCarcinogenesis occurs in elastin-rich tissues and leads to local inflammation and elastolytic proteinase release. This contributes to bioactive matrix fragment (Matrikine) accumulation like elastin degradation products (EDP) stimulating tumour cell invasive and metastatic properties. We previously demonstrate that EDPs exert protumoural activities through Hsp90 secretion to stabilised extracellular proteinases.MethodsEDP influence on cancer cell blebbing and extracellular vesicle shedding were examined with a videomicroscope coupled with confocal Yokogawa spinning disk, by transmission electron microscopy, scanning electron microscopy and confocal microscopy. The ribosomal protein SA (RPSA) elastin receptor was identified after affinity chromatography by western blotting and cell immunolocalisation. mRNA expression was studied using real-time PCR. SiRNA were used to confirm the essential role of RPSA.ResultsWe demonstrate that extracellular matrix degradation products like EDPs induce tumour amoeboid phenotype with cell membrane blebbing and shedding of extracellular vesicle containing Hsp90 and proteinases in the extracellular space. EDPs influence intracellular calcium influx and cytoskeleton reorganisation. Among matrikines, VGVAPG and AGVPGLGVG peptides reproduced EDP effects through RPSA binding.ConclusionsOur data suggests that matrikines induce cancer cell blebbing and extracellular vesicle release through RPSA binding, favouring dissemination, cell-to-cell communication and growth of cancer cells in metastatic sites.

A study on wound asepsis score for skin closure in surgical procedures

Acute wounds progress through the phases in an orderly fashion for normal healing to occur. Chronic wounds begin the healing process in a similar fashion; however, they have prolonged inflammatory phase in which there is significant destruction of the matrix elements caused by the release of proteolytic enzymes from the neutrophils. This was a comparative study in which patients were studied in two groups. One group comprised of incision closure with subcuticular 3-0 polyamide suture material and the other group comprised of closure with 2 - octylcyanoacrylate adhesive glue. The ASEPSIS score on 5th day shows a mean score of 0.12 for the adhesive glue group and a mean score of 0.28 for subcuticular skin suturing group (P=0.4). Here again there is a numerical difference in favor of adhesive glue group which n is statistically insignificant.

The Wrath of Pancreatic Enzymes: A Rare Case of Hematuria

Acute pancreatitis (AP) is a common gastrointestinal disease associated with many local complications including peripancreatic fluid collections, pseudocysts and necrosis. We present an unusual complication of AP: destruction of the ureter from pancreatic enzymes.A 60-year-old male with a history of former tobacco and alcohol abuse presented to the ED with a 3-day history of periumbilical pain and 1-day of hematuria. Vital signs on admission were within normal limits, physical exam was notable for periumbilical and suprapubic tenderness. Urinalysis showed a significant amount of blood, >100 rbc/hpf, 100mg/dl of protein. Lipase was 259 IU/L. Computed tomography (CT) without contrast showed significant peri-pancreatic stranding around the body and tail of the pancreas with small calcifications in the head. Subsequent CT urogram with contrast showed necrotizing tissue in the body of pancreas, a fluid collection adjacent to the left ureter and evidence of a ruptured left calyx without hydroureteronephrosis. A left ureteral stent and nephrostomy tube were placed. Patients course was complicated by DIC and ARDS, he was discharged 10 days later without any further episodes of hematuria. This case demonstrated multiple complications of severe pancreatitis including a rare manifestation of direct visceral damage. Pancreatic inflammation can have a local effect with involvement of the perirenal fascia in 82% of patients with severe pancreatitis. Release of proteolytic enzymes into the peripancreatic fat is suspected to cause inflammation of the retroperitoneal fascial planes. This local inflammation is noted as perirenal fat stranding on CT imaging. However, progression of this inflammation into direct ureteral damage is exceedingly rare and rupture is usually associated with hydronephrosis and ureteral obstruction. According to a 2009 review, only 27 cases have been reported in the literature. Our case is unique as there is ureteral injury causing rupture but no evidence of hydronephrosis on imaging. Furthermore, the majority of cases with hydronephrosis and ureteral injury are on the right side. Due to the evidence of calcifications in the pancreas on imaging, it is suspected that our patient had acute on chronic pancreatitis causing direct proteolytic injury to the ureter without any concurrent obstruction or hydronephrosis. The case serves as a reminder that two presenting complaints are often correlated to an underlying pathophysiology.1246_A.tif Figure 1: Severe pancreatitis with necrosis1246_B.tif Figure 2: Coronal MIP showing evidence of extravasation of contrast from ureter

Pure platelet rich plasma injections in the management of knee osteoarthritis

Introduction: Osteoarthritis is a degenerative joint disease. It is a slowly progressive disease that affects individuals from the fourth decade and continues to progress at a variable rate. Knee is the most common joint affected in this condition. Osteoarthritis of the knee is a common problem in India among the population above 40 years of age. Platelets have known roles in coagulation, inflammatory processes, and immunity modulation. Moreover, during degranulation, platelets release various cytokines and growth factors (vascular endothelial growth factor, platelet-derived growth factor, transforming growth factor-B, insulin growth factor-I, and hepatocyte growth factor) which promote angiogenesis, tissue remodelling, and wound healing. In addition, platelets can reduce painful symptoms by an unknown mechanism which is apparently dependent on the release of proteases with analgesic properties. Pure Platelet rich plasma is a rapid and cost effective method which decreases the symptoms of osteoarthritis for a variable period and results in better functional outcome. Materials and Methods: This is a prospective observational study conducted on 100 knees in 75 patients suffering from Osteoarthritis treated from Feb 2017 till Jan 2018, in the Department Of Orthopaedics, Dr D. Y. Patil Hospital, Pimpri, Pune. All the baseline characteristics of the patients, like demographics, symptoms, Kellegren and Lawrence criteria and functional outcome were recorded. The patients were treated at our hospital with Pure Platelet rich plasma and followed up for a minimum period of nine months. The follow-up information included range of motion, scoring, patient satisfaction and overall outcome of the procedure in terms of patient acceptance. The data was analyzed to determine the functional outcome at nine months.Results: There was satisfaction in terms of symptoms of pain and range of movements of the knee. 37% patients showed excellent prognosis, good and fair prognosis was showed by 30% of the patients while only 3% people showed poor prognosis after a follow up of nine months duration.Conclusion: Pure Platelet Rich Plasma was not only associated with excellent functional outcome and short term recovery in terms of patient satisfaction and range of movements of the knee with decrease in pain nine months post-procedure but also was found to be cost effective.

Dynamics of Transcription Regulation in Human Bone Marrow Myeloid Differentiation to Mature Blood Neutrophils

SummaryNeutrophils are short-lived blood cells that play a critical role in host defense against infections. To better comprehend neutrophil functions and their regulation, we provide a complete epigenetic overview, assessing important functional features of their differentiation stages from bone marrow-residing progenitors to mature circulating cells. Integration of chromatin modifications, methylation, and transcriptome dynamics reveals an enforced regulation of differentiation, for cellular functions such as release of proteases, respiratory burst, cell cycle regulation, and apoptosis. We observe an early establishment of the cytotoxic capability, while the signaling components that activate these antimicrobial mechanisms are transcribed at later stages, outside the bone marrow, thus preventing toxic effects in the bone marrow niche. Altogether, these data reveal how the developmental dynamics of the chromatin landscape orchestrate the daily production of a large number of neutrophils required for innate host defense and provide a comprehensive overview of differentiating human neutrophils.

Gambogic acid induces cell apoptosis through endoplasmic reticulum stress triggered inhibition of Akt signaling pathways in extranodal NK/T-cell lymphoma cells

As the chemotherapeutic resistance of extranodal NK/T-cell lymphoma (ENKTL) rises year by year, searching for novel chemoprevention compounds has become imminent. Gambogic acid (GA) has recently been shown to have anti-tumor effects, but its role and underling mechanism in ENKTL are rather elusive. In the present study, we showed that GA inhibited the cell growth and potently induced the apoptosis of ENKTL cells in vitro in a time- and concentration-dependent manner. Furthermore, GA induced cell death through endoplasmic reticulum stress (ERS) mediated suppression of Akt signaling pathways and finally the release of the caspase-3 proteases. Overall, our data provided evidences supporting GA as a potential therapeutic agent for ENKTL, which may facilitate further preclinical development of anti-tumor drugs.

Pro-inflammatory effects of e-cigarette vapour condensate on human alveolar macrophages

ObjectiveVaping may increase the cytotoxic effects of e-cigarette liquid (ECL). We compared the effect of unvaped ECL to e-cigarette vapour condensate (ECVC) on alveolar macrophage (AM) function.MethodsAMs were treated with...

PO-211 Profiling the alternative splicing landscape of senescent cells

IntroductionCellular senescence, defined by an irreversible cell cycle arrest in response to potentially oncogenic stimuli, has been described as a protective mechanism in tumourigenesis and a therapeutic target in cancer. The senescence-associated secretory phenotype (SASP) is a pro-inflammatory response by senescent cells involving the release of cytokines, chemokines, growth factors and proteases that, in a cancer progression context, may be beneficial by the elimination of senescent cells or deleterious when triggering angiogenesis, cell proliferation and epithelial-to-mesenchymal transition. Despite senescence’s importance in cancer and the suggested role of alternative splicing in its regulation, the transcriptional heterogeneity of senescent cells has, to our knowledge, been extensively characterised only at the gene expression level.Material and methodsNext-generation sequencing of RNA (RNA-seq) allows alternative splicing quantification with unprecedented precision. The inclusion level of an exon is commonly quantified by its percent-spliced-in (PSI) value, i.e. the proportion of RNA-seq reads providing evidence supporting its inclusion. However, a PSI ratio does not incorporate information about the number of reads used in the quantification of the cognate alternative splicing event, directly proportional to the precision of its estimate. Beta distributions can be exploited in modelling inclusion levels, using reads supporting exon inclusion and exclusion as surrogates of the distribution’s shape parameters. We employed a computational pipeline, based on fitted beta distributions, to accurately quantify and compare alternative splicing across different types of senescent cells, relying both on public and in-house RNA-seq datasets.Results and discussionsOur analyses reproducibly identified, at a transcriptome-wide level, the alternative splicing changes specifically related with replicative and different types of induced senescence in multiple types of cells. For instance, Ras-induced senescence appears to associate with alterations in the splicing of genes involved in the secretory pathway and intracellular trafficking.ConclusionDifferential splicing analyses based on beta distribution modelling contribute to elucidate the specific alternative splicing signatures of different types of senescent cells, providing insights for targeting senescence in cancer therapies.

Extracellular release of virulence factor major surface protease via exosomes in Leishmania infantum promastigotes

BackgroundThe Leishmania spp. protozoa are introduced into humans through a sand fly blood meal, depositing the infectious metacyclic promastigote form of the parasite into human skin. Parasites enter a variety of host cells, although a majority are found in macrophages where they replicate intracellularly during chronic leishmaniasis. Symptomatic leishmaniasis causes considerable human morbidity in endemic regions. The Leishmania spp. evade host microbicidal mechanisms partially through virulence-associated proteins such as the major surface protease (MSP or GP63), to inactivate immune factors in the host environment. MSP is a metalloprotease encoded by a tandem array of genes belonging to three msp gene classes, whose mRNAs are differentially expressed in different life stages of the parasite. Like other cells, Leishmania spp. release small membrane-bound vesicles called exosomes into their environment. The purpose of this study was to detect MSP proteins in exosomal vesicles of Leishmania spp. protozoa.MethodsUsing mass spectrometry data we determined the profile of MSP class proteins released in L. infantum exosomes derived from promastigotes in their avirulent procyclic (logarithmic) stage and virulent stationary and metacyclic stages. MSP protein isoforms belonging to each of the three msp gene classes could be identified by unique peptides.ResultsMetacyclic promastigote exosomes contained the highest, and logarithmic exosomes had the lowest abundance of total MSP. Among the MSP classes, MSPC class had the greatest variety of isoforms, but was least abundant in all exosomes. Nonetheless, all MSP classes were present at higher levels in exosomes released from stationary or metacyclic promastigotes than logarithmic promastigotes.ConclusionsThe data suggest the efficiency of exosome release may be more important than the identity of MSP isoform in determining the MSP content of Leishmania spp. exosomes.

Luteolin attenuates neutrophilic oxidative stress and inflammatory arthritis by inhibiting Raf1 activity

Neutrophils play a significant role in inflammatory tissue injury. Activated neutrophils produce reactive oxygen species (ROS), release proteases, and form neutrophil extracellular traps (NETs), significantly affecting the pathogenesis of inflammatory arthritis. We examined the therapeutic effects of luteolin, a flavone found in many plants, in neutrophilic inflammation and on acute inflammatory arthritis. Luteolin significantly inhibited superoxide anion generation, ROS production, and NET formation in human neutrophils. The increase in elastase release, CD11b expression, and chemotaxis was also inhibited by luteolin. Luteolin significantly suppressed phosphorylation of extracellular signal-regulated kinase (Erk) and mitogen-activated protein kinase kinase-1 (MEK-1), but not c-Jun N-terminal kinase (JNK) and p38 mitogen–activated protein kinase (MAPK). Analysis of the molecular mechanism further revealed that luteolin acts as a Raf-1 inhibitor. In mice with complete Freund’s adjuvant-induced arthritis, luteolin ameliorated neutrophil infiltration as well as the thickness of paw edema and ROS production. In conclusion, in addition to its known ROS scavenging effect, this study is the first to provide evidence that luteolin diminishes human neutrophil inflammatory responses by inhibiting Raf1-MEK-1-Erk. Our results focused on the importance of neutrophil activation in inflammatory tissue injury and offer opportunities for the development of luteolin’s therapeutic potential to attenuate neutrophilic inflammatory diseases.

Mitophagy in Intestinal Epithelial Cells Triggers Adaptive Immunity during Tumorigenesis

In colorectal cancer patients, a high density of cytotoxic CD8+ Tcells in tumors is associated with better prognosis. Using a Stat3 loss-of-function approach in two wnt/β-catenin-dependent autochthonous models of sporadic intestinal tumorigenesis, we unravel a complex intracellular process in intestinal epithelial cells (IECs) that controls the induction of a CD8+ Tcell based adaptive immune response. Elevated mitophagy in IECs causes iron(II)-accumulation in epithelial lysosomes, in turn, triggering lysosomal membrane permeabilization. Subsequent release of proteases into the cytoplasm augments MHC class I presentation and activation of CD8+ Tcells via cross-dressing of dendritic cells. Thus, our findings highlight a so-far-unrecognized link between mitochondrial function, lysosomal integrity, and MHC class I presentation in IECs and suggest that therapies triggering mitophagy or inducing LMP in IECs may prove successful in shifting the balance toward anti-tumor immunity in colorectal cancer.

Evaluation of the toxicity effects of silk fibroin on human lymphocytes and monocytes.

Silk fibroin nanoparticles (SFNPs) as a natural polymer have been utilized in biomedical applications such as suture, tissue engineering-based scaffolds, and drug delivery carriers. Since there is little data regarding the toxicity effects on different cells and tissues, we aimed to determine the toxicity mechanisms of SFNPs on human lymphocytes and monocytes based on reliable methods. Our results showed that SFNPs (0.5, 1, and 2mg/mL) induced oxidative stress via increasing reactive oxygen species production, mitochondrial membrane potential (∆Ψ) collapse, which was correlated to cytochrome c release and Adenosine diphosphate (ADP)/Adenosine tri phosphate (ATP) ratio increase as well as lysosomal as another toxicity mechanism, which led to cytosolic release of lysosomal digestive proteases, phosphor lipases, and apoptosis signaling. Taken together, these data suggested that SFNPs toxicity was associated with mutual mitochondrial/lysosomal cross-talk and oxidative stress on human lymphocytes and monocytes with activated apoptosis signaling.

An adjuvant-free mouse model of anaphylaxis to saline-soluble wheat protein

Abstract Wheat is a major food that can trigger life-threatening systemic anaphylaxis. Mechanism of sensitization to wheat anaphylaxis is not completely understood at present. For example, whether or not skin exposure to saline-soluble wheat protein (SSWP) can cause sensitization is unknown. Here we tested the hypothesis that transdermal exposure (TDE) to SSWP from durum wheat results in IgE response and clinical sensitization for anaphylaxis. Groups of Balb/c mice weaned onto a plant free diet received six TDEs with SSWP (1 mg/mouse/week) or saline over a 6 week period without adjuvant. Blood was analyzed for IgE responses by ELISA, and anaphylaxis and mucosal mast cell protease (mMCP-1) release upon challenge were quantified. Spleen tissue was analyzed for cytokines using a protein microarray system. Results showed significant IgE response, hypothermia shock response and elevation of mMCP-1 upon challenge. Spleen analysis showed significant elevations of IL-4/5/7/10/17B,E,F, and TSLP and significant reductions of IL-1a and IL-6. The following cytokines were not significantly altered: IL-1b/2/9/13/17A/22/23, IFNg and TNFa.

Murine neutrophils treated with alphaB-crystallin reduce IL-12p40 production by dendritic cells.

Neutrophils are essential in the fight against invading pathogens. They utilize antimicrobial effector mechanisms, such as phagocytosis, release of proteases and other antimicrobial products, robust oxidative bursts and neutrophil extracellular traps to combat infections. Neutrophils also modulate immune responses through the production of eicosanoids, cytokines and chemokines, as well as via direct communication with other immune cells. This system of high-intensity offense against pathogens is exquisitely balanced through regulation to limit damage to host tissue. Unfortunately, the control of neutrophils is not failproof. In cases of sterile injury, autoimmunity and even during an infection, neutrophils can cause tissue destruction and become detrimental to the host. For that reason, there is a need to find means to regulate the aberrant activation of these cells. We found that alphaB-crystallin (αBC), a heat-shock protein known to have anti-inflammatory abilities, affects certain properties of mouse neutrophils that subsequently influence the pro-inflammatory state of antigen-presenting cells (APCs). More specifically, αBC mediated small but significant increases in the levels of IL-10 and matrix metalloproteinase 8, and altered hydrogen peroxide secretion by stimulated neutrophils. Further, the heat-shock protein influenced the communication between neutrophils and dendritic cells by decreasing the production of pro-inflammatory cytokines, specifically IL-12p40, by the APCs. αBC could thus contribute to dampening neutrophil inflammatory responses by impacting the effect of neutrophils on other immune cells.

FIBROKINE™ Peptides: A Broad‐Spectrum of Anti‐Fibrotic Chemokine Peptides to Treat Organ Fibrosis

ObjectiveFibrotic diseases are associated with 45% of deaths in the United States but effective therapy does not exist, representing a major unmet medical need. The scope of clinical and etiological manifestations is diverse among fibrotic disorders, yet they share an underlying irritation that promotes the release of various growth factors, proteolytic enzymes, angiogenic factors, and fibrogenic cytokines/chemokines. Persistence of this process causes increased, excessive accumulation of extracellular matrix components and chronic inflammation that progressively alters the cellular and structural architecture of the tissue, decreasing functionality. With progression, these changes can eventually lead to organ dysfunction and death. It is well accepted that common fibrotic diseases such as cardiac fibrosis, scleroderma, idiopathic pulmonary fibrosis, diabetic nephropathy, liver cirrhosis, rheumatoid arthritis, atherosclerosis, and nepthritis share causative factors. Our group has recently designed and developed a class of chemokine‐derived FIBROKINE™ peptides that successfully target the underlying causes of common fibrotic diseases and are capable of halting, reversing, and/or eliminating fibrosis or the underlying molecular processes causing fibrosis.MethodsEvidence has shown the unique multifunctional role of chemokines and their ubiquitous modulation of fibrotic processes. In this study, we test our newly generated class of chemokine‐derived FIBROKINE™ peptides, developed by in silico prediction‐based functional peptide design. Our goal is to advance the mechanistic knowledge of how FIBROKINE™ peptides decrease fibrosis to improve therapy. The efficacy of several FIBROKINE™ peptides was tested via functional assays for cell motility, proliferation, apoptosis, metabolic activity, angiogenic effects, and altered gene expression and protein profiles. Our in vitro and ex vivo experimental testing systems included primary human and murine dermal fibroblasts, endothelial cells, keratinocytes, cardiac fibroblasts, and cardiomyocytes, in both direct and indirect co‐cultures as well as individually.ResultsWe found that FIBROKINE™ peptides acted as potent and efficient antagonists of both chemotaxis and cellular function induced by pro‐fibrotic chemokines. Among the broad spectrum of cellular affects, FIBROKINE™ peptides reduced the expression of α‐Smooth Muscle Actin and reduced mRNA and protein secretion levels of Collagen I, Laminin, Fibronectin, and Tenascin C in both dermal and cardiac fibroblasts after induction by pro‐fibrotic TGF‐β. Furthermore, FIBROKINE™ peptides inhibited the TGF‐β1‐mediated activation of epithelial–mesenchymal transition (EMT), in keratinocytes and dermal fibroblast co‐cultures, a known feature of fibrosis. Lastly, treatment of endothelial cells with FIBROKINE™ peptides significantly inhibited VEGF‐induced endothelial motility and tube formation in vitro, properties critical for angiogenesis.ConclusionOur data suggest FIBROKINE™ peptides mimic biological activities of natural chemokines on fibrosis‐inducing cell types. These data further reveal the effectiveness of the anti‐fibrotic design of FIBROKINE™ peptides as novel, targeted therapeutic solutions that are capable of treating fibrotic conditions through disruption of multiple disease‐causing mechanisms.Support or Funding InformationThis work was supported by grants from NIAMS (AR68317 CCY) and support in kind from University of Pittsburgh School of NursingThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Advances in the knowledge about human milk proteins

The mammary gland and maternal milk are the product of millions of years of evolution that resul ted in an optimal composition that sustains the growth and development of newborns and infants. Maternal milk supports the growth, adaptation and survival of this immature organism. Recent studies have detected 1606 different proteins in human milk, most of them synthesized in the acini of the glandular tissue while others originate from distant organs such as the lymphoid tissue and the digestive tract. Maternal milk enzymes modify its proteins and liberate peptides with antimicrobial, antihypertensive or stimulatory activities. This proteolytic activity occurs at specific sites in peptide chains. To prevent the extemporaneous activation of these proteolytic enzymes, that would result in inflammatory processes, maternal milk also contains inhibitory peptides that together with the stimulatory peptides conform a complex regulatory system. Some enzymes in maternal milk main tain their activity in the gastrointestinal tract of infants and compensate for the decreased activity of digestive tract enzymes in newborns. Thus, the milk enterokynase stimulates the release of pancreatic proteases as it induces the liberation of cholecystokynin/pancreozymin. The bile salt-activated lipase of human milk is activated in the duodenum by the infants' bile salts and partially compensates for the low levels of pancreatic lipase in newborns. These milk enzymes probably contribute to the nutrition of premature infants as they increase the availability of amino acids and peptides in their upper gastrointestinal tract; furthermore, as their intestinal epithelium is more permeable to peptides and partially digested protein this may help induce immune tolerance. The most relevant issues in the physiology and composition of the maternal milk are presented in this review.

Calcium signalling in the acinar environment of the exocrine pancreas: physiology and pathophysiology

Key points Ca2+ signalling in different cell types in exocrine pancreatic lobules was monitored simultaneously and signalling responses to various stimuli were directly compared.Ca2+ signals evoked by K+‐induced depolarization were recorded from pancreatic nerve cells. Nerve cell stimulation evoked Ca2+ signals in acinar but not in stellate cells.Stellate cells are not electrically excitable as they, like acinar cells, did not generate Ca2+ signals in response to membrane depolarization.The responsiveness of the stellate cells to bradykinin was markedly reduced in experimental alcohol‐related acute pancreatitis, but they became sensitive to stimulation with trypsin.Our results provide fresh evidence for an important role of stellate cells in acute pancreatitis. They seem to be a critical element in a vicious circle promoting necrotic acinar cell death. Initial trypsin release from a few dying acinar cells generates Ca2+ signals in the stellate cells, which then in turn damage more acinar cells causing further trypsin liberation. Physiological Ca2+ signals in pancreatic acinar cells control fluid and enzyme secretion, whereas excessive Ca2+ signals induced by pathological agents induce destructive processes leading to acute pancreatitis. Ca2+ signals in the peri‐acinar stellate cells may also play a role in the development of acute pancreatitis. In this study, we explored Ca2+ signalling in the different cell types in the acinar environment of the pancreatic tissue. We have, for the first time, recorded depolarization‐evoked Ca2+ signals in pancreatic nerves and shown that whereas acinar cells receive a functional cholinergic innervation, there is no evidence for functional innervation of the stellate cells. The stellate, like the acinar, cells are not electrically excitable as they do not generate Ca2+ signals in response to membrane depolarization. The principal agent evoking Ca2+ signals in the stellate cells is bradykinin, but in experimental alcohol‐related acute pancreatitis, these cells become much less responsive to bradykinin and then acquire sensitivity to trypsin. Our new findings have implications for our understanding of the development of acute pancreatitis and we propose a scheme in which Ca2+ signals in stellate cells provide an amplification loop promoting acinar cell death. Initial release of the proteases kallikrein and trypsin from dying acinar cells can, via bradykinin generation and protease‐activated receptors, induce Ca2+ signals in stellate cells which can then, possibly via nitric oxide generation, damage more acinar cells and thereby cause additional release of proteases, generating a vicious circle.

Neutrophil Adhesion Is a Prerequisite for Antibody-Mediated Proteolytic Tissue Damage in Experimental Models of Epidermolysis Bullosa Acquisita

Although uncontrolled proteolytic activity mediated by activated neutrophils is a major reason for tissue damage, therapeutic approaches using protease inhibitors are inefficient. Here, we investigated the role of the immune complex-induced neutrophil adhesion and protease release in tissue damage. We show both invitro and invivo that immune complex-mediated neutrophil adhesion to the target tissue depends on β2 integrins. Without affecting elastase or reactive oxygen species release, blocking of adhesion drastically inhibited tissue damage in an experimental model of autoantibody-mediated skin blistering disease. By using a cell-bound fluorescent resonance energy transfer-based elastase sensor, we detected elastase enzyme activity on the surface of adherent cells resistant to protease inhibitors. Inhibitor resistance was lost by CD18 blockade or deficiency invitro and invivo. Immune complex-induced neutrophil adhesion created an enclosed protected space between the cell and its target structure where proteinases and reactive oxygen species can execute their tissue-damaging effect. Because immune complex-induced neutrophil adhesion represents an indispensable step for tissue damage of many diseases, our findings may facilitate the development of strategies for the treatment of such disorders.