Publications by authors named "Sabrina Sonda"

35 Publications

CD8 T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naive precursor frequency and TCR promiscuity.

Immunity 2021 05 15;54(5):1066-1082.e5. Epub 2021 Apr 15.

Department of Infectious Diseases, Austin Hospital, Heidelberg, VIC 3084, Australia; Department of Medicine and Radiology, The University of Melbourne, Parkville, VIC 3000, Australia; Data Analytics Research and Evaluation (DARE) Centre, Austin Health and The University of Melbourne, Heidelberg, VIC 3084, Australia.

To better understand primary and recall T cell responses during coronavirus disease 2019 (COVID-19), it is important to examine unmanipulated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells. By using peptide-human leukocyte antigen (HLA) tetramers for direct ex vivo analysis, we characterized CD8 T cells specific for SARS-CoV-2 epitopes in COVID-19 patients and unexposed individuals. Unlike CD8 T cells directed toward subdominant epitopes (B7/N, A2/S, and A24/S) CD8 T cells specific for the immunodominant B7/N epitope were detected at high frequencies in pre-pandemic samples and at increased frequencies during acute COVID-19 and convalescence. SARS-CoV-2-specific CD8 T cells in pre-pandemic samples from children, adults, and elderly individuals predominantly displayed a naive phenotype, indicating a lack of previous cross-reactive exposures. T cell receptor (TCR) analyses revealed diverse TCRαβ repertoires and promiscuous αβ-TCR pairing within B7/NCD8 T cells. Our study demonstrates high naive precursor frequency and TCRαβ diversity within immunodominant B7/N-specific CD8 T cells and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses.
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http://dx.doi.org/10.1016/j.immuni.2021.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8049468PMC
May 2021

Serine administration as a novel prophylactic approach to reduce the severity of acute pancreatitis during diabetes in mice.

Diabetologia 2020 09 8;63(9):1885-1899. Epub 2020 May 8.

Swiss Hepato-Pancreato-Biliary Center, Department of Visceral and Transplantation Surgery, University Hospital Zurich, Zurich, Switzerland.

Aims/hypothesis: Compared with the general population, individuals with diabetes have a higher risk of developing severe acute pancreatitis, a highly debilitating and potentially lethal inflammation of the exocrine pancreas. In this study, we investigated whether 1-deoxysphingolipids, atypical lipids that increase in the circulation following the development of diabetes, exacerbate the severity of pancreatitis in a diabetic setting.

Methods: We analysed whether administration of an L-serine-enriched diet to mouse models of diabetes, an established method for decreasing the synthesis of 1-deoxysphingolipids in vivo, reduced the severity of acute pancreatitis. Furthermore, we elucidated the molecular mechanisms underlying the lipotoxicity exerted by 1-deoxysphingolipids towards rodent pancreatic acinar cells in vitro.

Results: We demonstrated that L-serine supplementation reduced the damage of acinar tissue resulting from the induction of pancreatitis in diabetic mice (average histological damage score: 1.5 in L-serine-treated mice vs 2.7 in the control group). At the cellular level, we showed that L-serine decreased the production of reactive oxygen species, endoplasmic reticulum stress and cellular apoptosis in acinar tissue. Importantly, these parameters, together with DNA damage, were triggered in acinar cells upon treatment with 1-deoxysphingolipids in vitro, suggesting that these lipids are cytotoxic towards pancreatic acinar cells in a cell-autonomous manner. In search of the initiating events of the observed cytotoxicity, we discovered that 1-deoxysphingolipids induced early mitochondrial dysfunction in acinar cells, characterised by ultrastructural alterations, impaired oxygen consumption rate and reduced ATP synthesis.

Conclusions/interpretation: Our results suggest that 1-deoxysphingolipids directly damage the functionality of pancreatic acinar cells and highlight that an L-serine-enriched diet may be used as a promising prophylactic intervention to reduce the severity of pancreatitis in the context of diabetes.
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http://dx.doi.org/10.1007/s00125-020-05156-xDOI Listing
September 2020

Akt1 signalling supports acinar proliferation and limits acinar-to-ductal metaplasia formation upon induction of acute pancreatitis.

J Pathol 2020 01 22;250(1):42-54. Epub 2019 Nov 22.

Swiss Hepato-Pancreato-Biliary Center, Department of Visceral and Transplantation Surgery, University Hospital, Zurich, Switzerland.

Molecular signalling mediated by the phosphatidylinositol-3-kinase (PI3K)-Akt axis is a key regulator of cellular functions. Importantly, alteration of the PI3K-Akt signalling underlies the development of different human diseases, thus prompting the investigation of the pathway as a molecular target for pharmacologic intervention. In this regard, recent studies showed that small molecule inhibitors of PI3K, the upstream regulator of the pathway, reduced the development of inflammation during acute pancreatitis, a highly debilitating and potentially lethal disease. Here we investigated whether a specific reduction of Akt activity, by using either pharmacologic Akt inhibition, or genetic inactivation of the Akt1 isoform selectively in pancreatic acinar cells, is effective in ameliorating the onset and progression of the disease. We discovered that systemic reduction of Akt activity did not protect the pancreas from initial damage and only transiently delayed leukocyte recruitment. However, reduction of Akt activity decreased acinar proliferation and exacerbated acinar-to-ductal metaplasia (ADM) formation, two critical events in the progression of pancreatitis. These phenotypes were recapitulated upon conditional inactivation of Akt1 in acinar cells, which resulted in reduced expression of 4E-BP1, a multifunctional protein of key importance in cell proliferation and metaplasia formation. Collectively, our results highlight the critical role played by Akt1 during the development of acute pancreatitis in the control of acinar cell proliferation and ADM formation. In addition, these results harbour important translational implications as they raise the concern that inhibitors of PI3K-Akt signalling pathways may negatively affect the regeneration of the pancreas. Finally, this work provides the basis for further investigating the potential of Akt1 activators to boost pancreatic regeneration following inflammatory insults. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5348DOI Listing
January 2020

The Zinc Transporter Zip7 Is Downregulated in Skeletal Muscle of Insulin-Resistant Cells and in Mice Fed a High-Fat Diet.

Cells 2019 07 1;8(7). Epub 2019 Jul 1.

College of Health and Medicine, School of Health Sciences, University of Tasmania, Hobart, Tasmania 7005, Australia.

Background: The zinc transporter Zip7 modulates zinc flux and controls cell signaling molecules associated with glucose metabolism in skeletal muscle. The present study evaluated the role of Zip7 in cell signaling pathways involved in insulin-resistant skeletal muscle and mice fed a high-fat diet.

Methods: Insulin-resistant skeletal muscle cells were prepared by treatment with an inhibitor of the insulin receptor, HNMPA-(AM)3 or palmitate, and Zip7 was analyzed along with pAkt, pTyrosine and Glut4. Similarly, mice fed normal chow (NC) or a high-fat diet (HFD) were also analyzed for protein expression of Glut4 and Zip7. An overexpression system for Zip7 was utilized to determine the action of this zinc transporter on several genes implicated in insulin signaling and glucose control.

Results: We identified that Zip7 is upregulated by glucose in normal skeletal muscle cells and downregulated in insulin-resistant skeletal muscle. We also observed (as expected) a decrease in pAkt and Glut4 in the insulin-resistant skeletal muscle cells. The overexpression of Zip7 in skeletal muscle cells led to the modulation of key genes involved in the insulin signaling axis and glucose metabolism including , , , , , , , and . In an mouse model, we identified a reduction in Glut4 and Zip7 in the skeletal muscle of mice fed a HFD compared to NC controls.

Conclusions: These data suggest that Zip7 plays a role in skeletal muscle insulin signaling and is downregulated in an insulin-resistant, and HFD state. Understanding the molecular mechanisms of Zip7 action will provide novel opportunities to target this transporter therapeutically for the treatment of insulin resistance and type 2 diabetes.
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http://dx.doi.org/10.3390/cells8070663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678147PMC
July 2019

Targeting the Zinc Transporter ZIP7 in the Treatment of Insulin Resistance and Type 2 Diabetes.

Nutrients 2019 Feb 15;11(2). Epub 2019 Feb 15.

College of Health and Medicine, School of Health Sciences, University of Tasmania, TAS 7005, Australia.

Type 2 diabetes mellitus (T2DM) is a disease associated with dysfunctional metabolic processes that lead to abnormally high levels of blood glucose. Preceding the development of T2DM is insulin resistance (IR), a disorder associated with suppressed or delayed responses to insulin. The effects of this response are predominately mediated through aberrant cell signalling processes and compromised glucose uptake into peripheral tissue including adipose, liver and skeletal muscle. Moreover, a major factor considered to be the cause of IR is endoplasmic reticulum (ER) stress. This subcellular organelle plays a pivotal role in protein folding and processes that increase ER stress, leads to maladaptive responses that result in cell death. Recently, zinc and the proteins that transport this metal ion have been implicated in the ER stress response. Specifically, the ER-specific zinc transporter ZIP7, coined the "gate-keeper" of zinc release from the ER into the cytosol, was shown to be essential for maintaining ER homeostasis in intestinal epithelium and myeloid leukaemia cells. Moreover, ZIP7 controls essential cell signalling pathways similar to insulin and activates glucose uptake in skeletal muscle. Accordingly, ZIP7 may be essential for the control of ER localized zinc and mechanisms that disrupt this process may lead to ER-stress and contribute to IR. Accordingly, understanding the mechanisms of ZIP7 action in the context of IR may provide opportunities to develop novel therapeutic options to target this transporter in the treatment of IR and subsequent T2DM.
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http://dx.doi.org/10.3390/nu11020408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6412268PMC
February 2019

Local hyperthyroidism promotes pancreatic acinar cell proliferation during acute pancreatitis.

J Pathol 2019 06 4;248(2):217-229. Epub 2019 Apr 4.

Swiss Hepato-Pancreato-Biliary Center, Department of Visceral and Transplantation Surgery, University Hospital, Zurich, Switzerland.

Proliferation of pancreatic acinar cells is a critical process in the pathophysiology of pancreatic diseases, because limited or defective proliferation is associated with organ dysfunction and patient morbidity. In this context, elucidating the signalling pathways that trigger and sustain acinar proliferation is pivotal to develop therapeutic interventions promoting the regenerative process of the organ. In this study we used genetic and pharmacological approaches to manipulate both local and systemic levels of thyroid hormones to elucidate their role in acinar proliferation following caerulein-mediated acute pancreatitis in mice. In addition, molecular mechanisms mediating the effects of thyroid hormones were identified by genetic and pharmacological inactivation of selected signalling pathways.In this study we demonstrated that levels of the thyroid hormone 3,3',5-triiodo-l-thyronine (T3) transiently increased in the pancreas during acute pancreatitis. Moreover, by using genetic and pharmacological approaches to manipulate both local and systemic levels of thyroid hormones, we showed that T3 was required to promote proliferation of pancreatic acinar cells, without affecting the extent of tissue damage or inflammatory infiltration.Finally, upon genetic and pharmacological inactivation of selected signalling pathways, we demonstrated that T3 exerted its mitogenic effect on acinar cells via a tightly controlled action on different molecular effectors, including histone deacetylase, AKT, and TGFβ signalling.In conclusion, our data suggest that local availability of T3 in the pancreas is required to promote acinar cell proliferation and provide the rationale to exploit thyroid hormone signalling to enhance pancreatic regeneration. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5247DOI Listing
June 2019

Serotonin uptake is required for Rac1 activation in Kras-induced acinar-to-ductal metaplasia in the pancreas.

J Pathol 2018 11 4;246(3):352-365. Epub 2018 Oct 4.

Department of Visceral and Transplant Surgery, University Hospital of Zurich, Zurich, Switzerland.

Pancreatic ductal adenocarcinoma (PDAC), which is the primary cause of pancreatic cancer mortality, is poorly responsive to currently available interventions. Identifying new targets that drive PDAC formation and progression is critical for developing alternative therapeutic strategies to treat this lethal malignancy. Using genetic and pharmacological approaches, we investigated in vivo and in vitro whether uptake of the monoamine serotonin [5-hydroxytryptamine (5-HT)] is required for PDAC development. We demonstrated that pancreatic acinar cells have the ability to readily take up 5-HT in a transport-mediated manner. 5-HT uptake promoted activation of the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for transdifferentiation of acinar cells into acinar-to-ductal metaplasia (ADM), a key determinant in PDAC development. Consistent with the central role played by Rac1 in ADM formation, inhibition of the 5-HT transporter Sert (Slc6a4) with fluoxetine reduced ADM formation both in vitro and in vivo in a cell-autonomous manner. In addition, fluoxetine treatment profoundly compromised the stromal reaction and affected the proliferation and lipid metabolism of malignant PDAC cells. We propose that Sert is a promising therapeutic target to counteract the early event of ADM, with the potential to stall the initiation and progression of pancreatic carcinogenesis. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5147DOI Listing
November 2018

Enhanced proliferation of pancreatic acinar cells in MRL/MpJ mice is driven by severe acinar injury but independent of inflammation.

Sci Rep 2018 06 20;8(1):9391. Epub 2018 Jun 20.

Swiss Hepato-Pancreato-Biliary Center, Department of Visceral and Transplantation Surgery, University Hospital, Zurich, Switzerland.

Adult pancreatic acinar cells have the ability to re-enter the cell cycle and proliferate upon injury or tissue loss. Despite this mitotic ability, the extent of acinar proliferation is often limited and unable to completely regenerate the injured tissue or restore the initial volume of the organ, thus leading to pancreatic dysfunction. Identifying molecular determinants of enhanced proliferation is critical to overcome this issue. In this study, we discovered that Murphy Roths Large (MRL/MpJ) mice can be exploited to identify molecular effectors promoting acinar proliferation upon injury, with the ultimate goal to develop therapeutic regimens to boost pancreatic regeneration. Our results show that, upon cerulein-induced acinar injury, cell proliferation was enhanced and cell cycle components up-regulated in the pancreas of MRL/MpJ mice compared to the control strain C57BL/6. Initial damage of acinar cells was exacerbated in these mice, manifested by increased serum levels of pancreatic enzymes, intra-pancreatic trypsinogen activation and acinar cell apoptosis. In addition, MRL/MpJ pancreata presented enhanced inflammation, de-differentiation of acinar cells and acinar-to-ductal metaplasia. Manipulation of inflammatory levels and mitogenic stimulation with the thyroid hormone 5,3-L-tri-iodothyronine revealed that factors derived from initial acinar injury rather than inflammatory injury promote the replicative advantage in MRL/MpJ mice.
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http://dx.doi.org/10.1038/s41598-018-27422-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6010442PMC
June 2018

Inhibition of Class I Histone Deacetylases Abrogates Tumor Growth Factor Expression and Development of Fibrosis during Chronic Pancreatitis.

Mol Pharmacol 2018 08 7;94(2):793-801. Epub 2018 Jun 7.

Swiss Hepato-Pancreato-Biliary Center, Department of Visceral and Transplantation Surgery, University Hospital (M.B., R.C., E.M., E.S., R.G., S.S.) and Center for Integrative Human Physiology (ZIHP), University of Zurich (R.G., S.S.), Zurich, Switzerland; and School of Health Sciences, College of Health and Medicine, University of Tasmania, Launceston, Australia (A.P.H., S.S.)

Pancreatic fibrosis is the hallmark of chronic pancreatitis, a highly debilitating disease for which there is currently no cure. The key event at the basis of pancreatic fibrosis is the deposition of extracellular matrix proteins by activated pancreatic stellate cells (PSCs). Transforming growth factor (TGF) is a potent profibrotic factor in the pancreas as it promotes the activation of PSC; thus, pharmacologic interventions that effectively reduce TGF expression harbor considerable therapeutic potential in the treatment of chronic pancreatitis. In this study, we investigated whether TGF expression is reduced by pharmacologic inhibition of the epigenetic modifiers histone deacetylases (HDACs). To address this aim, chronic pancreatitis was induced in C57BL/6 mice with serial injections of cerulein, and the selective class 1 HDAC inhibitor MS-275 was administered in vivo in a preventive and therapeutic manner. Both MS-275 regimens potently reduced deposition of extracellular matrix and development of fibrosis in the pancreas after 4 weeks of chronic pancreatitis. Reduced pancreatic fibrosis was concomitant with lower expression of pancreatic TGF and consequent reduced PSC activation. In search of the cell types targeted by the inhibitor, we found that MS-275 treatment abrogated the expression of TGF in acinar cells stimulated by cerulein treatment. Our study demonstrates that MS-275 is an effective antifibrotic agent in the context of experimental chronic pancreatitis and thus may constitute a valid therapeutic intervention for this severe disease.
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http://dx.doi.org/10.1124/mol.117.110924DOI Listing
August 2018

Class I histone deacetylase inhibition improves pancreatitis outcome by limiting leukocyte recruitment and acinar-to-ductal metaplasia.

Br J Pharmacol 2017 Nov 20;174(21):3865-3880. Epub 2017 Sep 20.

Swiss Hepato-Pancreato-Biliary Center, Department of Visceral and Transplantation Surgery, University Hospital of Zurich, Zurich, Switzerland.

Background And Purpose: Pancreatitis is a common inflammation of the pancreas with rising incidence in many countries. Despite improvements in diagnostic techniques, the disease is associated with high risk of severe morbidity and mortality and there is an urgent need for new therapeutic interventions. In this study, we evaluated whether histone deacetylases (HDACs), key epigenetic regulators of gene transcription, are involved in the development of the disease.

Experimental Approach: We analysed HDAC regulation during cerulein-induced acute, chronic and autoimmune pancreatitis using different transgenic mouse models. The functional relevance of class I HDACs was tested with the selective inhibitor MS-275 in vivo upon pancreatitis induction and in vitro in activated macrophages and primary acinar cell explants.

Key Results: HDAC expression and activity were up-regulated in a time-dependent manner following induction of pancreatitis, with the highest abundance observed for class I HDACs. Class I HDAC inhibition did not prevent the initial acinar cell damage. However, it effectively reduced the infiltration of inflammatory cells, including macrophages and T cells, in both acute and chronic phases of the disease, and directly disrupted macrophage activation. In addition, MS-275 treatment reduced DNA damage in acinar cells and limited acinar de-differentiation into acinar-to-ductal metaplasia in a cell-autonomous manner by impeding the EGF receptor signalling axis.

Conclusions And Implications: These results demonstrate that class I HDACs are critically involved in the development of acute and chronic forms of pancreatitis and suggest that blockade of class I HDAC isoforms is a promising target to improve the outcome of the disease.
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http://dx.doi.org/10.1111/bph.13984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5647184PMC
November 2017

Development of autoimmune pancreatitis is independent of CDKN1A/p21-mediated pancreatic inflammation.

Gut 2018 09 3;67(9):1663-1673. Epub 2017 Aug 3.

Department of Visceral and Transplantation Surgery, Swiss HPB Centre, University Hospital Zurich, Zurich, Switzerland.

Objective: Chronic pancreatitis (CP) and autoimmune pancreatitis (AIP) are characterised by different inflammatory processes. If pancreatic inflammation is a prerequisite for autoimmunity is still unclear. AIP is considered mostly a T cell-mediated disease; however, in induction of CP, macrophages play a pivotal role. p21-a member of cyclin-dependent kinase inhibitors-can influence inflammatory processes, in particular can regulate T cell activation and promote macrophage development. We therefore examined the role of p21-mediated inflammation in AIP.

Design: We intercrossed lymphotoxin (LT) overexpressing mice (Tg(Ela1-LTa,b))-a model to study AIP development-with p21-deficient mice. Furthermore, we characterised p21 expression in human AIP and non-AIP specimens.

Results: p21 deficiency in LT mice (LTp21) prevented early pancreatic injury and reduced inflammation. In acinar cells, diminished proliferation and abrogated activation of non-canonical nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) pathway was observed. In contrast, 12-month-old LT mice with and without p21 had similar inflammatory signatures and T-B cell infiltration. Interestingly, LT and LTp21 mice had comparable tertiary lymphoid organs (TLOs), autoantibodies and elevated IgG levels. However, acinar cell proliferation, acinar-to-ductal metaplasia and acinar non-canonical NF-κB pathway activation remained impaired in LTp21 pancreata.

Conclusions: Our findings indicate that p21 is crucial for pancreatic inflammation in LT-driven pancreatic injury. p21 is involved in early acinar secretion of inflammatory mediators that attract innate immune cells. However, p21 is not essential for humoral immune response, accountable for autoimmunity. Remarkably, p21 renders acinar cells less susceptible to proliferation and transdifferentiation. We therefore suggest that AIP can also develop independent of chronic inflammatory processes.
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http://dx.doi.org/10.1136/gutjnl-2016-313458DOI Listing
September 2018

Ibuprofen and diclofenac treatments reduce proliferation of pancreatic acinar cells upon inflammatory injury and mitogenic stimulation.

Br J Pharmacol 2018 01 16;175(2):335-347. Epub 2017 Jun 16.

Swiss Hepato-Pancreato-Biliary Center, Department of Visceral and Transplantation Surgery, University Hospital of Zurich, Zurich, Switzerland.

Background And Purpose: Nonsteroidal anti-inflammatory drugs (NSAIDs) are administered to manage the pain typically found in patients suffering from pancreatitis. NSAIDs also display anti-proliferative activity against cancer cells; however, their effects on normal, untransformed cells are poorly understood. Here, we evaluated whether NSAIDs inhibit the proliferation of pancreatic acinar cells during the development of acute pancreatitis.

Experimental Approach: The NSAIDs ibuprofen and diclofenac were administered to C57BL/6 mice after induction of pancreatitis with serial injections of cerulein. In addition, ibuprofen was administered concomitantly with 3,5,3-L-tri-iodothyronine (T3), which induces acinar cell proliferation in the absence of tissue inflammation. The development of pancreatic inflammation, acinar de-differentiation into metaplastic lesions and acinar proliferation were quantified by histochemical, biochemical and RT-PCR approaches.

Key Results: Therapeutic ibuprofen treatment selectively reduced pancreatic infiltration of activated macrophages in vivo, and M1 macrophage polarization and pro-inflammatory cytokine expression both in vivo and in vitro. Reduced macrophage activation was accompanied by reduced acinar de-differentiation into acinar-to-ductal metaplasia. Acinar proliferation was significantly impaired in the presence of ibuprofen and diclofenac, as demonstrated at both the level of proliferation markers and expression of cell cycle regulators. Ibuprofen also reduced acinar cell proliferation induced by mitogenic stimulation with T3, a treatment that does not elicit pancreatic inflammation.

Conclusions And Implications: Our study provides evidence that the NSAIDs ibuprofen and diclofenac inhibit pancreatic acinar cell division. This suggests that prolonged treatment with these NSAIDs may negatively affect the regeneration of the pancreas and further studies are needed to confirm these findings in a clinical setting.

Linked Articles: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.
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http://dx.doi.org/10.1111/bph.13867DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5758389PMC
January 2018

1-Deoxysphingolipid-induced neurotoxicity involves N-methyl-d-aspartate receptor signaling.

Neuropharmacology 2016 11 23;110(Pt A):211-222. Epub 2016 Mar 23.

Institute of Veterinary Physiology, Vetsuisse Faculty, University of Zurich, Switzerland; Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Switzerland. Electronic address:

1-Deoxysphingolipids (1-deoxySL) are atypical and neurotoxic sphingolipids formed by alternate substrate usage of the enzyme serine-palmitoyltransferase. Pathologically increased 1-deoxySL formation causes hereditary sensory and autosomal neuropathy type 1 (HSAN1) - a progressive peripheral axonopathy. However, the underlying molecular mechanisms by which 1-deoxySL acts are unknown. Herein we studied the effect of 1-deoxysphinganine (1-deoxySA) and its canonical counterpart sphinganine (SA) in aged cultured neurons comparing their outcome on cell survival and cytoskeleton integrity. 1-deoxySA caused rapid neuronal cytoskeleton disruption and modulated important cytoskeletal regulatory and associated components including Rac1, Ezrin and insulin receptor substrate 53. We show that 1-deoxySA is internalized and metabolized downstream to 1-deoxydihydroceramide since inhibition of ceramide synthase protected neurons from 1-deoxySA-mediated cell death. In addition, 1-deoxySA reduced protein levels of N-methyl-d-aspartate receptor (NMDAR) subunit GluN2B, the postsynaptic density protein 95 and induced cleavage of p35 to p25. Notably, blocking NMDAR activation by MK-801 or memantine significantly prevented 1-deoxySA neurotoxicity. Functional studies of differentiating primary neurons via the patch-clamp technique demonstrated that 1-deoxySA irreversibly depolarizes the neuronal membrane potential in an age-dependent manner. Notably, only neuronal cells that displayed functional NMDAR- and NMDA-induced whole-cell currents responded to 1-deoxySA treatment. Furthermore, pre-exposure to the non-competitive antagonist MK-801 blocked the current response of NMDA and glycine, as well as 1-deoxySA. We conclude that 1-deoxySA-induced neurotoxicity compromises cytoskeletal stability and targets NMDAR signaling in an age-dependent manner. Thus stabilization of cytoskeletal structures and/or inhibition of glutamate receptors could be a potential therapeutic approach to prevent 1-deoxySA-induced neurodegeneration.
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http://dx.doi.org/10.1016/j.neuropharm.2016.03.033DOI Listing
November 2016

Inactivation of TGFβ receptor II signalling in pancreatic epithelial cells promotes acinar cell proliferation, acinar-to-ductal metaplasia and fibrosis during pancreatitis.

J Pathol 2016 Feb 28;238(3):434-45. Epub 2015 Nov 28.

Swiss Hepato-Pancreato-Biliary Centre, Department of Visceral and Transplantation Surgery, University Hospital, Zurich, and Zurich Centre for Integrative Human Physiology (ZIHP), University of Zurich, Switzerland.

Determining signalling pathways that regulate pancreatic regeneration following pancreatitis is critical for implementing therapeutic interventions. In this study we elucidated the molecular mechanisms underlying the effects of transforming growth factor-β (TGFβ) in pancreatic epithelial cells during tissue regeneration. To this end, we conditionally inactivated TGFβ receptor II (TGFβ-RII) using a Cre-LoxP system under the control of pancreas transcription factor 1a (PTF1a) promoter, specific for the pancreatic epithelium, and evaluated the molecular and cellular changes in a mouse model of cerulein-induced pancreatitis. We show that TGFβ-RII signalling does not mediate the initial acinar cell damage observed at the onset of pancreatitis. However, TGFβ-RII signalling not only restricts acinar cell replication during the regenerative phase of the disease but also limits ADM formation in vivo and in vitro in a cell-autonomous manner. Analyses of molecular mechanisms underlying the observed phenotype revealed that TGFβ-RII signalling stimulates the expression of cyclin-dependent kinase inhibitors and intersects with the EGFR signalling axis. Finally, TGFβ-RII ablation in epithelial cells resulted in increased infiltration of inflammatory cells in the early phases of pancreatitis and increased activation of pancreatic stellate cells in the later stages of pancreatitis, thus highlighting a TGFβ-based crosstalk between epithelial and stromal cells regulating the development of pancreatic inflammation and fibrosis. Collectively, our data not only contribute to clarifying the cellular processes governing pancreatic tissue regeneration, but also emphasize the conserved role of TGFβ as a tumour suppressor, both in the regenerative process following pancreatitis and in the initial phases of pancreatic cancer.
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http://dx.doi.org/10.1002/path.4666DOI Listing
February 2016

Serotonin promotes acinar dedifferentiation following pancreatitis-induced regeneration in the adult pancreas.

J Pathol 2015 Dec 10;237(4):495-507. Epub 2015 Sep 10.

Swiss Hepato-pancreato-biliary Centre, Department of Visceral and Transplantation Surgery, University Hospital, Zurich, Switzerland.

The exocrine pancreas exhibits a distinctive capacity for tissue regeneration and renewal following injury. This regenerative ability has important implications for a variety of disorders, including pancreatitis and pancreatic cancer, diseases associated with high morbidity and mortality. Thus, understanding its underlying mechanisms may help in developing therapeutic interventions. Serotonin has been recognized as a potent mitogen for a variety of cells and tissues. Here we investigated whether serotonin exerts a mitogenic effect in pancreatic acinar cells in three regenerative models, inflammatory tissue injury following pancreatitis, tissue loss following partial pancreatectomy, and thyroid hormone-stimulated acinar proliferation. Genetic and pharmacological techniques were used to modulate serotonin levels in vivo. Acinar dedifferentiation and cell cycle progression during the regenerative phase were investigated over the course of 2 weeks. By comparing acinar proliferation in the different murine models of regeneration, we found that serotonin did not affect the clonal regeneration of mature acinar cells. Serotonin was, however, required for acinar dedifferentiation following inflammation-mediated tissue injury. Specifically, lack of serotonin resulted in delayed up-regulation of progenitor genes and delayed the formation of acinar-to-ductal metaplasia and defective acinar cell proliferation. We identified serotonin-dependent acinar secretion as a key step in progenitor-based regeneration, as it promoted acinar cell dedifferentiation and the recruitment of type 2 macrophages. Finally, we identified a regulatory Hes1-Ptfa axis in the uninjured adult pancreas, activated by zymogen secretion. Our findings indicated that serotonin plays a critical role in the regeneration of the adult pancreas following pancreatitis by promoting the dedifferentiation of acinar cells.
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http://dx.doi.org/10.1002/path.4595DOI Listing
December 2015

p21(WAF1) (/Cip1) limits senescence and acinar-to-ductal metaplasia formation during pancreatitis.

J Pathol 2015 Feb 6;235(3):502-14. Epub 2014 Nov 6.

Swiss Hepato-Pancreato-Biliary Centre, Department of Visceral and Transplantation Surgery, University Hospital, Zurich, Switzerland.

Trans-differentiation of pancreatic acinar cells into ductal-like lesions, a process defined as acinar-to-ductal metaplasia (ADM), is observed in the course of organ regeneration following pancreatitis. In addition, ADM is found in association with pre-malignant PanIN lesions and correlates with an increased risk of pancreatic adenocarcinoma (PDAC). Human PDAC samples show down-regulation of p21(WAF1) (/Cip1) , a key regulator of cell cycle and cell differentiation. Here we investigated whether p21 down-regulation is implicated in controlling the early events of acinar cell trans-differentiation and ADM formation. p21-mediated regulation of ADM formation and regression was analysed in vivo during the course of cerulein-induced pancreatitis, using wild-type (WT) and p21-deficient (p21(-/-) ) mice. Biochemical and immunohistochemical methods were used to evaluate disease progression over 2 weeks of the disease and during a recovery phase. We found that p21 was strongly up-regulated in WT acinar cells during pancreatitis, while it was absent in ADM areas, suggesting that p21 down-regulation is associated with ADM formation. In support of this hypothesis, p21(-/-) mice showed a significant increase in number and size of metaplasia. In addition, p21 over-expression in acinar cells reduced ADM formation in vitro, suggesting that the protein regulates the metaplastic transition in a cell-autonomous manner. p21(-/-) mice displayed increased expression and relocalization of β-catenin both during pancreatitis and in the subsequent recovery phase. Finally, loss of p21 was accompanied by increased DNA damage and development of senescence. Our findings are consistent with a gate-keeper role of p21 in acinar cells to limit senescence activation and ADM formation during pancreatic regeneration.
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http://dx.doi.org/10.1002/path.4440DOI Listing
February 2015

Deoxysphingolipids, novel biomarkers for type 2 diabetes, are cytotoxic for insulin-producing cells.

Diabetes 2014 Apr 30;63(4):1326-39. Epub 2013 Dec 30.

Division of Endocrinology, Diabetes and Clinical Nutrition, University Hospital Zurich, Zurich, Switzerland.

Irreversible failure of pancreatic β-cells is the main culprit in the pathophysiology of diabetes, a disease that is now a global epidemic. Recently, elevated plasma levels of deoxysphingolipids, including 1-deoxysphinganine, have been identified as a novel biomarker for the disease. In this study, we analyzed whether deoxysphingolipids directly compromise the functionality of insulin-producing Ins-1 cells and primary islets. Treatment with 1-deoxysphinganine induced dose-dependent cytotoxicity with senescent, necrotic, and apoptotic characteristics and compromised glucose-stimulated insulin secretion. In addition, 1-deoxysphinganine altered cytoskeleton dynamics, resulting in intracellular accumulation of filamentous actin and activation of the Rho family GTPase Rac1. Moreover, 1-deoxysphinganine selectively upregulated ceramide synthase 5 expression and was converted to 1-deoxy-dihydroceramides without altering normal ceramide levels. Inhibition of intracellular 1-deoxysphinganine trafficking and ceramide synthesis improved the viability of the cells, indicating that the intracellular metabolites of 1-deoxysphinganine contribute to its cytotoxicity. Analyses of signaling pathways identified Jun N-terminal kinase and p38 mitogen-activated protein kinase as antagonistic effectors of cellular senescence. The results revealed that 1-deoxysphinganine is a cytotoxic lipid for insulin-producing cells, suggesting that the increased levels of this sphingolipid observed in diabetic patients may contribute to the reduced functionality of pancreatic β-cells. Thus, targeting deoxysphingolipid synthesis may complement the currently available therapies for diabetes.
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http://dx.doi.org/10.2337/db13-1042DOI Listing
April 2014

Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole.

Mol Biol Cell 2013 Jun 24;24(12):1974-95. Epub 2013 Apr 24.

Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205, USA.

The obligate intracellular protozoan Toxoplasma gondii actively invades mammalian cells and, upon entry, forms its own membrane-bound compartment, named the parasitophorous vacuole (PV). Within the PV, the parasite replicates and scavenges nutrients, including lipids, from host organelles. Although T. gondii can synthesize sphingolipids de novo, it also scavenges these lipids from the host Golgi. How the parasite obtains sphingolipids from the Golgi remains unclear, as the PV avoids fusion with host organelles. In this study, we explore the host Golgi-PV interaction and evaluate the importance of host-derived sphingolipids for parasite growth. We demonstrate that the PV preferentially localizes near the host Golgi early during infection and remains closely associated with this organelle throughout infection. The parasite subverts the structure of the host Golgi, resulting in its fragmentation into numerous ministacks, which surround the PV, and hijacks host Golgi-derived vesicles within the PV. These vesicles, marked with Rab14, Rab30, or Rab43, colocalize with host-derived sphingolipids in the vacuolar space. Scavenged sphingolipids contribute to parasite replication since alterations in host sphingolipid metabolism are detrimental for the parasite's growth. Thus our results reveal that T. gondii relies on host-derived sphingolipids for its development and scavenges these lipids via Golgi-derived vesicles.
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http://dx.doi.org/10.1091/mbc.E12-11-0827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3681701PMC
June 2013

Introduction of caveolae structural proteins into the protozoan Toxoplasma results in the formation of heterologous caveolae but not caveolar endocytosis.

PLoS One 2012 14;7(12):e51773. Epub 2012 Dec 14.

Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health Baltimore, Maryland, United States of America.

Present on the plasma membrane of most metazoans, caveolae are specialized microdomains implicated in several endocytic and trafficking mechanisms. Caveolins and the more recently discovered cavins are the major protein components of caveolae. Previous studies reported that caveolar invaginations can be induced de novo on the surface of caveolae-negative mammalian cells upon heterologous expression of caveolin-1. However, it remains undocumented whether other components in the transfected cells participate in caveolae formation. To address this issue, we have exploited the protozoan Toxoplasma as a heterologous expression system to provide insights into the minimal requirements for caveogenesis and caveolar endocytosis. Upon expression of caveolin-1, Toxoplasma accumulates prototypical exocytic caveolae 'precursors' in the cytoplasm. Toxoplasma expressing caveolin-1 alone, or in conjunction with cavin-1, neither develops surface-located caveolae nor internalizes caveolar ligands. These data suggest that the formation of functional caveolae at the plasma membrane in Toxoplasma and, by inference in all non-mammalian cells, requires effectors other than caveolin-1 and cavin-1. Interestingly, Toxoplasma co-expressing caveolin-1 and cavin-1 displays an impressive spiraled network of membranes containing the two proteins, in the cytoplasm. This suggests a synergistic activity of caveolin-1 and cavin-1 in the morphogenesis and remodeling of membranes, as illustrated for Toxoplasma.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0051773PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3522706PMC
June 2013

Sphingolipid synthesis and scavenging in the intracellular apicomplexan parasite, Toxoplasma gondii.

Mol Biochem Parasitol 2013 Jan 16;187(1):43-51. Epub 2012 Dec 16.

Biophysical Sciences Institute, Department of Chemistry and School of Biological Sciences, University Science Laboratories, Durham DH1 3LE, UK.

Sphingolipids are essential components of eukaryotic cell membranes, particularly the plasma membrane, and are involved in a diverse array of signal transduction pathways. Mammals produce sphingomyelin (SM) as the primary complex sphingolipid via the well characterised SM synthase. In contrast yeast, plants and some protozoa utilise an evolutionarily related inositol phosphorylceramide (IPC) synthase to synthesise IPC. This activity has no mammalian equivalent and IPC synthase has been proposed as a target for anti-fungals and anti-protozoals. However, detailed knowledge of the sphingolipid biosynthetic pathway of the apicomplexan protozoan parasites was lacking. In this study bioinformatic analyses indicated a single copy orthologue of the putative SM synthase from the apicomplexan Plasmodium falciparum (the causative agent of malaria) was a bona fide sphingolipid synthase in the related model parasite, Toxoplasma gondii (TgSLS). Subsequently, TgSLS was indicated, by complementation of a mutant cell line, to be a functional orthologue of the yeast IPC synthase (AUR1p), demonstrating resistance to the well characterised AUR1p inhibitor aureobasidin A. In vitro, recombinant TgSLS exhibited IPC synthase activity and, for the first time, the presence of IPC was demonstrated in T. gondii lipid extracts by mass spectrometry. Furthermore, host sphingolipid biosynthesis was indicated to influence, but be non-essential for, T. gondii proliferation, suggesting that whilst scavenging does take place de novo sphingolipid synthesis may be important for parasitism.
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http://dx.doi.org/10.1016/j.molbiopara.2012.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3629565PMC
January 2013

The herpes simplex virus 1 U(S)3 regulates phospholipid synthesis.

Virology 2012 Oct 11;432(2):353-60. Epub 2012 Jul 11.

Institute of Veterinary Anatomy, University of Zürich, Switzerland.

Herpes simplex virus type 1 capsids bud at nuclear and Golgi membranes for envelopment by phospholipid bilayers. In the absence of U(S)3, nuclear membranes form multiple folds harboring virions that suggests disturbance in membrane turnover. Therefore, we investigated phospholipid metabolism in cells infected with the U(S)3 deletion mutant R7041(ΔU(S)3), and quantified membranes involved in viral envelopment. We report that (i) [(3)H]-choline incorporation into nuclear membranes and cytoplasmic membranes was enhanced peaking at 12 or 20 h post inoculation with wild type HSV-1 and R7041(ΔU(S)3), respectively, (ii) the surface area of nuclear membranes increased until 24 h of R7041(ΔU(S)3) infection forming folds that equaled ~45% of the nuclear surface, (iii) the surface area of viral envelopes between nuclear membranes equaled ~2400 R7041(ΔU(S)3) virions per cell, and (iv) during R7041(ΔU(S)3) infection, the Golgi complex expanded dramatically. The data indicate that U(S)3 plays a significant role in regulation of membrane biosynthesis.
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http://dx.doi.org/10.1016/j.virol.2012.06.020DOI Listing
October 2012

Serotonin regulates amylase secretion and acinar cell damage during murine pancreatitis.

Gut 2013 Jun 16;62(6):890-8. Epub 2012 May 16.

Department of Visceral and Transplantation Surgery, Swiss Hepato-Pancreato-Biliary Center, University Hospital, Zurich 8091, Switzerland.

Objective: Serotonin (5-hydroxytryptamine, 5-HT) is a potent bioactive molecule involved in a variety of physiological processes. In this study, the authors analysed whether 5-HT regulates zymogen secretion in pancreatic acinar cells and the development of pancreatic inflammation, a potentially lethal disease whose pathophysiology is not completely understood.

Methods: 5-HT regulation of zymogen secretion was analysed in pancreatic acini isolated from wild-type or tryptophan hydoxylase-1 knock-out (TPH1(-/-)) mice, which lack peripheral 5-HT, and in amylase-secreting pancreatic cell lines. Pancreatitis was induced by cerulein stimulation and biochemical and immunohistochemical methods were used to evaluate disease progression over 2 weeks.

Results: Absence and reduced intracellular levels of 5-HT inhibited the secretion of zymogen granules both ex vivo and in vitro and altered cytoskeleton dynamics. In addition, absence of 5-HT resulted in attenuated pro-inflammatory response after induction of pancreatitis. TPH1(-/-) mice showed limited zymogen release, reduced expression of the pro-inflammatory chemokine MCP-1 and minimal leucocyte infiltration compared with wild-type animals. Restoration of 5-HT levels in TPH1(-/-) mice recovered the blunted inflammatory processes observed during acute pancreatitis. However, cellular damage, inflammatory and fibrotic processes accelerated in TPH1(-/-) mice during disease progression.

Conclusions: These results identify a 5-HT-mediated regulation of zymogen secretion in pancreatic acinar cells. In addition, they demonstrate that 5-HT is required for the onset but not for the progression of pancreatic inflammation. These findings provide novel insights into the normal physiology of pancreatic acinar cells and into the pathophysiology of pancreatitis, with potential therapeutic implications.
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http://dx.doi.org/10.1136/gutjnl-2011-301724DOI Listing
June 2013

Herpes simplex virus 1 induces de novo phospholipid synthesis.

Virology 2012 Aug 5;429(2):124-35. Epub 2012 May 5.

Electron Microscopy, Institute of Veterinary Anatomy, University of Zürich, Switzerland.

Herpes simplex virus type 1 capsids bud at nuclear membranes and Golgi membranes acquiring an envelope composed of phospholipids. Hence, we measured incorporation of phospholipid precursors into these membranes, and quantified changes in size of cellular compartments by morphometric analysis. Incorporation of [³H]-choline into both nuclear and cytoplasmic membranes was significantly enhanced upon infection. [³H]-choline was also part of isolated virions even grown in the presence of brefeldin A. Nuclei expanded early in infection. The Golgi complex and vacuoles increased substantially whereas the endoplasmic reticulum enlarged only temporarily. The data suggest that HSV-1 stimulates phospholipid synthesis, and that de novo synthesized phospholipids are inserted into nuclear and cytoplasmic membranes to i) maintain membrane integrity in the course of nuclear and cellular expansion, ii) to supply membrane constituents for envelopment of capsids by budding at nuclear membranes and Golgi membranes, and iii) to provide membranes for formation of transport vacuoles.
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http://dx.doi.org/10.1016/j.virol.2012.04.004DOI Listing
August 2012

COX-2 is not required for the development of murine chronic pancreatitis.

Am J Physiol Gastrointest Liver Physiol 2011 Jun 3;300(6):G968-75. Epub 2011 Mar 3.

Swiss HPB Center, Pancreatitis Research Laboratory, Department of Visceral and Transplantation Surgery, University Hospital, Zurich, Switzerland.

Chronic pancreatitis is a severe inflammation of the pancreas associated with destruction of the parenchyma, fibrosis, and persistent abdominal pain. Cyclooxygenase-2 (COX-2) and COX-2-derived prostaglandins, key mediators of the inflammatory response, are elevated in patients with chronic pancreatitis. Previous studies investigated COX-2 as a therapeutic target. These reports showed a reduced pathology in COX-2-deficient mice with a better outcome. Here we compared the role of COX-2 in acute and chronic pancreatic inflammation using the same COX-2(-/-) mouse model of cerulein-induced pancreatitis. In a setting of acute pancreatitis, juvenile COX-2(-/-) mice exhibited a reduced histopathological score compared with wild-type littermates; on the contrary, adult mice did not show any difference in the development of the disease. Similarly, in a setting of chronic pancreatitis induced over a period of 4 wk, adult mice of the two strains showed comparable histological score and collagen deposition. However, the abundance of mRNAs coding for profibrotic genes, such as collagen, α-smooth muscle actin, and transforming growth factor-β was consistently lower in COX-2(-/-) mice. In addition, comparable histological scores and collagen deposition were observed in wild-type mice treated with a COX-2 inhibitor. We conclude that, in contrast to what was observed in the rat pancreatitis models, COX-2 has a limited and age-dependent effect on inflammatory processes in the mouse pancreas. These results suggest that COX-2 modulates the inflammatory process during the development of pancreatitis in a species-specific manner. Thus the pathophysiological roles of COX-2 and its therapeutic implications in patients with pancreatitis should be reexamined.
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http://dx.doi.org/10.1152/ajpgi.00497.2010DOI Listing
June 2011

The P-glycoprotein inhibitor GF120918 modulates Ca2+-dependent processes and lipid metabolism in Toxoplasma gondii.

PLoS One 2010 Apr 8;5(4):e10062. Epub 2010 Apr 8.

Institute of Parasitology, University of Zurich, Zurich, Switzerland.

Up-regulation of the membrane-bound efflux pump P-glycoprotein (P-gp) is associated with the phenomenon of multidrug-resistance in pathogenic organisms, including protozoan parasites. In addition, P-gp plays a role in normal physiological processes, however our understanding of these P-gp functions remains limited. In this study we investigated the effects of the P-gp inhibitor GF120918 in Toxoplasma gondii, a model apicomplexan parasite and an important human pathogen. We found that GF120918 treatment severely inhibited parasite invasion and replication. Further analyses of the molecular mechanisms involved revealed that the P-gp inhibitor modulated parasite motility, microneme secretion and egress from the host cell, all cellular processes known to depend on Ca2+ signaling in the parasite. In support of a potential role of P-gp in Ca2+-mediated processes, immunoelectron and fluorescence microscopy showed that T. gondii P-gp was localized in acidocalcisomes, the major Ca2+ storage in the parasite, at the plasma membrane, and in the intravacuolar tubular network. In addition, metabolic labeling of extracellular parasites revealed that inhibition or down-regulation of T. gondii P-gp resulted in aberrant lipid synthesis. These results suggest a crucial role of T. gondii P-gp in essential processes of the parasite biology and further validate the potential of P-gp activity as a target for drug development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0010062PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2851653PMC
April 2010

Glucosylceramide synthesis inhibition affects cell cycle progression, membrane trafficking, and stage differentiation in Giardia lamblia.

J Lipid Res 2010 Sep 24;51(9):2527-45. Epub 2010 Mar 24.

Institute of Parasitology, University of Zurich, Zurich, Switzerland.

Synthesis of glucosylceramide via glucosylceramide synthase (GCS) is a crucial event in higher eukaryotes, both for the production of complex glycosphingolipids and for regulating cellular levels of ceramide, a potent antiproliferative second messenger. In this study, we explored the dependence of the early branching eukaryote Giardia lamblia on GCS activity. Biochemical analyses revealed that the parasite has a GCS located in endoplasmic reticulum (ER) membranes that is active in proliferating and encysting trophozoites. Pharmacological inhibition of GCS induced aberrant cell division, characterized by arrest of cytokinesis, incomplete cleavage furrow formation, and consequent block of replication. Importantly, we showed that increased ceramide levels were responsible for the cytokinesis arrest. In addition, GCS inhibition resulted in prominent ultrastructural abnormalities, including accumulation of cytosolic vesicles, enlarged lysosomes, and clathrin disorganization. Moreover, anterograde trafficking of the encystations-specific protein CWP1 was severely compromised and resulted in inhibition of stage differentiation. Our results reveal novel aspects of lipid metabolism in G. lamblia and specifically highlight the vital role of GCS in regulating cell cycle progression, membrane trafficking events, and stage differentiation in this parasite. In addition, we identified ceramide as a potent bioactive molecule, underscoring the universal conservation of ceramide signaling in eukaryotes.
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http://dx.doi.org/10.1194/jlr.M003392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2918437PMC
September 2010

Epigenetic mechanisms regulate stage differentiation in the minimized protozoan Giardia lamblia.

Mol Microbiol 2010 Apr 1;76(1):48-67. Epub 2010 Feb 1.

Institute of Parasitology, University of Zürich, Zürich, Switzerland.

Histone modification is an important mechanism regulating both gene expression and the establishment and maintenance of cellular phenotypes during development. Regulation of histone acetylation via histone acetylases and deacetylases (HDACs) appears to be particularly crucial in determining gene expression patterns. In this study we explored the effect of HDAC inhibition on the life cycle of the human pathogen Giardia lamblia, a highly reduced parasitic protozoan characterized by minimized cellular processes. We found that the HDAC inhibitor FR235222 increased the level of histone acetylation and induced transcriptional regulation of approximately 2% of genes in proliferating and encysting parasites. In addition, our analyses showed that the levels of histone acetylation decreased during differentiation into cysts, the infective stage of the parasite. Importantly, FR235222 treatment during encystation reversed this histone hypo-acetylation and potently blocked the formation of cysts. These results provide the first direct evidence for epigenetic regulation of gene expression in this simple eukaryote. This suggests that regulation of histone acetylation is involved in the control of Giardia stage differentiation, and identifies epigenetic mechanisms as a promising target to prevent Giardia transmission.
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http://dx.doi.org/10.1111/j.1365-2958.2010.07062.xDOI Listing
April 2010

Neogenesis and maturation of transient Golgi-like cisternae in a simple eukaryote.

J Cell Sci 2009 Aug 21;122(Pt 16):2846-56. Epub 2009 Jul 21.

Institute of Parasitology, University of Zürich, Zürich, Switzerland.

The highly reduced protozoan parasite Giardia lamblia has minimal machinery for cellular processes such as protein trafficking. Giardia trophozoites maintain diverse and regulated secretory pathways but lack an identifiable Golgi complex. During differentiation to cysts, however, they produce specialized compartments termed encystation-specific vesicles (ESVs). ESVs are hypothesized to be unique developmentally regulated Golgi-like organelles dedicated to maturation and export of pre-sorted cyst wall proteins. Here we present a functional analysis of this unusual compartment by direct interference with the functions of the small GTPases Sar1, Rab1 and Arf1. Conditional expression of dominant-negative variants revealed an essential role of Sar1 in early events of organelle neogenesis, whilst inhibition of Arf1 uncoupled morphological changes and cell cycle progression from extracellular matrix export. The latter led to development of ;naked cysts', which lacked water resistance and thus infectivity. Time-lapse microscopy and photobleaching experiments showed that putative Golgi-like cisternae in Giardia develop into a network capable of exchanging soluble cargo at a high rate via dynamic, tubular connections, presumably to synchronize maturation. The minimized and naturally pulsed trafficking machinery for export of the cyst wall biopolymer in Giardia is a simple model for investigating basic principles of neogenesis and maturation of Golgi compartments.
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http://dx.doi.org/10.1242/jcs.049411DOI Listing
August 2009

Host cell P-glycoprotein is essential for cholesterol uptake and replication of Toxoplasma gondii.

J Biol Chem 2009 Jun 22;284(26):17438-48. Epub 2009 Apr 22.

Institute of Parasitology, University of Zurich, Zurich, Switzerland.

P-glycoprotein (P-gp) is a membrane-bound efflux pump that actively exports a wide range of compounds from the cell and is associated with the phenomenon of multidrug resistance. However, the role of P-gp in normal physiological processes remains elusive. Using P-gp-deficient fibroblasts, we showed that P-gp was critical for the replication of the intracellular parasite Toxoplasma gondii but was not involved in invasion of host cells by the parasite. Importantly, we found that the protein participated in the transport of host-derived cholesterol to the intracellular parasite. T. gondii replication in P-gp-deficient host cells not only resulted in reduced cholesterol content in the parasite but also altered its sphingolipid metabolism. In addition, we found that different levels of P-gp expression modified the cholesterol metabolism in uninfected fibroblasts. Collectively our findings reveal a key and previously undocumented role of P-gp in host-parasite interaction and suggest a physiological role for P-gp in cholesterol trafficking in mammalian cells.
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http://dx.doi.org/10.1074/jbc.M809420200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2719384PMC
June 2009

A sphingolipid inhibitor induces a cytokinesis arrest and blocks stage differentiation in Giardia lamblia.

Antimicrob Agents Chemother 2008 Feb 17;52(2):563-9. Epub 2007 Dec 17.

Institute of Parasitology, University of Zurich, Winterthurerstrasse 266a, CH-8057 Zurich, Switzerland.

Sphingolipid biosynthesis pathways have recently emerged as a promising target for therapeutic intervention against pathogens, including parasites. A key step in the synthesis of complex sphingolipids is the glucosylation of ceramide, mediated by glucosylceramide (GlcCer) synthase, whose activity can be inhibited by PPMP (1-phenyl-2-palmitoylamino-3-morpholino-1-propanol). In this study, we investigated whether PPMP inhibits the proliferation and differentiation of the pathogenic parasite Giardia lamblia, the major cause of parasite-induced diarrhea worldwide. PPMP was found to block in vitro parasite replication in a dose-dependent manner, with a 50% inhibitory concentration of 3.5 muM. The inhibition of parasite replication was irreversible at 10 muM PPMP, a concentration that did not affect mammalian cell metabolism. Importantly, PPMP inhibited the completion of cell division at a specific stage in late cytokinesis. Microscopic analysis of cells incubated with PPMP revealed the aberrant accumulation of cellular membranes belonging to the endoplasmic reticulum network in the caudal area of the parasites. Finally, PPMP induced a 90% reduction in G. lamblia differentiation into cysts, the parasite stage responsible for the transmission of the disease. These results show that PPMP is a powerful inhibitor of G. lamblia in vitro and that as-yet-uncharacterized sphingolipid biosynthetic pathways are potential targets for the development of anti-G. lamblia agents.
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http://dx.doi.org/10.1128/AAC.01105-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2224734PMC
February 2008