1-Deoxysphingolipids, Early Predictors of Type 2 Diabetes, Compromise the Functionality of Skeletal Myoblasts.

Metabolic dysfunction, dysregulated differentiation, and atrophy of skeletal muscle occur as part of a cluster of abnormalities associated with the development of Type 2 diabetes mellitus (T2DM). Recent interest has turned to the attention of the role of 1-deoxysphingolipids (1-DSL), atypical class of sphingolipids which are found significantly elevated in patients diagnosed with T2DM but also in the asymptomatic population who later develop T2DM. In vitro studies demonstrated that 1-DSL have cytotoxic properties and compromise the secretion of insulin from pancreatic beta cells. However, the role of 1-DSL on the functionality of skeletal muscle cells in the pathophysiology of T2DM still remains unclear. This study aimed to investigate whether 1-DSL are cytotoxic and disrupt the cellular processes of skeletal muscle precursors (myoblasts) and differentiated cells (myotubes) by performing a battery of in vitro assays including cell viability adenosine triphosphate assay, migration assay, myoblast fusion assay, glucose uptake assay, and immunocytochemistry. Our results demonstrated that 1-DSL significantly reduced the viability of myoblasts in a concentration and time-dependent manner, and induced apoptosis as well as cellular necrosis. Importantly, myoblasts were more sensitive to the cytotoxic effects induced by 1-DSL rather than by saturated fatty acids, such as palmitate, which are critical mediators of skeletal muscle dysfunction in T2DM. Additionally, 1-DSL significantly reduced the migration ability of myoblasts and the differentiation process of myoblasts into myotubes. 1-DSL also triggered autophagy in myoblasts and significantly reduced insulin-stimulated glucose uptake in myotubes. These findings demonstrate that 1-DSL directly compromise the functionality of skeletal muscle cells and suggest that increased levels of 1-DSL observed during the development of T2DM are likely to contribute to the pathophysiology of muscle dysfunction detected in this disease.

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

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.

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

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.

Local hyperthyroidism promotes pancreatic acinar cell proliferation during acute pancreatitis.

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 TGFß 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.

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

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.

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

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.

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

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 ß (TGFß) is a potent profibrotic factor in the pancreas as it promotes the activation of PSC; thus, pharmacologic interventions that effectively reduce TGFß expression harbor considerable therapeutic potential in the treatment of chronic pancreatitis. In this study, we investigated whether TGFß 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 TGFß 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 TGFß 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.

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

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.

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

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.

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

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.

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

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.

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

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-ß (TGFß) in pancreatic epithelial cells during tissue regeneration. To this end, we conditionally inactivated TGFß receptor II (TGFß-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 TGFß-RII signalling does not mediate the initial acinar cell damage observed at the onset of pancreatitis. However, TGFß-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 TGFß-RII signalling stimulates the expression of cyclin-dependent kinase inhibitors and intersects with the EGFR signalling axis. Finally, TGFß-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 TGFß-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 TGFß as a tumour suppressor, both in the regenerative process following pancreatitis and in the initial phases of pancreatic cancer.

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

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.

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

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 ß-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.

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

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.

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

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.

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

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.

Neospora caninum protein disulfide isomerase is involved in tachyzoite-host cell interaction.

We have previously shown that treatment of Neospora caninum tachyzoites with the aspartyl protease inhibitor pepstatin A reduces host cell invasion [Naguleswaran, A., Muller, N., Hemphill, A., 2003. Neospora caninum and Toxoplasma gondii: a novel adhesion/invasion assay reveals distinct differences in tachyzoite-host cell interactions. Exp. Parasitol. 104, 149-158]. Pepstatin A-affinity-chromatography led to the isolation of a major band of approximately 52 kDa which was identified as a homologue of a previously described Toxoplasma gondii putative protein disulfide isomerase (TgPDI) through tandem mass spectrometry. A BLAST search against N. caninum expressed sequence tags (ESTs) on the ApiDots server using TgPDI cDNA as query sequence revealed a 2251 bp PDI-like consensus (NcPDI), which shows 94% identity to the T. gondii homologue. In N. caninum tachyzoites, NcPDI was found mainly in the soluble hydrophilic fraction. Immunofluorescence showed that expression of NcPDI was dramatically down-regulated in the bradyzoite stage, and immunogold-EM on tachyzoites localised the protein to the cytoplasm, mostly in close vicinity to the nuclear membrane, to the micronemes, and to the parasite cell surface. However, NcPDI was absent in rhoptries and dense granules. Preincubation of tachyzoites with the sulfhydryl blocker 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoic acid (pCMBA), and with the PDI inhibitor bacitracin reduced adhesion of parasites to host cells. In addition, incubation of N. caninum tachyzoites with affinity-purified anti-NcPDI antibodies reduced host cell adhesion. PDIs catalyse the formation, reduction or isomerisation of disulfide bonds. Many major components of the adhesion and invasion machinery of apicomplexan parasites are cysteine-rich and dependent on correct folding via disulfide bond formation. Thus, our data points towards an important role for surface-associated NcPDI in Neospora-host cell interaction.

Pyridinylimidazole p38 mitogen-activated protein kinase inhibitors block intracellular Toxoplasma gondii replication.

Toxoplasma gondii is a medically important, obligate intracellular parasite. Little is known regarding factors that regulate its replication within cells. Such knowledge would further understanding of T. gondii pathogenesis, and might lead to novel therapeutic strategies. Mitogen-activated protein kinases (MAPKs) govern diverse cellular processes including proliferation and differentiation. We now show that treatment of T. gondii-infected cells with SB203580 or SB202190, substituted pyridinylimidazoles that are potent inhibitors of human p38 MAPK, inhibits intracellular T. gondii replication. Several independent experimental approaches suggest that the anti-proliferative effects of pyridinylimidazoles depend on direct action on tachyzoites, not the host cell: (i) selective inhibition of host p38 MAPK using recombinant adenoviruses had little effect on tachyzoite replication, (ii) pyridinylimidazole-treated tachyzoites developed abnormal morphology suggesting defective parasite division, and (iii) pyridinylimidazole-resistant mutant tachyzoites were developed through culture in progressively higher drug concentrations. We hypothesise that pyridinylimidazoles target a human p38 MAPK homologue in tachyzoites that regulates their replication. Phylogenetic data suggest that T. gondii likely encodes a p38 MAPK homologue, but such a homologue is absent from the incomplete Toxoplasma genomic data base. As all eukaryotic pathogens, including agents of malaria, leishmaniasis and trypanosomiasis encode endogenous MAPKs, drugs inhibiting endogenous MAPK activation may represent a novel, potentially broadly-acting class of anti-parasitic agents. Pyridinylimidazoles also represent tools to elucidate factors governing intracellular tachyzoite replication.