Publications by authors named "Purnima Bhat"

21 Publications

  • Page 1 of 1

A Confocal Microscopic Study on Percentage Penetration of Different Sealers into Dentin.

J Pharm Bioallied Sci 2021 Jun 5;13(Suppl 1):S725-S730. Epub 2021 Jun 5.

Vivid Dental Care, Pandeshwar, Mangalore, Karnataka, India.

Context: Three-dimensional seal of the root canal space has always been challenging. The incorporation of gutta-percha and sealer prevents microleakage by bacteria, ensuring a shielded root canal space. However, the penetration of sealer to various depths within the root canal has always been looked with curiosity.

Aims: Thus, the present study was undertaken to evaluate the percentage and average depth of penetration of Endoflas F. S., AH Plus, and Epiphany sealers into dentinal tubules among the coronal, middle, and apical thirds of the roots following obturation with a lateral compaction technique using Confocal Laser Scanning Microscopy.

Settings And Design: The study is an randomized control trial.

Subjects And Methods: Thirty sound central incisors were decoronated at the level of the cementoenamel junction. Working length determination was done followed by a meticulous cleaning, shaping, and under copious irrigation. The teeth were then randomly divided into three groups: Endoflas FS sealer, AH Plus sealer, and Epiphany sealer. On fluorescence treatment, the teeth were sectioned at the midpoint of coronal, middle, and apical third of each root and viewed under confocal laser scanning microscope.

Statistical Analysis Used: The results were analyzed using one-way ANOVA, and the significant difference between groups was analyzed with post hoc Tukey test.

Results: Epiphany sealer provided with better percentage and depth penetration in comparison to Endoflas FS and AH Plus sealers. Furthermore, the coronal third of the root had better percentage and sealer penetration than the middle and apical thirds.

Conclusions: Sealers tend to provide a firm bond between the tooth and the gutta-percha. They bind, lubricate, and seal the gutta-percha cones to fill the accessory canals. Within the limitations of this study, the superior flow and enhanced setting time Epihany sealers provide better percentage and depth of penetration than AH Plus and Endoflas FS.
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http://dx.doi.org/10.4103/jpbs.JPBS_646_20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8375863PMC
June 2021

Dimeric IgA is a specific biomarker of recent SARS-CoV-2 infection.

medRxiv 2021 Jul 1. Epub 2021 Jul 1.

Current tests for SARS-CoV-2 antibodies (IgG, IgM, IgA) cannot differentiate recent and past infections. We describe a point of care, lateral flow assay for SARS-CoV-2 dIgA based on the highly selective binding of dIgA to a chimeric form of secretory component (CSC), that distinguishes dIgA from monomeric IgA. Detection of specific dIgA uses a complex of biotinylated SARS-CoV-2 receptor binding domain and streptavidin-colloidal gold. SARS-CoV-2-specific dIgA was measured both in 112 cross-sectional samples and a longitudinal panel of 362 plasma samples from 45 patients with PCR-confirmed SARS-CoV-2 infection, and 193 discrete pre-COVID-19 or PCR-negative patient samples. The assay demonstrated 100% sensitivity from 11 days post-symptom onset, and a specificity of 98.2%. With an estimated half-life of 6.3 days, dIgA provides a unique biomarker for the detection of recent SARS-CoV-2 infections with potential to enhance diagnosis and management of COVID-19 at point-of-care.
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http://dx.doi.org/10.1101/2021.06.28.21259671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8259913PMC
July 2021

Promotion of gastrointestinal endoscopy in Sub-Saharan Africa: What is needed, and how can ESGE and WEO help?

Endosc Int Open 2021 Jul 17;9(7):E1001-E1003. Epub 2021 Jun 17.

Department of Transplantation Medicine, Faculty of Medicine, Oslo University Hospital - Rikshospitalet, Oslo, Norway. Co-chair of the Committee of Activities to Reach Africa - World Endoscopy Organization.

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http://dx.doi.org/10.1055/a-1495-5215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8211488PMC
July 2021

Impact of the COVID-19 pandemic on gastrointestinal endoscopy in Africa.

Endosc Int Open 2020 Aug 7;8(8):E1097-E1101. Epub 2020 Aug 7.

Endoscopy Unit, Nuovo Regina Margherita Hospital, Rome, Italy.

As with all other fields of medical practice, gastrointestinal endoscopy has been impacted by the COVID-19 pandemic. However, data on the impact of the pandemic in Africa, especially sub-Saharan Africa are lacking. A web-based survey was conducted by the International Working Group of the European Society for Gastrointestinal Endoscopy and the World Endoscopy Organization to determine the impact and effects the COVID-19 pandemic has had on endoscopists in African countries. Thirty-one gastroenterologists from 14 countries in north, central, and sub-Saharan Africa responded to the survey. The majority of respondents reduced their endoscopy volume considerably. Personal protective equipment including FFP-2 masks were available in almost all participating centers. Pre-endoscopy screening was performed as well. The COVID-19 pandemic has had a substantial impact on gastrointestinal endoscopy in most African countries; however, the impact may not have been as devastating as expected.
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http://dx.doi.org/10.1055/a-1210-4274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7413826PMC
August 2020

A Region of UNC-89 (Obscurin) Lying between Two Protein Kinase Domains Is a Highly Elastic Spring Required for Proper Sarcomere Organization.

J Mol Biol 2020 08 6;432(17):4799-4814. Epub 2020 Jul 6.

Department of Pathology, Emory University, Atlanta, GA 30322, USA. Electronic address:

In Caenorhabditis elegans, unc-89 encodes a set of giant multi-domain proteins (up 8081 residues) localized to the M-lines of muscle sarcomeres and required for normal sarcomere organization and whole-animal locomotion. Multiple UNC-89 isoforms contain two protein kinase domains. There is conservation in arrangement of domains between UNC-89 and its two mammalian homologs, obscurin and SPEG: kinase, a non-domain region of 647-742 residues, Ig domain, Fn3 domain and a second kinase domain. In all three proteins, this non-domain "interkinase region" has low sequence complexity, has high proline content, and lacks predicted secondary structure. We report that a major portion of this interkinase (571 residues out of 647 residues) when examined by single molecule force spectroscopy in vitro displays the properties of a random coil and acts as an entropic spring. We used CRISPR/Cas9 to create nematodes carrying an in-frame deletion of the same 571-residue portion of the interkinase. These animals display severe disorganization of all portions of the sarcomere in body wall muscle. Super-resolution microscopy reveals extra, short-A-bands lying close to the outer muscle cell membrane and between normally spaced A-bands. Nematodes with this in-frame deletion show defective locomotion and muscle force generation. We designed our CRISPR-generatedin-frame deletion to contain an HA tag at the N terminus of the large UNC-89 isoforms. This HA tag results in normal organization of body wall muscle, but approximately half the normal levels of the giant UNC-89 isoforms, dis-organization of pharyngeal muscle, small body size, and reduced muscle force, likely due to poor nutritional uptake.
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http://dx.doi.org/10.1016/j.jmb.2020.06.024DOI Listing
August 2020

Role of Endoscopy in Primary Sclerosing Cholangitis.

Clin Endosc 2021 Mar 8;54(2):193-201. Epub 2020 May 8.

Department of Transplantation Medicine, Oslo University Hospital, Oslo, Norway.

Primary sclerosing cholangitis (PSC) is a progressive disease of the bile ducts that usually results in chronic liver disease often requiring liver transplantation. Endoscopy remains crucial to the care of these patients, although magnetic resonance cholangiopancreatography has replaced endoscopic retrograde cholangiopancreatography (ERCP) as the primary imaging modality for diagnosis. For detection of dysplasia or cholangiocarcinoma, ERCP with intraductal sampling remains compulsory. Moreover, dominant strictures play an important part in the disease development, and management by balloon dilatation or stenting could contribute to long-term prognosis. In addition, endoscopy offers management for adverse events such as bile leaks and anastomotic strictures after liver transplantation. Finally, the special phenotype of inflammatory bowel disease associated with PSC as well as the frequent occurrence of portal hypertension mandates close follow-up with colonoscopy and upper endoscopy. With the emergence of novel techniques, the endoscopist remains a key member of the multidisciplinary team caring for PSC patients.
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http://dx.doi.org/10.5946/ce.2020.019-IDENDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8039754PMC
March 2021

Dysbiosis of the Duodenal Mucosal Microbiota Is Associated With Increased Small Intestinal Permeability in Chronic Liver Disease.

Clin Transl Gastroenterol 2019 08;10(8):e00068

Department of Gastroenterology and Hepatology, Princess Alexandra Hospital, Woolloongabba, Queensland, Australia.

Objectives: Chronic liver disease (CLD) is associated with both alterations of the stool microbiota and increased small intestinal permeability. However, little is known about the role of the small intestinal mucosa-associated microbiota (MAM) in CLD. The aim of this study was to evaluate the relationship between the duodenal MAM and both small intestinal permeability and liver disease severity in CLD.

Methods: Subjects with CLD and a disease-free control group undergoing routine endoscopy underwent duodenal biopsy to assess duodenal MAM by 16S rRNA gene sequencing. Small intestinal permeability was assessed by a dual sugar (lactulose: rhamnose) assay. Other assessments included transient elastography, endotoxemia, serum markers of hepatic inflammation, dietary intake, and anthropometric measurements.

Results: Forty-six subjects (35 with CLD and 11 controls) were assessed. In subjects with CLD, the composition (P = 0.02) and diversity (P < 0.01) of the duodenal MAM differed to controls. Constrained multivariate analysis and linear discriminate effect size showed this was due to Streptococcus-affiliated lineages. Small intestinal permeability was significantly higher in CLD subjects compared to controls. In CLD, there were inverse correlations between microbial diversity and both increased small intestinal permeability (r = -0.41, P = 0.02) and serum alanine aminotransferase (r = -0.35, P = 0.04). Hepatic stiffness was not associated with the MAM.

Discussion: In CLD, there is dysbiosis of the duodenal MAM and an inverse correlation between microbial diversity and small intestinal permeability.

Translational Impact: Strategies to ameliorate duodenal MAM dysbiosis may ameliorate intestinal barrier dysfunction and liver injury in CLD.
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http://dx.doi.org/10.14309/ctg.0000000000000068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736223PMC
August 2019

Human papillomavirus E7 oncoprotein expression by keratinocytes alters the cytotoxic mechanisms used by CD8 T cells.

Oncotarget 2018 Jan 14;9(5):6015-6027. Epub 2017 Dec 14.

University of Queensland Diamantina Institute, University of Queensland, Translational Research Institute, Brisbane, Qld, Australia.

Cervical cancer is a malignant transformation of keratinocytes initiated by the E7 oncoprotein of human papillomavirus (HPV). These tumors are characterized by keratinocyte hyperproliferation and are often infiltrated with activated CD8 T cells. HPV infection confers changes to gain immunological advantage to promote chronic infection, and these persist with malignant transformation. We investigated the relative importance of the many redundant mechanisms of cytotoxicity used by CD8 T cells to kill keratinocytes expressing HPV E7 oncoprotein using extended-duration time-lapse microscopy that allows examination of cell-to-cell interactions during killing. E7 expression by keratinocytes increased susceptibility to cell-mediated killing. However, while killing of non-transgenic keratinocytes was traditional, perforin-mediated, and caspase-dependent, E7-expression favored killing by perforin-independent, caspase-independent mechanisms. The roles of perforin, TNFα, IFNγ, Fas/FasL and PD1/PD-L1 were graded according to target cell survival to produce a hierarchy of killing mechanisms utilized in killing E7-expressing cells. TNFα was essential for perforin-mediated killing of E7-expressing cells, but not perforin-independent killing. IFNγ facilitated killing by Fas/FasL interaction, especially in the absence of perforin. Additionally, expression of E7 offered protection from killing by up regulation of PD-L1, Fas and FasL expression on keratinocytes promoting fight-back by target cells, resulting in effector cell death. This study shows that keratinocytes expressing E7 are highly susceptible to killing by CD8 T cells, but utilizing different armamentarium. Down-regulation of CD8 T cell cytotoxicity in HPV-related tumors may be due to suppression by E7-expressing keratinocytes. Immunotherapy for HPV-related cancers may be improved by suppression of PD-L1, or by suppression of FasL.
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http://dx.doi.org/10.18632/oncotarget.23210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5814191PMC
January 2018

Interferon-γ derived from cytotoxic lymphocytes directly enhances their motility and cytotoxicity.

Cell Death Dis 2017 06 1;8(6):e2836. Epub 2017 Jun 1.

The University of Queensland Diamantina Institute, Translational Research Institute, 37 Kent Street, Woolloongabba, QLD 4102, Australia.

Interferon gamma (IFNγ) is a key moderator of cell-mediated immunity with diverse, mainly pro-inflammatory actions on immunocytes and target tissue. Recent studies have shown it may enhance anti-tumor and antiviral effects of CD8 T cells. Here we investigate the mechanisms by which IFNγ mediates CD8 T-cell cytotoxic function. We show that in vivo, antigen-specific CD8 T cells that produce INFγ are necessary to effect rejection of skin grafts expressing OVA as a transgene in keratinocytes. The ability of CD8 T cells to produce IFNγ enhanced their ability to migrate to the site of antigen-presenting skin cells. By in vivo imaging, we show that CTL motility, particularly speed, during graft rejection was enhanced by locally available IFNγ. We then used a reductionist two-cell model of CTL effectors and keratinocyte targets to investigate the effects of locally available (paracrine) and CTL-producing (autocrine) IFNγ on the motility behavior and killing ability of the CTL. Using live-cell imaging by prolonged time-lapse microscopy of primary effector CD8 T cells and antigen-expressing primary keratinocyte targets, we show that CD8 T-cell cytotoxic function and motility is enhanced by locally available IFNγ. Conversely, deprivation of either autocrine or paracrine IFNγ, or blockade of IFNγ signaling to CTL markedly reduced their cytotoxic function, their kinematics, and effector cell survival. We conclude that in vitro and in vivo, autocrine production of IFNγ by CTL enhances their motility and promotes killing of primary target keratinocytes. The absolute need for local IFNγ to enable cytotoxic CD8 T-cell function is of significance for immunotherapy for chronic viral infection and for cancer.
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http://dx.doi.org/10.1038/cddis.2017.67DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5520949PMC
June 2017

mRNA Structural constraints on EBNA1 synthesis impact on in vivo antigen presentation and early priming of CD8+ T cells.

PLoS Pathog 2014 Oct 9;10(10):e1004423. Epub 2014 Oct 9.

QIMR Centre for Immunotherapy and Vaccine Development and Tumour Immunology, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.

Recent studies have shown that virally encoded mRNA sequences of genome maintenance proteins from herpesviruses contain clusters of unusual structural elements, G-quadruplexes, which modulate viral protein synthesis. Destabilization of these G-quadruplexes can override the inhibitory effect on self-synthesis of these proteins. Here we show that the purine-rich repetitive mRNA sequence of Epstein-Barr virus encoded nuclear antigen 1 (EBNA1) comprising G-quadruplex structures, limits both the presentation of MHC class I-restricted CD8(+) T cell epitopes by CD11c(+) dendritic cells in draining lymph nodes and early priming of antigen-specific CD8(+) T-cells. Destabilization of the G-quadruplex structures through codon-modification significantly enhanced in vivo antigen presentation and activation of virus-specific T cells. Ex vivo imaging of draining lymph nodes by confocal microscopy revealed enhanced antigen-specific T-cell trafficking and APC-CD8(+) T-cell interactions in mice primed with viral vectors encoding a codon-modified EBNA1 protein. More importantly, these antigen-specific T cells displayed enhanced expression of the T-box transcription factor and superior polyfunctionality consistent with the qualitative impact of translation efficiency. These results provide an important insight into how viruses exploit mRNA structure to down regulate synthesis of their viral maintenance proteins and delay priming of antigen-specific T cells, thereby establishing a successful latent infection in vivo. Furthermore, targeting EBNA1 mRNA rather than protein by small molecules or antisense oligonucleotides will enhance EBNA1 synthesis and the early priming of effector T cells, to establish a more rapid immune response and prevent persistent infection.
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http://dx.doi.org/10.1371/journal.ppat.1004423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4192603PMC
October 2014

The kinematics of cytotoxic lymphocytes influence their ability to kill target cells.

PLoS One 2014 6;9(5):e95248. Epub 2014 May 6.

The University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Queensland, Australia.

Cytotoxic lymphocytes (CTL) have been reported to show a range of motility patterns from rapid long-range tracking to complete arrest, but how and whether these kinematics affect their ability to kill target cells is not known. Many in vitro killing assays utilize cell lines and tumour-derived cells as targets, which may be of limited relevance to the kinetics of CTL-mediated killing of somatic cells. Here, live-cell microscopy is used to examine the interactions of CTL and primary murine skin cells presenting antigens. We developed a qualitative and quantitative killing assay using extended-duration fluorescence time-lapse microscopy coupled with large-volume objective software-based data analysis to obtain population data of cell-to-cell interactions, motility and apoptosis. In vivo and ex vivo activated antigen-specific cytotoxic lymphocytes were added to primary keratinocyte targets in culture with fluorometric detection of caspase-3 activation in targets as an objective determinant of apoptosis. We found that activated CTL achieved contact-dependent apoptosis of non-tumour targets after a period of prolonged attachment - on average 21 hours - which was determined by target cell type, amount of antigen, and activation status of CTL. Activation of CTL even without engagement of the T cell receptor was sufficient to mobilise cells significantly above baseline, while the addition of cognate antigen further enhanced their motility. Highly activated CTL showed markedly increased vector displacement, and velocity, and lead to increased antigen-specific target cell death. These data show that the inherent kinematics of CTL correlate directly with their ability to kill non-tumour cells presenting cognate antigen.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0095248PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4011687PMC
October 2015

Recipient nonhematopoietic antigen-presenting cells are sufficient to induce lethal acute graft-versus-host disease.

Nat Med 2011 Nov 29;18(1):135-42. Epub 2011 Nov 29.

The Queensland Institute of Medical Research, Brisbane, Australia.

The presentation pathways by which allogeneic peptides induce graft-versus-host disease (GVHD) are unclear. We developed a bone marrow transplant (BMT) system in mice whereby presentation of a processed recipient peptide within major histocompatibility complex (MHC) class II molecules could be spatially and temporally quantified. Whereas donor antigen presenting cells (APCs) could induce lethal acute GVHD via MHC class II, recipient APCs were 100-1,000 times more potent in this regard. After myeloablative irradiation, T cell activation and memory differentiation occurred in lymphoid organs independently of alloantigen. Unexpectedly, professional hematopoietic-derived recipient APCs within lymphoid organs had only a limited capacity to induce GVHD, and dendritic cells were not required. In contrast, nonhematopoietic recipient APCs within target organs induced universal GVHD mortality and promoted marked alloreactive donor T cell expansion within the gastrointestinal tract and inflammatory cytokine generation. These data challenge current paradigms, suggesting that experimental lethal acute GVHD can be induced by nonhematopoietic recipient APCs.
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http://dx.doi.org/10.1038/nm.2597DOI Listing
November 2011

Hepatocytes traffic and export hepatitis B virus basolaterally by polarity-dependent mechanisms.

J Virol 2011 Dec 21;85(23):12474-81. Epub 2011 Sep 21.

Ian Potter Hepatitis Research Laboratory, Macfarlane Burnet Institute for Medical Research and Public Health, 85 Commercial Rd., Melbourne 3004, Australia.

Viruses commonly utilize the cellular trafficking machinery of polarized cells to effect viral export. Hepatocytes are polarized in vivo, but most in vitro hepatocyte models are either nonpolarized or have morphology unsuitable for the study of viral export. Here, we investigate the mechanisms of trafficking and export for the hepadnaviruses hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) in polarized hepatocyte-derived cell lines and primary duck hepatocytes. DHBV export, but not replication, was dependent on the development of hepatocyte polarity, with export significantly abrogated over time as primary hepatocytes lost polarity. Using Transwell cultures of polarized N6 cells and adenovirus-based transduction, we observed that export of both HBV and DHBV was vectorially regulated and predominantly basolateral. Monitoring of polarized N6 cells and nonpolarized C11 cells during persistent, long-term DHBV infection demonstrated that newly synthesized sphingolipid and virus displayed significant colocalization and fluorescence resonance energy transfer, implying cotransportation from the Golgi complex to the plasma membrane. Notably, 15% of virus was released apically from polarized cells, corresponding to secretion into the bile duct in vivo, also in association with sphingolipids. We conclude that DHBV and, probably, HBV are reliant upon hepatocyte polarity to be efficiently exported and this export is in association with sphingolipid structures, possibly lipid rafts. This study provides novel insights regarding the mechanisms of hepadnavirus trafficking in hepatocytes, with potential relevance to pathogenesis and immune tolerance.
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http://dx.doi.org/10.1128/JVI.05344-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209395PMC
December 2011

Regulation of immune responses to HPV infection and during HPV-directed immunotherapy.

Immunol Rev 2011 Jan;239(1):85-98

The University of Queensland Diamantina Institute, Princess Alexandra Hospital, Brisbane, Australia.

The recent development of vaccines prophylactic against human papillomavirus (HPV) infection has the potential to reduce the incidence of cervical cancer globally by up to 70% over the next 40 years, if universal immunization is adopted. As these prophylactic vaccines do not alter the natural history of established HPV infection, immunotherapies to treat persistent HPV infection and associated precancers would be of benefit to assist with cervical cancer control. Efforts to develop immuno-therapeutic vaccines have been hampered by the relative non-immunogenicity of HPV infection, by immunoregulatory processes in skin, and by subversion of immune response induction and immune effector functions by papillomavirus proteins. This review describes HPV-specific immune responses induced by viral proteins, their regulation by host and viral factors, and highlights some conclusions from our own recent research.
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http://dx.doi.org/10.1111/j.1600-065X.2010.00966.xDOI Listing
January 2011

Myosin 2 maintains an open exocytic fusion pore in secretory epithelial cells.

Mol Biol Cell 2009 Mar 21;20(6):1795-803. Epub 2009 Jan 21.

School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, QLD 4072, Australia.

Many studies have implicated F-actin and myosin 2 in the control of regulated secretion. Most recently, evidence suggests a role for the microfilament network in regulating the postfusion events of vesicle dynamics. This is of potential importance as postfusion behavior can influence the loss of vesicle content and may provide a new target for drug therapy. We have investigated the role of myosin 2 in regulating exocytosis in secretory epithelial cells by using novel assays to determine the behavior of the fusion pore in individual granules. We immunolocalize myosin 2A to the apical region of pancreatic acinar cells, suggesting it is this isoform that plays a role in granule exocytosis. We further show myosin 2 phosphorylation increased on cell stimulation, consistent with a regulatory role in secretion. Importantly, in a single-cell, single-granule secretion assay, neither the myosin 2 inhibitor (-)-blebbistatin nor the myosin light chain kinase inhibitor ML-9 had any effect on the numbers of granules stimulated to fuse after cell stimulation. These data indicate that myosin 2, if it has any action on secretion, must be targeting postfusion granule behavior. This interpretation is supported by direct study of fusion pore opening in which we show that (-)-blebbistatin and ML-9 promote fusion pore closure and decrease fusion pore lifetimes. Our work now adds to a growing body of evidence showing that myosin 2 is an essential regulator of postfusion granule behavior. In particular, in the case of the secretory epithelial cells, myosin 2 activity is necessary to maintain fusion pore opening.
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http://dx.doi.org/10.1091/mbc.e08-10-1048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2655261PMC
March 2009

Vectorial entry and release of hepatitis A virus in polarized human hepatocytes.

J Virol 2008 Sep 25;82(17):8733-42. Epub 2008 Jun 25.

Ian Potter Hepatitis Research Laboratory, Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne 3004, Australia.

Hepatitis A virus (HAV) is an enterically transmitted virus that replicates predominantly in hepatocytes within the liver before excretion via bile through feces. Hepatocytes are polarized epithelial cells, and it has been assumed that the virus load in bile results from direct export of HAV via the apical domain of polarized hepatocytes. We have developed a subclone of hepatocyte-derived HepG2 cells (clone N6) that maintains functional characteristics of polarized hepatocytes but displays morphology typical of columnar epithelial cells, rather than the complex morphology that is typical of hepatocytes. N6 cells form microcolonies of polarized cells when grown on glass and confluent monolayers of polarized cells on semipermeable membranes. When N6 microcolonies were exposed to HAV, infection was restricted to peripheral cells of polarized colonies, whereas all cells could be infected in colonies of nonpolarized HepG2 cells (clone C11) or following disruption of tight junctions in N6 colonies with EGTA. This suggests that viral entry occurs predominantly via the basolateral plasma membrane, consistent with uptake of virus from the bloodstream after enteric exposure, as expected. Viral export was also found to be markedly vectorial in N6 but not C11 cells. However, rather than being exported from the apical domain as expected, more than 95% of HAV was exported via the basolateral domain of N6 cells, suggesting that virus is first excreted from infected hepatocytes into the bloodstream rather than to the biliary tree. Enteric excretion of HAV may therefore rely on reuptake and transcytosis of progeny HAV across hepatocytes into the bile. These studies provide the first example of the interactions between viruses and polarized hepatocytes.
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http://dx.doi.org/10.1128/JVI.00219-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2519658PMC
September 2008

Dynamic regulation of the large exocytotic fusion pore in pancreatic acinar cells.

Mol Biol Cell 2007 Sep 27;18(9):3502-11. Epub 2007 Jun 27.

Department of Pharmacology, University of Cambridge, Cambridge, CB2 1PD, United Kingdom.

Loss of granule content during exocytosis requires the opening of a fusion pore between the secretory granule and plasma membrane. In a variety of secretory cells, this fusion pore has now been shown to subsequently close. However, it is still unclear how pore closure is physiologically regulated and contentious as to how closure relates to granule content loss. Here, we examine the behavior of the fusion pore during zymogen granule exocytosis in pancreatic acinar cells. By using entry of high-molecular-weight dyes from the extracellular solution into the granule lumen, we show that the fusion pore has a diameter of 29-55 nm. We further show that by 5 min after granule fusion, many granules have a closed fusion pore with evidence indicating that pore closure is a prelude to endocytosis and that in granules with a closed fusion pore the chymotrypsinogen content is low. Finally, we show that latrunculin B treatment promotes pore closure, suggesting F-actin affects pore dynamics. Together, our data do not support the classical view in acinar cells that exocytosis ends with granule collapse. Instead, for many granules the fusion pore closes, probably as a transition to endocytosis, and likely involving an F-actin-dependent mechanism.
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http://dx.doi.org/10.1091/mbc.e07-01-0024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1951744PMC
September 2007

Hepatitis B virus translocates across a trophoblastic barrier.

J Virol 2007 Jul 18;81(13):7200-7. Epub 2007 Apr 18.

School of Biomedical Sciences, The University of Queensland, St. Lucia 4072, Australia.

Mother-infant transmission of hepatitis B virus (HBV) accounts for up to 30% of worldwide chronic infections. The mechanism and high-risk period of HBV transmission from mother to infant are unknown. Although largely prevented by neonatal vaccination, significant transmission continues to occur in high-risk populations. It is unclear whether HBV can traverse an intact epithelial barrier to infect a new host. Transplacental transmission of a number of viruses relies on transcytotic pathways across placental cells. We wished to determine whether infectious HBV can traverse a polarized trophoblast monolayer. We used a human placenta-derived cell line, BeWo, cultured on membranes as polarized monolayers, to model the maternal-fetal barrier. We assessed the effects of placental maturity and maternal immunoglobulin on viral transport. Intracellular viral trafficking pathways were investigated by confocal microscopy. Free HBV (and infectious duck hepatitis B virus) transcytosed across trophoblastic cells at a rate of 5% in 30 min. Viral transport occurred in microtubule-dependent endosomal vesicles. Additionally, confocal microscopy showed that the internalized virus traverses a monensin-sensitive endosomal compartment. Differentiation of the cytotrophoblasts to syncytiotrophoblasts resulted in a 25% reduction in viral transcytosis, suggesting that placental maturity may protect the fetus. Virus translocation was also reduced in the presence of HBV immunoglobulin. We show for the first time that transcytosis of infectious hepadnavirus can occur across a trophoblastic barrier early in gestation, with the risk of transmission being reduced by placental maturity and specific maternal antibody. This study suggests a mechanism by which mother-infant transmission may occur.
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http://dx.doi.org/10.1128/JVI.02371-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1933314PMC
July 2007
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