Publications by authors named "Michelle J Holder"

14 Publications

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Neutrophil Extracellular Traps Exert Potential Cytotoxic and Proinflammatory Effects in the Dental Pulp.

J Endod 2019 May 29;45(5):513-520.e3. Epub 2019 Mar 29.

Oral Biology, Birmingham Dental School and Hospital, College of Medical and Dental Sciences, Birmingham, United Kingdom. Electronic address:

Introduction: Neutrophil extracellular traps (NETs) are an important innate immune mechanism aimed at limiting the dissemination of bacteria within tissues and localizing antibacterial killing mechanisms. There is significant interest in the role of NETs in a range of infectious and inflammatory diseases; however, their role in diseased pulp has yet to be explored. Our aim was to determine their relevance to infected pulp and how their components affect human dental pulp cell (HDPC) responses.

Methods: Diseased pulp tissue was stained for the presence of extracellular DNA and elastase to detect the presence of NETs. Bacteria known to infect pulp were also assayed to determine their ability to stimulate NETs. Coculture studies and NET component challenge were used to determine the effect of extracellular NET release on HDPC viability and inflammatory response. NET-stimulated HDPC secretomes were assessed for their chemotactic activity for lymphocytes and macrophages.

Results: Data indicate that NETs are present in infected pulp tissue and whole NETs, and their histone components, particularly H2A, decreased HDPC viability and stimulated chemokine release, resulting in an attraction of lymphocyte populations.

Conclusions: NETs are likely important in pulpal pathogenesis with injurious and chronic inflammatory effects on HDPCs, which may contribute to disease progression. Macrophages are chemoattracted to NET-induced apoptotic HDPCs, facilitating cellular debris removal. NETs and histones may provide novel prognostic markers and/or therapeutic targets for pulpal diseases.
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http://dx.doi.org/10.1016/j.joen.2019.02.014DOI Listing
May 2019

The dark art of light measurement: accurate radiometry for low-level light therapy.

Lasers Med Sci 2016 May 10;31(4):789-809. Epub 2016 Mar 10.

Biomaterials Unit, School of Dentistry, College of Medical and Dental Sciences, University of Birmingham, St Chads Queensway, Birmingham, UK, B4 6NN.

Lasers and light-emitting diodes are used for a range of biomedical applications with many studies reporting their beneficial effects. However, three main concerns exist regarding much of the low-level light therapy (LLLT) or photobiomodulation literature; (1) incomplete, inaccurate and unverified irradiation parameters, (2) miscalculation of 'dose,' and (3) the misuse of appropriate light property terminology. The aim of this systematic review was to assess where, and to what extent, these inadequacies exist and to provide an overview of 'best practice' in light measurement methods and importance of correct light measurement. A review of recent relevant literature was performed in PubMed using the terms LLLT and photobiomodulation (March 2014-March 2015) to investigate the contemporary information available in LLLT and photobiomodulation literature in terms of reporting light properties and irradiation parameters. A total of 74 articles formed the basis of this systematic review. Although most articles reported beneficial effects following LLLT, the majority contained no information in terms of how light was measured (73%) and relied on manufacturer-stated values. For all papers reviewed, missing information for specific light parameters included wavelength (3%), light source type (8%), power (41%), pulse frequency (52%), beam area (40%), irradiance (43%), exposure time (16%), radiant energy (74%) and fluence (16%). Frequent use of incorrect terminology was also observed within the reviewed literature. A poor understanding of photophysics is evident as a significant number of papers neglected to report or misreported important radiometric data. These errors affect repeatability and reliability of studies shared between scientists, manufacturers and clinicians and could degrade efficacy of patient treatments. Researchers need a physicist or appropriately skilled engineer on the team, and manuscript reviewers should reject papers that do not report beam measurement methods and all ten key parameters: wavelength, power, irradiation time, beam area (at the skin or culture surface; this is not necessarily the same size as the aperture), radiant energy, radiant exposure, pulse parameters, number of treatments, interval between treatments and anatomical location. Inclusion of these parameters will improve the information available to compare and contrast study outcomes and improve repeatability, reliability of studies.
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http://dx.doi.org/10.1007/s10103-016-1914-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4851696PMC
May 2016

Inflammation and regeneration in the dentin-pulp complex: a double-edged sword.

J Endod 2014 Apr;40(4 Suppl):S46-51

Oral Biology, School of Dentistry, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.

Dental tissue infection and disease result in acute and chronic activation of the innate immune response, which is mediated by molecular and cellular signaling. Different cell types within the dentin-pulp complex are able to detect invading bacteria at all stages of the infection. Indeed, at relatively early disease stages, odontoblasts will respond to bacterial components, and as the disease progresses, core pulpal cells including fibroblasts, stems cells, endothelial cells, and immune cells will become involved. Pattern recognition receptors, such as Toll-like receptors expressed on these cell types, are responsible for detecting bacterial components, and their ligand binding leads to the activation of the nuclear factor-kappa B and p38 mitogen-activated protein (MAP) kinase intracellular signaling cascades. Subsequent nuclear translocation of the transcription factor subunits from these pathways will lead to proinflammatory mediator expression, including increases in cytokines and chemokines, which trigger host cellular defense mechanisms. The complex molecular signaling will result in the recruitment of immune system cells targeted at combating the invading microbes; however, the trafficking and antibacterial activity of these cells can lead to collateral tissue damage. Recent evidence suggests that if inflammation is resolved relatively low levels of proinflammatory mediators may promote tissue repair, whereas if chronic inflammation ensues repair mechanisms become inhibited. Thus, the effects of mediators are temporal context dependent. Although containment and removal of the infection are keys to enable dental tissue repair, it is feasible that the development of anti-inflammatory and immunomodulatory approaches, based on molecular, epigenetic, and photobiomodulatory technologies, may also be beneficial for future endodontic treatments.
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http://dx.doi.org/10.1016/j.joen.2014.01.021DOI Listing
April 2014

Enhancing the anti-lymphoma potential of 3,4-methylenedioxymethamphetamine ('ecstasy') through iterative chemical redesign: mechanisms and pathways to cell death.

Invest New Drugs 2012 Aug 18;30(4):1471-83. Epub 2011 Aug 18.

School of Immunity & Infection, The Medical School, Birmingham, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

While 3,4-methylenedioxymethamphetamine (MDMA/'ecstasy') is cytostatic towards lymphoma cells in vitro, the concentrations required militate against its translation directly to a therapeutic in vivo. The possibility of 'redesigning the designer drug', separating desired anti-lymphoma activity from unwanted psychoactivity and neurotoxicity, was therefore mooted. From an initial analysis of MDMA analogues synthesized with a modified α-substituent, it was found that incorporating a phenyl group increased potency against sensitive, Bcl-2-deplete, Burkitt's lymphoma (BL) cells 10-fold relative to MDMA. From this lead, related analogs were synthesized with the 'best' compounds (containing 1- and 2-naphthyl and para-biphenyl substituents) some 100-fold more potent than MDMA versus the BL target. When assessed against derived lines from a diversity of B-cell tumors MDMA analogues were seen to impact the broad spectrum of malignancy. Expressing a BCL2 transgene in BL cells afforded only scant protection against the analogues and across the malignancies no significant correlation between constitutive Bcl-2 levels and sensitivity to compounds was observed. Bcl-2-deplete cells displayed hallmarks of apoptotic death in response to the analogues while BCL2 overexpressing equivalents died in a caspase-3-independent manner. Despite lymphoma cells expressing monoamine transporters, their pharmacological blockade failed to reverse the anti-lymphoma actions of the analogues studied. Neither did reactive oxygen species account for ensuing cell death. Enhanced cytotoxic performance did however track with predicted lipophilicity amongst the designed compounds. In conclusion, MDMA analogues have been discovered with enhanced cytotoxic efficacy against lymphoma subtypes amongst which high-level Bcl-2--often a barrier to drug performance for this indication--fails to protect.
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http://dx.doi.org/10.1007/s10637-011-9730-5DOI Listing
August 2012

SLC6A4 expression and anti-proliferative responses to serotonin transporter ligands chlomipramine and fluoxetine in primary B-cell malignancies.

Leuk Res 2010 Aug 2;34(8):1103-6. Epub 2010 Apr 2.

MRC Centre for Immune Regulation, The Medical School, Vincent Drive, Birmingham B15 2TT, UK.

B-cell lines of diverse neoplastic origin express the serotonin transporter (SERT/SLC6A4) and growth arrest in response to SERT-ligands, including the antidepressants chlomipramine and fluoxetine. Here we detail SLC6A4 transcript (Q-PCR) and protein (FACS) expression in primary cells from patients with: chronic lymphocytic leukaemia; mantle cell lymphoma; follicular lymphoma; Burkitt's lymphoma; and diffuse large B-cell lymphoma. The ability of the SERT-binding antidepressants to impact the growth of these cells when sustained on CD154-transfected fibroblasts was also determined. The results reveal a broad spectrum of primary B-cell malignancies expressing SLC6A4 with a proportion additionally displaying growth arrest on SERT-ligand exposure.
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http://dx.doi.org/10.1016/j.leukres.2010.03.007DOI Listing
August 2010

Characterisation of the endogenous human peripheral serotonin transporter SLC6A4 reveals surface expression without N-glycosylation.

J Neuroimmunol 2008 Nov;204(1-2):75-84

Division of Immunity and Infection, The Medical School, University of Birmingham, Birmingham, 815 2TT, United Kingdom.

RT-PCR confirmed that human cell lines of diverse peripheral origins express transcripts for the serotonin transporter (sert/slc6a4). Molecular weights reported for the translated protein appear to be quite variable however. Here we compared directly immunoreactive protein generated from cloned sert transfected into HEK293 with that carried endogenously among the cell lines. The dominant glycosylated 85-95 kDa immunoreactive species contained in HEK-sert transfectants was poorly represented in any native cell: instead, discrete 70 and 60 kDa bands were universally detected. Biotinylation of lymphoid cells revealed that the endogenous non-glycosylated 60 kDa but not 70 kDa protein was available at the surface to access exogenous ligands.
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http://dx.doi.org/10.1016/j.jneuroim.2008.07.014DOI Listing
November 2008

Dynamic interplay between the neutral glycosphingolipid CD77/Gb3 and the therapeutic antibody target CD20 within the lipid bilayer of model B lymphoma cells.

Biochem Biophys Res Commun 2007 Apr 21;355(4):944-9. Epub 2007 Feb 21.

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.

The centroblast-specific differentiation marker CD77 (Gb(3)), is the receptor for Shiga-like toxin (SLT). The dynamic relationship between Gb(3)/CD77 and key B-cell membrane proteins was studied in Burkitt's lymphoma cells with a focus on CD20. Engagement of Gb(3)/CD77 with SLT-B reduced the amount of CD20 and CXCR4 available, but levels of BCR, MHC Class II, CD21, CD27 and CD54 remained unchanged. Cholesterol depletion promoted a decrease in the number of sites accessed by CD20, CXCR4 and Gb(3)/CD77 antibodies. Constitutive localisation of Gb(3)/CD77 to lipid rafts was unperturbed by either SLT-B binding or cholesterol depletion, whereas the opposite was true for CD20. The effects were specific to SLT-B, highlighted by the inability of cholera toxin B-subunit to alter CD20 availability. Thus, the binding of Gb(3)/CD77 by its cognate ligand transmits information within the lipid bilayer of model lymphoma cells to impact the behaviour of selective proteins, most notably CD20, via a mechanism influenced by the level of cholesterol within the membrane.
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http://dx.doi.org/10.1016/j.bbrc.2007.02.053DOI Listing
April 2007

Dopamine targets cycling B cells independent of receptors/transporter for oxidative attack: Implications for non-Hodgkin's lymphoma.

Proc Natl Acad Sci U S A 2006 Sep 28;103(36):13485-90. Epub 2006 Aug 28.

Medical Research Council Centre for Immune Regulation, Division of Immunity and Infection, The Medical School, University of Birmingham, Vincent Drive, Birmingham B15 2TT, United Kingdom.

Human B lymphocytes and derived lines from a spectrum of B cell malignancy were studied for expression of dopaminergic pathway components and for their cytostatic response to the catecholamine and related, potentially therapeutic compounds. Proliferating normal lymphocytes and dividing malignant clones rapidly arrested on exposure to dopamine in the low (
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http://dx.doi.org/10.1073/pnas.0605993103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1569189PMC
September 2006

Lymphoma cells protected from apoptosis by dysregulated bcl-2 continue to bind annexin V in response to B-cell receptor engagement: a cautionary tale.

Leuk Res 2006 Jan 1;30(1):77-80. Epub 2005 Aug 1.

MRC Centre for Immune Regulation, The Medical School, University of Birmingham, Vincent Drive B15 2TT, UK.

Translocation of phosphatidylserine (PS) from the inner to outer leaflet of the surface membrane lipid bilayer, a characteristic early event of cells entering the apoptotic program, is routinely assessed by the Ca(2+)-dependent binding of Annexin V (AV). Here, we show that lymphoma cells protected from apoptosis by expression of a bcl-2 transgene or by virtue of the t(14;18)(q32;q21) translocation continue to register enhanced AV binding in response to BCR crosslinking. Induced AV binding appeared BCR-selective in that it did not proceed in Bcl-2(high) cells in response to calcium ionophore or the antidepressant fluoxetine, each of which activate the full apoptotic program in Bcl-2(low) equivalents. AV-positive cells did increase on crosslinking the BCR co-receptor CD19, despite it being a completely non-apoptotic signal. These findings advise caution when interpreting studies where Annexin V binding is used as a sole, or major, indicator of apoptotic death among lymphoma B cells.
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http://dx.doi.org/10.1016/j.leukres.2005.05.018DOI Listing
January 2006

Close encounters of the monoamine kind: immune cells betray their nervous disposition.

Immunology 2005 Jul;115(3):289-95

MRC Centre for Immune Regulation, The Medical School, University of Vincent Drive, Birmingham B15 2TT, UK.

Here we review the evidence for immune cells expressing multiple components of the serotonergic and dopaminergic systems that are more commonly associated with the central nervous system (CNS). We discuss where and how peripheral encounters with these biogenic monoamines occur and posit reasons as to why the immune system would wish to deploy these pathways. A full taxonomy of serotonergic and dopaminergic constituents and their workings in component cells of the immune system should facilitate the formulation of novel therapeutic approaches in diseases characterized by immune dysfunction and potentially provide a range of surrogate peripheral markers for registering and monitoring disturbances within the CNS.
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http://dx.doi.org/10.1111/j.1365-2567.2005.02166.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1782168PMC
July 2005

The serotonin transporter (SLC6A4) is present in B-cell clones of diverse malignant origin: probing a potential anti-tumor target for psychotropics.

FASEB J 2005 Jul 3;19(9):1187-9. Epub 2005 May 3.

Division of Immunity and Infection, The Medical School, Birmingham, UK.

Following our previous description of the serotonin transporter (SERT) acting as a conduit to 5-hydroxytryptamine (5-HT)-mediated apoptosis, specifically in Burkitt's lymphoma, we now detail its expression among a broad spectrum of B cell malignancy, while exploring additional SERT substrates for potential therapeutic activity. SERT was readily detected in derived B cell lines with origins as diverse as B cell precursor acute lymphoblastic leukemia, mantle cell lymphoma, diffuse large B cell lymphoma, and multiple myeloma. Concentration and timecourse kinetics for the antiproliferative and proapoptotic activities of the amphetamine derivatives fenfluramine (an appetite suppressant) and 3,4-methylenedioxymethamphetamine (MDMA; "Ecstasy") revealed them as being similar to the endogenous indoleamine. A tricyclic antidepressant, clomipramine, instead mirrored the behavior of the selective serotonin reuptake inhibitor fluoxetine, both being effective in the low micromolar range. A majority of neoplastic clones were sensitive to one or more of the serotonergic compounds. Dysregulated bcl-2 expression, either by t(14;18)(q32;q21) translocation or its introduction as a constitutively active transgene, provided protection from proapoptotic but not antiproliferative outcomes. These data indicate a potential for SERT as a novel anti-tumor target for amphetamine analogs, while evidence is presented that the seemingly more promising antidepressants are likely impacting malignant B cells independently of the transporter itself.
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http://dx.doi.org/10.1096/fj.04-3477fjeDOI Listing
July 2005

Improved access to CD20 following B cell receptor cross-linking at Burkitt's lymphoma cell surfaces.

Leuk Res 2004 Nov;28(11):1197-202

MRC Centre for Immune Regulation, The Medical School, The University of Birmingham, Vincent Drive, B15 2TT, UK.

Here we report that B cell receptor (BCR) engagement rapidly improves the capacity of CD20 to be accessed by cognate antibody at model Burkitt's lymphoma cell surfaces. None of eight other surface molecules demonstrated such BCR-dependent enhancement of ligand binding while the quantity of accessible CD20 remained unchanged on either CD19 or CD40 engagement. Neither the actin-depolymerizing agent cytochalasin D nor inhibitors targeting signalling pathways associated with the BCR attenuated the CD20 increase that could be uncoupled from BCR endocytosis. Instead, a role for lipid rafts was indicated both from the inhibitory actions of cholesterol-sequestering methyl-beta-cyclodextrin and direct analysis of CD20 redistribution using sucrose density gradients and confocal microscopy. Whether such observations could find application in CD20-directed therapies where success can be compromised by otherwise low-level expression of target antigen is discussed.
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http://dx.doi.org/10.1016/j.leukres.2004.02.008DOI Listing
November 2004

Selective serotonin reuptake inhibitors directly signal for apoptosis in biopsy-like Burkitt lymphoma cells.

Blood 2003 Apr 19;101(8):3212-9. Epub 2002 Dec 19.

Medical Research Council Centre for Immune Regulation, School of Biosciences, University of Birmingham, Birmingham, United Kingdom.

Selective serotonin reuptake inhibitors (SSRIs) are the treatment of choice for clinical depression and a range of anxiety-related disorders. They are well tolerated over extended periods with more than 50 million people worldwide benefiting from their use. Here we show that 3 structurally distinct SSRIs--fluoxetine, paroxetine, and citalopram--act directly on Burkitt lymphoma (BL) cells to trigger rapid and extensive programmed cell death. SSRIs unexpectedly stimulated calcium flux, tyrosine phosphorylation, and down-regulation of the c-myc and nm23 genes in Burkitt lymphoma cells remaining faithful to the biopsy phenotype. Resultant SSRI-induced apoptosis was preceded by caspase activation, poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, DNA fragmentation, a loss of mitochondrial membrane potential, and the externalization of phosphatidylserine, and reversed by the overexpression of bcl-2. Normal peripheral blood mononuclear cells and tonsil B cells, whether resting or stimulated into cycle, were largely resistant to SSRI-induced death as were 5 non-BL lymphoid cell lines tested. We discuss these findings within the context of whether the SSRI class of antidepressants could find future application as potential therapeutics for the highly aggressive and-because of its association with AIDS-increasingly more common Burkitt lymphoma.
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http://dx.doi.org/10.1182/blood-2002-07-2044DOI Listing
April 2003

Population depletion activates autonomous CD154-dependent survival in biopsylike Burkitt lymphoma cells.

Blood 2002 May;99(9):3411-8

MRC Centre for Immune Regulation and the Institute for Cancer Studies, The University of Birmingham, Birmingham, United Kingdom.

Population size is governed through cells reacting to a variety of intrinsic and extrinsic cues. Tumors, while liberated from many of the homeostatic constraints placed on physiologic counterparts, can nonetheless remain subject to both social and environmental control. Burkitt lymphoma cells faithful to the biopsy phenotype were used to model the reliance of the colony, if any, on an inbuilt population sensor. Below a normally suicidal threshold number of cells, low picomolar quantities of exogenous CD40 ligand (CD40L/CD154) were found to sustain the clone without the discernible shift in phenotype that accompanies high CD40L encounter. Although CD154 was undetectable in populous cultures, message was induced as numbers became limiting. Correspondingly, attempts to neutralize endogenous CD40L activity failed to perturb cells at optimal densities but resulted in their marked decline as the critical threshold was approached. These data reveal an auto-inducible survival mechanism seemingly regulated through the monitoring of population size, a process somewhat akin to that of "quorum sensing" among gram-negative bacteria in which diffusible molecules provide a means of communication to coordinate gene expression with population density. This process could be activated as cells discern depletions in their community or when deprived of signals otherwise furnished within an appropriate environmental niche.
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http://dx.doi.org/10.1182/blood.v99.9.3411DOI Listing
May 2002