Potential interactions among single nucleotide polymorphisms in bone- and cartilage-related genes in skeletal malocclusions

Objective To investigate SNPs in bone‐ and cartilage‐related genes and their interaction in the aetiology of sagittal and vertical skeletal malocclusions. Settings and sample population This study included 143 patients and classified as follows: skeletal class I (n = 77), class II (n = 47) and class III (n = 19); maxillary retrusion (n = 39), protrusion (n = 52) and well‐positioned maxilla (n = 52); mandibular retrognathism (n = 50), prognathism (n = 50) and well‐positioned mandible (n = 43); normofacial (n = 72), dolichofacial (n = 55) and brachyfacial (n = 16). Materials and methods Steiner's ANB, SNA, SNB angles and Ricketts’ NBa‐PtGn angle were measured to determine the skeletal malocclusion and the vertical pattern. Nine SNPs in BMP2, BMP4, SMAD6, RUNX2, WNT3A and WNT11 were genotyped. Chi‐squared test was used to compare genotypes among the groups. Multifactor dimensionality reduction (MDR) and binary logistic regression analysis, both using gender and age as co‐variables, were also used. We performed Bonferroni correction for multiple testing. Results Significant associations at P < .05 were observed for SNPs rs1005464 (P = .042) and rs235768 (P = .021) in BMP2 with mandibular retrognathism and for rs59983488 (RUNX2) with maxillary protrusion (P = .04) as well as for rs708111 (WNT3A) with skeletal class III (P = .02; dominant model), rs1533767 (WNT11) with a brachyfacial skeletal pattern (P = .01, OR = 0.10; dominant model) and for rs3934908 (SMAD6) with prognathism (P = .02; recessive model). After the Bonferroni correction, none of the SNPs remained associated. The MDR predicted some interaction for skeletal class II, dolichofacial and brachyfacial phenotypes. Conclusion Our results suggest that SNPs in BMP2, BMP4, SMAD6, RUNX2, WNT3A and WNT11 could be involved in the aetiology of sagittal and vertical malocclusions

Redirecting T Cells to Ewing's Sarcoma Family of Tumors by a Chimeric NKG2D Receptor Expressed by Lentiviral Transduction or mRNA Transfection

We explored the possibility to target Ewing's sarcoma family of tumors (ESFT) by redirecting T cells. To this aim, we considered NKG2D-ligands (NKG2D-Ls) as possible target antigens. Detailed analysis of the expression of MICA, MICB, ULBP-1, -2, and -3 in fourteen ESFT cell lines revealed consistent expression of at least one NKG2D-L. Thus, for redirecting T cells, we fused a CD3ζ/CD28-derived signaling domain to the ectodomain of NKG2D, however, opposite transmembrane orientation of this signaling domain and NKG2D required inverse orientation fusion of either of them. We hypothesized that the particularly located C-terminus of the NKG2D ectodomain should allow reengineering of the membrane anchoring from a native N-terminal to an artificial C-terminal linkage. Indeed, the resulting chimeric NKG2D receptor (chNKG2D) was functional and efficiently mediated ESFT cell death triggered by activated T cells. Notably, ESFT cells with even low NKG2D-L expression were killed by CD8pos and also CD4pos cells. Both, mRNA transfection and lentiviral transduction resulted in high level surface expression of chNKG2D. However, upon target-cell recognition receptor surface levels were maintained by tranfected RNA only during the first couple of hours after transfection. Later, target-cell contact resulted in strong and irreversible receptor down-modulation, whereas lentivirally mediated expression of chNKG2D remained constant under these conditions. Together, our study defines NKG2D-Ls as targets for a CAR-mediated T cell based immunotherapy of ESFT. A comparison of two different methods of gene transfer reveals strong differences in the susceptibility to ligand-induced receptor down-modulation with possible implications for the applicability of RNA transfection

Influence of immune evasion mechanisms of cytomegalovirus on the efficiency of CAR-based adoptive immunotherapy

Ein vielversprechender Ansatz zur Behandlung von Infektionen mit dem humanen Zytomegalievirus (HCMV) nach Stammzelltransplantation ist die adoptive Immuntherapie mit virusspezifischen T-Zellen. Allerdings ist diese Therapie bei HCMV-seropositiven Patienten, die ein Transplantat von einem HCMV-seronegativen Donor erhalten haben, aufgrund der geringen Frequenz HCMV-spezifischer T-Zellen im Blut des Spenders nicht anwendbar. Alternativ könnten polyklonale T-Zellen infundiert werden, in welche die Virus-Spezifität durch Expression von chimären Antigenrezeptoren (CAR) eingebracht wurde. In der vorliegenden Arbeit wurde in vitro das Potential eines Ansatzes zur adoptiven Immuntherapie von HCMV-Infektionen untersucht, bei dem T-Zellen durch einen CAR gegen das Glykoprotein B des HCMV gerichtet werden. Die Funktionalität dieses CAR wurde bereits in Vorarbeiten demonstriert. Es stellte sich jedoch in dieser Arbeit heraus, dass CAR-modifizierte T-Zellen HCMV-infizierte Vorhautfibroblasten (HFF) nicht abtöten, obwohl antigenexprimierende nicht-infizierte Zielzellen effizient lysiert wurden. Eine Reduktion der Lyse von HFF ab dem 2. Tag nach der HCMV-Infektion wurde auch bei T-Zellrezeptor (TCR)-induzierter Aktivierung der T-Zellen beobachtet. Im Gegensatz zur CAR-vermittelten Erkennung konnte jedoch mit der TCR-vermittelten Erkennung eine deutliche Lyse von HCMV-infizierten HFF durch Verlängerung der Inkubationszeit erzielt werden. Eine reduzierte CAR- bzw- TCR-vermittelte Aktivierung der T-Zellen als Grund für die Lysehemmung konnte ausgeschlossen werden, da eine reduzierte Degranulation oder verringerte Zytokinfreisetzung nicht beobachtet wurde. Trotzdem konnte gezeigt werden, dass CAR-T-Zellen antiviral wirksam sein können, indem sie durch die sezernierten Zytokine TNF und IFN-γ die Ausbreitung der HCMV-Infektion effizient hemmen. Zur näheren Charakterisierung der beobachteten Lysehemmung wurde der Einfluss der anti-apoptotischen HCMV-kodierten Proteine UL36 und UL37x1 auf die T-Zellvermittelte Zytolyse untersucht. Tatsächlich konnte die Expression dieser viralen Proteine in HFF den durch CAR-exprimierende T-Zellen induzierten Zelltod teilweise reduzieren. Dabei konnte nicht nur die Todesrezeptor-vermittelte sondern auch die Perforin-vermittelte Apoptose durch die Expression beider Proteine gehemmt werden. Die vorliegende Arbeit beschreibt erstmals einen Immunevasionsmechanismus des HCMV, der auf einer Blockade der zytotoxischen T-Zellfunktionen beruht. Diese Blockade wird zum Teil durch die viralen Proteine UL36 und UL37x1 vermittelt und inhibiert die Lyse der infizierten Zellen durch CAR-modifizierte T-Zellen. Dennoch könnten CAR-modifizierte T-Zellen z.B. durch Sekretion von Zytokinen die Infektion eindämmen und so ein wirksames Therapeutikum darstellen.Restoring the virus-specific immunity by application of virus-specific T cells for adoptive therapy of human cytomegalovirus (HCMV) infections after stem cell transplantation is an attractive treatment option. However, this strategy is not applicable in the setting of a HCMV-seronegative patient and a HCMV-seropositive stem cell donor due to low frequencies of HCMV-specific T cells in the blood of seronegative donors. Alternatively, chimeric antigen receptors (CAR) can be used to introduce virus-specificity into a polyclonal T cell population. For this purpose, the potential of a CAR-based adoptive immunotherapy directed against the glycoprotein B of HCMV was investigated. The functionality of this HCMV-specific CAR in T cells has already been demonstrated. However, the present study revealed that CAR-induced lysis of HCMV-infected foreskin fibroblasts (HFF) is strongly reduced although lytic activity of CAR-modified T cells against antigen-expressing non-infected target cells was observed. Similarly, T cell receptor (TCR)-mediated lysis of HFF was inhibited by HCMV starting at day 2 after infection. In contrast to CAR-mediated recognition, TCR-mediated recognition of target cells allowed for inducing substantial cell death of HCMV-infected HFF by prolonging the incubation time. Notably, the observed reduction of the lysis of HCMV-infected cells was not associated with impairment of CAR-mediated or TCR-mediated activation of T cells, since effector functions like degranulation and cytokine release were not inhibited. Nevertheless, it was shown that CAR-expressing T cells could be potent mediators of antiviral activity by secretion of TNF und IFN-γ, which efficiently inhibit HCMV dissemination. Additionally, the underlying mechanism of the observed lysis inhibition was investigated by analyzing the impact of the HCMV-encoded anti-apoptotic proteins UL36 and UL37x1 on T cell-mediated lysis. Overexpression of these proteins could protect HFF from CAR-mediated lysis. Remarkably, expression of both proteins could not only inhibit death receptor-induced apoptosis but also perforin-mediated cell death. The thesis scrutinizes for the first time a mechanism of immune escape, which is based on efficient impairment of T cell cytotoxicity. This mechanism is partially mediated by the HCMV-encoded proteins UL36 and UL37x1 and abrogates the cytolytic activity of CAR-expressing T cells. Still, the administration of these CAR-T cells could be attractive since other effector functions like release of cytokines also contribute to the control of HCMV infection

Turning the tables on cytomegalovirus: targeting viral Fc receptors by CARs containing mutated CH2–CH3 IgG spacer domains

Abstract Background During infection with human cytomegalovirus (HCMV) several viral proteins occur on cell surfaces in high quantity. We thus pursue an HLA-independent approach for immunotherapy of HCMV using chimeric antigen receptors (CARs) and bispecific BiTE® antibody constructs. In this context, HCMV-encoded proteins that mediate viral immune evasion and bind human IgG might represent particularly attractive target antigens. Unlike in observations of similar approaches for HIV and hepatitis B and C viruses, however, HCMV-infected cells develop a striking resistance to cytotoxic effector functions at later stages of the replication cycle. In our study we therefore wanted to test two hypotheses: (1) CAR T cells can efficiently inhibit HCMV replication independently from cytotoxic effector functions, and (2) HCMV can be targeted by CH2–CH3 IgG spacer domains that contain mutations previously reported to prevent exhaustion and to rescue CAR T cell function in vivo. Methods Replication of GFP-encoding recombinant HCMV in fibroblasts in the presence and absence of supernatants from T cell co-cultures plus/minus cytokine neutralizing antibodies was analyzed by flow cytometry. CARs with wild type and mutated CH2–CH3 domains were expressed in human T cells by mRNA electroporation, and the function of the CARs was assessed by quantifying T cell cytokine secretion. Results We confirm and extend previous evidence of antiviral cytokine effects and demonstrate that CAR T cells strongly block HCMV replication in fibroblasts mainly by combined secretion of IFN-γ and TNF. Furthermore, we show that fibroblasts infected with HCMV strains AD169 and Towne starting from day 3 have a high capacity for binding of human IgG1 and also strongly activate T cells expressing a CAR with CH2–CH3 domain. Importantly, we further show that mutations in the CH2–CH3 domain of IgG1 and IgG4, which were previously reported to rescue CAR T cell function by abrogating interaction with endogenous Fc receptors (FcRs), still enable recognition of FcRs encoded by HCMV. Conclusions Our findings identify HCMV-encoded FcRs as an attractive additional target for HCMV immunotherapy by CARs and possibly bispecific antibodies. The use of specifically mutated IgG domains that bind to HCMV-FcRs without recognizing endogenous FcRs may supersede screening for novel binders directed against individual HCMV-FcRs

Morphometric characteristics of anencephalic skulls – A comparative study

Anencephaly is the most severe form of a neural tube defect resulting from the incomplete occlusion of the anterior neuropore in the fourth week of development and associated with a severely underdeveloped brain mass. As desmal ossification of the neurocranium is induced by the presence of soft tissues (brain), no bone develops as direct consequence of the missing brain. The cranial base, by contrast, is formed by chondral ossification, which is genetically determined, and thus present also in anencephaly. Morphometric characteristics of anencephalic skulls, however, have not yet been investigated in sufficient detail before. In this study we therefore comparatively assessed macroscopic morphological-anatomical and cephalometric CT data on structures and dimensions of 11 macerated anencephalic and 4 normal neonatal skulls highlighting skeletal morphological differences. The most striking results were the missing skullcap and the greatly changed morphology of the existing skull bones, which were reduced in size. The parameters of the skull base, the transverse orbital diameter and maxillary width were significantly smaller in anencephalic skulls. The morphology of the viscerocranium appeared similar to that of normal neonatal skulls. The results of this study can be used in diagnosis and skeletal classification for anencephaly. This can help identify bones that are incomplete, fragmented and taphonomically altered, which is often the case in historical and forensic studies. (C) 2020 Elsevier GmbH. All rights reserved

MOESM1 of Turning the tables on cytomegalovirus: targeting viral Fc receptors by CARs containing mutated CH2–CH3 IgG spacer domains

Additional file 1: Figure S1. Replication kinetics of HCMV in HFF. Shown is the number of infectious virus particles obtained from 1x106 HFF at different time points after infection with HCMV (AD169; MOI as indicated). (A) Infectious particles released into the supernatant. (B) Cell associated infectious particles obtained from centrifuged supernatant after ultrasonic homogenization of the HFF

Effects of mechanical strain on periodontal ligament fibroblasts in presence of Aggregatibacter actinomycetemcomitans lysate

Purpose Many adult orthodontic patients suffer from periodontitis, which is caused by oral pathogens such as the gram-negative Aggregatibacter actinomycetemcomitans (Agac). Like orthodontic tooth movement, periodontitis is associated with inflammation and alveolar bone remodelling thereby affecting orthodontic treatment. Interactions of both processes, however, are not sufficiently explored, particularly with regard to oxidative stress. Methods After preincubation with Agac lysate for 24 h periodontal ligament fibroblasts (PDLF) were either stretched or compressed for further 48 h simulating orthodontic forces in vitro. We analysed the expression of genes and proteins involved in the formation of reactive oxygen species (NOX-4, ROS) and nitric oxide (NOS-2), inflammation (TNF, IL-6, PTGS-2) and bone remodelling (OPG, RANKL). Results Agac lysate elevated the expression of NOX-4, NOS-2, inflammatory IL-6 and PTGS-2 and the bone-remodelling RANKL/OPG ratio during compressive, but not tensile mechanical strain. Agac lysate stimulated pressure-induced inflammatory signalling, whereas surprisingly ROS formation was reduced. Pressure-induced downregulation of OPG expression was inhibited by Agac lysate. Conclusions Agac lysate impact on the expression of genes and proteins involved in inflammation and bone remodelling as well as ROS formation, when PDLF were subjected to mechanical forces occurring during orthodontic tooth movement

Scientific Reports / A gB/CD3 bispecific BiTE antibody construct for targeting Human Cytomegalovirus-infected cells

Bispecific T cell engager (BiTE) antibody constructs are successfully used as cancer therapeutics. We hypothesized that this treatment strategy could also be applicable for therapy of human cytomegalovirus (HCMV) infection, since HCMV-encoded proteins are abundantly expressed on the surface of infected cells. Here we show that a BiTE antibody construct directed against HCMV glycoprotein B (gB) and CD3 efficiently triggers T cells to secrete IFN- and TNF upon co-culture with fibroblasts infected with HCMV strain AD169, Towne or Toledo. Titration of gB expression levels in non-infected cells confirmed that already low levels of gB are sufficient for efficient triggering of T cells in presence of the BiTE antibody construct. Comparison of redirecting T cells with the bispecific antibody versus a chimeric antigen receptor (CAR) based on the same scFv showed a similar sensitivity for gB expression. Although lysis of infected target cells was absent, the BiTE antibody construct inhibited HCMV replication by triggering cytokine production. Notably, even strongly diluted supernatants of the activated T cells efficiently blocked the replication of HCMV in infected primary fibroblasts. In summary, our data prove the functionality of the first BiTE antibody construct targeting an HCMV-encoded glycoprotein for inhibiting HCMV replication in infected cells.(VLID)471789

GTPase-specific binding to their effectors.

<p>A) Coomassie-stained SDS-gel of precipitates of pull down experiments from HEp2 cell lysate. Arrows indicate GST-Rac1/−Rac1 S71E (48 kDa) used as bait and a coprecipitated 190 kDa protein that was identified as IQGAP1 by MALDI-TOF/TOF analysis. B) The interaction of active, GTP-bound Rac1/Cdc42 and their active forms (Q61L) with specific effectors was analyzed by immunoblot analyses of precipitates from pull down assays. Non-specific binding was tested by GST-loaded glutathione beads as control. C) Representative input control of pull down analyses using constitutively active (Q61L) mutants of Rac1 and Cdc42 and their S71E mutants. D) Representative immunoblots of pull down precipitates showing the interaction of constitutively active Rac1 and Cdc42 and constitutively active S71E mutants with their effector proteins. Rac1 Q61L/S71E and Cdc42 Q61L/S71E did hardly bind to their specific effectors Sra-1 and N-WASP, respectively and to their common effector protein PAK1. Both phosphomimetic GTPases, however bound to their common effectors IQGAP and MRCK alpha, although to a lesser extent. The bars show the arithmetic mean value ± SD of densitometrical evaluation of 3 independent experiments.</p