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Article ; Online: Synergistic targeting of the PI3K/mTOR and MAPK/ERK pathways in Merkel cell carcinoma.

Temblador, Arturo / Topalis, Dimitrios / Andrei, Graciela / Snoeck, Robert

Tumour virus research

2022 Volume 14, Page(s) 200244

Abstract: Merkel cell carcinoma (MCC) is an aggressive type of skin cancer, which is caused either by integration of the oncogenic Merkel cell polyomavirus (MCPyV) or by accumulation of UV-light induced mutations. Since the response to immune-checkpoint inhibitors ...

Abstract	Merkel cell carcinoma (MCC) is an aggressive type of skin cancer, which is caused either by integration of the oncogenic Merkel cell polyomavirus (MCPyV) or by accumulation of UV-light induced mutations. Since the response to immune-checkpoint inhibitors is limited, new therapeutic agents need to be explored. Previous studies have shown that MCC cell lines and xenografts are sensitive to MLN0128, a dual mTOR1/2 inhibitor. Prompted by these results and considering that the PI3K/mTOR and MAPK/ERK pathways are the most commonly deregulated pathways in cancer, the combination of MLN0128 with the MEK1/2 inhibitor trametinib was investigated. Importantly, the combined targeting showed to be synergistic in MCC cell lines and induced alterations in the protein levels of downstream elements of the targeted pathways. This synergistic activity implies a reduction in the dose of each inhibitor necessary to reach the same effect that when used as single agents. Therefore, this is a promising approach to improve the clinical management of MCC and to overcome the limited efficacy of single drug regimens owed to the appearance of toxicity or drug resistance.
Language	English
Publishing date	2022-08-22
Publishing country	Netherlands
Document type	Journal Article
ISSN	2666-6790
ISSN (online)	2666-6790
DOI	10.1016/j.tvr.2022.200244
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Article: Résistance des virus herpes simplex aux antiviraux.

Burrel, Sonia / Topalis, Dimitrios / Boutolleau, David

Virologie (Montrouge, France)

2020 Volume 24, Issue 5, Page(s) 325–342

Abstract: Herpes simplex virus (HSV) infections remain an important cause of morbidity among immunocompromised patients, such as transplant recipients and human immunodeficiency virus [HIV]-infected individuals. Only few antiviral drugs are available to treat HSV ... ...

Title translation	Herpes simplex virus resistance to antivirals.
Abstract	Herpes simplex virus (HSV) infections remain an important cause of morbidity among immunocompromised patients, such as transplant recipients and human immunodeficiency virus [HIV]-infected individuals. Only few antiviral drugs are available to treat HSV infections: (val)acyclovir, foscarnet, and cidofovir. Prophylactic and curative antiviral treatments administered during prolonged periods among patients with altered T-cell immunity may lead to the emergence of HSV resistance to antivirals, contributing to a challenging therapeutic management of viral infection. The persistence of herpetic lesions after 10 days of well-conducted antiviral therapy is suggestive of viral resistance. Resistance to antivirals can be detected using genotypic methods (identifications of antiviral resistance-associated mutations by sequencing genes encoding viral proteins involved in the mechanism of action of antivirals) or phenotypic methods (measure of antiviral drug concentration inhibiting 50% of viral replication in cell culture). The prevalence of HSV resistance to acyclovir is below 1% in immunocompetent individuals, except those with herpetic keratitis for whom prevalence can reach 7%, and varies from 3.5% to 11% in immunocompromised patients. Adverse effects and the absence of eradication of viral latent infection constitute other limits to the use of antiviral drugs. New antiviral compounds undergoing clinical trials and novel potential viral targets seem very promising to enlarge the panel of efficient compounds to treat HSV infections.
MeSH term(s)	Acyclovir/pharmacology ; Acyclovir/therapeutic use ; Antiviral Agents/pharmacology ; Antiviral Agents/therapeutic use ; Foscarnet/therapeutic use ; Herpes Simplex/drug therapy ; Humans ; Simplexvirus
Chemical Substances	Antiviral Agents ; Foscarnet (364P9RVW4X) ; Acyclovir (X4HES1O11F)
Language	French
Publishing date	2020-11-12
Publishing country	France
Document type	Journal Article
ZDB-ID	2118387-9
ISSN	1950-6961 ; 1267-8694
ISSN (online)	1950-6961
ISSN	1267-8694
DOI	10.1684/vir.2020.0864
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Article ; Online: Photobiological hazards in shielded metal arc welding.

Gourzoulidis, G A / Bouroussis, C A / Achtipis, A / Kazasidis, M / Pantelis, D / Markoulis, A / Konstantakopoulos, I / Topalis, F V

Physica medica : PM : an international journal devoted to the applications of physics to medicine and biology : official journal of the Italian Association of Biomedical Physics (AIFB)

2023 Volume 106, Page(s) 102520

Abstract: Purpose: Photobiological hazards caused by artificial optical radiation are assessed for the most commonly used arc welding technique, the SMAW (Shielded Metal Arc Welding), which operates with a hand-held system and is widely used both at occupational ... ...

Abstract	Purpose: Photobiological hazards caused by artificial optical radiation are assessed for the most commonly used arc welding technique, the SMAW (Shielded Metal Arc Welding), which operates with a hand-held system and is widely used both at occupational and domestic environments, expanding our previous investigation of a robotic arc welding process. Methods: The complex exposure limits of the emitted blue and visible light, ultraviolet and infrared, are assessed through the European Directive 2006/25/EC, using three dedicated sensors set to measure irradiance from various typical welding procedures in the controlled environment (currents, electrodes, etc.) of a welding laboratory. In this sense, field measurements are employed, applying existing policies. Occupational limits are also applicable to the domestic welding. Results: Apart from sub-minute overexposures, which were detected in every spectral band, overexposures in the order of one second were also observed at very close distances, which are common at complex working environments. Investigation of the initial welding procedure, which is often performed without the use of Personal Protective Equipment, revealed exposure of the order of the corresponding limit. Conclusions: Simulation of a 'bad' welding procedure revealed increased exposure, indicating the importance of training in the occupational environment. Concern for the exposure of near-by workers (working a few meters away from the welding point) is also crucial. Future work needs to incorporate more welding techniques and measurements from original workplaces, in order to set the basis for an integrated risk assessment and provide valuable information concerning occupational diseases.
MeSH term(s)	Humans ; Occupational Exposure/analysis ; Ultraviolet Rays ; Welding/methods ; Metals ; Light
Chemical Substances	Metals
Language	English
Publishing date	2023-01-05
Publishing country	Italy
Document type	Journal Article
ZDB-ID	1122650-x
ISSN	1724-191X ; 1120-1797
ISSN (online)	1724-191X
ISSN	1120-1797
DOI	10.1016/j.ejmp.2022.102520
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Article: Organotypic Epithelial Raft Cultures as a Three-Dimensional In Vitro Model of Merkel Cell Carcinoma.

Temblador, Arturo / Topalis, Dimitrios / van den Oord, Joost / Andrei, Graciela / Snoeck, Robert

Cancers

2022 Volume 14, Issue 4

Abstract: Merkel cell carcinoma (MCC) is a rare type of skin cancer for which an in vitro model is still lacking. MCC tumorigenesis is associated either with the integration of Merkel cell polyomavirus into the host genome, or with the accumulation of somatic ... ...

Abstract	Merkel cell carcinoma (MCC) is a rare type of skin cancer for which an in vitro model is still lacking. MCC tumorigenesis is associated either with the integration of Merkel cell polyomavirus into the host genome, or with the accumulation of somatic mutations upon chronic exposure to UV light. Transgenic animals expressing the viral oncoproteins, which are constitutively expressed in virus-related MCC, do not fully recapitulate MCC. Although cell-line-derived xenografts have been established for the two subtypes of MCC, they still present certain limitations. Here, we generated organotypic epithelial raft cultures (OERCs) of MCC by using primary human keratinocytes and both virus-positive and virus-negative MCC cell lines. The primary human keratinocytes and the tumor cells were grown on top of a dermal equivalent. Histological and immunohistochemical examination of the rafts confirmed the growth of MCC cells. Furthermore, gene expression analysis revealed differences in the expression profiles of the distinct tumor cells and the keratinocytes at the transcriptional level. In summary, considering the limited availability of patient samples, OERCs of MCC may constitute a suitable model for evaluating the efficacy and selectivity of new drug candidates against MCC; moreover, they are a potential tool to study the oncogenic mechanisms of this malignancy.
Language	English
Publishing date	2022-02-21
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2527080-1
ISSN	2072-6694
ISSN	2072-6694
DOI	10.3390/cancers14041091
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Article: Poxviruses Bearing DNA Polymerase Mutations Show Complex Patterns of Cross-Resistance.

Andrei, Graciela / Fiten, Pierre / Krečmerová, Marcela / Opdenakker, Ghislain / Topalis, Dimitrios / Snoeck, Robert

Biomedicines

2022 Volume 10, Issue 3

Abstract: Despite the eradication of smallpox four decades ago, poxviruses continue to be a threat to humans and animals. The arsenal of anti-poxvirus agents is very limited and understanding mechanisms of resistance to agents targeting viral DNA polymerases is ... ...

Abstract	Despite the eradication of smallpox four decades ago, poxviruses continue to be a threat to humans and animals. The arsenal of anti-poxvirus agents is very limited and understanding mechanisms of resistance to agents targeting viral DNA polymerases is fundamental for the development of antiviral therapies. We describe here the phenotypic and genotypic characterization of poxvirus DNA polymerase mutants isolated under selective pressure with different acyclic nucleoside phosphonates, including HPMPC (cidofovir), cHPMPC, HPMPA, cHPMPA, HPMPDAP, HPMPO-DAPy, and PMEO-DAPy, and the pyrophosphate analogue phosphonoacetic acid. Vaccinia virus (VACV) and cowpox virus drug-resistant viral clones emerging under drug pressure were characterized phenotypically (drug-susceptibility profile) and genotypically (DNA polymerase sequencing). Different amino acid changes in the polymerase domain and in the 3'-5' exonuclease domain were linked to drug resistance. Changes in the 3'-5' domain emerged earlier than in the polymerase domain when viruses acquired a combination of mutations. Our study highlights the importance of poxvirus DNA polymerase residues 314, 613, 684, 688, and 851, previously linked to drug resistance, and identified several novel mutations in the 3'-5' exonuclease domain (M313I, F354L, D480Y) and in the DNA polymerase domain (A632T, T831I, E856K, L924F) associated with different drug-susceptibility profiles. Furthermore, a combination of mutations resulted in complex patterns of cross-resistance. Modeling of the VACV DNA polymerase bearing the newly described mutations was performed to understand the effects of these mutations on the structure of the viral enzyme. We demonstrated the emergence of drug-resistant DNA polymerase mutations in complex patterns to be considered in case such mutations should eventually arise in the clinic.
Language	English
Publishing date	2022-03-01
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2720867-9
ISSN	2227-9059
ISSN	2227-9059
DOI	10.3390/biomedicines10030580
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Article ; Online: Treatment of Rat Lymphedema by Propeller Lymphatic Tissue Flap Combined with Nanofibrillar Collagen Scaffolds.

Dionyssiou, Dimitrios / Nguyen, Dung / Topalis, Anastasios / Deptula, Peter / Paukshto, Michael / Zaitseva, Tatiana / Demiri, Efterpi / Cheva, Angeliki / Rockson, Stanley

Journal of reconstructive microsurgery

2023 Volume 40, Issue 2, Page(s) 145–155

Abstract: Background: The aim of our study was to evaluate a new propeller vascularized lymphatic tissue flap (pVLNT) combined with aligned nanofibrillar collagen scaffolds (CS) (BioBridge) in reducing lymphedema in the rat lymphedema model.: Methods: ... ...

Abstract	Background: The aim of our study was to evaluate a new propeller vascularized lymphatic tissue flap (pVLNT) combined with aligned nanofibrillar collagen scaffolds (CS) (BioBridge) in reducing lymphedema in the rat lymphedema model. Methods: Unilateral left hindlimb lymphedema was created in 15 female Sprague-Dawley rats following inguinal and popliteal lymph nodes (LN) resection and radiation. An inguinal pVLNT was elevated from the contralateral groin and transferred through a skin tunnel to the affected groin. Four collagen threads were attached to the flap and inserted in the hindlimb at the subcutaneous level in a fan shape. The three study groups consisted of group A (control), group B (pVLNT), and group C (pVLNT + CS). Volumetric analysis of both hindlimbs was performed using micro-computed tomography imaging before the surgery (at initial time point) and then at 1 and 4 months, postoperatively, and the relative volume difference (excess volume) was measured for each animal. Lymphatic drainage was assessed by indocyanine green (ICG) fluoroscopy for number and morphology of new collectors and the time required for ICG to move from injection point to the midline. Results: Four months after the induction of lymphedema, an increased relative volume difference remained in group A (5.32 ± 4.74%), while there was a significant relative volume reduction in group B (-13.39 ± 8.55%) and an even greater reduction in group C (-14.56 ± 5.04%). ICG fluoroscopy proved the functional restoration of lymphatic vessels and viability of pVLNT in both B and C groups. Notably, only group C demonstrated statistically significant improvements in lymphatic pattern/morphology and in the number of lymphatic collectors as compared with the control group A. Conclusion: The pedicle lymphatic tissue flap combined with SC is an effective procedure for the treatment of lymphedema in rats. It can be easily translated into treatment of humans' lower and upper limb lymphedema and further clinical studies are warranted.
MeSH term(s)	Humans ; Rats ; Female ; Animals ; X-Ray Microtomography ; Rats, Sprague-Dawley ; Lymphedema/surgery ; Lymph Nodes ; Lymphatic Vessels/surgery ; Collagen
Chemical Substances	Collagen (9007-34-5)
Language	English
Publishing date	2023-05-04
Publishing country	United States
Document type	Journal Article
ZDB-ID	605983-1
ISSN	1098-8947 ; 0743-684X ; 0743-684X
ISSN (online)	1098-8947 ; 0743-684X
ISSN	0743-684X
DOI	10.1055/a-2086-0269
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Article ; Online: A Herpes Simplex Virus 1 DNA Polymerase Multidrug Resistance Mutation Identified in a Patient With Immunodeficiency and Confirmed by Gene Editing.

Schalkwijk, Hanna Helena / Georgala, Aspasia / Gillemot, Sarah / Temblador, Arturo / Topalis, Dimitri / Wittnebel, Sebastian / Andrei, Graciela / Snoeck, Robert

The Journal of infectious diseases

2023 Volume 228, Issue 11, Page(s) 1505–1515

Abstract: Background: Herpes simplex virus 1 can cause severe infections in individuals who are immunocompromised. In these patients, emergence of drug resistance mutations causes difficulties in infection management.: Methods: Seventeen herpes simplex virus 1 ...

Abstract	Background: Herpes simplex virus 1 can cause severe infections in individuals who are immunocompromised. In these patients, emergence of drug resistance mutations causes difficulties in infection management. Methods: Seventeen herpes simplex virus 1 isolates were obtained from orofacial/anogenital lesions in a patient with leaky severe combined immunodeficiency over 7 years, before and after stem cell transplantation. Spatial/temporal evolution of drug resistance was characterized genotypically-with Sanger and next-generation sequencing of viral thymidine kinase (TK) and DNA polymerase (DP)-and phenotypically. CRISPR/Cas9 was used to introduce the novel DP Q727R mutation, and dual infection-competition assays were performed to assess viral fitness. Results: Isolates had identical genetic backgrounds, suggesting that orofacial/anogenital infections derived from the same virus lineage. Eleven isolates proved heterogeneous TK virus populations by next-generation sequencing, undetectable by Sanger sequencing. Thirteen isolates were acyclovir resistant due to TK mutations, and the Q727R isolate additionally exhibited foscarnet/adefovir resistance. Recombinant Q727R mutant virus showed multidrug resistance and increased fitness under antiviral pressure. Conclusions: Long-term follow-up of a patient with severe combined immunodeficiency revealed virus evolution and frequent reactivation of wild-type and TK mutant strains, mostly as heterogeneous populations. The DP Q727R resistance phenotype was confirmed with CRISPR/Cas9, a useful tool to validate novel drug resistance mutations.
MeSH term(s)	Humans ; Antiviral Agents/pharmacology ; Antiviral Agents/therapeutic use ; Herpesvirus 1, Human ; Herpes Simplex/drug therapy ; Severe Combined Immunodeficiency/drug therapy ; Gene Editing ; Drug Resistance, Viral/genetics ; Acyclovir/pharmacology ; Acyclovir/therapeutic use ; Mutation ; DNA-Directed DNA Polymerase/genetics ; Immunologic Deficiency Syndromes ; Drug Resistance, Multiple ; Thymidine Kinase/genetics ; Thymidine Kinase/therapeutic use
Chemical Substances	Antiviral Agents ; Acyclovir (X4HES1O11F) ; DNA-Directed DNA Polymerase (EC 2.7.7.7) ; Thymidine Kinase (EC 2.7.1.21)
Language	English
Publishing date	2023-05-08
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	3019-3
ISSN	1537-6613 ; 0022-1899
ISSN (online)	1537-6613
ISSN	0022-1899
DOI	10.1093/infdis/jiad184
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Article: CRISPR/Cas9 Editing of the Polyomavirus Tumor Antigens Inhibits Merkel Cell Carcinoma Growth In Vitro.

Temblador, Arturo / Topalis, Dimitrios / Andrei, Graciela / Snoeck, Robert

Cancers

2019 Volume 11, Issue 9

Abstract: Merkel cell carcinoma (MCC) is an aggressive type of skin cancer whose main causative agent is Merkel cell polyomavirus (MCPyV). MCPyV is integrated into the genome of the tumor cells in most MCCs. Virus-positive tumor cells constitutively express two ... ...

Abstract	Merkel cell carcinoma (MCC) is an aggressive type of skin cancer whose main causative agent is Merkel cell polyomavirus (MCPyV). MCPyV is integrated into the genome of the tumor cells in most MCCs. Virus-positive tumor cells constitutively express two viral oncoproteins that promote cell growth: the small (sT) and the large (LT) tumor antigens (TAs). Despite the success of immunotherapies in patients with MCC, not all individuals respond to these treatments. Therefore, new therapeutic options continue to be investigated. Herein, we used CRISPR/Cas9 to target the viral oncogenes in two virus-positive MCC cell lines: MS-1 and WAGA. Frameshift mutations introduced in the target sequence upon repair of the Cas9-induced DNA break resulted in decreased LT protein levels, which subsequently impaired cell proliferation, caused cell cycle arrest, and led to increased apoptosis. Importantly, a virus-negative non-MCC cell line (HEK293T) remained unaffected, as well as those cells expressing a non-targeting single-guide RNA (sgRNA). Thus, we presumed that the noted effects were not due to the off-target activity of the TAs-targeting sgRNAs. Additionally, WAGA cells had altered levels of cellular proteins involved in cell cycle regulation, supporting the observed cell cycle. Taken together, our findings provide evidence for the development of a CRISPR/Cas9-based therapeutic option for virus-positive MCC.
Language	English
Publishing date	2019-08-28
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2527080-1
ISSN	2072-6694
ISSN	2072-6694
DOI	10.3390/cancers11091260
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Article ; Online: Viral fitness of MHV-68 viruses harboring drug resistance mutations in the protein kinase or thymidine kinase.

Trompet, Erika / Topalis, Dimitrios / Gillemot, Sarah / Snoeck, Robert / Andrei, Graciela

Antiviral research

2020 Volume 182, Page(s) 104901

Abstract: Murine γ-herpesvirus-68 (MHV-68), genetically and biologically related to human γ-herpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, can be easily propagated in vitro allowing drug resistance studies. Previously, we described ... ...

Abstract	Murine γ-herpesvirus-68 (MHV-68), genetically and biologically related to human γ-herpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, can be easily propagated in vitro allowing drug resistance studies. Previously, we described specific changes in MHV-68 protein kinase (PK) or thymidine kinase (TK) associated with resistance to various purine or pyrimidine nucleoside analogues, respectively. To investigate how specific TK and PK mutations affect viral replication capacity, we performed dual infection competition assays in which wild-type and drug-resistant virus compete in absence or presence of antivirals in Vero cells. The composition of the mixed viral population was analyzed using next-generation sequencing and relative fitness of seven MHV-68 PK or TK mutants was calculated based on the frequency of viral variants at the time of infection and after 5-days growth. A MHV-68 mutant losing the PK function due to a 2-nucleotide deletion was less fit than the wild-type virus in absence of antivirals, consistent with the essential role of viral PKs during lytic replication, but overgrew the wild-type virus under pressure of purine nucleosides. TK mutant viruses, with frameshift or missense mutations, grew equal to wild-type virus in absence of antivirals, in accordance with the viral TK function only being essential in non-replicating or in TK-deficient cells, but were more fit when treated with pyrimidine nucleosides. Moreover, TK missense mutant viruses also increased fitness under pressure of antivirals other than pyrimidine nucleosides, indicating that MHV-68 TK mutations might influence viral fitness by acting on cellular and/or viral functions that are unrelated to nucleoside activation.
Language	English
Publishing date	2020-08-05
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	306628-9
ISSN	1872-9096 ; 0166-3542
ISSN (online)	1872-9096
ISSN	0166-3542
DOI	10.1016/j.antiviral.2020.104901
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Article: Occupational electromagnetic spectrum hazards and the significance of artificial optical radiation: country report for Greece.

Gourzoulidis, George A / Karabetsos, Efthymios / Bourousis, Constantinos / Tyrakis, Charilaos / Flouris, Andreas D / Maris, Thomas G / Topalis, Frangiskos V

La Medicina del lavoro

2022 Volume 113, Issue 2, Page(s) e2022016

Abstract: Background: The electromagnetic spectrum spans over an enormous range from 0 up to more than 1020 Hz in the deep ionizing region, significant exposures exist in specific occupational environments. Between the ionizing and the electromagnetic fields (EMF) ...

Abstract	Background: The electromagnetic spectrum spans over an enormous range from 0 up to more than 1020 Hz in the deep ionizing region, significant exposures exist in specific occupational environments. Between the ionizing and the electromagnetic fields (EMF) part of the spectrum, the 'optical radiation' (OR) region has specific properties. Comparative and concise evaluation enables action prioritization. Methods: Following the transposition and implementation periods of the artificial optical radiation (AOR) and EMF European Directives, the Hellenic Ministry of Labour in collaboration with the Greek Atomic Energy Commission (EEAE) and the National Technical University of Athens, conducted thorough occupational exposure investigation in Greece. Using dedicated measuring equipment and procedures, the majority of EMF emitting installations in Greece and also AOR emitting installations including arc welding, lasers and PC monitors has been assessed. Results: Measurement results from occupational settings reveal that it is the non-coherent metal arc welding AOR that can pose even sub-second overexposures. Rare EMF overexposures are manageable and EMF concern is not justified. Maintenance procedures demand proper attention. Preliminary laser safety assessment reveals OHS gaps and potential eye and skin hazards. Blue light exposure from computer monitors is well below safety limits. Conclusions: This electromagnetic spectrum risk assessment conducted in Greece enables the justification of the real occupational hazards, in this sense: i) EMF exposure assessment has to be concentrated to maintenance procedures; ii) AOR measuring setups are challenging and standardized measurement procedures are missing, and iii) AOR overexposures from arc welding pose significant eye and skin hazards.
MeSH term(s)	Electromagnetic Fields/adverse effects ; Greece ; Humans ; Occupational Exposure ; Radiation Exposure
Language	English
Publishing date	2022-04-26
Publishing country	Italy
Document type	Journal Article
ZDB-ID	123678-7
ISSN	0025-7818
ISSN	0025-7818
DOI	10.23749/mdl.v113i2.12636
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