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1.
Cancers (Basel) ; 13(19)2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34638268

RESUMO

Clinical translation of novel immunotherapeutic strategies such as chimeric antigen receptor (CAR) T-cells in acute myeloid leukemia (AML) is still at an early stage. Major challenges include immune escape and disease relapse demanding for further improvements in CAR design. To overcome such hurdles, we have invented the switchable, flexible and programmable adaptor Reverse (Rev) CAR platform. This consists of T-cells engineered with RevCARs that are primarily inactive as they express an extracellular short peptide epitope incapable of recognizing surface antigens. RevCAR T-cells can be redirected to tumor antigens and controlled by bispecific antibodies cross-linking RevCAR T- and tumor cells resulting in tumor lysis. Remarkably, the RevCAR platform enables combinatorial tumor targeting following Boolean logic gates. We herein show for the first time the applicability of the RevCAR platform to target myeloid malignancies like AML. Applying in vitro and in vivo models, we have proven that AML cell lines as well as patient-derived AML blasts were efficiently killed by redirected RevCAR T-cells targeting CD33 and CD123 in a flexible manner. Furthermore, by targeting both antigens, a Boolean AND gate logic targeting could be achieved using the RevCAR platform. These accomplishments pave the way towards an improved and personalized immunotherapy for AML patients.

2.
Pain ; 162(9): 2349-2365, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34448751

RESUMO

ABSTRACT: Endometriosis (ENDO) and interstitial cystitis/bladder pain syndrome (IC/BPS) are chronic pain conditions for which better treatments are urgently needed. Development of new therapies with proven clinical benefit has been slow. We have conducted a review of existing preclinical in vivo models for ENDO and IC/BPS in rodents, discussed to what extent they replicate the phenotype and pain experience of patients, as well as their relevance for translational research. In 1009 publications detailing ENDO models, 41% used autologous, 26% syngeneic, 18% xenograft, and 11% allogeneic tissue in transplantation models. Intraperitoneal injection of endometrial tissue was the subcategory with the highest construct validity score for translational research. From 1055 IC/BPS publications, most interventions were bladder centric (85%), followed by complex mechanisms (8%) and stress-induced models (7%). Within these categories, the most frequently used models were instillation of irritants (92%), autoimmune (43%), and water avoidance stress (39%), respectively. Notably, although pelvic pain is a hallmark of both conditions and a key endpoint for development of novel therapies, only a small proportion of the studies (models of ENDO: 0.5%-12% and models of IC/BPS: 20%-44%) examined endpoints associated with pain. Moreover, only 2% and 3% of publications using models of ENDO and IC/BPS investigated nonevoked pain endpoints. This analysis highlights the wide variety of models used, limiting reproducibility and translation of results. We recommend refining models so that they better reflect clinical reality, sharing protocols, and using standardized endpoints to improve reproducibility. We are addressing this in our project Innovative Medicines Initiative-PainCare/Translational Research in Pelvic Pain.


Assuntos
Cistite Intersticial , Endometriose , Cistite Intersticial/terapia , Feminino , Humanos , Dor Pélvica/terapia , Reprodutibilidade dos Testes , Pesquisa Médica Translacional
3.
J Cyst Fibros ; 2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34419414

RESUMO

BACKGROUND: Riociguat is a first-in-class soluble guanylate cyclase stimulator for which preclinical data suggested improvements in cystic fibrosis transmembrane conductance regulator (CFTR) function. METHODS: This international, multicenter, two-part, Phase II study of riociguat enrolled adults with cystic fibrosis (CF) homozygous for Phe508del CFTR. Part 1 was a 28-day, randomized, double-blind, placebo-controlled study in participants not receiving CFTR modulator therapy. Twenty-one participants were randomized 1:2 to placebo or oral riociguat (0.5 mg three times daily [tid] for 14 days, increased to 1.0 mg tid for the subsequent 14 days). The primary and secondary efficacy endpoints were change in sweat chloride concentration and percent predicted forced expiratory volume in 1 second (ppFEV1), respectively, from baseline to Day 14 and Day 28 with riociguat compared with placebo. RESULTS: Riociguat did not alter CFTR activity (change in sweat chloride) or lung function (change in ppFEV1) at doses up to 1.0 mg tid after 28 days. The most common drug-related adverse event (AE) was headache occurring in three participants (21%); serious AEs occurred in one participant receiving riociguat (7%) and one participant receiving placebo (14%). This safety profile was consistent with the underlying disease and the known safety of riociguat for its approved indications. CONCLUSIONS: The Rio-CF study was terminated due to lack of efficacy and the changing landscape of CF therapeutic development. The current study⁠, within its limits of a small sample size, did not provide evidence that riociguat could be a valid treatment option for CF. CLINICAL TRIAL REGISTRATION NUMBER: NCT02170025.

4.
Drug Dev Ind Pharm ; 46(11): 1753-1762, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33006298

RESUMO

OBJECTIVE: Probiotic bacteria, such as different lactobacilli strains, have successfully been used to treat gingivitis and periodontitis or caries. By formulating probiotics as orodispersible tablet (ODT), the benefits of this dosage form could be utilized. Without any further measures, the probiotic bacteria will be eliminated too fast from the intended site of action, the oral mucosa. The use of mucoadhesive granules, composed of mucoadhesive polymer and probiotics, is a promising strategy to prolong the contact time between lactobacilli and oral mucosa without delaying disintegration. METHODS: Three common mucoadhesive polymers, anionic Carbopol 971P NF, nonionic Metolose 65SH50 and cationic chitosan were included into tablets either by direct compression (DC) or after granulation with the probiotics. Disintegration, mucoadhesion of the tablets, and storage stability of the probiotics were characterized. RESULTS: By incorporating a sufficient amount of polymer superior probiotic mucoadhesion could be achieved. All formulations based on granulated probiotics and mucoadhesive polymer fulfilled the Food and Drug Administration (FDA) acceptance level for disintegration of orodispersible tablets. These formulations exhibited excellent storage stability under refrigerated conditions over 30 months. Interestingly, ODTs including Carbopol 971P NF still proved superior mucoadhesion after long-term storage, whereas the mucoadhesive effect of Metolose 65SH50 and chitosan declined markedly. CONCLUSIONS: The results of this study suggest that Carbopol 971P NF was the most appropriate polymer for a probiotic mucoadhesive ODT.


Assuntos
Polímeros/química , Probióticos , Adesividade , Administração Bucal , Mucosa Bucal/efeitos dos fármacos , Comprimidos
5.
Oncoimmunology ; 9(1): 1785608, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32923149

RESUMO

Chimeric antigen receptor (CAR) T cells show remarkable therapeutic effects in some hematological malignancies. However, CAR T cells can also cause life-threatening side effects. In order to minimize off-target and on-target/off-tumor reactions, improve safety, enable controllability, provide high flexibility, and increase tumor specificity, we established a novel humanized artificial receptor platform termed RevCARs. RevCAR genes encode for small surface receptors lacking any antigen-binding moiety. Steering of RevCAR T cells occurs via bispecific targeting molecules (TMs). The small size of RevCAR-encoding genes allows the construction of polycistronic vectors. Here, we demonstrate that RevCAR T cells efficiently kill tumor cells, can be steered by TMs, flexibly redirected against multiple targets, and used for combinatorial targeting following the "OR" and "AND" gate logic.


Assuntos
Receptores de Antígenos Quiméricos , Terapia Baseada em Transplante de Células e Tecidos , Imunoterapia Adotiva , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Linfócitos T
6.
ACS Chem Biol ; 15(9): 2331-2337, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32786258

RESUMO

We report on using the synthetic aminoadamantane-CH2-aryl derivatives 1-6 as sensitive probes for blocking M2 S31N and influenza A virus (IAV) M2 wild-type (WT) channels as well as virus replication in cell culture. The binding kinetics measured using electrophysiology (EP) for M2 S31N channel are very dependent on the length between the adamantane moiety and the first ring of the aryl headgroup realized in 2 and 3 and the girth and length of the adamantane adduct realized in 4 and 5. Study of 1-6 shows that, according to molecular dynamics (MD) simulations and molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) calculations, all bind in the M2 S31N channel with the adamantyl group positioned between V27 and G34 and the aryl group projecting out of the channel with the phenyl (or isoxazole in 6) embedded in the V27 cluster. In this outward binding configuration, an elongation of the ligand by only one methylene in rimantadine 2 or using diamantane or triamantane instead of adamantane in 4 and 5, respectively, causes incomplete entry and facilitates exit, abolishing effective block compared to the amantadine derivatives 1 and 6. In the active M2 S31N blockers 1 and 6, the phenyl and isoxazolyl head groups achieve a deeper binding position and high kon/low koff and high kon/high koff rate constants, compared to inactive 2-5, which have much lower kon and higher koff. Compounds 1-5 block the M2 WT channel by binding in the longer area from V27-H37, in the inward orientation, with high kon and low koff rate constants. Infection of cell cultures by influenza virus containing M2 WT or M2 S31N is inhibited by 1-5 or 1-4 and 6, respectively. While 1 and 6 block infection through the M2 block mechanism in the S31N variant, 2-4 may block M2 S31N virus replication in cell culture through the lysosomotropic effect, just as chloroquine is thought to inhibit SARS-CoV-2 infection.


Assuntos
Adamantano/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/prevenção & controle , Canais Iônicos/antagonistas & inibidores , Sondas Moleculares/química , Proteínas da Matriz Viral/antagonistas & inibidores , Adamantano/análogos & derivados , Adamantano/química , Adamantano/metabolismo , Betacoronavirus/efeitos dos fármacos , Sítios de Ligação , COVID-19 , Células Cultivadas , Cloroquina/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/prevenção & controle , Variação Genética , Humanos , Vírus da Influenza A/química , Vírus da Influenza A/genética , Influenza Humana/tratamento farmacológico , Cinética , Sondas Moleculares/metabolismo , Pandemias/prevenção & controle , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/prevenção & controle , Ligação Proteica , SARS-CoV-2 , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos
7.
Onco Targets Ther ; 13: 5515-5527, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606767

RESUMO

Introduction: Since epithelial growth factor receptor (EGFR) overexpression is linked to a variety of malignancies, it is an attractive target for immune therapy including chimeric antigen receptor (CAR)-engineered T cells. Unfortunately, CAR T cell therapy harbors the risk of severe, even life-threatening side effects. Adaptor CAR T cell platforms such as the previously described UniCAR system might be able to overcome these problems. In contrast to conventional CARs, UniCAR T cells are per se inert. Their redirection towards target cells occurs only in the presence of a tumor-specific target molecule (TM). TMs are bifunctional molecules being able to recognize a tumor-associated antigen and to cross-link the CAR T cell via a peptide epitope recognized by the UniCAR domain. Materials and Methods: Here, we compare αEGFR TMs: a nanobody (nb)-based αEGFR TM derived from the camelid αEGFR antibody 7C12 with a murine and humanized single-chain fragment variable (scFv) based on the clinically used antibody Cetuximab®. Results: In principle, both the nb- and scFv-based TM formats are able to redirect UniCAR T cells to eliminate EGFR-expressing tumor cells in an antigen-specific and TM-dependent manner. However, the scFv-based αEGFR TM was significantly superior to the nb-based TM especially with respect to lysis of tumor cells. Discussion: Improved efficiency of the scFv-based TM allowed the redirection of UniCAR T cells towards tumor cells expressing high as well as low EGFR levels in comparison to nb-based αEGFR TMs.

8.
Oncoimmunology ; 9(1): 1743036, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32426176

RESUMO

Induction or selection of radioresistant cancer (stem) cells following standard radiotherapy is presumably one of the major causes for recurrence of metastatic disease. One possibility to prevent tumor relapse is the application of targeted immunotherapies including, e.g., chimeric antigen receptor (CAR) T cells. In light of long-term remissions, it is highly relevant to clarify whether radioresistant cancer cells are susceptible to CAR T cell-mediated killing. To answer this question, we evaluated the anti-tumor activity of the switchable universal chimeric antigen receptor (UniCAR) system against highly radioresistant head and neck squamous cell carcinoma cells both in vitro and in vivo. Following specific UniCAR T cell engagement via EGFR or CD98 target modules, T cell effector mechanisms were induced including secretion of pro-inflammatory cytokines, up-regulation of granzyme B and perforin, as well as T cell proliferation. CD98- or EGFR-redirected UniCAR T cells further possess the capability to efficiently lyse radioresistant tumor cells. Observed anti-tumor effects were comparable to those against the radiosensitive parental cell lines. Finally, redirected UniCAR T cells significantly inhibited the growth of radioresistant cancer cells in immunodeficient mice. Taken together, our obtained data underline that the UniCAR system is able to overcome radioresistance. Thus, it represents an attractive technology for the development of combined radioimmunotherapeutic approaches that might improve the outcome of patients with metastatic radioresistant tumor diseases.


Assuntos
Imunoterapia Adotiva , Neoplasias , Animais , Humanos , Camundongos , Recidiva Local de Neoplasia , Neoplasias/radioterapia , Neoplasias/terapia , Tolerância a Radiação , Receptores de Antígenos Quiméricos , Linfócitos T
9.
Oncoimmunology ; 8(9): e1621676, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31428518

RESUMO

Adoptive transfer of chimeric antigen receptor (CAR)-equipped T cells have demonstrated astonishing clinical efficacy in hematological malignancies recently culminating in the approval of two CAR T cell products. Despite this tremendous success, CAR T cell approaches have still achieved only moderate efficacy against solid tumors. As a major obstacle, engineered conventional T cells (Tconvs) face an anti-inflammatory, hostile tumor microenvironment often infiltrated by highly suppressive regulatory T cells (Tregs). Thus, potent CAR T cell treatment of solid tumors requires efficient activation of Tconvs via their engrafted CAR to overcome Treg-mediated immunosuppression. In that regard, selecting an optimal intracellular signaling domain might represent a crucial step to achieve best clinical efficiency. To shed light on this issue and to investigate responsiveness to Treg inhibition, we engrafted Tconvs with switchable universal CARs (UniCARs) harboring intracellularly the CD3ζ domain alone or in combination with costimulatory CD28 or 4-1BB. Our studies reveal that UniCAR ζ-, and UniCAR BB/ζ-engineered Tconvs are strongly impaired by activated Tregs, whereas UniCARs providing CD28 costimulation overcome Treg-mediated suppression both in vitro and in vivo. Compared to UniCAR ζ- and UniCAR BB/ζ-modified cells, UniCAR 28/ζ-armed Tconvs secrete significantly higher amounts of Th1-related cytokines and, furthermore, levels of these cytokines are elevated even upon exposure to Tregs. Thus, in contrast to 4-1BB costimulation, CD28 signaling in UniCAR-transduced Tconvs seems to foster a pro-inflammatory milieu, which contributes to enhanced resistance to Treg suppression. Overall, our results may have significant implications for CAR T cell-based immunotherapies of solid tumors strongly invaded by Tregs.

10.
Eur J Pharm Biopharm ; 139: 240-245, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30946916

RESUMO

Orodispersible tablets (ODTs) are a convenient dosage form and a recent trend in formulation development. The fast disintegration is accompanied by rapid removal of the active principle and the excipients from the mouth due to saliva flow and swallowing. Probiotic bacteria are a promising strategy to fight disease with bacterial aetiology in the mouth, but a certain residence time in the oral cavity is inevitable to exert their positive effects. The addition of a mucoadhesive polymer, like hydroxypropyl methylcellulose (HPMC), is an auspicious strategy to prolong this residence time. Nevertheless, the disintegration time of the tablets should still meet the acceptance level from the FDA (<30 s). To reach intimate contact of bacteria and mucoadhesive polymer on the one hand and to support fast disintegration on the other hand, granulation of probiotic bacteria and mucoadhesive HPMC with a methacrylic acid copolymer was performed first. Moreover, high mucoadhesion could be obtained because bacteria and mucoadhesive polymer could interact more strongly with the mucosa after the ODT disintegrated and the methacrylic acid copolymer dissolved in the pH neutral saliva.


Assuntos
Excipientes/química , Derivados da Hipromelose/química , Mucosa Bucal/metabolismo , Probióticos/administração & dosagem , Saliva/metabolismo , Adesividade , Administração Bucal , Animais , Disponibilidade Biológica , Química Farmacêutica , Composição de Medicamentos/métodos , Lactobacillus paracasei , Lactobacillus plantarum , Ácidos Polimetacrílicos/química , Probióticos/farmacocinética , Solubilidade , Suínos , Comprimidos , Fatores de Tempo
11.
Reprod Sci ; 26(4): 523-531, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-29806538

RESUMO

BAY 1158061 is a potent monoclonal prolactin (PRL) receptor antibody, blocking PRL receptor (PRLR)-mediated signaling in a noncompetitive manner, which was tested in a randomized, placebo-controlled multiple dose study in postmenopausal women. The objective was to investigate safety, tolerability, pharmacokinetic characteristics, and effects of BAY 1158061 on serum PRL level. The study consisted of 4 parallel groups receiving up to 3 subcutaneous (sc) administrations of BAY 1158061 or placebo in 2 different dosing regimens. Twenty-nine healthy postmenopausal women were randomized and treated with BAY 1158061 or placebo: 30 mg at 14-day interval (7 participants), 60 mg at 28-day interval (8 participants), 90 mg at 14-day interval (7 participants), and placebo (7 participants). To keep the blinding, all randomized participants received sc injections biweekly (14-day interval) on 3 occasions in the lower abdomen. The PRLR antibody showed a favorable safety and tolerability profile in postmenopausal women with no distinct differences in occurrence of adverse events in BAY 1158061 or placebo-treated participants. BAY 1158061 displayed low immunogenicity with low titers of antidrug antibodies and absence of neutralizing antidrug antibodies. Pharmacokinetics were characterized by slow absorption after sc administration with median peak plasma concentrations 7 to 11 days after first dose and about 2-fold accumulation after repeated dosing every 2 weeks. The apparent mean elimination half-life was 9 to 16 days. The PRL concentration-time profiles over 24 hours showed no differences between verum- and placebo-treated participants. Based on the data obtained, BAY 1158061 is considered a good candidate for further development in endometriosis or other PRL-mediated disease conditions.


Assuntos
Anticorpos Monoclonais/farmacologia , Receptores da Prolactina/antagonistas & inibidores , Anticorpos Monoclonais/sangue , Esquema de Medicação , Endometriose/prevenção & controle , Feminino , Humanos , Injeções Subcutâneas , Pessoa de Meia-Idade , Pós-Menopausa , Receptores da Prolactina/imunologia
12.
Int J Pharm ; 551(1-2): 141-147, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30194013

RESUMO

Oromucosal drug delivery is necessary when a local effect in the oral cavity is required. Bioadhesive formulations should be advantageous because a larger fraction of the active principal is retained at the site of action allowing for an enhanced and prolonged effect. Despite a variety of mucoadhesion test systems being described in literature, none of these in-vitro tests does relate to physiological conditions in the oral cavity and suites for the testing of complete dosage forms, e.g. tablets. The novel mucoadhesion test is based on porcine buccal mucosa. Constituents, osmolality and pH of the used artificial saliva was as close as possible to physiologic conditions and the flushing rate was equivalent to the physiological secretion rate of saliva. Suitability of the novel test was evaluated with ODTs with live probiotic bacteria as the active principle. With the novel test system, it was shown that in the presence of mucoadhesive polymers (Carbopol®, Metolose® and chitosan) up to 36 % of the probiotic bacteria adhered to the mucosa corresponding to a two- or threefold increase compared to tablets without such polymers. Furthermore, the influence of the tablet size (contact area) on the mucoadhesive properties could be resolved.


Assuntos
Resinas Acrílicas/administração & dosagem , Quitosana/administração & dosagem , Derivados da Hipromelose/administração & dosagem , Mucosa Bucal/química , Probióticos/administração & dosagem , Adesividade , Administração Bucal , Animais , Suínos , Comprimidos
13.
Methods Mol Biol ; 1836: 159-183, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30151573

RESUMO

To infect host cells, viruses have to gain access to the intracellular compartment. The infection process starts with the attachment of viruses to the cell surface. Then a complex series of events, highly dynamic, tightly intricate, and often hard to investigate, follows. This includes virus displacement at the plasma membrane, binding to receptors, signaling, internalization, and release of the viral genome and material into the cytosol. In the past decades, the emergence of sensitive, accurate fluorescence-based technologies has opened new perspectives of investigations in the field. Visualization of single viral particles in fixed and living cells as well as quantification of each virus entry step has been made possible. Here we describe the procedure to fluorescently label viral particles. We also illustrate how to use this powerful tool to decipher the entry of viruses with the most recent fluorescence-based techniques such as high-speed confocal and total internal reflection microscopy, flow cytometry, and fluorimetry.


Assuntos
Corantes Fluorescentes , Coloração e Rotulagem , Vírion/metabolismo , Internalização do Vírus , Fenômenos Fisiológicos Virais , Animais , Linhagem Celular , Citometria de Fluxo , Corantes Fluorescentes/química , Humanos , Microscopia de Fluorescência
14.
ACS Med Chem Lett ; 9(3): 198-203, 2018 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-29541360

RESUMO

Recently, the binding kinetics of a ligand-target interaction, such as the residence time of a small molecule on its protein target, are seen as increasingly important for drug efficacy. Here, we investigate these concepts to explain binding and proton blockage of rimantadine variants bearing progressively larger alkyl groups to influenza A virus M2 wild type (WT) and M2 S31N protein proton channel. We showed that resistance of M2 S31N to rimantadine analogues compared to M2 WT resulted from their higher koff rates compared to the kon rates according to electrophysiology (EP) measurements. This is due to the fact that, in M2 S31N, the loss of the V27 pocket for the adamantyl cage resulted in low residence time inside the M2 pore. Both rimantadine enantiomers have similar channel blockage and binding kon and koff against M2 WT. To compare the potency between the rimantadine variants against M2, we applied approaches using different mimicry of M2, i.e., isothermal titration calorimetry and molecular dynamics simulation, EP, and antiviral assays. It was also shown that a small change in an amino acid at site 28 of M2 WT, which does not line the pore, seriously affects M2 WT blockage kinetics.

15.
Toxins (Basel) ; 10(2)2018 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-29385106

RESUMO

In May 2017, at least 12 dogs showed signs of acute neurotoxicosis after swimming in or drinking from Lake Tegel, a mesotrophic lake in Berlin, Germany, and several of the affected dogs died shortly afterwards despite intensive veterinary treatment. Cyanobacterial blooms were not visible at the water surface or the shorelines. However, detached and floating water moss (Fontinalis antipyretica) with high amounts of Tychonema sp., a potential anatoxin-a (ATX) producing cyanobacterium, was found near the beaches where the dogs had been swimming and playing. Necropsies of two of the dogs revealed no specific lesions beside the anamnestic neurotoxicosis. ATX was detected in concentrations up to 8700 µg L-1 in the stomach contents, while other (neuro)toxic substances were not found. In the aqueous fraction of Fontinalis/Tychonema clumps sampled after the casualties, ATX was found in concentrations up to 1870 µg L-1. This is the first report of a dense population of Tychonema sp. in stands of Fontinalis resulting in high ATX contents. This case emphasizes the need for further investigation of potentially toxic, non-bloom forming cyanobacteria in less eutrophic water bodies and underlines the novel challenge of developing appropriate surveillance schemes for respective bathing sites.


Assuntos
Síndromes Neurotóxicas/etiologia , Síndromes Neurotóxicas/veterinária , Tropanos/toxicidade , Poluentes da Água/toxicidade , Animais , Berlim , Cianobactérias , Cães , Evolução Fatal , Feminino , Lagos
16.
Antiviral Res ; 152: 53-57, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29427675

RESUMO

Therapy or prophylaxis of herpes simplex virus type 2 (HSV-2) infections with the nucleoside analog aciclovir (ACV) can lead to the emergence of drug-resistant HSV-2 strains, particularly in immunocompromised patients. In this context, multiple amino acid (aa) changes can accumulate in the ACV-converting viral thymidine kinase (TK) which hampers sequence-based diagnostics significantly. In this study, the so far unknown or still doubted relevance of several individual aa changes for drug resistance in HSV-2 was clarified. For this purpose, ten recombinant fluorescent HSV-2 strains differing in the respective aa within their TK were constructed using the bacterial artificial chromosome (BAC) pHSV2(MS)Lox. Similar TK expression levels and similar replication behavior patterns were demonstrated for the mutants as compared to the unmodified BAC-derived HSV-2 strain. Subsequently, the resulting strains were tested for their susceptibility to ACV as well as penciclovir (PCV) in parallel to a modified cytopathic effect (CPE) inhibition assay and by determining the relative fluorescence intensity (quantified using units, RFU) as a measure for the viral replication capacity. While aa changes Y53N and R221H conferred ACV resistance with cross-resistance to PCV, the aa changes G25A, G39E, T131M, Y133F, G150D, A157T, R248W, and L342W maintained a susceptible phenotype against both antivirals. The CPE inhibition assay and the measurement of relative fluorescence intensity yielded comparable results for the phenotypic testing of recombinant viruses. The latter test showed some technical advantages. In conclusion, the significance of single aa changes in HSV-2 TK on ACV/PCV resistance was clarified by the construction and phenotypic testing of recombinant viral strains. This was facilitated by the fluorescence based method.


Assuntos
Antivirais/farmacologia , Farmacorresistência Viral , Herpes Simples/virologia , Herpesvirus Humano 2/efeitos dos fármacos , Herpesvirus Humano 2/enzimologia , Timidina Quinase/genética , Proteínas Virais/genética , Aciclovir/análogos & derivados , Aciclovir/farmacologia , Guanina , Herpesvirus Humano 2/genética , Humanos , Mutação , Timidina Quinase/metabolismo , Proteínas Virais/metabolismo
17.
J Clin Virol ; 95: 61-65, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28886462

RESUMO

BACKGROUND: Genotypic resistance testing of varicella-zoster virus (VZV) strains to antivirals is of high relevance in immunocompromised patients with VZV reactivations unresponsive to therapy. However, the knowledge on mutations associated with natural gene polymorphism or resistance is limited. OBJECTIVES: To examine the genotype of the thymidine kinase (TK) and DNA polymerase (pol) of unselected clinical VZV isolates collected between 1984 and 2014 and to verify the phenotype related to novel amino acid (aa) substitutions. STUDY DESIGN: The TK and DNA pol genes of 169 VZV isolates were analyzed by amplification and sequencing. Sequences were compared to that of the reference strain Dumas. The phenotype to acyclovir and other antivirals was examined in isolates with novel aa substitutions using modified plaque reduction assay. RESULTS: In the TK of four strains, four different aa substitutions were detected, apart from the known change S288L that was present in all strains compared to Dumas. All four substitutions have hitherto not been described in the literature and were phenotypically classified as natural gene polymorphisms although two out of them (S51L, K186R) were localized in conserved gene centers. The DNA pol of 34 isolates exhibited 19 different substitutions, 14 out of them were novel, and two (R753K, V777I) were within conserved gene regions. Again, these changes were characterized as natural gene polymorphisms. CONCLUSIONS: Non-synonymous mutations in VZV TK or DNA pol conferring natural gene polymorphism are rare events. Nevertheless, the phenotypic characterization of 18 novel polymorphisms can help to provide a better identification of resistance mutations.


Assuntos
DNA Polimerase Dirigida por DNA/genética , Herpesvirus Humano 3/genética , Polimorfismo Genético , Timidina Quinase/genética , Infecção pelo Vírus da Varicela-Zoster/virologia , Aciclovir/uso terapêutico , Adolescente , Adulto , Substituição de Aminoácidos , Antivirais/uso terapêutico , Criança , Pré-Escolar , Farmacorresistência Viral/genética , Feminino , Genótipo , Herpesvirus Humano 3/enzimologia , Herpesvirus Humano 3/isolamento & purificação , Humanos , Lactente , Masculino , Mutação , Fenótipo , Análise de Sequência de DNA , Adulto Jovem
18.
Plant Reprod ; 30(3): 141-146, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28695277

RESUMO

KEY MESSAGE: Size limits on molecular movement among female gametes. Cellular decisions can be influenced by information communicated from neighboring cells. Communication can occur via signaling or through the direct transfer of molecules. Movement of RNAs and proteins has frequently been observed among symplastically connected plant cells. In flowering plants, the female gametes, the egg cell and central cell, are closely apposed within the female gametophyte. Here we investigated the ability of fluorescently labeled dyes and small RNAs to move from the Arabidopsis thaliana central cell to the egg apparatus following microinjection. These results define a size limit of at least 20 kDa for symplastic movement between the two gametes, somewhat larger than that previously observed in Torenia fournieri. Our results indicate that symplastic connectivity in Arabidopsis thaliana changes after fertilization and suggest that prior to fertilization mechanisms are in place to facilitate small RNA movement from the central cell to the egg cell and synergids.


Assuntos
Arabidopsis/metabolismo , Óvulo Vegetal/metabolismo , Arabidopsis/crescimento & desenvolvimento , Comunicação Celular , Endosperma/metabolismo , Corantes Fluorescentes/metabolismo , Microinjeções , Tamanho da Partícula , Polinização , RNA/metabolismo , Sementes/metabolismo
19.
Front Microbiol ; 8: 205, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28261167

RESUMO

Viral neuraminidases are an established drug target to combat influenza. Severe complications observed in influenza patients are primarily caused by secondary infections with e.g., Streptococcus pneumoniae. These bacteria engage in a lethal synergism with influenza A viruses (IAVs) and also express neuraminidases. Therefore, inhibitors with dual activity on viral and bacterial neuraminidases are expected to be advantageous for the treatment of influenza infections. Here we report on the discovery and characterization of diazenylaryl sulfonic acids as dual inhibitors of viral and Streptococcus pneumoniae neuraminidase. The initial hit came from a virtual screening campaign for inhibitors of viral neuraminidases. For the most active compound, 7-[2-[4-[2-[4-[2-(2-hydroxy-3,6-disulfo-1-naphthalenyl)diazenyl]-2-methylphenyl]diazenyl]-2-methylphenyl]diazenyl]-1,3-naphthalenedisulfonic acid (NSC65847; 1), the Ki-values measured in a fluorescence-based assay were lower than 1.5 µM for both viral and pneumococcal neuraminidases. The compound also inhibited N1 virus variants containing neuraminidase inhibitor resistance-conferring substitutions. Via enzyme kinetics and nonlinear regression modeling, 1 was suggested to impair the viral neuraminidases and pneumococcal neuraminidase with a mixed-type inhibition mode. Given its antiviral and antipneumococcal activity, 1 was identified as a starting point for the development of novel, dual-acting anti-infectives.

20.
ACS Med Chem Lett ; 8(2): 145-150, 2017 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-28217261

RESUMO

Recent findings from solid state NMR (ssNMR) studies suggested that the (R)-enantiomer of rimantadine binds to the full M2 protein with higher affinity than the (S)-enantiomer. Intrigued by these findings, we applied functional assays, such as antiviral assay and electrophysiology (EP), to evaluate the binding affinity of rimantadine enantiomers to the M2 protein channel. Unexpectedly, no significant difference was found between the two enantiomers. Our experimental data based on the full M2 protein function were further supported by alchemical free energy calculations and isothermal titration calorimetry (ITC) allowing an evaluation of the binding affinity of rimantadine enantiomers to the M2TM pore. Both enantiomers have similar channel blockage, affinity, and antiviral potency.

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