FLIM reveals alternative EV-mediated cellular up-take pathways of paclitaxel

In response to physiological and artificial stimuli, cells generate nano-scale extracellular vesicles (EVs) by encapsulating biomolecules in plasma membrane-derived phospholipid envelopes. These vesicles are released to bodily fluids, hence acting as powerful endogenous mediators in intercellular signaling. EVs provide a compelling alternative for biomarker discovery and targeted drug delivery, but their kinetics and dynamics while interacting with living cells are poorly understood. Here we introduce a novel method, fluorescence lifetime imaging microscopy (FLIM) to investigate these interaction attributes. By FLIM, we show distinct cellular uptake mechanisms of different EV subtypes, exosomes and microvesicles, loaded with anti-cancer agent, paclitaxel. We demonstrate differences in intracellular behavior and drug release profiles of paclitaxel-containing EVs. Exosomes seem to deliver the drug mostly by endocytosis while microvesicles enter the cells by both endocytosis and fusion with cell membrane. This research offers a new real-time method to investigate EV kinetics with living cells, and it is a potential advancement to complement the existing techniques. The findings of this study improve the current knowledge in exploiting EVs as next-generation targeted drug delivery systems.Peer reviewe

Cryopreservation of raspberry cultivar accessions bred in Russia from the VIR <i>in vitro</i> collection

Cryobanks use plant cryocollections for long-term preservation of crops which cannot be preserved in seed collections. These are vegetatively propagated crops, accessions of species which form either a small amount of seeds, or recalcitrant seeds. Shoot tips (apexes) of in vitro plants are used for cryopreservation for most berry crops, therefore maintenance of in vitro collections is very important. The VIR in vitro collection includes 150 accessions of Rubus L. species, 85 of them are raspberry cultivars, 59 of which were bred in Russia. These cultivars reflect a wide ecogeographic diversity. Among them, there are raspberry cultivars created at the end of the 19th – first half of the 20th centuries, including cultivars bred by I.V. Michurin and by the pioneer of northern horticulture V.V. Spirin. More than half of national raspberry varieties (33) are listed in the State Register for Selection Achievements Admitted for Usage. Raspberry cultivars from Russian breeding programs have a very limited representation in foreign genebanks. The first aim of the present work was cryopreservation of mostly folk and old Russian raspberry cultivars received by VIR from 1925 till 1950 and their transfer into the cryobank. The second aim of the work was to monitor post-cryogenic regeneration of raspberry cultivars transferred to the cryobank earlier. A modified protocol of the droplet vitrification method by “DV-biotech” was used for cryopreservation of shoot tips of in vitro plants of 10 raspberry cultivars (7 of which are folk and old Russian ones) from the VIR in vitro collection. Post-cryogenic regeneration was evaluated for 17 raspberry cultivars preserved in the cryobank from one to five years. Ten raspberry cultivars (900 apexes) with an average mean post-cryoregenic regeneration value of 38.2±3.0% determined in control tests, were placed in the cryobank for long-term storage. A statistically significant effect of the genotype on the viability of explants after cryopreservation was noted, while the post-cryogenic regeneration was genotype insensitive. Additionally, levels of post-cryogenic regeneration were evaluated for 17 raspberry cultivars (296 apexes) preserved in the cryobank from one to five years. Post-cryogenic regeneration within the 20-70% range was displayed by four raspberry cultivars preserved in the cryobank for one year, and for 8 cultivars conserved there from three to five years post-cryogenic regeneration was within the 10-50% range. According to the results of monitoring, regeneration displayed by 12 raspberry cultivars was within the 10-70% range, which can be considered as a reliable rate of apex preservation in liquid nitrogen vapors in the VIR cryobank. Monitoring of the post-cryogenic regeneration of the raspberry accessions preserved in the VIR cryobank and cryopreservation of new raspberry cultivars will be continued

Structure and Dynamics of Thermosensitive pDNA Polyplexes Studied by Time-Resolved Fluorescence Spectroscopy

Combining multiple stimuli-responsive functionalities into the polymer design is an attractive approach to improve nucleic acid delivery. However, more in-depth fundamental understanding how the multiple functionalities in the polymer structures are influencing polyplex formation and stability is essential for the rational development of such delivery systems. Therefore, in this study the structure and dynamics of thermosensitive polyplexes were investigated by tracking the behavior of labeled plasmid DNA (pDNA) and polymer with time-resolved fluorescence spectroscopy using fluorescence resonance energy transfer (FRET). The successful synthesis of a heterofunctional poly(ethylene glycol) (PEG) macroinitiator containing both an atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain-transfer (RAFT) initiator is reported. The use of this novel PEG macroinitiator allows for the controlled polymerization of cationic and thermosensitive linear triblock copolymers and labeling of the chain-end with a fluorescent dye by maleimide-thiol chemistry. The polymers consisted of a thermosensitive poly(N-isopropylacrylamide) (PNIPAM, N), hydrophilic PEG (P), and cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA, D) block, further referred to as NPD. Polymer block D chain-ends were labeled with Cy3, while pDNA was labeled with FITC. The thermosensitive NPD polymers were used to prepare pDNA polyplexes, and the effect of the N/P charge ratio, temperature, and composition of the triblock copolymer on the polyplex properties were investigated, taking nonthermosensitive PD polymers as the control. FRET was observed both at 4 and 37 degrees C, indicating that the introduction of the thermosensitive PNIPAM block did not compromise the polyplex structure even above the polymer's cloud point. Furthermore, FRET results showed that the NPD- and PD-based polyplexes have a less dense core compared to polyplexes based on cationic homopolymers (such as PEI) as reported before. The polyplexes showed to have a dynamic character meaning that the polymer chains can exchange between the polyplex core and shell. Mobility of the polymers allow their uniform redistribution within the polyplex and this feature has been reported to be favorable in the context of pDNA release and subsequent improved transfection efficiency, compared to nondynamic formulations.Peer reviewe

Features of systemic cytokine production in Behcet's disease associated with uveitis without ocular lesions

Behcet's disease (BD) is a systemic disease underlyed by chronic vasculitis. Hyperactivity of innate and adaptive immunity plays important role in its pathogenesis. Uveitis occurs in 30-70% of the patients, often recurring and reducing visual function. The objective of our work was to study the features of systemic production of immune mediators in BD patients, depending on presence and activity of uveitis. 116 BD patients were divided into 3 groups: (1) 41 patients with active uveitis (UA), (2) 64 subjects with uveitis remission (UR), (3) 11 uveitis-free BD patients (WU). Control group (CG) comprised 34 conditionally healthy people. Detection rate (%) and contents (pg/ml) were measured for IL-1β IL-2, IL-4, IL-5, IL-6, IL-12p70, IL-13, IL-18, IFNγ, CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, CCL11/Eotaxin, СXCL1/GRO-α, CXCL8/IL-8, CXCL10/IP-10, CXCL12/SDF-1α, GM-CSF, TNFα in blood serum by means of multiplex analysis using MAGPIX analyzer (Luminex Corp., USA), Procarta Plex “Human Th1/Th2&amp;Chemokine Panel 20 plex” kits (Bioscience, Austria). TGF-P1, TGF-P2 levels were assayed by ELISA-test (“Vfector-Best”). All the BD patients showed high detection rates of CXCL1/GRO-α (but not its level) in comparison with CG. Detection rate and levels of IL-6, IL-8 were increased in 1st and 2nd BD groups, compared to CG. In UR, unlike UA and WU groups, IL-4 was detected more often than in CG. WU patients showed increased detection rate of only CXCL1/GRO -α. When compared with UA, WU patients had lower serum concentrations of IFNγ, MCP-1, IP-10, MIP-1a, SDF-1α, TGF-β1; UR patients also showed decreased serum levels of IL-18, Eotaxin, GRO-α, RANTES, TGF-β2. Our results indicate the importance of angiogenic and proinflammatory chemokines and cytokines in pathogenesis of BD uveitis, as well as imbalanced production of various immunomediators. Higher detection rates and levels of IL-6 and IL-8 in UA and UR patients may result from weak persistent intraocular inflammation, even upon relief of clinical symptoms, thus, probably, requiring therapeutic correction

EFFECT OF HUMAN HERPES VIRUS REACTIVATION ON SYSTEMIC CYTOKINE PRODUCTION IN PATIENTS WITH BEHCET’S DISEASE AND UVEITIS

Behcet’s disease (BD) is a systemic autoinflammatory-autoimmune disease (chronic systemic vasculitis) of unknown etiology, almost 70% of patients develop uveitis. BD pathogenesis is complex, human herpesviruses (HHV) play an important role among infectious trigger factors. Ability of herpesviruses to modulate cytokine production and evade host’s immune response is known.Aim of the study was to assess the effect of Herpes simplex virus type 1, Herpes simplex virus type 2, Cytomegalovirus, Epstein-Barr virus on systemic levels of chemokines, pro- and anti-inflammatory cytokines in BD with and without uveitis. Serum samples were collected from 116 BD patients chronically infected with HHV and examined in ELISA-test for markers of HHV reactivation (IgG-antibodies to immediate early HSV antigens 1, 2 and CMV, early EBV antigen). Concentration of IL-1β, IFNγ, MCP-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-12p70, IL-13, IL-18, TNFα, GMCSF, Eotaxin, GRO-α, IP-10, MIP-1α, MIP-1β, SDF-1α, RANTES detected in multiplex analysis. TGF-β1, TGF-β2 were measured in ELISA-test. Depending on presence and activity of uveitis 3 groups of patients with BD were identified: group 1 – active uveitis, group 2 – remission of uveitis, group 3 – BD without ocular manifestations. After serological study 2 subgroups were highlighted in each group: a) patients with antibody markers of reactivation of at least one HHV, b) patients chronically infected with HHV, without serological signs of reactivation. Mean level and detection rate of cytokines and chemokines in patients with active uveitis (1a, 1b) and in remission (2a, 2b) were compared with patients without eye damage (3a, 3b). Chronic HHV infection (subgroup “b”) was compared with reactivation (subgroup “a”). A significant increase of MCP-1/ CCL2, MIP-1α/CCL3, MIP-1β/CCL4, RANTES/CCL5, IP-10, SDF-1α chemokines in serum, as well as IFNγ, TGF-β1, and TGF-β2 was observed in patients with uveitis (regardless of their activity) and HHV reactivation compared to patients without uveitis. Our data indicate that systemic production of cytokines and chemokines in BD patients and uveitis could be affected by the activity of chronic herpesvirus infections, and the greatest changes are related to chemokines

Проблема усталости у пациентов с системной красной волчанкой, по данным российской когорты РЕНЕССАНС

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with a wide range of clinical manifestations. Numerous observations and surveys of patients have shown that the most common symptom of SLE is fatigue complaints in 51 to 90% of patients.Objective: to determine the significance of fatigue in the general health status of RENAISSANCE cohort patients with SLE who were hospitalized in the Clinic, V.A. Nasonova Research Institute of Rheumatology.Patients and methods. The investigation included SLE patients aged 18 years and older who met the 2012 SLICC criteria. The standard examination accepted in the management of patients with SLE was made. Disease activity was determined by SLEDAI-2K; irreversible lesions in various organs were identified using the SLICC damage index. The SF-36 and the LupusQoL questionnaires were used to assess health-related quality of life (HRQOL) and the FACIT-Fatigue scale was applied to measure fatigue.Results and discussion. The investigation enrolled 328 patients, mainly women (91%); the mean age was 34.4±11.5 years; the duration of the disease was 106.3±97.9 months. In this group, moderate and high disease activities (SLEDAI-2K scores of 6–10 and 11–19, respectively) were observed at approximately the same frequency. At the time of inclusion, more than half (56.5%) of the patients already had various irreversible organ lesions. At Visit 1, the FACIT-Fatigue scale showed that fatigue was present in 148 (45%) of the 328 patients. According to the presence of fatigue, the patients were divided into two groups. Group 1 included 148 patients with fatigue; Group 2 consisted of 180 patients without fatigue. The Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) and anti-DNA antibody levels were significantly higher in the fatigue group (p=0.01 and p=0.02, respectively); the patients also had decreased HRQOL according to 7 LupusQol domains (p&lt;0.001). The patients with fatigue were significantly more likely to receive intravenous glucocorticoids and rituximab. At 12 months after the start of treatment, the patients with fatigue were found to have a statistically significant reduction in disease activity, as well as normalization of anti-DNA antibody levels, improvements in HRQOL according to the LupusQol domains, and less severity of fatigue according to the FACIT-Fatigue scale.Conclusion. Fatigue was detected in almost half (45–53%) of SLE patients. It is associated with a higher disease activity by SLEDAI-2K and with a high anti-DNA antibody level. The patients with fatigue are observed to have an obvious worsening of HRQOL according to all LupusQol domains.Системная красная волчанка (СКВ) – хроническое аутоиммунное заболевание с широким спектром клинических проявлений. Как показали многочисленные наблюдения и опросы пациентов, наиболее распространенным симптомом СКВ является усталость, на которую жалуются от 51 до 90% больных.Цель настоящего исследования – определение значения усталости в общем состоянии больных СКВ когорты РЕНЕССАНС, госпитализированных в клинику ФГБНУ «Научно-исследовательский институт ревматологии» им. В.А. Насоновой.Пациенты и методы. В исследование включали больных СКВ, соответствующих критериям SLICC 2012 г., в возрасте 18 лет и старше. Проводили стандартное обследование, принятое при ведении пациентов с СКВ. Активность заболевания определяли по индексу SLEDAI-2K, необратимые повреждения различных органов – с помощью индекса повреждения (ИП) SLICC. Для оценки качества жизни, связанного со здоровьем (КЖСЗ), использовали опросники SF-36 и LupusQoL, усталости – шкалу FACIT-Fatigue.Результаты и обсуждение. В исследование включено 328 больных, преимущественно женщин (91%), средний возраст – 34,4±11,5 года, длительность заболевания – 106,3±97,9 мес. В данной группе примерно с одинаковой частотой наблюдалась умеренная (SLEDAI-2K 6–10 баллов) и высокая (SLEDAI-2K 11–19 баллов) активность заболевания. На момент включения в исследование более чем у половины пациентов (56,5%) уже имелись различные необратимые органные повреждения. Во время визита 1 усталость по шкале FACIT-Fatigue выявлена у 148 (45%) из 328 пациентов. В зависимости от наличия усталости пациенты были разделены на две группы. В 1-ю группу вошли 148 пациентов с усталостью, во 2-ю – 180 пациентов без усталости. Индекс активности SLEDAI-2K и уровень антител к ДНК были значимо выше в группе пациентов с усталостью (p=0,01 и p=0,02 соответственно); у них также установлено снижение КЖСЗ по 7 шкалам опросника LupusQol (p&lt;0,001). Пациенты с усталостью достоверно чаще получали глюкокортикоиды внутривенно и ритуксимаб. Через 12 мес после начала лечения у пациентов с усталостью отмечены статистически значимые снижение активности заболевания, нормализация уровня антител к ДНК, улучшение КЖСЗ по шкалам LupusQol, уменьшение выраженности усталости по шкале FACIT-Fatigue.Заключение. Усталость выявлена практически у половины (45–53%) пациентов с СКВ. Она ассоциируется с более высокой активностью заболевания по индексу SLEDAI-2K и высоким уровнем антител к ДНК. У пациентов с усталостью наблюдается выраженное ухудшение КЖСЗ по всем шкалам опросника LupusQol

Comparative analysis of anxiety-depressive spectrum disorders in patients with rheumatic diseases

Research objective - comparative analysis of incidence and structure of anxiety-depressive spectrum disorders (ADD) in patients with various rheumatic diseases (RD). Materials and methods. 613 patients with RD were enrolled in the study: 180 with a reliable diagnosis of systemic lupus erythematosus (SLE), 128 with rheumatoid arthritis (RA), 110 with systemic sclerosis (SSc), 115 with Behcet's disease (BD), 80 with primary Sjögren's syndrome (pSS). Female prevailed in all groups (95% of patients with pSS, 88,2% - SSc, 87,2% - RA, 85,5% of SLE) except BD patients (70% male). The mean age was 42.3±1.54 years and was lower in patients with BD (33.3±0.98 years) and SLE (34.6±0.93 years) compared to patients with SSc (49.9±2.47 years), RA (47.4±0.99 years) and pSS (46.2±2.3 years). The mean RD duration was 130,0±8,65 months and was more at BD - 148,5±10,4 months, pSS - 141,6±8,92 months, RA - 138,4±10,1months, and less at SLE - 134,9±8,8 months and SSc - 87,0±5,04 months. The mean SLE activity index SLEDAI was 9,13±0,63 points (high), RA (DAS28) - 5,26±0,17 points (high), BD (BDCAF) - 3,79±0,2 points (moderate) and SSc by G. Valentini - 1,1±0,20 points (moderate). Glucocorticoids took 100% of patients with pSS, 91,1% - SLE, 90% - SSc, 87% - BD and 67,2% - RA patients; conventional disease modifying anti-rheumatic drugs (cDMARDs) took 90% of patients with SSc, 84% - BD, 79,6% - RA, 68% - pSS, 40,6% - SLE. Biologic DMARDs took 32% of patients with RA, 17,4% - BD, 7,3% - SSc and 7,2% - SLE. Mental disorders were diagnosed by psychiatrist as a result of screening by the hospital anxiety and depression scale (HADS) and in semi-structured interview in accordance with the ICD-10/ DSM-IV. The severity of depression was evaluated by Montgomery-Asberg Depression Rating Scale (MADRS) and anxiety - by Hamilton Anxiety Rating Scale (HAM-A). Projective psychological methods were used for cognitive impairment detection. Results. Screening of depressive disorders (HADS-D≥8) was positive in 180 (29,4%) patients with RD, including 74 (41%) patients with SLE, 38 (35%) - SSc, 29 (23%) - RA, 23 (20%) - BD and 16 (20%) - pSS; anxiety disorders (HADS-A≥8) - in 272 (44,4%) patients, including 66 (52%) patients with RA, 40 (50%) - pSS, 77 (43%) - SLE, 45 (41%) - SSc and 44 (38%) - BD. In accordance with the ICD-10/ DSM-IV depressive disorders have been identified in 389 (63%) patients, including 94 (73%) patients with RA, 71 (64,5%) - SSc, 69 (60%) - BD, 90 (50%) - SLE and 39 (49%) - pSS; anxiety disorders - in 377 (61,5%) patients, including 20 (25%) patients with pSS, 44 (24,5%) - SLE, 29 (23%) - RA, 20 (17%) - BD and 7 (6,4%) - SSc. Conclusion. Anxiety-depressive spectrum disorders are typical for most patients with RA, SLE, SSc, pSS and BD. ADDs diagnosis in RD patients with the use of the HADS did not reveal a significant proportion. To obtain objective data on the frequency and structure of ADDs, psychopathological and clinical psychological diagnosis is necessary

Genome analysis of E. coli isolated from Crohn's disease patients

© 2017 The Author(s). Background: Escherichia coli (E. coli) has been increasingly implicated in the pathogenesis of Crohn's disease (CD). The phylogeny of E. coli isolated from Crohn's disease patients (CDEC) was controversial, and while genotyping results suggested heterogeneity, the sequenced strains of E. coli from CD patients were closely related. Results: We performed the shotgun genome sequencing of 28 E. coli isolates from ten CD patients and compared genomes from these isolates with already published genomes of CD strains and other pathogenic and non-pathogenic strains. CDEC was shown to belong to A, B1, B2 and D phylogenetic groups. The plasmid and several operons from the reference CD-associated E. coli strain LF82 were demonstrated to be more often present in CDEC genomes belonging to different phylogenetic groups than in genomes of commensal strains. The operons include carbon-source induced invasion GimA island, prophage I, iron uptake operons I and II, capsular assembly pathogenetic island IV and propanediol and galactitol utilization operons. Conclusions: Our findings suggest that CDEC are phylogenetically diverse. However, some strains isolated from independent sources possess highly similar chromosome or plasmids. Though no CD-specific genes or functional domains were present in all CD-associated strains, some genes and operons are more often found in the genomes of CDEC than in commensal E. coli. They are principally linked to gut colonization and utilization of propanediol and other sugar alcohols

Адаптация русскоязычной версии индекса активности системной красной волчанки EASY-BILAG

   Assessment of systemic lupus erythematosus (SLE) activity is important to determine the efficacy of treatment and to decide on further therapy. Russian-language versions of activity indices are needed to meet the needs of Russian-speaking patients and clinicians and to facilitate the use of these tools in clinical practice. An important step in this direction is the adaptation of the EASY-BILAG index, which is used to more accurately assess disease activity and select appropriate therapy in patients with SLE.   Оценка активности системной красной волчанки (СКВ) имеет важное значение для определения эффективности лечения и принятия решения о дальнейшей терапии. Русскоязычные версии индексов активности необходимы для удовлетворения потребностей русскоязычной популяции пациентов и клиницистов и облегчения использования этих инструментов в клинической практике. Одним из важных шагов в этом направлении является адаптация индекса Easy-BILAG, которая служит более точной оценке активности заболевания и адекватному подбору терапии у пациентов с СКВ