Dynamic processes such as diffusion and binding/unbinding of macromolecules (e.g. growth factors or nutrients) are crucial parameters for the design and application of effective artificial tissue materials. Here, dynamics of selected macromolecules were studied in two different composite tissue engineering scaffolds containing an electrospun nanofiber mesh (polycaprolactone or hydrophobically plasma modified polyvinylalcohol-chitosan) encapsulated in agarose hydrogels by a conventional approach fluorescence recovery after photobleaching (FRAP) and a novel technique, raster image correlation spectroscopy (RICS). The two approaches are compared, and it is shown that FRAP is unable to determine processes occurring at low molecular concentrations, especially accurately separating binding/unbinding from diffusion, and its results depend on the concentration of the studied molecules. RICS measures processes of single molecules and, because of its multiple adjustable timescales, can distinguish whether diffusion or binding controls molecular movement and separates fast diffusion from slow transient binding. In addition, RICS provides a robust read-out parameter quantifying binding affinity. Finally, the combination of FRAP and RICS helps to characterize diffusion and binding of macromolecules in tested artificial tissues better, and therefore predicts the behavior of biologically active molecules in these materials for medical applications.

• MeSH
• difuze MeSH
• nanovlákna MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.

• MeSH
• buněčná membrána chemie   MeSH
• konfokální mikroskopie metody   MeSH
• membránové proteiny analýza   MeSH
• počítačové zpracování obrazu metody   MeSH
• rostlinné buňky chemie   MeSH
• spektrální analýza metody   MeSH
• tabák chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

OBJECTIVE: Patients with deep-seated arteriovenous malformations (AVMs) have a higher rate of unfavorable outcome and lower rate of nidus obliteration after primary stereotactic radiosurgery (SRS). The aim of this study was to evaluate and quantify the effect of AVM location on repeat SRS outcomes. METHODS: This retrospective, multicenter study involved 505 AVM patients managed with repeat, single-session SRS. The endpoints were nidus obliteration, hemorrhage in the latency period, radiation-induced changes (RICs), and favorable outcome. Patients were split on the basis of AVM location into the deep (brainstem, basal ganglia, thalamus, deep cerebellum, and corpus callosum) and superficial cohorts. The cohorts were matched 1:1 on the basis of the covariate balancing score for volume, eloquence of location, and prescription dose. RESULTS: After matching, 149 patients remained in each cohort. The 5-year cumulative probability rates for favorable outcome (probability difference -18%, 95% CI -30.9 to -5.8%, p = 0.004) and AVM obliteration (probability difference -18%, 95% CI -30.1% to -6.4%, p = 0.007) were significantly lower in the deep AVM cohort. No significant differences were observed in the 5-year cumulative probability rates for hemorrhage (probability difference 3%, 95% CI -2.4% to 8.5%, p = 0.28) or RICs (probability difference 1%, 95% CI -10.6% to 11.7%, p = 0.92). The median time to delayed cyst formation was longer with deep-seated AVMs (deep 62 months vs superficial 12 months, p = 0.047). CONCLUSIONS: AVMs located in deep regions had significantly lower favorable outcomes and obliteration rates compared with superficial lesions after repeat SRS. Although the rates of hemorrhage in the latency period and RICs in the two cohorts were comparable, delayed cyst formation occurred later in patients with deep-seated AVMs.

• MeSH
• dospělí MeSH
• intrakraniální arteriovenózní malformace * chirurgie   radioterapie   MeSH
• kohortové studie MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• radiochirurgie * metody   MeSH
• reoperace MeSH
• retrospektivní studie MeSH
• výsledek terapie MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH

OBJECTIVE: The purpose of this study was to describe the long-term outcomes and associated risks related to repeat stereotactic radiosurgery (SRS) for persistent arteriovenous malformations (AVMs) in pediatric patients. METHODS: Under the auspices of the International Radiosurgery Research Foundation, this retrospective multicenter study analyzed pediatric patients who underwent repeat, single-session SRS between 1987 and 2022. The primary outcome variable was a favorable outcome, defined as nidus obliteration without hemorrhage or neurological deterioration. Secondary outcomes included rates and probabilities of hemorrhage, radiation-induced changes (RICs), and cyst or tumor formation. RESULTS: The cohort included 83 pediatric patients. The median patient age was 11 years at initial SRS and 15 years at repeat SRS. Fifty-seven children (68.7%) were managed exclusively using SRS, and 42 (50.6%) experienced hemorrhage prior to SRS. Median AVM diameter and volume were substantially different between the first (25 mm and 4.5 cm3, respectively) and second (16.5 mm and 1.6 cm3, respectively) SRS, while prescription dose and isodose line remained similar. At the 5-year follow-up evaluation from the second SRS, nidus obliteration was achieved in 42 patients (50.6%), with favorable outcome in 37 (44.6%). The median time to nidus obliteration and hemorrhage was 35.5 and 38.5 months, respectively. The yearly cumulative probability of favorable outcome increased from 2.5% (95% CI 0.5%-7.8%) at 1 year to 44% (95% CI 32%-55%) at 5 years. The probability of achieving obliteration followed a similar pattern and reached 51% (95% CI 38%-62%) at 5 years. The 5-year risk of hemorrhage during the latency period after the second SRS reached 8% (95% CI 3.2%-16%). Radiographically, 25 children (30.1%) had RICs, but only 5 (6%) were symptomatic. Delayed cyst formation occurred in 7.2% of patients, with a median onset of 47 months. No radiation-induced neoplasia was observed. CONCLUSIONS: The study results showed nidus obliteration in most pediatric patients who underwent repeat SRS for persistent AVMs. The risks of symptomatic RICs and latency period hemorrhage were quite low. These findings suggest that repeat radiosurgery should be considered when treating pediatric patients with residual AVM after prior SRS. Further study is needed to define the role of repeat SRS more fully in this population.

• MeSH
• cysty * MeSH
• dítě MeSH
• intrakraniální arteriovenózní malformace * diagnostické zobrazování   radioterapie   komplikace   MeSH
• krvácení komplikace   chirurgie   MeSH
• lidé MeSH
• následné studie MeSH
• radiochirurgie * škodlivé účinky   metody   MeSH
• retrospektivní studie MeSH
• výsledek terapie MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH

The epidermal growth factor receptor (EGFR) is a rational target of anticancer therapies due to its overexpression in a variety of malignant epithelial tumors. Nevertheless, this antigen is also present in normal tissues. Consequently, monoclonal antibodies which selectively bind to EGFR-overexpressing tumors will be choice drug candidates for development of radioimmunoconjugates (RIC). Nimotuzumab (h-R3) and trastuzumab are monoclonal antibodies (mAbs) which would preferentially target tissues with EGFR and HER2 overexpression, respectively. In this chapter, we describe preparation and evaluation of the targeting properties of RIC formed by (177)Lu/(90)Y and monoclonal antibodies which selectively target EGFR- and HER2/c-neu-overexpressing tissues. mAbs were labeled with n.c.a. (177)Lu/(90)Y using bifunctional chelating agents. RIC binding properties and toxicity were evaluated in vitro using cell lines with varying antigen expression. In vivo tumor targeting properties of RIC were evaluated in mice bearing colorectal (SNU-C2B) and A431 tumor xenografts. RICs were prepared with specific activities up to 2 GBq/mg without significant loss in biological activity. (90)Y-h-R3/trastuzumab increased cell growth inhibition compared with unmodified mAbs or (90)YCl(3) alone in cell lines with overexpression of the target antigen. (177)Lu-h-R3 showed significantly higher uptake in A431 (22.8 ± 3.1% ID/g) than in SNU-C2B (8.8 ± 4.1% ID/g) xenografts at 72 h post injection, indicating strong association between tumor uptake and EGFR expression levels.

• MeSH
• erbB receptory antagonisté a inhibitory   MeSH
• imunokonjugáty farmakokinetika   terapeutické užití   MeSH
• izotopové značení MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• lutecium terapeutické užití   MeSH
• monoklonální protilátky terapeutické užití   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• potkani Wistar MeSH
• radioimunoterapie MeSH
• radioizotopy ytria terapeutické užití   MeSH
• radionuklidy terapeutické užití   MeSH
• receptor erbB-2 antagonisté a inhibitory   MeSH
• xenogenní modely - testy protinádorové aktivity MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

UNLABELLED: Here we investigated the effect of disruption of plasma membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility in HEK293 cells stably expressing TRH-R-eGFP fusion protein (VTGP cells). Detailed analysis by fluorescence recovery after photobleaching (FRAP) in bleached spots of different sizes indicated that cholesterol depletion did not result in statistically significant alteration of mobile fraction of receptor molecules (Mf). The apparent diffusion coefficient (Dapp) was decreased, but this decrease was detectable only under the special conditions of screening and calculation of FRAP data. Analysis of mobility of receptor molecules by raster image correlation spectroscopy (RICS) did not indicate any significant difference between control and cholesterol-depleted cells. Results of our FRAP and RICS experiments may be collectively interpreted in terms of a "membrane fence" model which regards the plasma membrane of living cells as compartmentalized plane where lateral diffusion of membrane proteins is limited to restricted areas by cytoskeleton constraints. Hydrophobic interior of plasma membrane, studied by steady-state and time-resolved fluorescence anisotropy of hydrophobic membrane probe DPH, became substantially more "fluid" and chaotically organized in cholesterol-depleted cells. Decrease of cholesterol level impaired the functional coupling between the receptor and the cognate G proteins of Gq/G11 family. IN CONCLUSION: the presence of an unaltered level of cholesterol in the plasma membrane represents an obligatory condition for an optimum functioning of TRH-R signaling cascade. The decreased order and increased fluidity of hydrophobic membrane interior suggest an important role of this membrane area in TRH-R-Gq/G11α protein coupling.

• MeSH
• algoritmy MeSH
• buněčná membrána chemie   metabolismus   MeSH
• cholesterol metabolismus   MeSH
• difenylhexatrien chemie   metabolismus   MeSH
• difuze MeSH
• fluorescenční polarizace MeSH
• FRAP MeSH
• HEK293 buňky MeSH
• kinetika MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• proteiny vázající GTP - alfa-podjednotky Gq-G11 metabolismus   MeSH
• receptory thyroliberinu chemie   genetika   metabolismus   MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• zelené fluorescenční proteiny chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cells continuously communicate with the surrounding environment employing variety of signaling molecules and pathways to integrate and transport the information in the cell. An example of signaling initiation is binding of extracellular ligand to its receptor at the plasma membrane. This initializes enzymatic reactions leading to the formation of bi- or multimolecular signaling complexes responsible for the regulation or progress of signal transduction. Here, we describe three imaging techniques enabling detection of individual signaling molecules, their complexes, and clusters in human cells. Described imaging techniques require only basic microscopy systems available in the majority of current biomedical research centers but apply advanced data processing. First, total internal reflection fluorescence microscopy (TIRFM) variant of wide-field fluorescence microscopy for imaging highly dynamic clusters is described. Second, superresolution localization microscopy techniques-photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM)-recently enabled to achieve higher resolution with precision limit of about 20 nm in fixed samples. The developments toward live cell superresolution imaging are indicated. Third, raster image correlation spectroscopy (RICS) employed for molecular diffusion and binding analysis explains the advantages and hurdles of this novel method. Presented techniques provide a new level of detail one can learn about higher organization of signaling events in human cells.

• MeSH
• buněčná membrána metabolismus   MeSH
• buněčný tracking metody   MeSH
• fluorescenční barviva MeSH
• fluorescenční mikroskopie metody   MeSH
• interpretace obrazu počítačem metody   MeSH
• Jurkat buňky MeSH
• lidé MeSH
• ligandy MeSH
• mezibuněčná komunikace MeSH
• signální transdukce MeSH
• zelené fluorescenční proteiny MeSH
• zobrazování trojrozměrné MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Use of a reduced intensity conditioning (RIC) regimen has now become standard practice among older or more infirmed stem cell transplantation candidates. Encouraging outcome in this population has led to the question of whether RIC should replace standard myeloablative conditioning (MAC) regimens. This review will summarize the available outcomes data comparing RIC and MAC approaches to stem cell transplantation in adult patients with acute myeloid leukemia, myelodysplastic syndrome (MDS) and acute lymphoid leukemia. RECENT FINDINGS: There are currently no completed prospective randomized controlled studies comparing outcomes of RIC to MAC. The best insight into differences in outcome comes from large registry-based retrospective studies. These studies demonstrate that the use of RIC is associated with a reduction in transplant-related mortality but an increased risk of disease relapse. As a result, for patients undergoing stem cell transplantation in remission, disease free and overall survival are similar. SUMMARY: The current retrospective data provide justification for the use of RIC regimens in all adult stem cell transplant candidates with acute leukemia in remission and MDS. More definitive conclusions regarding differences between the MAC and RIC approach to stem cell transplantation await results of ongoing prospective randomized trials.

• MeSH
• akutní lymfatická leukemie chirurgie   MeSH
• akutní myeloidní leukemie chirurgie   MeSH
• lidé MeSH
• myelodysplastické syndromy chirurgie   MeSH
• transplantace kmenových buněk metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

This retrospective study by the European Society for Blood and Marrow Transplantation analyzed the outcome of 2224 patients with myelofibrosis (MF) who underwent allogeneic stem cell transplantation (allo-SCT) between 2000 and 2014; 781 (35%) underwent myeloablative conditioning (MAC) and 1443 (65%) reduced-intensity conditioning (RIC). Median patient age was 52.9 years (range, 18 to 74 years) and 57.5 years (range, 21 to 76 years) in the MAC and RIC cohorts, respectively. Donor type was similar: matched sibling donors (MAC, 317 [41%]; RIC, 552 [38%]) and unrelated donors (MAC, 464 [59%]; RIC, 891 [62%]). Median time to both neutrophil and platelet (>20 × 109/L) engraftment did not differ between cohorts. Rates of grade II to IV acute GVHD were 28% (MAC) and 31% (RIC; P = NS). Cumulative chronic GVHD rates (limited/extensive) were 22%/27% (MAC) and 19%/31% (RIC; P = .10). Cumulative incidences of nonrelapse mortality (NRM) at 1, 3, and 5 years were 25.5%, 32.2%, and 34.6% (MAC) and 26.3%, 32.8%, and 34.4% (RIC), respectively. There was a trend toward a higher relapse rate with RIC regimens compared with MAC (P = .08); rates at 1, 3, and 5 years were 10.9%, 17.2%, and 20.1% (MAC) and 14%, 19.7%, and 23.2% (RIC), respectively. No significant difference in 5-year probabilities of overall survival (OS) was noted: MAC (53.0%; 95% confidence interval [CI], 49.1% to 56.9%) and RIC (51.0%; 95% CI, 48.3% to 53.7%); P = .78. Regarding the composite end point of GVHD-free/relapse-free survival (GRFS), the unadjusted Kaplan-Meier estimate of 5-year GRFS was 32.4% (95% CI, 29.0% to 36.1%) in the MAC group and 26.1% (95% CI, 23.9% to 28.2%) in the RIC group (P = .001). In the MAC cohort, multivariable analysis confirmed worse OS and NRM with older age (>50 years), using an unrelated donor and a Karnofsky Performance Status of 80 or less. For the RIC cohort, worse OS and NRM were associated with age 60 to 70 years compared with younger recipients, use of a mismatched donor, and poor performance status. In conclusion, although similar OS rates existed for both cohorts overall, this study suggests that MAC should still be used for younger individuals suitable for such an approach due to a trend toward less relapse and an overall suggested advantage of improved GRFS, albeit this should be examined in a more homogeneous cohort. RIC allo-SCT still offers significant survival advantage in the older, fitter MF allograft patient, and optimization to reduce significant relapse and NRM rates is required.

• MeSH
• alografty MeSH
• chronická nemoc MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• míra přežití MeSH
• mladiství MeSH
• přežití bez známek nemoci MeSH
• primární myelofibróza mortalita   terapie   MeSH
• příprava pacienta k transplantaci * MeSH
• retrospektivní studie MeSH
• senioři MeSH
• společnosti lékařské MeSH
• transplantace hematopoetických kmenových buněk * MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• Geografické názvy
• Evropa MeSH

PURPOSE: The optimal dose intensity for conditioning prior to allogeneic hematopoietic stem cell transplantation (alloHSCT) for chronic lymphocytic leukemia (CLL) is unknown. METHODS: We retrospectively compared outcomes of patients who received a first alloHCST after non-myeloablative (NMA) and reduced intensity conditioning (RIC). Data of 432 patients with a median age of 55 years were included, of which 86 patients underwent NMA and 346 RIC. RESULTS: The median follow-up after alloHSCT was 4.3 years. Compared to the RIC group, more NMA patients had purine-analog-sensitive disease, were in complete remission and received matched related donor transplantation. After RIC, the probabilities for 5-year OS, EFS, CIR, and NRM were 46%, 38%, 28%, and 35% and after NMA the respective probabilities were 52%, 43%, 25%, and 32%. In multivariate analysis, remission status prior to conditioning but not RIC versus NMA conditioning had a significant impact on CIR, EFS, and OS. CONCLUSION: Presumed higher anti-leukemic activity of RIC versus NMA conditioning did not translate into better outcomes after alloHSCT, but better remission status prior to conditioning did. Effective pathway inhibitor-based salvage therapies combined with NMA conditioning might thus represent the most attractive contemporary approach for alloHSCT for patients with CLL.

• MeSH
• busulfan aplikace a dávkování   MeSH
• chronická lymfatická leukemie mortalita   patologie   terapie   MeSH
• cyklofosfamid aplikace a dávkování   MeSH
• dospělí MeSH
• homologní transplantace MeSH
• incidence MeSH
• indukce remise MeSH
• kombinovaná terapie MeSH
• lidé středního věku MeSH
• lidé MeSH
• melfalan aplikace a dávkování   MeSH
• míra přežití MeSH
• mladý dospělý MeSH
• následné studie MeSH
• nemoc štěpu proti hostiteli epidemiologie   mortalita   MeSH
• příprava pacienta k transplantaci mortalita   MeSH
• prognóza MeSH
• protokoly protinádorové kombinované chemoterapie terapeutické užití   MeSH
• retrospektivní studie MeSH
• senioři MeSH
• transplantace hematopoetických kmenových buněk mortalita   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH