Glucocorticoids are potent anti-inflammatory drugs, although their use is associated with severe side effects. Loading glucocorticoids into suitable nanocarriers can significantly reduce these undesirable effects. Macrophages play a crucial role in inflammation, making them strategic targets for glucocorticoid-loaded nanocarriers. The main objective of this study is to develop a glucocorticoid-loaded PLGA nanocarrier specifically targeting liver macrophages, thereby enabling the localized release of glucocorticoids at the site of inflammation. Dexamethasone acetate (DA)-loaded PLGA nanospheres designed for passive macrophage targeting are synthesized using the nanoprecipitation method. Two types of PLGA NSs in the size range of 100-300 nm are prepared, achieving a DA-loading efficiency of 19 %. Sustained DA release from nanospheres over 3 days is demonstrated. Flow cytometry analysis using murine bone marrow-derived macrophages demonstrates the efficient internalization of fluorescent dye-labeled PLGA nanospheres, particularly into pro-inflammatory macrophages. Significant down-regulation in pro-inflammatory cytokine genes mRNA is observed without apparent cytotoxicity after treatment with DA-loaded PLGA nanospheres. Subsequent experiments in mice confirm liver macrophage-specific nanospheres accumulation following intravenous administration using in vivo imaging, flow cytometry, and fluorescence microscopy. Taken together, the data show that the DA-loaded PLGA nanospheres are a promising drug-delivery system for the treatment of inflammatory liver diseases.
- MeSH
- Anti-Inflammatory Agents pharmacology chemistry MeSH
- Dexamethasone * pharmacology chemistry analogs & derivatives MeSH
- Liver * drug effects metabolism MeSH
- Polylactic Acid-Polyglycolic Acid Copolymer * chemistry MeSH
- Macrophages * drug effects metabolism MeSH
- Mice MeSH
- Nanospheres * chemistry MeSH
- Drug Carriers chemistry pharmacology MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: Diffuse midline glioma, H3 K27-altered (DMG) is a fatal tumour that arises in the midline structures of the brain. When located in the pons, it is more commonly referred to as diffuse intrinsic pontine glioma (DIPG). DMG/DIPG is usually diagnosed when children are < 10 years, and it has a median overall survival of < 12 months after diagnosis. Radiological imaging is still the gold standard for DIPG diagnosis while the use of biopsy procedures led to our knowledge on its biology, such as with the identification of the canonical histone H3K27M mutation. However, the need to improve survival encourages the development of non-invasive, fast and inexpensive assays on biofluids for optimizing molecular diagnoses in DMG/DIPG. Here, we propose a rapid, new, imaging and epigenetics-based approach to diagnose DMG/DIPG in the plasma of paediatric patients. METHODS: A total of 20 healthy children (mean age: 10.5 years) and 24 children diagnosed with DMG/DIPG (mean age: 8.5 years) were recruited. Individual histones (H2A, H2B, H3, H4, macroH2A1.1 and macroH2A1.2), histone dimers and nucleosomes were assayed in biofluids by means of a new advanced flow cytometry ImageStream(X)-adapted method. RESULTS: We report a significant increase in circulating histone dimers and tetramers (macroH2A1.1/H2B versus control: p value < 0.0001; macroH2A1.2/H2B versus control: p value < 0.0001; H2A/H2B versus control: p value < 0.0001; H3/H4 versus control: p value = 0.008; H2A/H2B/H3/H4 versus control: p value < 0.0001) and a significant downregulation of individual histones (H2B versus control: p value < 0.0001; H3 versus control: p value < 0.0001; H4 versus control: p value < 0.0001). Moreover, histones were also detectable in the cerebrospinal fluid (CSF) of patients with DMG/DIPG and in the supernatant of SF8628, OPBG-DIPG002 and OPBG-DIPG004 DMG/DIPG cell lines, with patterns mostly similar to each other, but distinct compared to blood plasma. CONCLUSIONS: In summary, we identified circulating histone signatures able to detect the presence of DMG/DIPG in biofluids of children, using a rapid and non-invasive ImageStream(X)-based imaging technology, which may improve diagnosis and benefit the patients.
- MeSH
- Diffuse Intrinsic Pontine Glioma genetics diagnosis blood MeSH
- Child MeSH
- Epigenesis, Genetic MeSH
- Glioma genetics diagnosis blood pathology diagnostic imaging MeSH
- Histones * genetics metabolism blood MeSH
- Humans MeSH
- Adolescent MeSH
- Mutation MeSH
- Biomarkers, Tumor blood MeSH
- Brain Stem Neoplasms genetics diagnosis blood diagnostic imaging pathology metabolism MeSH
- Child, Preschool MeSH
- Check Tag
- Child MeSH
- Humans MeSH
- Adolescent MeSH
- Male MeSH
- Child, Preschool MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
INTRODUCTION: Central nervous system (CNS) involvement in diffuse large B-cell lymphoma (DLBCL) is a rare but serious condition requiring accurate diagnostics. Cerebrospinal fluid (CSF) analysis plays a crucial role, particularly in cases where biopsy is not feasible, and imaging is inconclusive. AREAS COVERED: Chemical markers have limitations, particularly in low-cellularity samples. Novel molecular techniques, including circulating tumor DNA (ctDNA) analysis and microRNAs (miRNAs), are gaining prominence for their ability to detect gene mutations at diagnosis and monitor minimal residual disease during follow-up. The sensitivity and specificity of genetic mutations, particularly MYD88 L265P, in combination with interleukin-10 (IL-10) levels, are discussed. The literature search methodology involved reviewing relevant studies and clinical data.This review examines both traditional and emerging methods for CSF analysis in diagnosing CNS involvement in DLBCL. Conventional approaches such as cytomorphology, flow cytometry, and biochemical markers have limitations, particularly in low-cellularity samples. Novel molecular techniques, including ctDNA analysis and miRNAs, are gaining prominence for their ability to detect gene mutations at diagnosis and monitor minimal residual disease during follow-up. The sensitivity and specificity of genetic mutations, particularly MYD88 L265P, in combination with interleukin-10 (IL-10) levels, are discussed. The literature search methodology involved reviewing relevant studies and clinical data. EXPERT OPINION: Advancements in CSF biomarker analysis are improving the diagnosis of CNS lymphoma, aiding early detection and personalized treatment approaches. However, further research and broader clinical validation are necessary for their routine implementation.
- MeSH
- Circulating Tumor DNA cerebrospinal fluid genetics MeSH
- Molecular Diagnostic Techniques methods MeSH
- Lymphoma, Large B-Cell, Diffuse * diagnosis cerebrospinal fluid genetics pathology MeSH
- Interleukin-10 genetics cerebrospinal fluid MeSH
- Humans MeSH
- Meningeal Neoplasms * diagnosis cerebrospinal fluid genetics MeSH
- MicroRNAs genetics cerebrospinal fluid MeSH
- Mutation MeSH
- Myeloid Differentiation Factor 88 genetics MeSH
- Biomarkers, Tumor * cerebrospinal fluid genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
BACKGROUND: Diagnosing primary or secondary CNS lymphoma (CNSL) is a clinical challenge due to the limitations of standard biopsy and imaging procedures despite established guidelines. Therefore, accurate biomarkers and analytical methods that are convenient for practical routine use are needed to diagnose and manage these aggressive lymphomas effectively. We evaluated the utility of minimally invasive circulating tumor DNA (ctDNA) detection in a prospective real-world scenario, moving this approach closer to clinical practice. METHODS: A total of 164 plasma, cerebrospinal fluid (CSF), and tumor samples from 56 CNSL patients were collected to analyze tumor DNA by the diagnostic next-generation sequencing (NGS) panel LYNX, enabling simultaneous analysis of gene variants, chromosomal aberrations, and antigen receptor rearrangements in targeted regions. RESULTS: The well-known genetic heterogeneity of CNSL was refined with integrative molecular data, showing the most frequent MYD88, PIM1, and KMT2D mutations and a broad spectrum of chromosomal aberrations, reflecting high genomic complexity. The multi-target approach achieved a substantially higher detection rate of CNS infiltration (90%) than tracking a single variant in gene MYD88 (46%). CSF clearly surpasses plasma if applying a routine (non-ultrasensitive) NGS approach and allows for more reliable evidence of CNS involvement than conventional flow cytometry (91% vs. 21%, p < 0.001). Parallel analysis of tumor DNA in both cell-free and cellular DNA from CSF makes the probability of primary or secondary CNS malignancy detection even higher. CONCLUSIONS: Our prospective, tissue-agnostic approach highlights the feasibility of ctDNA sequencing by a commonplace and affordable method, offering higher sensitivity to detect CNS infiltration with lymphoma than standard cell-analyzing techniques. We accentuate the benefit of a multi-target NGS approach and adequate CSF sampling to obtain satisfactory diagnostic yield. Less invasive liquid biopsy testing by comprehensive NGS complements standard procedures in the diagnostics and management of CNSL patients, especially when encountering limitations.
- Publication type
- Journal Article MeSH
STUDY QUESTION: Can oocyte functionality be assessed by observing changes in their intracytoplasmic lipid droplets (LDs) profiles? SUMMARY ANSWER: Lipid profile changes can reliably be detected in human oocytes; lipid changes are linked with maternal age and impaired developmental competence in a mouse model. WHAT IS KNOWN ALREADY: In all cellular components, lipid damage is the earliest manifestation of oxidative stress (OS), which leads to a cascade of negative consequences for organelles and DNA. Lipid damage is marked by the accumulation of LDs. We hypothesized that impaired oocyte functionality resulting from aging and associated OS could be assessed by changes in LDs profile, hereafter called lipid fingerprint (LF). STUDY DESIGN, SIZE, DURATION: To investigate if it is possible to detect differences in oocyte LF, we subjected human GV-stage oocytes to spectroscopic examinations. For this, a total of 48 oocytes derived from 26 young healthy women (under 33 years of age) with no history of infertility, enrolled in an oocyte donation program, were analyzed. Furthermore, 30 GV human oocytes from 12 women were analyzed by transmission electron microscopy (TEM). To evaluate the effect of oocytes' lipid profile changes on embryo development, a total of 52 C57BL/6 wild-type mice and 125 Gnpat+/- mice were also used. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human oocytes were assessed by label-free cell imaging via coherent anti-Stokes Raman spectroscopy (CARS). Further confirmation of LF changes was conducted using spontaneous Raman followed by Fourier transform infrared (FTIR) spectroscopies and TEM. Additionally, to evaluate whether LF changes are associated with developmental competence, mouse oocytes and blastocysts were evaluated using TEM and the lipid dyes BODIPY and Nile Red. Mouse embryonic exosomes were evaluated using flow cytometry, FTIR and FT-Raman spectroscopies. MAIN RESULTS AND THE ROLE OF CHANCE: Here we demonstrated progressive changes in the LF of oocytes associated with the woman's age consisting of increased LDs size, area, and number. LF variations in oocytes were detectable also within individual donors. This finding makes LF assessment a promising tool to grade oocytes of the same patient, based on their quality. We next demonstrated age-associated changes in oocytes reflected by lipid peroxidation and composition changes; the accumulation of carotenoids; and alterations of structural properties of lipid bilayers. Finally, using a mouse model, we showed that LF changes in oocytes are negatively associated with the secretion of embryonic exosomes prior to implantation. Deficient exosome secretion disrupts communication between the embryo and the uterus and thus may explain recurrent implantation failures in advanced-age patients. LIMITATIONS, REASONS FOR CAUTION: Due to differences in lipid content between different species' oocytes, the developmental impact of lipid oxidation and consequent LF changes may differ across mammalian oocytes. WIDER IMPLICATIONS OF THE FINDINGS: Our findings open the possibility to develop an innovative tool for oocyte assessment and highlight likely functional connections between oocyte LDs and embryonic exosome secretion. By recognizing the role of oocyte LF in shaping the embryo's ability to implant, our original work points to future directions of research relevant to developmental biology and reproductive medicine. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by National Science Centre of Poland, Grants: 2021/41/B/NZ3/03507 and 2019/35/B/NZ4/03547 (to G.E.P.); 2022/44/C/NZ4/00076 (to M.F.H.) and 2019/35/N/NZ3/03213 (to Ł.G.). M.F.H. is a National Agency for Academic Exchange (NAWA) fellow (GA ULM/2019/1/00097/U/00001). K.F. is a Diamond Grant fellow (Ministry of Education and Science GA 0175/DIA/2019/28). The open-access publication of this article was funded by the Priority Research Area BioS under the program "Excellence Initiative - Research University" at the Jagiellonian University in Krakow. The authors declare no competing interest. TRIAL REGISTRATION NUMBER: N/A.
- MeSH
- Adult MeSH
- Embryonic Development physiology MeSH
- Humans MeSH
- Lipid Droplets metabolism MeSH
- Lipid Metabolism MeSH
- Mice, Inbred C57BL * MeSH
- Mice MeSH
- Oocytes * metabolism MeSH
- Oxidative Stress MeSH
- Spectrum Analysis, Raman MeSH
- Aging metabolism MeSH
- Microscopy, Electron, Transmission MeSH
- Maternal Age MeSH
- Animals MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Prostate cancer (PC) is an aggressive cancer that can progress rapidly and eventually become castrate-resistant prostate cancer (CRPC). Stage IV metastatic castrate-resistant prostate cancer (mCRPC) is an incurable late-stage cancer type with a low 5-year overall survival rate. Targeted therapeutics such as antibody-drug conjugates (ADCs) based on high-affinity monoclonal antibodies and potent drugs conjugated via smart linkers are being developed for PC management. Conjugating further with in vitro or in vivo imaging agents, ADCs can be used as antibody-theranostic conjugates (ATCs) for diagnostic and image-guided drug delivery. In this study, we have developed a novel ATC for PSMA (+) PC therapy utilizing (a) anti-PSMA 5D3 mAb, (b) Aurora A kinase inhibitor, MLN8237, and (c) for the first time using tetrazine (Tz) and trans-cyclooctene (TCO) click chemistry-based conjugation linker (CC linker) in ADC development. The resulting 5D3(CC-MLN8237)3.2 was labeled with suitable fluorophores for in vitro and in vivo imaging. The products were characterized by SDS-PAGE, MALDI-TOF, and DLS and evaluated in vitro by optical imaging, flow cytometry, and WST-8 assay for cytotoxicity in PSMA (+/-) cells. Therapeutic efficacy was determined in human PC xenograft mouse models following a designed treatment schedule. After the treatment study animals were euthanized, and toxicological studies, complete blood count (CBC), blood clinical chemistry analysis, and H&E staining of vital organs were conducted to determine side effects and systemic toxicities. The IC50 values of 5D3(CC-MLN8237)3.2-AF488 in PSMA (+) PC3-PIP and PMSA (-) PC3-Flu cells are 8.17 nM and 161.9 nM, respectively. Pure MLN8237 shows 736.9 nM and 873.4 nM IC50 values for PC3-PIP and PC3-Flu cells, respectively. In vivo study in human xenograft mouse models confirmed high therapeutic efficacy of 5D3(CC-MLN8237)3.2-CF750 with significant control of PSMA (+) tumor growth with minimal systemic toxicity in the treated group compared to PSMA (-) treated and untreated groups. Approximately 70% of PSMA (+) PC3-PIP tumors did not exceed the threshold of the tumor size in the surrogate Kaplan-Meyer analysis. The novel ATC successfully controlled the growth of PSMA (+) tumors in preclinical settings with minimal systemic toxicities. The therapeutic efficacy and favorable safety profile of novel 5D3(CC-MLN8237)3.2 ATC demonstrates their potential use as a theranostic against aggressive PC.
- Publication type
- Journal Article MeSH
Introduction: Pediatric brain tumours (PBT) are one of the most common malignancies during childhood, with variable severity according to the location and histological type. Certain types of gliomas, such a glioblastoma and diffuse intrinsic pontine glioma (DIPG), have a much higher mortality than ependymoma and medulloblastoma. Early detection of PBT is essential for diagnosis and therapeutic interventions. Liquid biopsies have been demonstrated using cerebrospinal fluid (CSF), mostly restricted to cell free DNA, which display limitations of quantity and integrity. In this pilot study, we sought to demonstrate the detectability and robustness of cell free histones in the CSF. Methods: We collected CSF samples from a pilot cohort of 8 children with brain tumours including DIPG, medulloblastoma, glioblastoma, ependymoma and others. As controls, we collected CSF samples from nine children with unrelated blood malignancies and without brain tumours. We applied a multichannel flow imaging approach on ImageStream(X) to image indiviual histone or histone complexes on different channels. Results: Single histones (H2A, macroH2A1.1, macroH2A1.2 H2B, H3, H4 and histone H3 bearing the H3K27M mutation), and histone complexes are specifically detectable in the CSF of PBT patients. H2A and its variants macroH2A1.1/macroH2A1/2 displayed the strongest signal and abundance, together with disease associated H3K27M. In contrast, mostly H4 is detectable in the CSF of pediatric patients with blood malignancies. Discussion: In conclusion, free histones and histone complexes are detectable with a strong signal in the CSF of children affected by brain tumours, using ImageStream(X) technology and may provide additive diagnostic and predictive information.
- Publication type
- Journal Article MeSH
Prostate cancer (PCa) tops the list of cancer-related deaths in men worldwide. Prostate-specific membrane antigen (PSMA) is currently the most prominent PCa biomarker, as its expression levels are robustly enhanced in advanced stages of PCa. As such, PSMA targeting is highly efficient in PCa imaging as well as therapy. For the latter, PSMA-positive tumors can be targeted directly by using small molecules or macromolecules with cytotoxic payloads or indirectly by engaging the immune system of the host. Here we describe the engineering, expression, purification, and biological characterization of bispecific T-cell engagers (BiTEs) that enable targeting PSMA-positive tumor cells by host T lymphocytes. To this end, we designed the 5D3-αCD3 BiTE as a fusion of single-chain fragments of PSMA-specific 5D3 and anti-CD3 antibodies. Detailed characterization of BiTE was performed by a combination of size-exclusion chromatography, differential scanning fluorimetry, and flow cytometry. Expressed in insect cells, BiTE was purified in monodisperse form and retained thermal stability of both functional parts and nanomolar affinity to respective antigens. 5D3-αCD3's efficiency and specificity were further evaluated in vitro using PCa-derived cell lines together with peripheral blood mononuclear cells isolated from human blood. Our data revealed that T-cells engaged via 5D3-αCD3 can efficiently eliminate tumor cells already at an 8 pM BiTE concentration in a highly specific manner. Overall, the data presented here demonstrate that the 5D3-αCD3 BiTE is a candidate molecule of high potential for further development of immunotherapeutic modalities for PCa treatment.
- Publication type
- Journal Article MeSH
Live cells act as biological lenses and can be employed as real-world optical components in bio-hybrid systems. Imaging at nanoscale, optical tweezers, lithography and also photonic waveguiding are some of the already proven functionalities, boosted by the advantage that cells are fully biocompatible for intra-body applications. So far, various cell types have been studied for this purpose, such as red blood cells, bacterial cells, stem cells and yeast cells. White Blood Cells (WBCs) play a very important role in the regulation of the human body activities and are usually monitored for assessing its health. WBCs can be considered bio-lenses but, to the best of our knowledge, characterization of their optical properties have not been investigated yet. Here, we report for the first time an accurate study of two model classes of WBCs (i.e., monocytes and lymphocytes) by means of a digital holographic microscope coupled with a microfluidic system, assuming WBCs bio-lens characteristics. Thus, quantitative phase maps for many WBCs have been retrieved in flow-cytometry (FC) by achieving a significant statistical analysis to prove the enhancement in differentiation among sphere-like bio-lenses according to their sizes (i.e., diameter d) exploiting intensity parameters of the modulated light in proximity of the cell optical axis. We show that the measure of the low intensity area (S: Iz
- MeSH
- Holography * methods MeSH
- Humans MeSH
- Lymphocytes MeSH
- Microscopy * methods MeSH
- Monocytes MeSH
- Optics and Photonics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
