Lysosomal sequestration of weak base drugs has been identified as one of the stress-related mechanisms that trigger in vitro lysosomal biogenesis controlled by transcription factor EB (TFEB). Whether such mechanism can induce lysosomal biogenesis in vivo is unknown. In this study, we addressed the question whether prolonged treatment with sunitinib (SUN) in patients with advanced renal cell carcinoma (n = 22) and with imatinib (IM) in those with gastrointestinal stromal tumor (n = 6) could induce lysosomal biogenesis in leukocytes. Lysosomal biogenesis was monitored using immunoblotting of three lysosomal membrane proteins: lysosome-associated membrane proteins 1 and 2 (LAMP1 and LAMP2) and vacuolar H+-ATPase, B2 subunit (ATP6V1B2). Present results indicate that prolonged treatment with SUN affects LAMP1 and LAMP2 expression only marginally in most patients. In contrast, changes in ATP6V1B2 expression were marked and resembled irregular oscillations. Very similar changes in the expression of lysosomal membrane proteins were also found in IM-treated patients. Conclusion: prolonged treatment of cancer patients with SUN and IM did not induce leucocyte lysosomal biogenesis but dramatically affected expression of ATP6V1B2.
- Klíčová slova
- B2 subunit, Imatinib, LAMP1, LAMP2, Lysosmal membrane proteins, Sunitinib, Vacuolar H+-ATPase,
- MeSH
- gastrointestinální stromální tumory farmakoterapie metabolismus MeSH
- imatinib mesylát terapeutické užití MeSH
- inhibitory proteinkinas terapeutické užití MeSH
- karcinom z renálních buněk farmakoterapie metabolismus MeSH
- leukocyty metabolismus MeSH
- lidé MeSH
- lyzozomy metabolismus MeSH
- membránové glykoproteiny asociované s lyzozomy metabolismus MeSH
- sunitinib terapeutické užití MeSH
- transkripční faktory BHLH-Zip metabolismus MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- imatinib mesylát MeSH
- inhibitory proteinkinas MeSH
- membránové glykoproteiny asociované s lyzozomy MeSH
- sunitinib MeSH
- transkripční faktory BHLH-Zip MeSH
PURPOSE: In multiple oncological settings, expression of the coinhibitory ligand PD-L1 by malignant cells and tumor infiltration by immune cells expressing coinhibitory receptors such as PD-1, CTLA4, LAG-3, or TIM-3 conveys prognostic or predictive information. Conversely, the impact of these features of the tumor microenvironment on disease outcome among high-grade serous carcinoma (HGSC) patients remains controversial. EXPERIMENTAL DESIGN: We harnessed a retrospective cohort of 80 chemotherapy-naïve HGSC patients to investigate PD-L1 expression and tumor infiltration by CD8+ T cells, CD20+ B cells, DC-LAMP+ dendritic cells as well as by PD-1+, CTLA4+, LAG-3+, and TIM-3+ cells in relation with prognosis and function orientation of the tumor microenvironment. IHC data were complemented with transcriptomic and functional studies on a second prospective cohort of freshly resected HGSC samples. In silico analysis of publicly available RNA expression data from 308 HGSC samples was used as a confirmatory approach. RESULTS: High levels of PD-L1 and high densities of PD-1+ cells in the microenvironment of HGSCs were strongly associated with an immune contexture characterized by a robust TH1 polarization and cytotoxic orientation that enabled superior clinical benefits. Moreover, PD-1+TIM-3+CD8+ T cells presented all features of functional exhaustion and correlated with poor disease outcome. However, although PD-L1 levels and tumor infiltration by TIM-3+ cells improved patient stratification based on the intratumoral abundance of CD8+ T cells, the amount of PD-1+ cells failed to do so. CONCLUSIONS: Our data indicate that PD-L1 and TIM-3 constitute prognostically relevant biomarkers of active and suppressed immune responses against HGSC, respectively.
- MeSH
- antigen CTLA-4 imunologie metabolismus MeSH
- antigeny CD274 imunologie metabolismus MeSH
- buněčný receptor 2 viru hepatitidy A imunologie metabolismus MeSH
- CD8-pozitivní T-lymfocyty imunologie MeSH
- dospělí MeSH
- epiteliální ovariální karcinom imunologie metabolismus patologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- membránové glykoproteiny asociované s lyzozomy imunologie metabolismus MeSH
- míra přežití MeSH
- nádorové biomarkery imunologie metabolismus MeSH
- nádorové proteiny imunologie metabolismus MeSH
- prognóza MeSH
- regulace genové exprese u nádorů * MeSH
- retrospektivní studie MeSH
- senioři MeSH
- serózní cystadenokarcinom imunologie metabolismus patologie MeSH
- tumor infiltrující lymfocyty imunologie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- Názvy látek
- antigen CTLA-4 MeSH
- antigeny CD274 MeSH
- buněčný receptor 2 viru hepatitidy A MeSH
- CD274 protein, human MeSH Prohlížeč
- CTLA4 protein, human MeSH Prohlížeč
- HAVCR2 protein, human MeSH Prohlížeč
- LAMP3 protein, human MeSH Prohlížeč
- membránové glykoproteiny asociované s lyzozomy MeSH
- nádorové biomarkery MeSH
- nádorové proteiny MeSH
Novel synthetic approaches for the development of multimodal imaging agents with high chemical stability are demonstrated. The magnetic cores are based on La0.63Sr0.37MnO3 manganite prepared as individual grains using a flux method followed by additional thermal treatment in a protective silica shell allowing to enhance their magnetic properties. The cores are then isolated and covered de novo with a hybrid silica layer formed through the hydrolysis and polycondensation of tetraethoxysilane and a fluorescent silane synthesized from rhodamine, piperazine spacer, and 3-iodopropyltrimethoxysilane. The aminoalkyltrialkoxysilanes are strictly avoided and the resulting particles are hydrolytically stable and do not release dye. The high colloidal stability of the material and the long durability of the fluorescence are reinforced by an additional silica layer on the surface of the particles. Structural and magnetic studies of the products using XRD, TEM, and SQUID magnetometry confirm the importance of the thermal treatment and demonstrate that no mechanical treatment is required for the flux-synthesized manganite. Detailed cell viability tests show negligible or very low toxicity at concentrations at which excellent labeling is achieved. Predominant localization of nanoparticles in lysosomes is confirmed by immunofluorescence staining. Relaxometric and biological studies suggest that the functionalized nanoparticles are suitable for imaging applications.
- Klíčová slova
- Cell labeling, Dual probes, MRI, Magnetic nanoparticles, Manganites, Molten salt synthesis, Silica coating,
- MeSH
- fibroblasty cytologie metabolismus MeSH
- fluorescence MeSH
- fluorescenční protilátková technika MeSH
- HeLa buňky MeSH
- Jurkat buňky MeSH
- kultivované buňky MeSH
- kůže cytologie metabolismus MeSH
- lanthan chemie MeSH
- lidé MeSH
- magnetická rezonanční tomografie MeSH
- magnetické nanočástice chemie MeSH
- membránové glykoproteiny asociované s lyzozomy imunologie metabolismus MeSH
- monoklonální protilátky imunologie MeSH
- oxid křemičitý chemie MeSH
- povrchové vlastnosti MeSH
- průtoková cytometrie MeSH
- silany chemie MeSH
- sloučeniny manganu chemie MeSH
- stroncium chemie MeSH
- transmisní elektronová mikroskopie MeSH
- velikost částic MeSH
- viabilita buněk MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- LAMP1 protein, human MeSH Prohlížeč
- lanthan MeSH
- magnetické nanočástice MeSH
- manganite MeSH Prohlížeč
- membránové glykoproteiny asociované s lyzozomy MeSH
- monoklonální protilátky MeSH
- oxid křemičitý MeSH
- silany MeSH
- sloučeniny manganu MeSH
- stroncium MeSH
- tetraethoxysilane MeSH Prohlížeč
NK cells represent a potential tool for adoptive immunotherapy against tumors. Membrane-bound Hsp70 acts as a tumor-specific marker enhancing NK cell activity. Using flow cytometry the effect of in vitro stimulation with IL-2 or IL-15 alone or in combination with Hsp70-derived 14-mer peptide (TKD) on cell surface expression of NK activatory receptors (CD16, NKG2D, NKG2C, NKp46, NKp44, NKp30, KIR2DL4, DNAM-1, and LAMP1) and NK inhibitory receptors (NKG2A, KIR2DL2/L3, LIR1/ILT-2, and NKR-P1A) in healthy individuals was studied. Results were expressed as the percentage of receptor expressing cells and the amount of receptor expressed by CD3(-)CD56(+) cellular population. CD94, NKG2D, NKp44, NKp30, KIR2DL4, DNAM-1, LAMP1, NKG2A, and NKR-P1A were upregulated after the stimulation with IL-2 or IL-15 alone or in combination with TKD. KIR2DL2/L3 was upregulated only by IL-15 and IL-15/TKD. Concurrently, an increase in a number of NK cells positive for CD94, NKp44, NKp30, KIR2DL4, and LAMP1 was observed. IL-15 and IL-15/TKD caused also cell number rise positive for KIR2DL2/L3 and NKR-P1A. Cell number positive for NKG2C and NKG2A was increased only by IL-2 and IL-2/TKD. The diverse effect of IL-2 or IL-15 w or w/o TKD on cell surface expression was observed in CD16, NKp46, and LIR1/ILT-2.
- MeSH
- buňky NK účinky léků metabolismus MeSH
- diferenciační antigeny T-lymfocytů metabolismus MeSH
- interleukin-15 farmakologie MeSH
- interleukin-2 farmakologie MeSH
- kultivované buňky MeSH
- lektinové receptory NK-buněk - podrodina B metabolismus MeSH
- lektinové receptory NK-buněk - podrodina C metabolismus MeSH
- lektinové receptory NK-buněk - podrodina K metabolismus MeSH
- leukocyty mononukleární účinky léků metabolismus MeSH
- lidé MeSH
- membránové glykoproteiny asociované s lyzozomy metabolismus MeSH
- peptidy chemie farmakologie MeSH
- proteiny tepelného šoku HSP70 chemie MeSH
- receptor 1 spouštějící přirozenou cytotoxicitu metabolismus MeSH
- receptor 2 spouštějící přirozenou cytotoxicitu metabolismus MeSH
- receptor 3 spouštějící přirozenou cytotoxicitu metabolismus MeSH
- receptory IgG metabolismus MeSH
- receptory KIR2DL2 MeSH
- receptory KIR2DL4 metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- diferenciační antigeny T-lymfocytů MeSH
- interleukin-15 MeSH
- interleukin-2 MeSH
- KIR2DL2 protein, human MeSH Prohlížeč
- KIR2DL4 protein, human MeSH Prohlížeč
- KLRB1 protein, human MeSH Prohlížeč
- KLRC2 protein, human MeSH Prohlížeč
- KLRK1 protein, human MeSH Prohlížeč
- LAMP1 protein, human MeSH Prohlížeč
- lektinové receptory NK-buněk - podrodina B MeSH
- lektinové receptory NK-buněk - podrodina C MeSH
- lektinové receptory NK-buněk - podrodina K MeSH
- membránové glykoproteiny asociované s lyzozomy MeSH
- NCR1 protein, human MeSH Prohlížeč
- NCR2 protein, human MeSH Prohlížeč
- NCR3 protein, human MeSH Prohlížeč
- peptidy MeSH
- proteiny tepelného šoku HSP70 MeSH
- receptor 1 spouštějící přirozenou cytotoxicitu MeSH
- receptor 2 spouštějící přirozenou cytotoxicitu MeSH
- receptor 3 spouštějící přirozenou cytotoxicitu MeSH
- receptory IgG MeSH
- receptory KIR2DL2 MeSH
- receptory KIR2DL4 MeSH
- T Lineage-Specific Activation Antigen 1 MeSH
AIMS: To identify reliable criteria with which to improve the diagnosis of lysosomal acid lipase (LAL) deficiency of the cholesterol ester storage disease (CESD) type in liver biopsies. METHODS AND RESULTS: We analysed a series of 16 liver biopsies of LAL deficiency of the CESD type confirmed by enzyme testing and DNA sequencing. The biopsy appearances were compared with those of biopsies of other causes of fatty liver. A predominantly microvesicular steatosis in CESD patients could not be reliably distinguished from other causes of fatty liver with cytosolic lipid accumulation in fixed paraffin-embedded tissues routinely stained with haematoxylin and eosin. The presence of luminal (cathepsin D) and membrane lysosomal markers [lysosomal-associated membrane protein (LAMP)1, LAMP2, and lysosomal integral membrane protein 2] around the lipid vacuoles facilitated the diagnosis of CESD in fixed paraffin-embedded material. Additional diagnostic clues included autofluorescent detection of ceroid induction in storage macrophages and the absence of lipopigment in hepatocytes. Stored liquid crystals of cholesteryl esters, which are associated with Maltese cross-type birefringence, were best appreciated in unfixed biopsy samples. CONCLUSIONS: The pathological diagnosis of CESD requires a high index of suspicion, and can be rapidly and effectively appreciated at the light microscopy level, even in routine fixed paraffin-embedded liver samples with immuohistochemical staining for lysosomal markers.
- MeSH
- diferenciální diagnóza MeSH
- dítě MeSH
- játra metabolismus patologie MeSH
- kathepsin D metabolismus MeSH
- kohortové studie MeSH
- kojenec MeSH
- lidé MeSH
- membránové glykoproteiny asociované s lyzozomy metabolismus MeSH
- membránový protein 2 asociovaný s lyzozomy MeSH
- metabolismus lipidů MeSH
- nealkoholová steatóza jater MeSH
- nemoc ze střádání esterů cholesterolu diagnóza metabolismus patologie MeSH
- předškolní dítě MeSH
- Wolmanova nemoc diagnóza metabolismus patologie MeSH
- ztučnělá játra diagnóza metabolismus patologie MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- předškolní dítě MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- CTSD protein, human MeSH Prohlížeč
- kathepsin D MeSH
- LAMP1 protein, human MeSH Prohlížeč
- LAMP2 protein, human MeSH Prohlížeč
- membránové glykoproteiny asociované s lyzozomy MeSH
- membránový protein 2 asociovaný s lyzozomy MeSH
Danon disease (DD) is a monogenic X-linked disorder characterized by cardiomyopathy, skeletal myopathy and variable degrees of intellectual disability. DD develops due to mutations in the gene encoding lysosomal-associated membrane protein 2 (LAMP2). We report on a family exhibiting the clinical phenotype comprising of hypertrophic cardiomyopathy and ventricular pre-excitation, myopia and mild myopathy in two male patients and cardiomyopathy and myopia in a female patient. The diagnosis of DD in this family was based on the assessment of the clinical phenotypes and the absence of LAMP2 in skeletal and/or cardiac muscle biopsy specimens. Sequence analysis of the LAMP2 gene and its mRNA revealed a novel LAMP2 mutation (c.940delG) in all three patients. Approximately 25% of the female patient's cardiomyocytes were LAMP2 positive apparently due to the unfavorable skewing of X chromosome inactivation. We further performed qualitative LAMP2 immunohistochemistry on peripheral white blood cells using the smear technique and revealed the absence of LAMP2 in the male patients. LAMP2 expression was further assessed in granulocytes, CD4+ and CD8+ T lymphocytes, CD20+ B lymphocytes, CD14+ monocytes and CD56+ natural killer cells by quantitative polychromatic flow cytometry. Whereas the male DD patients lacked LAMP2 in all WBC populations, the female patient expressed LAMP2 in 15.1% and 12.8% of monocytes and granulocytes, respectively. LAMP2 expression ratiometrics of highly vs. weakly expressing WBC populations discriminated the DD patients from the healthy controls. WBCs are thus suitable for initial LAMP2 expression testing when DD is a differential diagnostic option. Moreover, flow cytometry represents a quantitative method to assess the skewing of LAMP2 expression in female heterozygotes. Because LAMP2 is a major protein constituent of the membranes of a number of lysosome-related organelles, we also tested the exocytic capacity of the lytic granules from CD8+ T lymphocytes in the patient samples. The degranulation triggered by a specific stimulus (anti-CD3 antibody) was normal. Therefore, this process can be considered LAMP2 independent in human T cells. The c.940delG mutation results in a putatively truncated protein (p.A314QfsX32), which lacks the transmembrane domain and the cytosolic tail of the wild-type LAMP2. We tested whether this variant becomes exocytosed because of a failure in targeting to late endosomes/lysosomes. Western blotting of cardiac muscle, WBCs and cultured skin fibroblasts (and their culture media) showed no intra- or extracellular truncated LAMP2. By comparing the expression pattern and intracellular targeting in cultured skin fibroblasts of normal LAMP2 isoforms (A, B and C) tagged with green fluorescent protein (GFP) and the A314Qfs32-GFP fusion, we found that the A314Qfs32-GFP protein is not even expressed. These observations suggest that the truncated protein is unstable and is co-translationally or early post-translationally degraded.
- MeSH
- CD4-pozitivní T-lymfocyty metabolismus MeSH
- CD8-pozitivní T-lymfocyty metabolismus patologie MeSH
- dospělí MeSH
- fibroblasty metabolismus MeSH
- glykogenóza typu IIb krev diagnóza genetika patologie MeSH
- heterozygot MeSH
- hypertrofická kardiomyopatie genetika patologie MeSH
- leukocyty MeSH
- lidé MeSH
- membránové glykoproteiny asociované s lyzozomy nedostatek genetika metabolismus MeSH
- membránový protein 2 asociovaný s lyzozomy MeSH
- mutace * MeSH
- myokard metabolismus MeSH
- posttranslační úpravy proteinů MeSH
- průtoková cytometrie metody MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- práce podpořená grantem MeSH
- Názvy látek
- LAMP2 protein, human MeSH Prohlížeč
- membránové glykoproteiny asociované s lyzozomy MeSH
- membránový protein 2 asociovaný s lyzozomy MeSH
