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| 2. |
- Baltz, Niklas, et al.
(författare)
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Advancing nasal formulation characterization: Considerations toward a robust and precise method to determine the mass fraction below 10 μm in nasal products
- 2024
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Ingår i: Aerosol Science and Technology. - 0278-6826 .- 1521-7388. ; In Press
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Tidskriftsartikel (refereegranskat)abstract
- The expanding role of the nasal route in drug administration for local and systemic treatments has prompted the need for precise delivery methods to ensure efficacy and patient safety. This study addresses the challenges of evaluating the mass fraction below 10 μm in nasal products, a crucial factor in assessing lung deposition of drugs of nasal formulations. Current regulatory guidelines advocate for this assessment, yet a standardized compendial methodology is lacking. To fill this gap, we comprehensively examined several methods to determine a robust approach for quantifying the mass fraction below 10 μm in nasal products. As model formulation, a commercial nasal product (aqueous solution of sodium cromoglycate) was utilized. The assessment of mass fractions below 10 μm necessitates considerations like general handling, precise assessment of delivered dose, robust recovery, and appropriate impactor size analysis techniques. The choice of impactor and of inlet for size analysis may significantly influence the generated results. In this regard, the study highlights the necessity for careful impactor and inlet selection to ensure accurate measurements. In the course of this, the Kiel Nasal Inlet (KNI) had been designed to optimize nasal product testing, addressing the shortcomings of existing inlets. The KNI performed well across different laboratories and reproducible between impactor types. For the determination of mass fraction below 10 μm, the Fast Screening Impactor is the preferred choice of the authors.
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| 3. |
- Bankell, Elisabeth, et al.
(författare)
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LL-37-induced caspase-independent apoptosis is associated with plasma membrane permeabilization in human osteoblast-like cells
- 2021
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Ingår i: Peptides. - : Elsevier BV. - 0196-9781 .- 1873-5169. ; 135
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Tidskriftsartikel (refereegranskat)abstract
- The host defense peptide LL-37 is active against both gram-positive and gram-negative bacteria, but it has also been shown to reduce human host cell viability. However, the mechanisms behind LL-37-induced human host cell cytotoxicity are not yet fully understood. Here, we assess if LL-37-evoked attenuation of human osteoblast-like MG63 cell viability is associated with apoptosis, and if the underlying mechanism may involve LL-37-induced plasma membrane permeabilization. MG63 cell viability and plasma membrane permeabilization were investigated by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and by measuring lactate dehydrogenase (LDH) release, respectively. Apoptosis was assessed by the terminal deoxynucleotidyl dUTP nick end labeling (TUNEL) assay and Annexin V flow cytometry, and caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage were determined by Western blot. LL-37 (4 and 10 μM) reduced both cell number and cell viability, and these effects were associated with a pro-apoptotic effect demonstrated by positive TUNEL staining and Annexin V flow cytometry. LL-37-induced apoptosis was not coupled to either caspase-3 or PARP cleavage, suggesting that LL-37 causes caspase-independent apoptosis in MG63 cells. Both LL-37 and the well-known plasma membrane permeabilizer Triton X-100 reduced cell viability and stimulated LDH release. Triton X-100-treated cells showed positive TUNEL staining, and the detergent accumulated cells in late apoptosis/necrosis. Similar to LL-37, Triton X-100 caused no PARP cleavage. We conclude that LL-37 promotes caspase-independent apoptosis, and that this effect seems coupled to plasma membrane permeabilization in human MG63 cells.
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| 4. |
- Aidoukovitch, Alexandra, et al.
(författare)
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Antimicrobial peptide LL-37 and its pro-form, hCAP18, in desquamated epithelial cells of human whole saliva
- 2020
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Ingår i: European Journal of Oral Sciences. - : Wiley. - 0909-8836 .- 1600-0722. ; 128:1, s. 1-6
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Tidskriftsartikel (refereegranskat)abstract
- The antimicrobial peptide LL-37 is active against oral bacteria and has been demonstrated to be present in human saliva, but its distribution in different fractions of saliva is not known. LL-37 is formed from its intracellular pro-form, hCAP18, in an extracellular enzymatic reaction catalyzed by proteinase 3 and kallikrein 5. Here, we prepared cell-containing and cell-free fractions of unstimulated human whole saliva by centrifugation after depolymerization of mucins with dithiothreitol, and measured the levels of hCAP18/LL-37 in these fractions using ELISA. Cellular expression of hCAP18/LL-37 was determined by western blotting and immunocytochemistry. The ELISA analyses demonstrated that both cells and cell-free saliva contained hCAP18/LL-37. Western blot analysis of cell-pellet homogenates showed a strong band corresponding to hCAP18 at the correct molecular weight and a weak band corresponding to LL-37. Phase-contrast and light microscopy revealed that the cells consisted of desquamated epithelial cells. These cells expressed cytoplasmic immunoreactivity for hCAP18/LL-37. The peripheral part of the cytoplasm, corresponding to the plasma membrane, was particularly rich in hCAP18/LL-37 immunoreactivity. No immunoreactivity was observed after omission of the primary antibody. We conclude that desquamated epithelial cells of human whole saliva contain antimicrobial hCAP18/LL-37, suggesting that these cells may take part in the innate immune system by harboring and releasing these peptides.
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| 5. |
- Aidoukovitch, Alexandra, et al.
(författare)
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Vitamin D triggers hCAP18/LL-37 production : Implications for LL-37-induced human osteoblast cytotoxicity
- 2024
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Ingår i: Biochemical and Biophysical Research Communications. - 0006-291X. ; 712-713
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Tidskriftsartikel (refereegranskat)abstract
- The human cathelicidin LL-37 shows activity against microorganisms, but it is also cytotoxic to host cells. The CAMP gene codes for the LL-37 precursor hCAP18 which is processed extracellularly to active LL-37. It has previously been shown that vitamin D stimulates CAMP gene activity, but less information is available demonstrating that vitamin D also can increase hCAP18/LL-37 protein production. Here, we show with RT-qPCR that a physiological concentration of vitamin D (50 nM) enhances CAMP mRNA levels by about 170 times in human THP-1 monocyte cells. Stimulation with 50 nM vitamin D increases hCAP18/LL-37 protein contents 3–4 times in THP-1 cell lysates demonstrated by both dot blot analysis and ELISA applying two different hCAP18/LL-37 antibodies. Treatment with the proteasome inhibitor MG132 enhances hCAP18/LL-37 levels, suggesting that turnover of hCAP18/LL-37 protein is regulated by the proteasome. The hCAP18/LL-37 concentration in vitamin D-stimulated THP-1 cells corresponds to 1.04 μM LL-37. Interestingly, synthetic LL-37, at this concentration, reduces viability of human osteoblast-like MG63 cells, whereas the THP-1 cells are less sensitive as demonstrated by the MTT assay. In summary, we show that vitamin D enhances hCAP18/LL-37 production, and that this effect can be of physiological/pathophysiological relevance for LL-37-induced human osteoblast toxicity.
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| 6. |
- Bankell, Elisabeth, et al.
(författare)
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The antimicrobial peptide LL-37 triggers release of apoptosis-inducing factor and shows direct effects on mitochondria
- 2022
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Ingår i: Biochemistry and Biophysics Reports. - : Elsevier BV. - 2405-5808. ; 29
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Tidskriftsartikel (refereegranskat)abstract
- The human antimicrobial peptide LL-37 permeabilizes the plasma membrane of host cells, but LL-37-induced direct effects on mitochondrial membrane permeability and function has not been reported. Here, we demonstrate that LL-37 is rapidly (within 20 min) internalized by human osteoblast-like MG63 cells, and that the peptide co-localizes with MitoTracker arguing for accumulation in mitochondria. Subcellular fractionation and Western blot disclose that stimulation with LL-37 (8 μM) for 2 h triggers release of the mitochondrial protein apoptosis-inducing factor (AIF) to the cytosol, whereas LL-37 causes no release of cytochrome C oxidase subunit IV of the inner mitochondrial membrane, suggesting that LL-37 affects mitochondrial membrane permeability in a specific manner. Next, we investigated release of AIF and cytochrome C from isolated mitochondria by measuring immunoreactivity by dot blot. The media of mitochondria treated with LL-37 (8 μM) for 2 h contained 50% more AIF and three times more cytochrome C than that of control mitochondria, showing that LL-37 promotes release of both AIF and cytochrome C. Moreover, in vesicles reflecting mitochondrial membrane lipid composition, LL-37 stimulates membrane permeabilization and release of tracer molecules. We conclude that LL-37 is rapidly internalized by MG63 cells and accumulates in mitochondria, and that the peptide triggers release of pro-apoptotic AIF and directly affects mitochondrial membrane structural properties.
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| 7. |
- Baravdish, George, 1964-, et al.
(författare)
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Brain Tumour Evolution Backwards in Time via Reaction-Diffusion Models and Sobolev Regularisation
- 2024
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Ingår i: Modelling and Computational Approaches for Multi-scale Phenomena in Cancer Research. - London : World Scientific. - 9781800614376
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Bokkapitel (refereegranskat)abstract
- Evolution of brain tumours backwards in time is studied using well-established brain tumour growth models being semilinear parabolic equations of reaction-diffusion type. To run the models backwards, the tumour cell density data at a fixed (final) time is used, rendering an inverse ill-posed problem. This problem is recast as the minimisation of a cost functional matching the data against the solution at a final time of a forward parabolic model having the initial cell density as a control function. Regularisation is incorporated via penalising terms involving Sobolev norms. Mathematical properties of the semilinear parabolic equations are shown in Sobolev-Bochner spaces including uniqueness of a solution to the inverse problem. Differentiability of the control-to-state map is established rendering a sensitivity problem. The derivative of the cost functional is calculated and the adjoint state is derived via the Lagrange formalism. A non-linear conjugate gradient method (NCG) is presented for the minimisation. Numerical realisation of the minimisation on the BraTS'20 dataset is included using a standard finite difference discretisation of the space and time derivatives, showing that tumour evolution backwards in time can be accomplished and that the initial tumour cell density can be reconstructed. Comparison is done with a non-linear Landweber method.
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| 8. |
- Baravdish, George, 1964-, et al.
(författare)
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Generalizations of p-Laplace operator for image enhancement : Part 2
- 2020
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Ingår i: Communications on Pure and Applied Analysis. - Springfield, MO, United States : American Institute of Mathematical Sciences. - 1534-0392 .- 1553-5258. ; 19:7, s. 3477-3500
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Tidskriftsartikel (refereegranskat)abstract
- We have in a previous study introduced a novel elliptic operator Delta((p,q))u = vertical bar del u vertical bar(q) Delta(1)u + (p - 1) vertical bar del u vertical bar(p-2)Delta(infinity) u, p >= 1, q >= 0, as a generalization of the p-Laplace operator. In this paper, we establish the well-posedness of the parabolic equation u(t) =vertical bar del u vertical bar(1-q) Delta((1+q,q)), where q = q(vertical bar del u vertical bar) is continuous and has range in [0, 1], in the framework of viscosity solutions. We prove the consistency and convergence of the numerical scheme of finite differences of this parabolic equation. Numerical simulations shows the advantage of this operator applied to image enhancement.
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| 9. |
- Baravdish, George, et al.
(författare)
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Identifying a response parameter in a model of brain tumour evolution under therapy
- 2023
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Ingår i: IMA Journal of Applied Mathematics. - : Oxford University Press. - 0272-4960 .- 1464-3634. ; 88:2, s. 378-404
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Tidskriftsartikel (refereegranskat)abstract
- A nonlinear conjugate gradient method is derived for the inverse problem of identifying a treatment parameter in a nonlinear model of reaction-diffusion type corresponding to the evolution of brain tumours under therapy. The treatment parameter is reconstructed from additional information about the tumour taken at a fixed instance of time. Well-posedness of the direct problems used in the iterative method is outlined as well as uniqueness of a solution to the inverse problem. Moreover, the parameter identification is recasted as the minimization of a Tikhonov type functional and the existence of a minimizer to this functional is shown. Finite-difference discretization of the space and time derivatives are employed for the numerical implementation. Numerical simulations on full 3D brain data are included showing that information about a spacewise-dependent treatment parameter can be recovered in a stable way.
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| 10. |
- Baravdish, George, 1964-, et al.
(författare)
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On a new singular and degenerate extension of the p-Laplace operator
- 2024
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Ingår i: Nonlinear Analysis. - : PERGAMON-ELSEVIER SCIENCE LTD. - 0362-546X .- 1873-5215. ; 244
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Tidskriftsartikel (refereegranskat)abstract
- We study a novel degenerate and singular elliptic operator Δ˜(τ,χ) defined by Δ˜(τ,χ)u=τ(x,Du)(|Du|Δ1u+χ(x,Du)Δ∞u), where the singular weights τ(x,s)>0 and χ(x,s)≥0 are continuous functions on Ω×Rn∖{0}. The operator Δ˜(τ,χ) is an extension of Δ(p,q)u=|Du|qΔ1u+(p−1)|Du|p−2Δ∞u,p≥1,q≥0, introduced by the authors in Baravdishet al. (2020), which in turn is an extension of the p-Laplace operator Δp. We establish the well-posedness of the Neumann boundary value problem for the parabolic equation ut=Δ˜(τ,χ)u in the framework of viscosity solutions. For the solution u, the weight χ controls the evolution along the tangential and the normal directions, respectively, on the level surface of u. The weight τ controls the total speed of the evolution of u. We also prove the consistency and the convergence of the numerical scheme for the finite differences method of the parabolic equation above. Numerical simulations show that our novel nonlinear operator Δ˜(τ,χ) gives better results than both the Perona–Malik (Perona and Malik, 1990) and total variation (TV) methods (Chan and Shen, 2005) when applied to image enhancement.
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