Extraction and Fractionation of Bioactives from Dipsacus fullonum L. Leaves and Evaluation of Their Anti-Borrelia Activity

Lyme disease (LD) is a tick-borne bacterial disease that is caused by Borrelia burgdorferi. Although acute LD is treated with antibiotics, it can develop into relapsing chronic form caused by latent forms of B. burgdorferi. This leads to the search for phytochemicals against resistant LD. Therefore, this study aimed to evaluate the activity of Dipsacus fullonum L. leaves extract (DE) and its fractions against stationary phase B. burgdorferi in vitro. DE showed high activity against stationary phase B. burgdorferi (residual viability 19.8 ± 4.7%); however, it exhibited a noticeable cytotoxicity on NIH cells (viability 20.2 ± 5.2%). The iridoid-glycoside fraction showed a remarkable anti-Borrelia effect and reduced cytotoxicity. The iridoid-glycoside fraction was, therefore, further purified and showed to contain two main bioactives—sylvestrosides III and IV, that showed a considerable anti-Borrelia activity being the least toxic to murine fibroblast NIH/3T3 cells. Moreover, the concentration of sylvestrosides was about 15% of DE, endorsing the feasibility of purification of the compounds from D. fullonum L. leaves.

Peroxiredoxin 6 Applied after Exposure Attenuates Damaging Effects of X-ray Radiation in 3T3 Mouse Fibroblasts

Although many different classes of antioxidants have been evaluated as radioprotectors, none of them are in widespread clinical use because of their low efficiency. The goal of our study was to evaluate the potential of the antioxidant protein peroxiredoxin 6 (Prdx6) to increase the radioresistance of 3T3 fibroblasts when Prdx6 was applied after exposure to 6 Gy X-ray. In the present study, we analyzed the mRNA expression profiles of genes associated with proliferation, apoptosis, cellular stress, senescence, and the production of corresponding proteins from biological samples after exposure of 3T3 cells to X-ray radiation and application of Prdx6. Our results suggested that Prdx6 treatment normalized p53 and NF-κB/p65 expression, p21 levels, DNA repair-associated genes (XRCC4, XRCC5, H2AX, Apex1), TLR expression, cytokine production (TNF-α and IL-6), and apoptosis, as evidenced by decreased caspase 3 level in irradiated 3T3 cells. In addition, Prdx6 treatment reduced senescence, as evidenced by the decreased percentage of SA-β-Gal positive cells in cultured 3T3 fibroblasts. Importantly, the activity of the NRF2 gene, an important regulator of the antioxidant cellular machinery, was completely suppressed by irradiation but was restored by post-irradiation Prdx6 treatment. These data support the radioprotective therapeutic efficacy of Prdx6.

Cell proliferative properties of Forcespinning® nopal composite nanofibers

In this study, Forcespinning® was used to produce nanofibers composed of Opuntia cochenillifera, “nopal,” mucilage (N) extract, chitosan (CH), and pullulan (PL) (N/CH/PL). These nopal-incorporating nanofibers were examined for their ability to sustain adhesion and proliferation of mouse embryonic fibroblast (NIH 3T3) cells. After a 6-day incubation period, N/CH/PL nanofibers displayed robust cell proliferation, with continued cell growth after an extended incubation period of 14 days. These results demonstrate that natural bioactive compounds can be combined with biodegradable polymers to provide an enhanced environment for cell growth, suggesting potential natural active ingredients as alternatives in wound dressings.

Augmenting Efficacy of T-Cell Bispecific Antibodies in AML through a Tumor Stroma-Targeted 4-1BB Agonist

Bispecific antibodies represent a promising treatment option for acute myeloid leukemia (AML). We have recently described a novel T-cell bispecific antibody (TCB) targeting the intracellular tumor antigen Wilms tumor 1 (WT1) in the context of HLA-A*02 (Augsberger et al. Blood 2021). Based on these findings a multicenter first-in-human clinical trial was initiated in relapse/refractory AML (NCT04580121). Possible immune escape mechanisms against T-cell based immunotherapy are provided by the tumor microenvironment (TME) of the bone marrow by co-inhibition of T cells or stromal cells shielding leukemic cells from immune effector cells. To overcome the immunosuppressive effect of the TME and to enhance T-cell responses, we evaluated the combination of the WT1-TCB with an antibody fusion protein that targets a stromal antigen (Fibroblast-activation protein; FAP) and provides a positive costimulatory signal (4-1BBL) to T cells. FAP is upregulated on cancer-associated fibroblasts after remodulation of the bone marrow niche by leukemic cells, and the FAP specificity of the molecule therefore provides T-cell co-stimulation tightly restricted to the tumor niche. Efficacy of the combination (WT1-TCB + FAP-4-1BBL antibody fusion protein) was evaluated in co-culture assays over 4 days with primary HLA-A*02 + AML cells, healthy donor (HD) T cells and three NIH-3T3 fibroblast cell lines. NIH-3T3 cell lines were genetically modified to express low and high levels of FAP, respectively. Wild-type NIH-3T3 cells were included as control. Additionally, a control (Ctrl)-TCB and a Ctrl-4-1BBL antibody fusion protein recognizing a non-tumor target derived from the human germline repertoire were included. Enhancement of T-cell mediated cytotoxicity by the FAP-4-1BBL antibody fusion protein was evaluated by (1) specific lysis of primary AML cells, (2) upregulation of the T-cell activation markers CD25 and 4-1BB, (3) T-cell expansion calculated as fold change compared to day 0, and (4) Granzyme B-expression which was evaluated by intracellular staining. After 4 days of co-culture, with an E:T ratio of 1:2, we observed a mean specific lysis of 55.1±8.2% (±SEM; n=4) of primary AML cells mediated by HD T cells and WT1-TCB. Notably, this was reduced to 19.4±5.9% (±SEM; n=4) in the presence of NIH-3T3 cells. However, AML cell lysis was restored by the addition of the FAP-4-1BBL antibody fusion protein in the presence of high FAP expressing NIH-3T3 cells (mean specific lysis: 62.8±7.3%; ±SEM; n=4). Concomitantly, the FAP-4-1BBL antibody fusion protein led to increased expression of the activation molecules CD25 (MFI ratio: 22.1±5.3 vs. 10.4±1.3; ±SEM; n=4) and 4-1BB (MFI ratio: 10.4±6.0 vs. 2.1±0.3; ±SEM; n=4) on CD3 + T cells. Furthermore, lysis was accompanied by increased frequencies of granzyme B expressing T cells (45.0±2.5% vs. 16.1±5.3%; n=3). Importantly, the FAP-4-1BBL antibody fusion protein led to improved T-cell proliferation, especially of CD8 + T cells (fold change on day 4 vs day 0: 5.7±2.2 vs. 1.0±0.3; ±SEM; n=4). Overall similar observations were made in the presence of low FAP expressing NIH-3T3 cells. Taken together, we have established an in vitro model system mimicking the immunoprotective bone marrow TME using NIH-3T3 cells resulting in impaired AML cell lysis. Providing additional T-cell co-stimulation by a tumor-stroma targeted 4-1BB agonist, however, restored WT1-TCB-mediated cytotoxicity of primary AML cells in the presence of FAP expressing cell lines. Importantly, the combination overcame the immunosuppressive effect of the NIH-3T3 cells on T cells as further demonstrated by improved T-cell activation and expansion. The tumor-stroma targeted 4-1BB agonist therefore represents a promising combinatorial approach to enhance T-cell activity at the local tumor site and warrants further investigations in an in vivo model system. Disclosures Pulko: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Claus: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Buecklein: Pfizer: Consultancy, Honoraria, Speakers Bureau; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; Miltenyi: Research Funding; BMS/Celgene: Consultancy, Research Funding; Amgen: Consultancy, Honoraria. Umana: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Klein: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Subklewe: Novartis: Consultancy, Research Funding, Speakers Bureau; Klinikum der Universität München: Current Employment; Roche: Research Funding; Seattle Genetics: Consultancy, Research Funding; Pfizer: Consultancy, Speakers Bureau; Janssen: Consultancy; Takeda: Speakers Bureau; MorphoSys: Research Funding; Miltenyi: Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau.

A pre-formulation study of tetracaine loaded in optimized nanostructured lipid carriers

Tetracaine (TTC) is a local anesthetic broadly used for topical and spinal blockade, despite its systemic toxicity. Encapsulation in nanostructured lipid carriers (NLC) may prolong TTC delivery at the site of injection, reducing such toxicity. This work reports the development of NLC loading 4% TTC. Structural properties and encapsulation efficiency (%EE > 63%) guided the selection of three pre-formulations of different lipid composition, through a 23 factorial design of experiments (DOE). DLS and TEM analyses revealed average sizes (193–220 nm), polydispersity (< 0.2), zeta potential |− 21.8 to − 30.1 mV| and spherical shape of the nanoparticles, while FTIR-ATR, NTA, DSC, XRD and SANS provided details on their structure and physicochemical stability over time. Interestingly, one optimized pre-formulation (CP-TRANS/TTC) showed phase-separation after 4 months, as predicted by Raman imaging that detected lack of miscibility between its solid (cetyl palmitate) and liquid (Transcutol) lipids. SANS analyses identified lamellar arrangements inside such nanoparticles, the thickness of the lamellae been decreased by TTC. As a result of this combined approach (DOE and biophysical techniques) two optimized pre-formulations were rationally selected, both with great potential as drug delivery systems, extending the release of the anesthetic (> 48 h) and reducing TTC cytotoxicity against Balb/c 3T3 cells.

Electrochemical Anodic Synthesis and Analysis of TiO2 Nanotubes for Biomedical Applications

Nowadays, titanium and alloy materials are encouraged for biomedical applications. Fabrication of the passive layer over the titanium materials is limited. Typically, a plain titanium sample is not suitable for bioimplant applications because the adhesion of biological elements like blood cells, tissues, and bones is poor. The use of surface-modified titanium resolves this issue. Surface modifications on titanium by electrochemical methods are simple and cost-effective. The addition of water to the ethylene-based electrolyte-enhanced the oxidation process to increase the length of the nanotubes. Surface morphological analysis shows that the length of the nanotubes has been increased, nanoindentation analysis delivers that increasing the length has been increased the hardness level, and corrosion analysis indicates that the length of nanotubes encouraged the corrosion resistance. Potentiodynamic polarization, Bode and Nyquist plots were models fit analyzed with equivalent electrical circuits. Sample cell viability was characterized with NIH-3T3 cells using an inverted microscopy analyzer.

In-vitro Wound Healing Properties of Commiphora swynnertonii Resinous Extracts

Wound healing is a complex multicellular process involving many cell types which include; inflammatory cells, endothelial cells, fibroblasts and keratinocytes. The process involves an orderly sequence of events with four overlapping phases namely; haemostasis, inflammatory, proliferation and remodeling phases. The process can be facilitated by the use of wound healing agents including herbal remedies from plants. In this study the main objective was to evaluate the in vitro wound healing activity of the resin obtained from Commiphora swynnertonii (C.swynnertonii). First the NIH -3T3 cells viability were evaluated using (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl Tetrazolium Bromide (MTT) assay. Then the wound scratch assay model was used to evaluate cellular proliferation, closure of the wound and release of matrix metalloproteinase enzymes. Results indicate differences in mean cell viability between different concentrations within 24 hours of incubation. The highest viability was recorded at the concentration of 1% (v/v). The in-vitro wound scratch assay showed positive NIH - 3T3 cells proliferation on the wound area and cells migration when compared with control group (without treatment) at 0 and 24 hours. In addition, C. swynnertonii was able to stimulate secretion of MMP-2 release from NIH - 3T3 cells. MMP-2 is an important enzyme for extracellular matrix remodeling during wound healing suggesting that C. swynnertonii promotes wound healing by stimulating cell proliferation and production of MMP-2 in a mechanism that is currently not known.