Ketogenic Diet Protects from Experimental Colitis in a Mouse Model Regardless of Dietary Fat Source.

While ketogenic diets (KDs) may have potential as adjunct treatments for gastrointestinal diseases, there is little knowledge on how the fat source of these diets impacts intestinal health. The objective of this study was to investigate how the source of dietary fat of KD influences experimental colitis. We fed nine-week-old male C57BL/6J mice (n = 36) with a low-fat control diet or KD high either in saturated fatty acids (SFA-KD) or polyunsaturated linoleic acid (LA-KD) for four weeks and then induced colitis with dextran sodium sulfate (DSS). To compare the diets, we analyzed macroscopic and histological changes in the colon, intestinal permeability to fluorescein isothiocyanate-dextran (FITC-dextran), and the colonic expression of tight junction proteins and inflammatory markers. While the effects were more pronounced with LA-KD, both KDs markedly alleviated DSS-induced histological lesions. LA-KD prevented inflammation-related weight loss and the shortening of the colon, as well as preserved Il1b and Tnf expression at a healthy level. Despite no significant between-group differences in permeability to FITC-dextran, LA-KD mitigated changes in tight junction protein expression. Thus, KDs may have preventive potential against intestinal inflammation, with the level of the effect being dependent on the dietary fat source.

Choosing T-cell sources determines CAR-T cell activity in neuroblastoma.

Introduction: The clinical success of chimeric antigen receptor-modified T cells (CAR-T cells) for hematological malignancies has not been reproduced for solid tumors, partly due to the lack of cancer-type specific antigens. In this work, we used a novel combinatorial approach consisting of a versatile anti-FITC CAR-T effector cells plus an FITC-conjugated neuroblastoma (NB)-targeting linker, an FITC-conjugated monoclonal antibody (Dinutuximab) that recognizes GD2. Methods: We compared cord blood (CB), and CD45RA-enriched peripheral blood leukapheresis product (45RA) as allogeneic sources of T cells, using peripheral blood (PB) as a control to choose the best condition for anti-FITC CAR-T production. Cells were manufactured under two cytokine conditions (IL-2 versus IL-7+IL-15+IL-21) with or without CD3/CD28 stimulation. Immune phenotype, vector copy number, and genomic integrity of the final products were determined for cell characterization and quality control assessment. Functionality and antitumor capacity of CB/45RA-derived anti-FITC CAR-T cells were analyzed in co-culture with different anti-GD2-FITC labeled NB cell lines. Results: The IL-7+IL-15+IL-21 cocktail, in addition to co-stimulation signals, resulted in a favorable cell proliferation rate and maintained less differentiated immune phenotypes in both CB and 45RA T cells. Therefore, it was used for CAR-T cell manufacturing and further characterization. CB and CD45RA-derived anti-FITC CAR-T cells cultured with IL-7+IL-15+IL-21 retained a predominantly naïve phenotype compared with controls. In the presence of the NB-FITC targeting, CD4+ CB-derived anti-FITC CAR-T cells showed the highest values of co-stimulatory receptors OX40 and 4-1BB, and CD8+ CAR-T cells exhibited high levels of PD-1 and 4-1BB and low levels of TIM3 and OX40, compared with CAR-T cells form the other sources studied. CB-derived anti-FITC CAR-T cells released the highest amounts of cytokines (IFN-γ and TNF-α) into co-culture supernatants. The viability of NB target cells decreased to 30% when co-cultured with CB-derived CAR-T cells during 48h. Conclusion: CB and 45RA-derived T cells may be used as allogeneic sources of T cells to produce CAR-T cells. Moreover, ex vivo culture with IL-7+IL-15+IL-21 could favor CAR-T products with a longer persistence in the host. Our strategy may complement the current use of Dinutuximab in treating NB through its combination with a targeted CAR-T cell approach.

Preventative Effects of Milk Fat Globule Membrane Ingredients on DSS-Induced Mucosal Injury in Intestinal Epithelial Cells.

The goblet cells of the gastrointestinal tract (GIT) produce glycoproteins called mucins that form a protective barrier from digestive contents and external stimuli. Recent evidence suggests that the milk fat globule membrane (MFGM) and its milk phospholipid component (MPL) can benefit the GIT through improving barrier function. Our objective was to compare the effects of two digested MFGM ingredients with or without dextran sodium sulfate (DSS)-induced barrier stress on mucin proteins. Co-cultured Caco-2/HT29-MTX intestinal cells were treated with in vitro digests of 2%, 5%, and 10% (w/v) MFGM or MPL alone for 6 h or followed by challenge with 2.5% DSS (6 h). Transepithelial electrical resistance and fluorescein isothiocyanate (FITC)-dextran (FD4) permeability measurements were used to measure changes in barrier integrity. Mucin characterization was performed using a combination of slot blotting techniques for secreted (MUC5AC, MUC2) and transmembrane (MUC3A, MUC1) mucins, scanning electron microscopy (SEM), and periodic acid Schiff (PAS)/Alcian blue staining. Digested MFGM and MPL prevented a DSS-induced reduction in secreted mucins, which corresponded to the prevention of DSS-induced increases in FD4 permeability. SEM and PAS/Alcian blue staining showed similar visual trends for secreted mucin production. A predictive bioinformatic approach was also used to identify potential KEGG pathways involved in MFGM-mediated mucosal maintenance under colitis conditions. This preliminary in silico evidence, combined with our in vitro findings, suggests the role of MFGM in inducing repair and maintenance of the mucosal barrier.

Confocal laser endomicroscopy as predictive biomarker of clinical and endoscopic efficacy of vedolizumab in ulcerative colitis: The DETECT study.

AIMS: In patients with ulcerative colitis (UC), no biomarker is available to help the physician to choose the most suitable biotherapy. The primary objective of this pilot study was to assess the feasibility of identification of α4ß7- and TNF-expressing cells, to predict the response to vedolizumab using confocal laser endoscopy (CLE). METHODS: Patients with moderate-to-severe UC, naïve of biotherapy, received vedolizumab. Clinical evaluation was performed at each infusion. Endoscopic evaluation was performed before inclusion and at week 22. Fresh colonic biopsies were stained using FITC-labelled vedolizumab and Alexa fluor-labelled adalimumab and ex vivo dual-band CLE images were acquired. Blood samples were collected to measure trough concentrations of vedolizumab and to determine absolute counts of T and B cells subpopulations, NK cells and monocytes. RESULTS: Nineteen patients were enrolled in the study and received at least one dose of vedolizumab. Clinical remission and endoscopic improvement were observed in 58% of whom 5 patients (45%) had an endoscopic subscore of 0. In terms of clinical response and remission, endoscopic improvement and histologic response, FITC-conjugated vedolizumab staining tended to be higher in responder patients compared to non-responders at week 22. A threshold value of 6 positive FITC-vedolizumab staining areas detected by CLE seemed informative to discriminate the responders and non-responders. The results were similar in terms of clinical remission and endoscopic improvement with a sensitivity of 78% and a specificity of 85% (p = 0.05). Trough concentrations and blood immune cells were not associated with responses to vedolizumab. CONCLUSION: This pilot study demonstrate that dual-band CLE is feasible to detect α4ß7- and TNF-expressing cells. Positive α4ß7 staining seems to be associated with clinical and endoscopic remission in UC patients treated by anti-α4ß7-integrin, subject to validation by larger-scale studies. Clinical-trial.gov: NCT02878083.

Gelatin but not type I collagen promotes bacteria phagocytosis in PMA-treated U937 human lymphoma cells.

PURPOSE: Besides comprising scaffolding, extracellular matrix components modulate many biological processes including inflammation and cell differentiation. We previously found precoating cell plates with extracellular matrix collagen I, or its denatured product gelatin, causes aggregation of macrophage-like human lymphoma U937 cells, which are induced to differentiation by phorbol myristate treatment. In the present study, we investigated the influence of gelatin or collagen I precoating on the bacteria phagocytosis in PMA-stimulated U937 cells. MATERIALS AND METHODS: Colony forming units of phagocytosed bacteria, Giemsa-staining of cells with phagocytosed bacteria, confocal microscopic and flow cytometric analysis of cells with phagocytosed FITC-labeled bacteria and non-bioactive latex beats were conducted. RESULTS: Gelatin precoating enhances the phagocytosis of both Gram-negative and positive bacteria, as shown by the increased colony forming units of bacteria phagocytosed by cells, and increased intracellular bacteria observed after Giemsa-staining. But collagen I has no marked influence. Confocal microscopy reveals that both live and dead FITC-bacteria were phagocytosed more in the cells with gelatin-coating but not collagen-coating. Of note, both gelatin and collagen I coating had no influence on the phagocytosis of non-bioactive latex beads. Since gelatin-coating increases autophagy but collagen I has no such impact, we are curious about the role of autophagy. Inhibiting autophagy reduced the phagocytosis of bacteria, in cells with gelatin-coating, while stimulating autophagy enhanced phagocytosis. CONCLUSION: This study finds the bacteria-phagocytosis stimulatory effect of gelatin in PMA-treated U937 cells and reveals the positive regulatory role of autophagy, predicting the potential use of gelatin products in anti-bacterial therapy.

Hyaluronan-based nano-formulation with mesoporous silica enhances the anticancer efficacy of phloroglucinol against gastrointestinal cancers.

Gastrointestinal cancers are one among the most frequently reported cancers where colorectal and gastric cancers ranks third leading cause of cancer related death worldwide. Phloroglucinol, a well-known therapeutic agent for cancer, where its usage has been limited due to its poor water solubility and bioavailability. Hence, our study aims to synthesize and characterize Hyaluronan grafted phloroglucinol loaded Mesoporous silica nanoparticles (MSN-PG-HA). Our nano-formulation hasn't shown any teratogenic effect on Zebrafish embryos, no hemolysis and toxic effect with normal fibroblast cells with a maximum concentration of 300 µg/mL. The cumulative drug release profile of MSN-PG-HA showed a maximum drug release of 96.9 % with 5 mM GSH under redox responsive drug release, which is crucial for targeting cancer cells. In addition, the MSN-PG-HA nanoparticles showed significant a cytotoxic effect against HCT-116, AGS and SW-620 with IC50 values of 86.5 µg/mL, 80.65 µg/mL and 109.255 µg/mL respectively. Also, the cellular uptake assay has shown an increased uptake of FITC-labeled-MSN-PG-HA by HA-receptor mediated endocytosis than FITC-labeled-MSN-PG without HA modification in CD44+ gastrointestinal cancer cell lines. The ability of MSN-PG-HA to target CD44+ cells was further exploited for its application in cancer stem cell research utilizing in silico analysis with various stem cell pathway related targets, in which PG showed higher binding affinity with Gli 1 and the simulation studies proving its effectiveness in disrupting the protein structure. Thus, the findings of our study with nano-formulation are safe and non-toxic to recommend for targeted drug delivery against gastrointestinal cancers as well as its affinity towards cancer stem cell pathway related proteins proving to be a significant formulation for cancer stem cell research.

Liver and spleen predominantly mediate calciprotein particle clearance in a rat model of chronic kidney disease.

Calciprotein particles (CPPs) provide an efficient mineral buffering system to prevent the complexation of phosphate and calcium in the circulation. However, in chronic kidney disease (CKD), the phosphate load exceeds the mineral buffering capacity, resulting in the formation of crystalline CPP2 particles. CPP2 have been associated with cardiovascular events and mortality. Moreover, CPP2 have been demonstrated to induce calcification in vitro. In this study, we examined the fate of CPP2 in a rat model of CKD. Calcification was induced in Sprague-Dawley rats by 5/6 nephrectomy (5/6-Nx) combined with a high-phosphate diet. Control rats received sham surgery and high-phosphate diet. Twelve weeks after surgery, kidney failure was significantly induced in 5/6-Nx rats as determined by enhanced creatinine and urea plasma levels and abnormal kidney histological architecture. Subsequently, radioactive and fluorescent (FITC)-labeled CPP2 ([89Zr]Zr-CPP2-FITC) were injected intravenously to determine clearance in vivo. Using positron emission tomography scans and radioactive biodistribution measurements, it was demonstrated that [89Zr]Zr-CPP2-FITC are mainly present in the liver and spleen in both 5/6-Nx and sham rats. Immunohistochemistry showed that [89Zr]Zr-CPP2-FITC are predominantly taken up by Kupffer cells and macrophages. However, [89Zr]Zr-CPP2-FITC could also be detected in hepatocytes. In the different parts of the aorta and in the blood, low values of [89Zr]Zr-CPP2-FITC were detectable, independent of the presence of calcification. CPP2 are cleared rapidly from the circulation by the liver and spleen in a rat model of CKD. In the liver, Kupffer cells, macrophages, and hepatocytes contribute to CPP2 clearance.NEW & NOTEWORTHY Calciprotein particles (CPPs) buffer calcium and phosphate in the blood to prevent formation of crystals. In CKD, increased phosphate levels may exceed the buffering capacity of CPPs, resulting in crystalline CPPs that induce calcification. This study demonstrates that labeled CPPs are predominantly cleared from the circulation in the liver by Kupffer cells, macrophages, and hepatocytes. Our results suggest that targeting liver CPP clearance may reduce the burden of crystalline CPP in the development of vascular calcification.

Intracellular presence of Helicobacter pylori antigen and genes within gastric and vaginal Candida.

BACKGROUND: Helicobacter pylori infections are generally acquired during childhood and affect half of the global population, but its transmission route remains unclear. It is reported that H. pylori can be internalized into Candida, but more evidence is needed for the internalization of H. pylori in human gastrointestinal Candida and vaginal Candida. METHODS: Candida was isolated from vaginal discharge and gastric mucosa biopsies. We PCR-amplified and sequenced H. pylori-specific genes from Candida genomic DNA. Using optical and immunofluorescence microscopy, we identified and observed bacteria-like bodies (BLBs) in Candida isolates and subcultures. Intracellular H. pylori antigen were detected by immunofluorescence using Fluorescein isothiocyanate (FITC)-labeled anti-H. pylori IgG antibodies. Urease activity in H. pylori internalized by Candida was detected by inoculating with urea-based Sabouraud dextrose agar, which changed the agar color from yellow to pink, indicating urease activity. RESULTS: A total of 59 vaginal Candida and two gastric Candida strains were isolated from vaginal discharge and gastric mucosa. Twenty-three isolates were positive for H. pylori 16S rDNA, 12 were positive for cagA and 21 were positive for ureA. The BLBs could be observed in Candida cells, which were positive for H. pylori 16S rDNA, and were viable determined by the LIVE/DEAD BacLight Bacterial Viability kit. Fluorescein isothiocyanate (FITC)-conjugated antibodies could be reacted specifically with H. pylori antigen inside Candida cells by immunofluorescence. Finally, H. pylori-positive Candida remained positive for H. pylori 16S rDNA even after ten subcultures. Urease activity of H. pylori internalized by Candida was positive. CONCLUSION: In the form of BLBs, H. pylori can internalize into gastric Candida and even vaginal Candida, which might have great significance in its transmission and pathogenicity.

Glucose-lightened upconversion nanoprobes for accurate cellular-discrimination based on Warburg effect.

Accurate cellular-recognition based disease therapy is of significance for precision medicine. However, except of specific antibody-coupling strategy, very few probes have been reported to efficiently discriminate normal cells and lesion cells through cellular microenvironment. Herein, we proposed a glucose selectively-lightened upconversion nanoprobe to recognize cancer cells from a pile of normal cells based on Warburg effect, that indicated a heightened demand for glucose intake for cancer cells. The nanoprobes were constructed by mesoporous silica-coated upconversion nanoparticles (UCNP@mSiO2) with the crucial incorporation of a glucose-responsive modality, benzoboric acid (BA)-modified fluorescein molecules (FITC-BA). In cancer cells, the presence of elevated glucose concentrations triggered the transformation of FITC-BA to FITC-Glucose to recover nanoprobes' luminescence, however, the nanoprobes exhibited a shielded luminescent effect in healthy cells. To validate the hypothesis of accurate cellular-discrimination, a photodynamic therapy modality, riboflavin, with a specific ratio were also loaded into above UCNP@mSiO2 nanoprobes for effective production of reactive oxygen species to kill cells. It was found that 97.8% of cancer cells were cleaned up, but normal cells retained a nearly 100% viability after 10 min laser illumination. By leveraging the metabolic disparity from Warburg effect, the nanoprobes offer a highly accurate cellular discrimination, and significantly mitigate "off-target" damage commonly associated with conventional therapies.

Interleukin 17A Contributes to Blood-Brain Barrier Disruption of Hypothalamic Paraventricular Nucleus in Rats With Myocardial Infarction.

BACKGROUND: Elevated inflammatory cytokines in the periphery have been identified as active contributors to neuroinflammation and sympathetic overactivity in heart failure (HF). Yet, the exact mechanisms by which these cytokines breach the blood-brain barrier (BBB) to exert their effects on the brain remain elusive. Interleukin 17A has been linked to BBB disruption in various neurologic disorders, and its levels were significantly augmented in circulation and the brain in HF. The present study aimed to determine whether the BBB integrity was compromised within the hypothalamic paraventricular nucleus (PVN), and if so, whether interleukin 17A contributes to BBB disruption in myocardial infarction-induced HF. METHODS AND RESULTS: Male Sprague-Dawley rats underwent coronary artery ligation to induce HF or sham surgery. Some HF rats received bilateral PVN microinjections of an interleukin 17 receptor A small interfering RNA or a scrambled small interfering RNA adeno-associated virus. Four weeks after coronary artery ligation, the permeability of the BBB was evaluated by intracarotid injection of fluorescent dyes (fluorescein isothiocyanate-dextran 10 kDa+rhodamine-dextran 70 kDa). Compared with sham-operated rats, HF rats exhibited an elevated extravasation of fluorescein isothiocyanate-dextran 10 kDa within the PVN but not in the brain cortex. The plasma interleukin 17A levels were positively correlated with fluorescein isothiocyanate 10 kDa extravasation in the PVN. The expression of caveolin-1, a transcytosis marker, was augmented, whereas the expression of tight junction proteins was diminished in HF rats. Interleukin 17 receptor A was identified within the endothelium of PVN microvessels. Treatment with interleukin 17 receptor A small interfering RNA led to a significant attenuation of fluorescein isothiocyanate 10 kDa extravasation in the PVN and reversed expression of caveolin-1 and tight junction-associated proteins in the PVN. CONCLUSIONS: Collectively, these data indicate that BBB permeability within the PVN is enhanced in HF and is likely attributable to increased interleukin 17A/interleukin 17 receptor A signaling in the BBB endothelium, by promoting caveolar transcytosis and degradation of tight junction complexes.

Topical Application of Cell-Penetrating Peptide Modified Anti-VEGF Drug Alleviated Choroidal Neovascularization in Mice.

Background: Age-related macular degeneration (AMD) stands as the foremost cause of irreversible central vision impairment, marked by choroidal neovascularization (CNV). The prevailing clinical approach to AMD treatment relies on intravitreal injections of anti-vascular endothelial growth factor (VEGF) drugs. However, this method is encumbered by diverse complications, prompting exploration of non-invasive alternatives such as ocular administration via eye drops for anti-VEGF therapy. Methods: Two complexes, 5-FITC-CPP-Ranibizumab (5-FCR) and 5-FITC-CPP-Conbercept (5-FCC), were synthesized by incorporating the anti-VEGF drugs Ranibizumab (RBZ) or Conbercept (CBC) with cell-penetrating peptide (CPP). Circular dichroism spectrum (CD) facilitated complexes characterization. Eye drops was utilized to address laser-induced CNV in mice. Fluorescein fundus angiography (FFA) observe the CNV lesion, while FITC-dextran and IB4 dual fluorescent staining, along with hematoxylin-eosin (HE) staining, assessed in lesion size. Tissue immunofluorescence examined CD31 and VEGF expression in choroidal/retinal pigment epithelial (RPE) tissues. Biocompatibility and biosafety of 5-FCR and 5-FCC was evaluated through histological examination of various organs or cell experiments. Results: Both 5-FCR and 5-FCC exhibited favorable biocompatibility and safety profiles. VEGF-induced migration of Human umbilical vein endothelial cells (HUVECs) significantly decreased post-5-FCR/5-FCC treatment. Additionally, both complexes suppressed VEGF-induced tube formation in HUVECs. FFA results revealed a significant improvement in retinal exudation in mice. Histological examination unveiled the lesion areas in the 5-FCR and 5-FCC groups showed a significant reduction compared to the control group. Similar outcomes were observed in histological sections of the RPE-choroid-sclera flat mounts. Conclusion: In this study, utilizing the properties of CPP and two anti-VEGF drugs, we successfully synthesized two complexes, 5-FCR and 5-FCC, through a straightforward approach. Effectively delivering the anti-VEGF drugs to the target area in a non-invasive manner, suppressing the progression of laser-induced CNV. This offers a novel approach for the treatment of wet AMD.

Enhanced detection of acrylamide using a versatile solid-state upconversion sensor through spectral and visual analysis.

Acrylamide (AM) generally forms in high-temperature processes and has been classified as a potential carcinogen. In this study, we put forward a maneuverable solid-state luminescence sensor using polydimethylsiloxane (PDMS) as the matrix coupled with upconversion nanoparticles as the indicator. The core-shell upconversion nanoparticles emitting cyan light were uniformly encapsulated in PDMS. Then it was further modified with complementary DNA of AM aptamer. The nanocrystalline fluorescein isothiocyanate isomer (FITC), coupled with AM aptamer, was attached to the surface of PDMS. FITC effectively quenched the upconversion luminescence through fluorescence resonance energy transfer (FRET). The introduction of AM resulted in preferentially bound to aptamer caused the separation of the quencher and the donor, and led to luminescence recovery. The developed sensor was applied for both spectral and visual monitoring, demonstrating a detection limit (LOD) of 1.00 nM and 1.07 nM, respectively. Importantly, in the actual foodstuffs detection, there is no obvious difference between the results of this study and the standard method, which indicates the developed method has good accuracy. Therefore, this solid-state sensor has the potential for on-site detection using a smartphone device and an Android application.

Unraveling Interaction of Rhenium-188 Microspheres with Primary Hepatic Cancer Cell: A Breakthrough Study.

Introduction: Hepatocellular carcinoma is a prevalent contributor to global mortality rates. The main palliative treatments are trans-arterial chemoembolization and selective intra-arterial radionuclide therapy. Methods: A novel freeze-dried nonradioactive microsphere kit formulation has been developed, and the behavior and therapeutic potential of 188Re microspheres have been assessed. The microspheres were labeled with fluorescein isothiocyanate (FITC) and 188ReO4-. The uptake of FITC microspheres by HepG2 cells was examined at various time intervals. The impact of 188Re microspheres on cell viability and the mode of cell death were investigated with HepG2 cells using MTT and Annexin FITC-V/propidium iodide (PI) apoptosis assay. Results: The labeling efficiency of microspheres was more than 99% with FITC and 188ReO4-. The maximum uptake of FITC microspheres by HepG2 cells was achieved at 6 h. The exposure to 188Re microspheres has shown a decrease in cellular viability from 77.81% ± 0.015% to 42.03% ± 0.148% at 192 h of incubation (∼11 half-lives). The cellular uptake of 188Re microspheres was 0.255-0.901 MBq. These values were concordant with Annexin FITC-V/PI apoptosis assay. At 192 h, 53.28% ± 0.01% of cells entered the apoptotic phase after treatment with 188Re microspheres, and only 39.34% ± 0.02% of cells remained viable. However, in the cells treated with 188ReO4- alone, 74.86% ± 0.005% of cells were viable, and only 24.75% ± 0.577% of cells were in the early apoptotic phase at 192 h. Conclusion: The data revealed that 188Re microspheres treatment led to significant growth inhibition in HepG2 cells compared with 188ReO4-.

Erythrocyte membrane-coated nanocarriers modified by TGN for Alzheimer's disease.

Alzheimer's disease (AD) is an aging-related neurodegenerative disease, and the main pathological feature was ß-amyloid protein (Aß) deposition. Recently, bioactive materials-based drug delivery system has been widely investigated for the treatment of AD. In this study, we developed a red blood cells (RBC) membrane-coated polycaprolactone (PCL) nanoparticles (NPs) loading with a therapeutic agent for AD, curcumin (Cur). A functional peptide TGNYKALHPHN (TGN) was conjugated to the surface of membrane for blood-brain barrier (BBB) transport (TGN-RBC-NPs-Cur). TGN peptide can be recognized by receptors on the BBB and has great potential for brain transport. To confirm the targeted delivery of Cur to the brain, a cell co-culturing immortalized human cerebral microvascular endothelial cells and human brain astrocytes glioblastoma (hCMEC/D3 and U-118MG) in vitro model was established. As a result, the BBB transporting ratio of TGN-RBC-NPs-FITC was 29.64% at 12 h which was approximately eight-fold than RBC-NPs-FITC. The improvement of drug accumulation in the AD lesion was confirmed by the NPs modified with the BBB-penetrating peptide in the fluorescence imaging and quantitative analysis with UPLC-MS/MS in vivo. The neuroprotective effects were evaluated with new object recognition behavioral test, in vitro AD cell model, dendritic spine stain, GFAP and IBA1 immunofluorescence stain. The spatial learning and memory abilities of the AD model mice treated with TGN-RBC-NPs-Cur were obviously enhanced compared with the AD control mice and were also better than Cur at the same dosage. These results were consistent with the values of protection index of rat adrenal pheochromocytoma cells (PC12 cells) treated by Aß25-35. TGN-RBC-NPs-Cur increased the dendritic segments densities and restrained activation of microglia and astrocytes of AD mice, as well as reversed cognitive function of AD mice. All of the results demonstrated TGN-RBC-NPs-Cur a promising therapeutic strategy for delaying the progression of AD by designing biomimetic nanosystems to deliver drugs into the brain.

The Dual Luminescence Lifetime pH/Oxygen Sensor: Evaluation of Applicability for Intravital Analysis of 2D- and 3D-Cultivated Human Endometrial Mesenchymal Stromal Cells.

The oxygenation of cells and tissues and acidification of the cellular endolysosomal system are among the major factors that ensure normal functioning of an organism and are violated in various pathologies. Recording of these parameters and their changes under various conditions is an important task for both basic research and clinical applications. In the present work, we utilized internalizable dual pH/O2 lifetime sensor (Ir-HSA-FITC) based on the covalent conjugation of human serum albumin (HSA) with fluorescein isothiocyanate (FITC) as pH sensor and an orthometalated iridium complex as O2 sensor. The probe was tested for simultaneous detection of acidification level and oxygen concentration in endolysosomes of endometrial mesenchymal stem/stromal cells (enMSCs) cultivated as 2D monolayers and 3D spheroids. Using a combined FLIM/PLIM approach, we found that due to high autofluorescence of enMSCs FITC lifetime signal in control cells was insufficient to estimate pH changes. However, using flow cytometry and confocal microscopy, we managed to detect the FITC signal response to inhibition of endolysosomal acidification by Bafilomycin A1. The iridium chromophore phosphorescence was detected reliably by all methods used. It was demonstrated that the sensor, accumulated in endolysosomes for 24 h, disappeared from proliferating 2D enMSCs by 72 h, but can still be recorded in non-proliferating spheroids. PLIM showed high sensitivity and responsiveness of iridium chromophore phosphorescence to experimental hypoxia both in 2D and 3D cultures. In spheroids, the phosphorescence signal was detected at a depth of up to 60 µm using PLIM and showed a gradient in the intracellular O2 level towards their center.

Tamoxifen induces eryptosis through calcium accumulation and oxidative stress.

Chemotherapy-related anemia is a major obstacle in anticancer therapy. Tamoxifen (TAM) is an antiestrogen prescribed for breast cancer patients with hemolytic potential and apoptotic properties in nucleated cells. However, the eryptotic activity of TAM has hitherto escaped the efforts of investigators. RBCs from apparently healthy volunteers were treated with 1-50 µM of TAM for 24 h at 37 °C. Hemoglobin leakage and LDH, AST, and AChE activities were photometrically determined while K+, Na+, and Mg2+ were detected by ion-selective electrode. Flow cytometry was used to identify eryptotic cells by annexin-V-FITC, intracellular Ca2+ by Fluo4/AM, sell size and morphology by FSC and SSC signals, respectively, and oxidative stress by H2DCFDA. Whole blood was also exposed to 30 µM of TAM for 24 h at 37 °C to examine the toxicity of TAM to WBCs and platelets. TAM caused Ca2+-independent, dose-responsive hemolysis accompanied by K+, LDH, and AST leakage without improving the mechanical stability of RBCs in hypotonic environments. TAM treatment also increased the proportion of cells positive for annexin-V-FITC, Fluo4, and DCF, along with diminished FSC and SSC signals and AChE activity. Notably, TAM toxicity was aggravated by sucrose but abrogated by vitamin C, PEG 8000, and urea. Moreover, TAM exhibited distinct cytotoxic profiles against leukocytes and platelets. TAM-induced eryptosis is characterized by breakdown of membrane asymmetry, inhibition of AChE activity, Ca2+ accumulation, cell shrinkage, and oxidative stress. Vitamin C, PEG 8000, and urea may hold promise to subvert the undesirable toxic effects of TAM on RBCs.

Dynamic ultrasound molecular-targeted imaging of senescence in evaluation of lapatinib resistance in HER2-positive breast cancer.

BACKGROUND: Prolonged treatment of HER2+ breast cancer with lapatinib (LAP) causes cellular senescence and acquired drug resistance, which often associating with poor prognosis for patients. We aim to explore the correlation between cellular senescence and LAP resistance in HER2+ breast cancer, screen for molecular marker of reversible senescence, and construct targeted nanobubbles for ultrasound molecular imaging to dynamically evaluate LAP resistance. METHODS AND RESULTS: In this study, we established a new cellular model of reversible cellular senescence using LAP and HER2+ breast cancer cells and found that reversible senescence contributed to LAP resistance in HER2+ breast cancer. Then, we identified ecto-5'-nucleotidase (NT5E) as a marker of reversible senescence in HER2+ breast cancer. Based on this, we constructed NT5E-targeted nanobubbles (NT5E-FITC-NBs) as a new molecular imaging modality which could both target reversible senescent cells and be used for ultrasound imaging. NT5E-FITC-NBs showed excellent physical and imaging characteristics. As an ultrasound contrast agent, NT5E-FITC-NBs could accurately identify reversible senescent cells both in vitro and in vivo. CONCLUSIONS: Our data demonstrate that cellular senescence-based ultrasound-targeted imaging can identify reversible senescence and evaluate LAP resistance effectively in HER2+ breast cancer cells, which has the potential to improve cancer treatment outcomes by altering therapeutic strategies ahead of aggressive recurrences.

Cardiopulmonary bypass in a rat model may shorten the lifespan of stored red blood cells by activating caspase-3.

BACKGROUND: Red blood cell transfusion is required for many types of surgery against cardiovascular disease, and the function of transfused cells appears to decline over time. The present study examined whether transfusion also reduces red blood cell lifespan in a rat model. MATERIAL AND METHODS: Bypass in rats were established by connecting a roll pump to the femoral artery and vein. Then FITC-labeled stored red blood cells from rats were transfused in the animals, and the cells in circulation were counted after transfusion. In separate experiments, stored red blood cells were incubated with bypass plasma in vitro, and the effects of incubation were assessed on cell morphology, redox activity, ATP level, caspase-3 activity, and phosphatidylserine exposure on the cell surface. These in vivo and in vitro experiments were also performed after pretreating the stored red blood cells with the caspase-3 inhibitor Z-DEVD-FMK. RESULTS: Bypass significantly decreased the number of circulating FITC-labeled stored red blood cells and increased the proportions of monocytes, neutrophils and splenic macrophages that had phagocytosed the red blood cells. In vitro, bypass plasma altered the morphology of red blood cells and increased oxidative stress, caspase-3 activity and phosphatidylserine exposure, while decreasing ATP level. Pretreating stored red blood cells with Z-DEVD-FMK attenuated the effects of bypass on caspase-3 activity, but not oxidative stress, in stored red blood cells. DISCUSSION: Bypass appears to shorten the lifespan of stored red blood cells, at least in part by activating caspase-3 in the cells.

Camelid-derived CD38 antibody successfully circumvents epitope blockade by the therapeutic anti-CD38 antibody daratumumab.

OBJECTIVES: To evaluate the efficacy of an anti-CD38 nanobody to detect plasma cells in a flow cytometry myeloma minimal residual disease (MRD) panel in patients treated with daratumumab and other immunotherapies. METHODS: Twenty-three bone marrow samples from as many patients were collected during or at the end of daratumumab treatment cycles. A 5-tube, 8-color flow cytometry MRD panel was performed. Dotplots were reviewed, and the median fluorescence intensity (MFI) was calculated. RESULTS: Patients' ages ranged from 45 to 77 years, and the cohort was made up of 13 men and 10 women who had undergone 2 to 24 cycles of daratumumab therapy at the time of myeloma MRD testing. In all 23 cases, therapeutic use of daratumumab impaired pathologists' ability to measure CD38 on plasma cells when using a conventional murine monoclonal antibody (anti-CD38 fluorescein isothiocyanate [FITC], clone T16; Beckman Coulter). In 21 of the 23 cases, the measurement of CD38 was restored when the anti-CD38 nanobody was employed. Compared with anti-CD38 FITC, the anti-CD38 Alexa Fluor 488 nanobody (Beckman Coulter) produced higher MFI and allowed measurement of a higher frequency of discernable plasma cells. CONCLUSIONS: The camelid-derived CD38 antibody successfully circumvents the steric inhibition of CD38 that the therapeutic use of daratumumab imparts and facilitates myeloma MRD plasma cell detection.

Layered double hydroxides - poloxamer 188 nanocomposites based on exfoliation reassembling for improved cellular uptake and controlled delivery of methotrexate.

Exploitation of advanced methotrexate (MTX) delivery with nanocomposites has important clinical application value. Poloxamer 188 micelle and layered double hydroxide loaded with MTX (LDH-MTX) by exfoliation reassembling were used to prepare LDH-MTX-poloxamer 188 nanocomposites with good dispersibility and efficient cellular uptake for controlled drug delivery. The LDH-MTX-poloxamer 188 nanocomposites with sphere-like morphology, of which the average hydrodynamic diameter was <100 nm, were shown to have better dispersion state than naked LDH-MTX. Importantly, the LDH-MTX-poloxamer 188 nanocomposites could achieve significant sustained drug release and have obvious pH dependent responsive release ability. In addition, these nanocomposites also exhibited long-term and excellent in vitro antitumor efficacy as opposed to pure MTX or LDH-MTX as evident from cell viability. More interestingly, compared to pure FITC used to simulate MTX, LDH nanocomposites labeled with FITC were considered to have better cell adhesion through cell uptake. Therefore, the studied nanocomposites of LDH-MTX-poloxamer 188 can be further used as a new advanced MTX delivery nanovehicles with desired properties in future therapeutic aspects.