ß-Adrenoceptor Agonists Attenuate Thrombin-Induced Impairment of Human Lung Endothelial Cell Barrier Function and Protect the Lung Vascular Barrier during Resuscitation from Hemorrhagic Shock.

ß-adrenoceptor (ß-AR) agonists are known to antagonize thrombin-induced impairment (TII) of bovine and ovine lung endothelial barrier function. The effects of adrenoceptor agonists and other vasoactive agents on human lung microvascular endothelial cell (HULEC-5a) barrier function upon thrombin exposure have not been studied. Furthermore, it is unknown whether the in vitro effects of adrenoceptor agonists translate to lung protective effects in vivo. We observed that epinephrine, norepinephrine, and phenylephrine enhanced normal and prevented TII of HULEC-5a barrier function. Arginine vasopressin and angiotensin II were ineffective. α1B-, α2A/B-, and ß1/2-ARs were detectable in HULEC-5a by RT-PCR. Propranolol but not doxazosin blocked the effects of all adrenoceptor agonists. Phenylephrine stimulated ß2-AR-mediated Gαs activation with 13-fold lower potency than epinephrine. The EC50 to inhibit TII of HULEC-5a barrier function was 1.8 ± 1.9 nM for epinephrine and >100 nM for phenylephrine. After hemorrhagic shock and fluid resuscitation in rats, Evans blue extravasation into the lung increased threefold (p < 0.01 vs. sham). Single low-dose (1.8 µg/kg) epinephrine administration at the beginning of resuscitation had no effects on blood pressure and reduced Evans blue extravasation by 60% (p < 0.05 vs. vehicle). Our findings confirm the effects of ß-adrenoceptor agonists in HULEC-5a and suggest that low-dose ß-adrenoceptor agonist treatment protects lung vascular barrier function after traumatic hemorrhagic shock.

Detection and Quantification of Programmed Cell Death in Chlamydomonas reinhardtii: The Example of S-Nitrosoglutathione.

Chlamydomonas (Chlamydomonas reinhardtii) is a unicellular model alga that has been shown to undergo programmed cell death (PCD) that can be triggered in response to different stresses. We have recently shown that Chlamydomonas is particularly well suited to the study and quantification of PCD. We have shown for the first time that S-nitrosoglutathione (GSNO), a nitric oxide (NO) donor, is able to induce PCD and can be used as a study system in Chlamydomonas. In this article, we provide a simple and robust protocol for quantifying GSNO-induced PCD, which can be adapted to any other treatment. We explain how to detect NO production in the cell following GSNO treatment. We show how PCD can be identified simply by analyzing the degradation profile of genomic DNA. We also provide an easy and reproducible cell death quantification protocol, which makes it possible to follow the course of PCD over time and highlight very fine differences in the number of affected cells between different samples. Key features • Use of S-nitrosoglutathione (GSNO) as a means to study programmed cell death (PCD) in Chlamydomonas. • Discrimination of PCD vs. necrosis. • In vivo determination of NO production in the cell. • A simple, robust protocol for PCD quantification.

Development of [177Lu]Lu-LNC1010 for peptide receptor radionuclide therapy of nasopharyngeal carcinoma.

PURPOSE: Somatostatin Receptor 2 (SSTR2)-targeted radiopharmaceutical [68Ga]Ga-DOTATATE has potential advantages in the diagnosis of nasopharyngeal carcinoma (NPC). This study introduces a novel long-lasting SSTR2 analogue, LNC1010, based on DOTATATE, a truncated Evans blue-binding moiety, and a polyethylene-glycol linker. We hypothesised that peptide receptor radionuclide therapy (PRRT) is more effective with [177Lu]Lu-LNC1010 than with [177Lu]Lu-DOTATATE in treating metastatic NPC. METHODS: We assessed binding characteristics of LNC1010 in vitro using C666-1 NPC cells and in-vivo pharmacokinetics of [68Ga]Ga/[177Lu]Lu-LNC1010 in C666-1 NPC xenografts via PET and SPECT imaging, biodistribution studies, and PRRT, and compared them with [68Ga]Ga/[177Lu] Lu-labelled DOTATATE. Furthermore, a proof-of-concept approach for imaging and therapy was conducted in a patient with metastatic NPC. RESULTS: LNC1010 exhibited strong uptake and specific affinity for SSTR2 in C666-1 NPC cells. PET and SPECT imaging demonstrated higher uptake and longer tumour retention of [68Ga]Ga/[177Lu]Lu-LNC1010 than [68Ga]Ga/[177Lu]Lu-DOTATATE in C666-1 NPC xenografts, indicating its suitability for PRRT applications in NPCs. Biodistribution studies confirmed the higher uptake and prolonged retention of [177Lu]Lu-LNC1010 than [177Lu]Lu-DOTATATE. In preclinical PRRT studies, [177Lu]Lu-LNC1010 showed greater inhibition of tumour growth in C666-1 NPC xenografts than [177Lu]Lu-DOTATATE. In a subsequent pilot clinical study, PRRT with [177Lu]Lu-LNC1010 achieved favourable therapeutic and negligible side effects in a patient with metastatic NPC. CONCLUSION: [177Lu]Lu-LNC1010 demonstrated increased tumour uptake and prolonged retention in SSTR2-positive NPCs, with superior anti-tumour efficacy to that of [177Lu]Lu-DOTATATE in preclinical studies. These findings suggest that PRRT with [177Lu]Lu-LNC1010 is a promising treatment for advanced NPC, extending the clinical scope of PRRT beyond neuroendocrine tumours.

Blood-brain barrier disruption and edema formation due to prolonged starvation in wild-type mice.

INTRODUCTION: Different types of diseases have been treated by restricted caloric intake or fasting. Although during this long time, fasting protective measures, for example, supplements, are given to the patients to protect vital organs such as the liver and kidney, little attention is given to the brain. The current research aims to investigate hypoglycemia due to prolonged fasting disrupts blood-brain barrier (BBB) in mice. MATERIALS AND METHODS: Immunohistochemistry (IHC) and in situ hybridization (ISH) techniques were used to examine the expression of different genes. Evans blue extravasation and wet-dry technique were performed to evaluate the integrity of BBB and the formation of brain edema, respectively. RESULTS: We confirmed that hypoglycemia affected mice fasting brain by examining the increased expression of glucose transporter protein 1 and hyperphosphorylation of tau protein. We subsequently found downregulated expression of some genes, which are involved in maintaining BBB such as vascular endothelial growth factor (VEGF) in astrocytes and claudin-5 (a vital component of BBB) and VEGF receptor (VEGFR1) in endothelial cells by ISH. We also found that prolonged fasting caused the brain endothelial cells to express lipocalin-2, an inflammatory marker of brain endothelial cells. We performed Evans blue extravasation to show more dye was retained in the brain of fasted mice than in control mice as a result of BBB disruption. Finally, wet-dry method showed that the brain of prolonged fasted mice contained significantly higher amount of water confirming the formation of brain edema. Therefore, special attention should be given to the brain during treatment with prolonged fasting for various diseases. CONCLUSIONS: Our results demonstrated that hypoglycemia due to prolonged fasting disrupts BBB and produces brain edema in wild-type mice, highlighting the importance of brain health during treatment with prolonged fasting.

Corrigendum: HSA-nanobinders crafted from bioresponsive prodrugs for combined cancer chemoimmunotherapy-an in vitro exploration.

[This corrects the article DOI: 10.3389/fchem.2024.1378233.].

Exploring the Impact of Irradiation on Glioblastoma Blood-Brain-Barrier Permeability: Insights from Dynamic-Contrast-Enhanced-MRI and Histological Analysis.

(1) Background: Glioblastoma (GB) presents a formidable challenge in neuro-oncology due to its aggressive nature, limited treatment options, and poor prognosis. The blood-brain barrier (BBB) complicates treatment by hindering drug delivery to the tumor site, particularly to the infiltrative cells in the margin of the tumor, which are mainly responsible for tumor recurrence. Innovative strategies are therefore needed to enhance drug delivery in the margins of the tumor. This study explores whether irradiation can enhance BBB permeability by assessing hemodynamic changes and the distribution of contrast agents in the core and the margins of GB tumors. (2) Methods: Mice grafted with U-87MG cells were exposed to increasing irradiation doses. The distribution of contrast agents and hemodynamic parameters was evaluated using both non-invasive magnetic resonance imaging (MRI) techniques with gadolinium-DOTA as a contrast agent and invasive histological analysis with Evans blue, a fluorescent vascular leakage marker. Diffusion-MRI was also used to assess cytotoxic effects. (3) Results: The histological study revealed a complex dose-dependent effect of irradiation on BBB integrity, with increased vascular leakage at 5 Gy but reduced leakage at higher doses (10 and 15 Gy). However, there was no significant increase in the diffusion of Gd-DOTA outside the tumor area by MRI. (4) Conclusions: The increase in BBB permeability could be an interesting approach to enhance drug delivery in glioblastoma margins for low irradiation doses. In this model, DCE-MRI analysis was of limited value in assessing the BBB opening in glioblastoma after irradiation.

HSA-nanobinders crafted from bioresponsive prodrugs for combined cancer chemoimmunotherapy-an in vitro exploration.

Introduction: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer still lacking effective treatment options. Chemotherapy in combination with immunotherapy can restrict tumor progression and repolarize the tumor microenvironment towards an anti-tumor milieu, improving clinical outcome in TNBC patients. The chemotherapeutic drug paclitaxel has been shown to induce immunogenic cell death (ICD), whereas inhibitors of the indoleamine 2,3- dioxygenase 1 (IDO1) enzyme, whose expression is shared in immune regulatory and tumor cells, have been revealed to enhance the anti-tumor immune response. However, poor bioavailability and pharmacokinetics, off-target effects and hurdles in achieving therapeutic drug concentrations at the target tissue often limit the effectiveness of combination therapies. Methods: This work describes the development of novel biomimetic and carrier-free nanobinders (NBs) loaded with both paclitaxel and the IDO1 inhibitor NLG919 in the form of bioresponsive and biomimetic prodrugs. A fine tuning of the preparation conditions allowed to identify NB@5 as the most suitable nanoformulation in terms of reproducibility, stability and in vitro effectiveness. Results and discussion: Our data show that NB@5 effectively binds to HSA in cell-free experiments, demonstrating its protective role in the controlled release of drugs and suggesting the potential to exploit the protein as the endogenous vehicle for targeted delivery to the tumor site. Our study successfully proves that the drugs encapsulated within the NBs are preferentially released under the altered redox conditions commonly found in the tumor microenvironment, thereby inducing cell death, promoting ICD, and inhibiting IDO1.

Safety-related outcomes for patients with a tracheostomy and the use of flexible endoscopic evaluation of swallowing (FEES) for assessment and management of swallowing: A systematic review.

PURPOSE: The purpose of this systematic review was to examine safety-related outcomes for patients with tracheostomy after flexible endoscopic evaluation of swallowing (FEES) to assess and manage their swallow, when compared to other non-instrumental swallow assessments such as clinical swallowing examination (CSE) and/or a modified Evans blue dye test (MEBDT). METHOD: Three databases were searched for articles referring to safety-related outcome data for adults with a tracheostomy, who underwent FEES and CSE and/or MEBDT. Articles were screened using predefined inclusion/exclusion criteria. RESULT: The search strategy identified 2097 articles; following abstract and full-text screening, seven were included for review. The summary of evidence found low to very low certainty that FEES was associated with improved outcomes across swallow safety, physiological outcomes, tracheostomy cannulation duration, functional outcomes, and detection of upper airway pathologies. CONCLUSION: This systematic review demonstrated low to very low certainty evidence from seven heterogeneous studies with low sample sizes that incorporating FEES may be associated with improved safety-related outcomes. There is less evidence supporting the accuracy of other swallow assessments conducted at the point of care (i.e. CSE and MEBDT). Future research requires studies with larger sample sizes and routine reporting of safety-related outcomes with use of FEES.

Glypican-1-targeted antibody-drug conjugate inhibits the growth of glypican-1-positive glioblastoma.

Glioblastoma is the deadliest form of brain tumor. The presence of the blood-brain barrier (BBB) significantly hinders chemotherapy, necessitating the development of innovative treatment options for this tumor. This report presents the in vitro and in vivo efficacy of an antibody-drug conjugate (ADC) that targets glypican-1 (GPC1) in glioblastoma. The GPC1-ADC was created by conjugating a humanized anti-GPC1 antibody (clone T2) with monomethyl auristatin E (MMAE) via maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl linkers. Immunohistochemical staining analysis of a glioblastoma tissue microarray revealed that GPC1 expression was elevated in more than half of the cases. GPC1-ADC, when bound to GPC1, was efficiently and rapidly internalized in glioblastoma cell lines. It inhibited the growth of GPC1-positive glioma cell lines by inducing cell cycle arrest in the G2/M phase and triggering apoptosis in vitro. We established a heterotopic xenograft model by subcutaneously implanting KALS-1 and administered GPC1-ADC intravenously. GPC1-ADC significantly inhibited tumor growth and increased the number of mitotic cells. We also established an orthotopic xenograft model by intracranially implanting luciferase-transfected KS-1-Luc#19. After injecting Evans blue and resecting brain tissues, dye leakage was observed in the implantation area, confirming BBB disruption. We administered GPC1-ADC intravenously and measured the luciferase activity using an in vivo imaging system. GPC1-ADC significantly inhibited tumor growth and extended survival. In conclusion, GPC1-ADC demonstrated potent intracranial activity against GPC1-positive glioblastoma in an orthotopic xenograft model. These results indicate that GPC1-ADC could represent a groundbreaking new therapy for treating glioblastoma beyond the BBB.

Characteristics of the New Mast Cell-Rich Nodal Structure in the Rat Skin Surface.

Background: : Acupuncture, practiced for millennia, lacks a clear anatomical definition for acupoints. A prevailing theory suggests that acupoints overlap with skin areas with higher mast cell density. Skin spots stained with intravenously infused Evans blue (EB), indicative of neurogenic inflammation, have recently been posited as acupoints in rats. Objectives: : To demonstrate the concordance between EB-reactive skin spots and mast cell-enriched acupoints. Methods: : We employed staining and RNA-seq analysis to delineate the morphological characteristics and gene expression profiles of EB-reactive skin spots in rats. Results: : EB infusion revealed a novel nodal structure on the rat skin surface, visible to the naked eye, with dimensions of approximately 1 mm in both diameter and height. Around 30 such nodes were identified on one side of the abdominal area, spaced roughly 3 mm apart, excluding the linea alba. RNA-seq analysis indicated that the gene expression patterns within these nodes markedly differed from both non-nodal skin areas and lymph nodes. Histological examination using toluidine blue revealed a significantly greater mast cell count in the nodes than in non-nodal skin regions. Additionally, the nodes stained positively with Alcian blue and Hemacolor, reagents known to mark primo vascular tissues. Conclusion: : Our findings suggest that EB-reactive nodes are indeed rich in mast cells. Further research is warranted to establish these skin nodes as surface primo nodes.

Modulation of the capillary leakage by exogenous albumin in a rat model of endothelial glycocalyx damage.

BACKGROUND: Endothelial glycocalyx (EG) plays a crucial role in maintaining the plasma proteins within the intravascular space. OBJECTIVE: We studied whether exogenous albumin protects the EG in an experimental model of EG enzymatic damage in rats. METHODS: Rats were divided into three groups of 10 animals that received (1) Evans blue (2) Evans blue + hyaluronidase, or (3) Evans blue + hyaluronidase + 20% human albumin via the tail vein. Spectrophotometric analysis was performed 2 h later to quantify the leakage of Evans blue-labeled albumin into the heart, lungs, brain, kidneys, liver, small intestine, spleen, and skeletal muscle. RESULTS: Administration of hyaluronidase numerically increased the capillary leakage of Evans blue in all examined tissues. Co-administration of albumin decreased the leakage of albumin in all tissues except the heart. In the lungs, the ratio between the absorbance and dry organ weight decreased from 5.3 ± 2.4 to 1.7 ± 0.5 (mean ± SD) (P <  0.002), and in the liver, the absorbance decreased from 2.2 ± 0.7 to 1.5 ± 0.4 (P <  0.011). CONCLUSION: Exogenous albumin decreased the capillary leakage of albumin which was interpreted as a sign of maintained EG integrity.

Localized Increased Permeability of Blood-Brain Barrier for Antibody Conjugates in the Cuprizone Model of Demyelination.

The development of new neurotherapeutics depends on appropriate animal models being chosen in preclinical studies. The cuprizone model is an effective tool for studying demyelination and remyelination processes in the brain, but blood-brain barrier (BBB) integrity in the cuprizone model is still a topic for debate. Several publications claim that the BBB remains intact during cuprizone-induced demyelination; others demonstrate results that could explain the increased BBB permeability. In this study, we aim to analyze the permeability of the BBB for different macromolecules, particularly antibody conjugates, in a cuprizone-induced model of demyelination. We compared the traditional approach using Evans blue injection with subsequent dye extraction and detection of antibody conjugates using magnetic resonance imaging (MRI) and confocal microscopy to analyze BBB permeability in the cuprizone model. First, we validated our model of demyelination by performing T2-weighted MRI, diffusion tensor imaging, quantitative rt-PCR to detect changes in mRNA expression of myelin basic protein and proteolipid protein, and Luxol fast blue histological staining of myelin. Intraperitoneal injection of Evans blue did not result in any differences between the fluorescent signal in the brain of healthy and cuprizone-treated mice (IVIS analysis with subsequent dye extraction). In contrast, intravenous injection of antibody conjugates (anti-GFAP or non-specific IgG) after 4 weeks of a cuprizone diet demonstrated accumulation in the corpus callosum of cuprizone-treated mice both by contrast-enhanced MRI (for gadolinium-labeled antibodies) and by fluorescence microscopy (for Alexa488-labeled antibodies). Our results suggest that the methods with better sensitivity could detect the accumulation of macromolecules (such as fluorescent-labeled or gadolinium-labeled antibody conjugates) in the brain, suggesting a local BBB disruption in the demyelinating area. These findings support previous investigations that questioned BBB integrity in the cuprizone model and demonstrate the possibility of delivering antibody conjugates to the corpus callosum of cuprizone-treated mice.

Assessment of Hyperosmolar Blood-Brain Barrier Opening in Glioblastoma via Histology with Evans Blue and DCE-MRI.

BACKGROUND: While the blood-brain barrier (BBB) is often compromised in glioblastoma (GB), the perfusion and consequent delivery of drugs are highly heterogeneous. Moreover, the accessibility of drugs is largely impaired in the margins of the tumor and for infiltrating cells at the origin of tumor recurrence. In this work, we evaluate the value of methods to assess hemodynamic changes induced by a hyperosmolar shock in the core and the margins of a tumor in a GB model. METHODS: Osmotic shock was induced with an intracarotid infusion of a hypertonic solution of mannitol in mice grafted with U87-MG cells. The distribution of fluorescent dye (Evans blue) within the brain was assessed via histology. Dynamic contrast-enhanced (DCE)-MRI with an injection of Gadolinium-DOTA as the contrast agent was also used to evaluate the effect on hemodynamic parameters and the diffusion of the contrast agent outside of the tumor area. RESULTS: The histological study revealed that the fluorescent dye diffused much more largely outside of the tumor area after osmotic shock than in control tumors. However, the study of tumor hemodynamic parameters via DCE-MRI did not reveal any change in the permeability of the BBB, whatever the studied MRI parameter. CONCLUSIONS: The use of hypertonic mannitol infusion seems to be a promising method to increase the delivery of compounds in the margins of GB. Nevertheless, the DCE-MRI analysis method using gadolinium-DOTA as a contrast agent seems of limited value for determining the efficacy of opening the BBB in GB after osmotic shock.

Application of low-intensity ultrasound by opening blood-brain barrier for enhanced brain-targeted drug delivery.

Brain-targeted drug delivery has been a research hotspot, and substantial amount of related studies were already translated into standard therapy and put into clinical use. However, low effective rate retains a huge challenge for brain disease. Because, the blood-brain barrier (BBB) protects brain from pathogenic molecules and tightly controls the process of molecular transportation, which gives rise to poor-liposoluble drugs or molecules with high molecular weight cannot permeate the barrier to exert treating effect. There is an ongoing process to dig out more methods for efficient brain-targeted drug delivery. Besides modified chemical methods such as prodrugs design and brain-targeted nanotechnology, physical methods as a novel initiative could enhance the treatment effect for brain disease. In our study, the influence of low-intensity ultrasound on transient opening BBB and the related applications were explored. A medical ultrasound therapeutic device (1 MHz) was used on heads of mice at different intensities and for different treating time. Evans blue was used as a model to exhibit the permeability of the BBB after subcutaneous injection. Three types of intensities (0.6, 0.8, and 1.0 W/cm2) and duration times (1, 3, and 5 min) of ultrasound were respectively investigated. It was found that the combinations of 0.6 W/cm2/1 min, 0.6 W/cm2/3 min, 0.6 W/cm2/5 min, 0.8 W/cm2/1 min, and 1.0 W/cm2/1 min could open the BBB sufficiently with significant Evans blue staining in the brain. Brain pathological analysis showed structural change on moderate degree was found on cerebral cortex after ultrasound and could recovered rapidly. There are no obvious changes in the behavior of mice after ultrasound processing. More importantly, the BBB recovered quickly at 12 h after ultrasound application with complete BBB structure and unbroken tight junction, suggesting that ultrasound was safe to apply for brain-targeted drug delivery. Proper use of local ultrasound on the brain is a promising technique to open the BBB and enhance brain-targeted delivery.

Development of [177Lu]Lu-LNC1003 for radioligand therapy of prostate cancer with a moderate level of PSMA expression.

PURPOSE: Evans blue as an albumin binder has been widely used to improve pharmacokinetics and enhance tumor uptake of radioligands, including prostate-specific membrane antigen (PSMA) targeting agents. The goal of this study is to develop an optimal Evans blue-modified radiotherapeutic agent that could maximize the absolute tumor uptake and tumor absorbed dose thus the therapeutic efficacy to allow treatment of tumors even with moderate level of PSMA expression. METHODS: [177Lu]Lu-LNC1003 was synthesized based on PSMA-targeting agent and Evans blue. Binding affinity and PSMA targeting specificity were verified through cell uptake and competition binding assay in 22Rv1 tumor model that has moderate level of PSMA expression. SPECT/CT imaging and biodistribution studies in 22Rv1 tumor-bearing mice were performed to evaluate the preclinical pharmacokinetics. Radioligand therapy studies were conducted to systematically assess the therapeutic effect of [177Lu]Lu-LNC1003. RESULTS: LNC1003 showed high binding affinity (IC50 = 10.77 nM) to PSMA in vitro, which was comparable with that of PSMA-617 (IC50 = 27.49 nM) and EB-PSMA-617 (IC50 = 7.91 nM). SPECT imaging of [177Lu]Lu-LNC1003 demonstrated significantly improved tumor uptake and retention as compared with [177Lu]Lu-EB-PSMA and [177Lu]Lu-PSMA-617, making it suitable for prostate cancer therapy. Biodistribution studies further confirmed the remarkably higher tumor uptake of [177Lu]Lu-LNC1003 (138.87 ± 26.53%ID/g) over [177Lu]Lu-EB-PSMA-617 (29.89 ± 8.86%ID/g) and [177Lu]Lu-PSMA-617 (4.28 ± 0.25%ID/g) at 24 h post-injection. Targeted radioligand therapy results showed noteworthy inhibition of 22Rv1 tumor growth after administration of a single dose of 18.5 MBq [177Lu]Lu-LNC1003. There was no obvious antitumor effect after [177Lu]Lu-PSMA-617 treatment under the same condition. CONCLUSION: In this study, [177Lu]Lu-LNC1003 was successfully synthesized with high radiochemical purity and stability. High binding affinity and PSMA targeting specificity were identified in vitro and in vivo. With greatly enhanced tumor uptake and retention, [177Lu]Lu-LNC1003 has the potential to improve therapeutic efficacy using significantly lower dosages and less cycles of 177Lu that promises clinical translation to treat prostate cancer with various levels of PSMA expression.

Effect of Sublethal Concentrations of Zinc Oxide Nanoparticles on Bacillus cereus.

Zinc oxide nanoparticles (ZnONPs), which are produced on a large scale, pose a potential threat to various environments because they can interact with the microbial populations found in them. Bacteria that are widespread in soil, water, and plant material include the Bacillus cereus group, which plays an important role in biodegradation and the nutrient cycle and is a major factor determining ecological balance. This group includes, among others, the foodborne pathogen B. cereus sensu stricto (herein referred to as B. cereus). The aim of this study was a comprehensive assessment of the effects of commercially available ZnONPs on B. cereus. The MIC (minimum inhibitory concentration) for B. cereus was 1.6 mg/mL, and the MBC (minimum bactericidal concentration) was 1.8 mg/mL. Growth of B. cereus was inhibited by a concentration of ZnONPs lower than or equal to MIC50. Concentrations from 0.2 to 0.8 mg/mL inhibited the growth of these bacteria in liquid media, induced symptoms of oxidative stress, and stimulated an environmental stress response in the form of biofilm and endospore formation. In addition, ZnONPs negatively affected the ability of the bacteria to break down the azo dye Evans Blue but enhanced the antimicrobial properties of phenolic compounds. Sublethal concentrations of ZnONPs generally decreased the activity of B. cereus cells, especially in the presence of phenolics, which indicates their potential toxicological impact, but at the same time they induced universal defence responses in these cells, which in the case of potential pathogens can hinder their removal.

A decade of blood-brain barrier permeability assays: Revisiting old traumatic brain injury rat data for new insights and experimental design.

Increased microvascular permeability at the level of the blood-brain barrier (BBB) often leads to vasogenic brain edema following traumatic brain injury (TBI). These pathologic conditions compromise the integrity of the neurovascular unit resulting in severe brain dysfunction. To quantify this permeability and assess ionic equillibrium, preclinical researchers have relied on the use of various molecular weight permeable dyes such as Evans Blue that normally cannot enter the brain parenchyma under homeostatic conditions. Evans Blue, the most cited of the molecular weight dyes, has reported reproducibility issues because of harsh extraction processes, suboptimal detection via absorbance, and wide excitation fluorescence spectra associated with the dye. Our laboratory group transitioned to Alexa Fluor 680, a far-red dye with improved sensitivity compared to Evans Blue and thus improved reproducibility to alleviate this issue. To evaluate our reproducibility and increase the rigor of our experimental design, we retrospectively analyzed our controlled cortical impact (CCI) experiments over the past 10 years to evaluate effect size with larger samples and potential sources of variability. All of our BBB permeability experiments were performed with Male, Sprague Dawley rats weighing between 225 and 300 g. Historically, Sprague Dawleys were randomly divided into treatment groups: SHAM, CCI, and a stem cell-based treatment from years 2007-2020. The assessment of microvascular hyperpermeability were evaluated by comparing the mean at minimum threshold, area at 1 k-2 k, and intensity density obtained from Alexa Fluor 680 permeability data. Studies utilizing Evans Blue were further compared by tip depth, diameter size, and the hemisphere of injury. Statistical evaluation utilizing the G Power software analysis did not yield a significant difference in sample size comparing experimental groups for Evans Blue and Alexa Fluor 680 analyzed brain tissue. Our analysis also demonstrated a trend in that recent studies (years 2018-2020) have yielded more compact sample sizes between experimental groups in Alexa Fluor 680 analyzed rats. This retrospective study further revealed that Alexa Fluor 680 image analysis provides greater sensitivity to BBB permeability following TBI in comparison to Evans Blue. Significant differences in sample size were not detected between Evans Blue and Alexa Fluor 680; there were significant differences found throughout year to year analysis at the lower range of thresholds. SUMMARY STATEMENT: This work provides a comparative analysis of BBB permeability assay techniques after CCI model of injury in rats.

A Single-Arm, Low-Dose, Prospective Study of 177Lu-EB-PSMA Radioligand Therapy in Patients with Metastatic Castration-Resistant Prostate Cancer.

We aimed to investigate the safety and therapeutic efficacy of radioligand therapy (RLT) of 177Lu-EB-prostate-specific membrane antigen (PSMA) in patients with metastatic castration-resistant prostate cancer. Methods: Thirty men with progressive metastatic castration-resistant prostate cancer previously treated with taxane-based chemotherapy and second-generation androgen deprivation therapy were enrolled. All patients received up to 3 cycles of approximately 2.0 GBq (55 mCi) of 177Lu-EB-PSMA per cycle at 8-wk intervals. The primary endpoint was therapeutic safety, including changes in hematologic status, liver function, and renal function. An additional primary endpoint was therapeutic efficacy, including prostate-specific antigen (PSA) response and molecular imaging response. The secondary endpoints were PSA progression-free survival (PFS) and overall survival (OS). Another endpoint was patient-reported health-related quality of life. Results: From January 2019 to December 2021, 30, 22, and 11 patients received 1, 2, or 3 cycles of 177Lu-EB-PSMA RLT, respectively. During the entire follow-up period, 33.3% of patients experienced grade 3 hematologic adverse events. Seventeen (56.7%) patients achieved a PSA reduction of at least 50%. The median PSA PFS was 4.6 mo (95% CI, 2.7-6.5 mo), and the median OS was 12.6 mo (95% CI, 8.1-17.1 mo). A higher whole-body PSMA SUVmean correlated with a better PSA response, higher baseline alkaline phosphatase and larger total PSMA-positive tumor volume were associated with worse PSA PFS, and the existence of visceral metastases and higher PSA value at baseline were significant prognosticators of worse OS. Health-related quality-of-life outcomes improved significantly after 177Lu-EB-PSMA RLT. Conclusion: RLT based on approximately 2.0 GBq of 177Lu-EB-PSMA for up to 3 cycles may achieve a PSA response and hematologic toxicity comparable to those from 7.4-GBq doses of 177Lu-PSMA-617 for up to 4-6 cycles. Further studies with more cycles of 177Lu-EB-PSMA RLT are needed to evaluate the potential benefits in terms of PFS and OS.

Enhancing Techniques for Determining Inflammatory Edema Formation and Neutrophil Accumulation in Murine Skin.

Inflammatory edema formation and polymorphonuclear leukocyte (neutrophil) accumulation are common components of cutaneous vascular inflammation, and their assessment is a powerful investigative and drug development tool but typically requires independent cohorts of animals to assess each. We have established the use of a mathematical formula to estimate the ellipsoidal-shaped volume of the edematous wheal or bleb after intradermal injections of substances in mice pretreated intravenously with Evans blue dye (which binds to plasma albumin) to act as an edema marker. Whereas previous extraction of Evans blue dye with formamide is suitable for all strains of mice, we report this quicker and more reliable assessment of edema volume in situ. This therefore allows neutrophil accumulation to be assessed from the same mouse using the myeloperoxidase assay. Importantly, we examined the influence of Evans blue dye on the spectrometry readout at the wavelength at which myeloperoxidase activity is measured. The results indicate that it is feasible to quantify edema formation and neutrophil accumulation in the same mouse skin site. Thus, we show techniques that can assess edema formation and neutrophil accumulation at the same site in the same mouse, allowing paired measurements and reducing the total use of mice by 50%.

Cognitive Impairments and blood-brain Barrier Damage in a Mouse Model of Chronic Cerebral Hypoperfusion.

Chronic cerebral hypoperfusion (CCH) is commonly involved in various brain diseases. Tight junction proteins (TJs) are key components constituting the anatomical substrate of the blood-brain barrier (BBB). Changes in cognitive function and BBB after CCH and their relationship need further exploration. To investigate the effect of CCH on cognition and BBB, we developed a bilateral common carotid artery stenosis (BCAS) model in Tie2-GFP mice. Mice manifested cognitive impairments accompanied with increased microglia after the BCAS operation. BCAS mice also exhibited increased BBB permeability at all time points set from D1 to D42. Furthermore, BCAS mice showed reduced expression of TJs 42 d after the operation. In addition, correct entrances of mice in radial arm maze test had a moderate negative correlation with EB extravasation. Our data suggested that BCAS could lead to cognitive deficits, microglia increase and BBB dysfunction characterized by increased BBB permeability and reduced TJs expression level. BBB permeability may be involved in the cognitive impairments induced by CCH.