Single inhalation exposure to polyamide micro and nanoplastic particles impairs vascular dilation without generating pulmonary inflammation in virgin female Sprague Dawley rats.

BACKGROUND: Exposure to micro- and nanoplastic particles (MNPs) in humans is being identified in both the indoor and outdoor environment. Detection of these materials in the air has made inhalation exposure to MNPs a major cause for concern. One type of plastic polymer found in indoor and outdoor settings is polyamide, often referred to as nylon. Inhalation of combustion-derived, metallic, and carbonaceous aerosols generate pulmonary inflammation, cardiovascular dysfunction, and systemic inflammation. Additionally, due to the additives present in plastics, MNPs may act as endocrine disruptors. Currently there is limited knowledge on potential health effects caused by polyamide or general MNP inhalation. OBJECTIVE: The purpose of this study is to assess the toxicological consequences of a single inhalation exposure of female rats to polyamide MNP during estrus by means of aerosolization of MNP. METHODS: Bulk polyamide powder (i.e., nylon) served as a representative MNP. Polyamide aerosolization was characterized using particle sizers, cascade impactors, and aerosol samplers. Multiple-Path Particle Dosimetry (MPPD) modeling was used to evaluate pulmonary deposition of MNPs. Pulmonary inflammation was assessed by bronchoalveolar lavage (BAL) cell content and H&E-stained tissue sections. Mean arterial pressure (MAP), wire myography of the aorta and uterine artery, and pressure myography of the radial artery was used to assess cardiovascular function. Systemic inflammation and endocrine disruption were quantified by measurement of proinflammatory cytokines and reproductive hormones. RESULTS: Our aerosolization exposure platform was found to generate particles within the micro- and nano-size ranges (thereby constituting MNPs). Inhaled particles were predicted to deposit in all regions of the lung; no overt pulmonary inflammation was observed. Conversely, increased blood pressure and impaired dilation in the uterine vasculature was noted while aortic vascular reactivity was unaffected. Inhalation of MNPs resulted in systemic inflammation as measured by increased plasma levels of IL-6. Decreased levels of 17ß-estradiol were also observed suggesting that MNPs have endocrine disrupting activity. CONCLUSIONS: These data demonstrate aerosolization of MNPs in our inhalation exposure platform. Inhaled MNP aerosols were found to alter inflammatory, cardiovascular, and endocrine activity. These novel findings will contribute to a better understanding of inhaled plastic particle toxicity.

Development of a Device-Assisted Nerve-Regeneration Procedure in Disruptive Lesions of the Brachial Plexus.

BACKGROUND: We describe the development of a new surgical procedure to be used in the treatment of disruptive brachial plexus (BP) lesions. It is centered on an artificial device designed to assist nerve regeneration by providing a confined and protected environment. Nerve fibers can repair inside the device, while the adverse massive scar-tissue formation is limited to the outside of the device. METHODS: Steps in the development of the procedure were (1) definition of the rationale, (2) design of the device, (3) choice of an in vivo translational model, (4)refinement of the surgical procedure, and (5) performance of an in vivo pilot study as a proof of concept. An interdisciplinary team from several laboratories was involved in this work over a period of 6 years. RESULTS: Results showed the absence of significant scar tissue in the regenerate and the presence of myelinated fibers aligned proximodistally between the stumps. This surgical approach can be seen not only as a definitive treatment but also as an early examination and stabilization before some different surgery will be later performed. It may also be used as additional protection for traditional surgery like end-to-end coaptation. CONCLUSIONS: We conclude that the availability of a suitable device-assisted early treatment, even if not to be considered definitive, could help in addressing the BP lesions at an earlier stage and this may improve the final outcome. Our evidence justifies further experimentation on this approach.

A high affinity and specificity anti-HER2 single-domain antibody (VHH) that targets trastuzumab's epitope with versatile biochemical, biological, and medical applications.

In the past decade, various single-domain antibodies from llamas, also known as VHH or nanobody, have been discovered with applications in tumor imaging and cancer therapy. However, the potential application of anti-HER2 VHHs as a diagnostic tool suitable for ELISA, flow cytometry, cell imaging, bispecific antibody engineering, and immunohistochemistry has not been fully elucidated. To investigate this potential, HER2 antigen was expressed in HEK293 F cells, purified, and used to immunize llama. Using phage display, anti-HER2 VHHs with high affinity and specificity were isolated, sequenced, and constructed with a Histag and c-Myc tag. The constructed anti-HER2 VHHs were then expressed in E. coli, purified, and evaluated for their use in ELISA, flow cytometry, cell imaging, and immunohistochemistry. The affinities of the anti-HER2 VHHs toward the HER2 antigen were determined using biolayer interferometry. Furthermore, the binding sites of the anti-HER2 VHHs were evaluated by epitope mapping and in silico modeling and docking. Here, we report the sequence of an anti-HER2 VHH with high affinity (sub-nanomolar), specificity, and selectivity. This VHH binds to the same epitope as trastuzumab and can be utilized to generate bispecific antibodies or used as a diagnostic tool to differentiate HER2+ from HER2- antigens on plates, cells, and tissues. This discovery has broad applications in biochemical, biological, and medical sciences.

Isothiocyanate-rich moringa seed extract reduces skin inflammation in mouse ear edema model.

Background: Moringa (Moringa oleifera Lam.) seed extract (MSE) and its primary bioactive compound, moringa isothiocyanate-1(MIC-1), mitigate inflammation, oxidative stress, diabetes, and cancer in the in vivo rodent models following oral application. Purpose: To investigate the topical anti-inflammatory activity of MSE and purified MIC-1 in a TPA-induced mouse ear edema model. Study Design: The present study elucidates the topical anti-inflammatory effects and mechanisms of action of MSE, containing 38% of MIC-1 and purified MIC-1 using a mouse ear edema model utilizing 12-O-tetradecanoylphorbol-13-acetate (TPA), as the pro-inflammatory agent. Methods: A time-dependent and dose-dependent response was determined by pretreating CD-1 mice with various doses of MSE and MIC-1, positive control, dexamethasone, or vehicle control, followed by TPA, and the subsequent difference in ear thickness was measured using digital Vernier calipers. The effective doses of MSE and MIC-1were then selected to evaluate the change in weight of the ears using 6 mm biopsy punches and the results were confirmed by microscopy. Inflammatory markers were quantified with Luminex multiplex immunoassay. Results: MSE and MIC-1 were effective in a dose-dependent manner in a TPA-induced ear edema model, causing a reduction in ear thickness and a 48% and 49% decrease in ear punch weight, respectively. MSE and MIC-1 also caused a reduction in the levels of cytokine and chemokines, interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and keratinocyte chemoattractant (KC) in the ear tissue. MSE and MIC-1 reduced IL-6 expression by 84% and 78%, MCP1 by 74% and 73%, and KC by 56% and 43%, respectively. Additionally, the anti-inflammatory effect of MSE and MIC-1 was confirmed by hematoxylin and eosin (H&E) staining, used to assess the thickness of the ear swelling. MSE significantly reduced the thickness of the ears by 20% compared to TPA. Conclusion: These results reveal the topical anti-inflammatory properties of MSE, and MIC-1 likely transmitted via the nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor-kappa B (NF-κB) pathways as mentioned in previous studies. This work also suggests therapeutic uses of MSE and/or MIC-1 for skin inflammation.

Ingested Polystyrene Nanospheres Translocate to Placenta and Fetal Tissues in Pregnant Rats: Potential Health Implications.

Recent studies in experimental animals found that oral exposure to micro- and nano-plastics (MNPs) during pregnancy had multiple adverse effects on outcomes and progeny, although no study has yet identified the translocation of ingested MNPs to the placenta or fetal tissues, which might account for those effects. We therefore assessed the placental and fetal translocation of ingested nanoscale polystyrene MNPs in pregnant rats. Sprague Dawley rats (N = 5) were gavaged on gestational day 19 with 10 mL/kg of 250 µg/mL 25 nm carboxylated polystyrene spheres (PS25C) and sacrificed after 24 h. Hyperspectral imaging of harvested placental and fetal tissues identified abundant PS25C within the placenta and in all fetal tissues examined, including liver, kidney, heart, lung and brain, where they appeared in 10-25 µm clusters. These findings demonstrate that ingested nanoscale polystyrene MNPs can breach the intestinal barrier and subsequently the maternal-fetal barrier of the placenta to access the fetal circulation and all fetal tissues. Further studies are needed to assess the mechanisms of MNP translocation across the intestinal and placental barriers, the effects of MNP polymer, size and other physicochemical properties on translocation, as well as the potential adverse effects of MNP translocation on the developing fetus.

Pregnane X receptor exacerbates nonalcoholic fatty liver disease accompanied by obesity- and inflammation-prone gut microbiome signature.

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease due to the current epidemics of obesity and diabetes. The pregnane X receptor (PXR) is a xenobiotic-sensing nuclear receptor known for trans-activating liver genes involved in drug metabolism and transport, and more recently implicated in energy metabolism. The gut microbiota can modulate the host xenobiotic biotransformation and contribute to the development of obesity. While the male sex confers a higher risk for NAFLD than women before menopause, the mechanism remains unknown. We hypothesized that the presence of PXR promotes obesity by modifying the gut-liver axis in a sex-specific manner. Male and female C57BL/6 (wild-type/WT) and PXR-knockout (PXR-KO) mice were fed control or high-fat diet (HFD) for 16-weeks. Serum parameters, liver histopathology, transcriptomic profiling, 16S-rDNA sequencing, and bile acid (BA) metabolomics were performed. PXR enhanced HFD-induced weight gain, hepatic steatosis and inflammation especially in males, accompanied by PXR-dependent up-regulation in hepatic genes involved in microbial response, inflammation, oxidative stress, and cancer; PXR-dependent increase in intestinal Firmicutes/Bacteroides ratio (hallmark of obesity) and the pro-inflammatory Lactobacillus, as well as a decrease in the anti-obese Allobaculum and the anti-inflammatory Bifidobacterum, with a PXR-dependent reduction of beneficial BAs in liver. The resistance to NAFLD in females may be explained by PXR-dependent decrease in pro-inflammatory bacteria (Ruminococcus gnavus and Peptococcaceae). In conclusion, PXR exacerbates hepatic steatosis and inflammation accompanied by obesity- and inflammation-prone gut microbiome signature, suggesting that gut microbiome may contribute to PXR-mediated exacerbation of NAFLD.

The role of maternal high fat diet on mouse pup metabolic endpoints following perinatal PFAS and PFAS mixture exposure.

Per- and polyfluoroalkyl substances (PFAS) are a family of chemicals that are ubiquitous in the environment. Some of these chemicals, such as perfluorooctanesulfonic acid (PFOS), perfluorohexanesulfonate (PFHxS) and perfluorooctanoic acid (PFOA), are found in human sera and have been shown to cause liver steatosis and reduce postnatal survival and growth in rodents. The purpose of this work is to evaluate the impact of diet and PFAS exposure to mouse dam (mus musculus) on the risk to pup liver and metabolism endpoints later in life, as well as evaluate PFAS partitioning to pups. Timed-pregnant dams were fed a standard chow diet or 60 % kcal high fat diet (HFD). Dams were administered either vehicle, 1 mg/kg PFOA, 1 mg/kg PFOS, 1 mg/kg PFHxS, or a PFAS mixture (1 mg/kg of each PFOA, PFOS, and PFHxS) daily via oral gavage from gestation day 1 until postnatal day (PND) 20. At PND 21, livers of dams and 2 pups of each sex were evaluated for lipid changes while remaining pups were weaned to the same diet as the dam for an additional 10 weeks. Dam and pup serum at PND 21 and PND 90 were also evaluated for PFAS concentration, alanine aminotransferase (ALT), leptin and adiponectin, and glycosylated hemoglobin A1c. Perinatal exposure to a HFD, as expected, increased pup body weight, maternal liver weight, pup liver triglycerides, pup serum ALT, and pup serum leptin. PFOA and the PFAS mixture increased liver weights, and. treatment with all three compounds increased liver triglycerides. The maternal HFD increased dam and pup serum PFAS levels, however, was protective against PFOA-induced increase in serum ALT and observed increases in liver triglycerides. The PFAS mixture had very distinct effects when compared to single compound treatment, suggesting some cumulative effects, particularly when evaluating PFAS transfer from dam to pup. This data highlights the importance of diet and mixtures when evaluating liver effect of PFAS and PFAS partitioning.

Acute feeding suppression and toxicity of raspberry ketone [4-(4-hydroxyphenyl)-2-butanone] in mice.

Raspberry ketone (RK; [4-(4-hydroxyphenyl)-2-butanone]) is used by the food and cosmetic industry as a flavoring agent. RK is also marketed as a dietary supplement for weight maintenance and appetite control. The purpose of the study was to characterize the acute feeding suppression with RK (64-640 mg/kg) by oral gavage in male and female C57BL/6J mice. Cumulative 24 h food intake was reduced at 200 mg/kg (24% feeding suppression) in males and reliably reduced at 640 mg/kg (49-77% feeding suppression). Feeding suppression was not associated with pica behavior over the range of doses or conditioned taste aversion. In a separate experiment, a single oral gavage of RK (640 mg/kg) resulted in approximate 43% mortality rate (6 out 14 male mice) within 2 days. Atrophy of white adipose tissue, splenic abnormalities, and thymus involution were noted after 2-4 days after oral gavage RK. Total white blood cell count, lymphocytes, monocytes, eosinophils were significantly lower, while mean red blood cells, hemoglobin, and hematocrit were significantly higher with RK treatment. Our findings indicated a dose-dependent feeding suppression with acute RK, but doses that reliable suppress food intake are associated with pathological changes.

4D-Printed Transformable Tube Array for High-Throughput 3D Cell Culture and Histology.

3D cell cultures are rapidly emerging as a promising tool to model various human physiologies and pathologies by closely recapitulating key characteristics and functions of in vivo microenvironment. While high-throughput 3D culture is readily available using multi-well plates, assessing the internal microstructure of 3D cell cultures still remains extremely slow because of the manual, laborious, and time-consuming histological procedures. Here, a 4D-printed transformable tube array (TTA) using a shape-memory polymer that enables massively parallel histological analysis of 3D cultures is presented. The interconnected TTA can be programmed to be expanded by 3.6 times of its printed dimension to match the size of a multi-well plate, with the ability to restore its original dimension for transferring all cultures to a histology cassette in order. Being compatible with microtome sectioning, the TTA allows for parallel histology processing for the entire samples cultured in a multi-well plate. The test result with human neural progenitor cell spheroids suggests a remarkable reduction in histology processing time by an order of magnitude. High-throughput analysis of 3D cultures enabled by this TTA has great potential to further accelerate innovations in various 3D culture applications such as high-throughput/content screening, drug discovery, disease modeling, and personalized medicine.

Coencapsulation of ISCs and MSCs Enhances Viability and Function of both Cell Types for Improved Wound Healing.

INTRODUCTION: We previously demonstrated that insulin secreting cells (ISCs) accelerate healing of chronic wounds, and it is known that mesenchymal stem cells (MSCs) also accelerate wound healing. Here, we report that the combination of both cell types coencapsulated into a synthetic hydrogel dressing accelerates chronic wound healing 3 × faster than control and 2 × faster than each cell type delivered singly. Specifically, insulin released by ISCs activates the PI3/Akt pathway, which is vital to the function and survival of MSCs. MSCs in turn improve the viability and function of ISCs. MATERIALS AND METHODS: MSCs and/or rat islet tumor RIN-m cells were encapsulated into polyethylene glycol diacrylate hydrogel sheets and applied to 1 cm2 full thickness excisional wounds on the dorsa of genetically diabetic male mice (BKS.Cg-m +/+Leprdb/J) in accordance with protocols approved by the Rutgers IACUC. Encapsulated cell viability was assessed using a LIVE/DEAD® Viability/Cytotoxicity Kit. Akt phosphorylation, insulin, VEGF, and TGF-ß1 secretion were assessed by ELISA. Animals were sacrificed on postoperative days 14 and 28 and wound tissue was collected for histological and western blot analysis. RESULTS: ISC:MSC combination groups had the highest levels of every secreted product and phosphorylated Akt, and closed wounds in 14 days, ISC-only or MSC-only groups closed wounds in 28 days, control groups closed wounds in 40 days. Further, ISC:MSC groups healed without intermediate scab or scar. CONCLUSIONS: Combining MSCs with ISCs results in a more robust healing response than singly delivered cells, warranting further investigation of coencapsulation for MSC therapies.

Bioengineered adipose-derived stem cells for targeted enzyme-prodrug therapy of ovarian cancer intraperitoneal metastasis.

The objective of this study was to develop a stem cell-based system for targeted suicide gene therapy of recurrent, metastatic, and unresectable ovarian cancer. Malignant cells were obtained from the ascites of a patient with advanced recurrent epithelial ovarian cancer (named OVASC-1). Cancer cells were characterized to determine the percentages of drug-resistant ALDH+ cells, MDR-1/ABCG2 overexpressing cells, and cancer stem-like cells. The sensitivity and resistance of the OVASC-1 cells and spheroids to the metabolites of three different enzyme/prodrug systems were assessed, and the most effective one was selected. Adipose-derived stem cells (ASCs) were genetically engineered to express recombinant secretory human carboxylesterase-2 and nanoluciferase genes for simultaneous disease therapy and quantitative imaging. Bioluminescent imaging, magnetic resonance imaging and immuno/histochemistry results show that the engineered ASCs actively targeted and localized at both tumor stroma and necrotic regions. This created the unique opportunity to deliver drugs to not only tumor supporting cells in the stroma, but also to cancer stem-like cells in necrotic/hypoxic regions. The statistical analysis of intraperitoneal OVASC-1 tumor burden and survival rates in mice shows that the administration of the bioengineered ASCs in combination with irinotecan prodrug in the designed sequence and timeline eradicated all intraperitoneal tumors and provided survival benefits. In contrast, treatment of the drug-resistant OVASC-1 tumors with cisplatin/paclitaxel (standard-of-care) did not have any statistically significant benefit. The histopathology and hematology results do not show any toxicity to major peritoneal organs. Our toxicity data in combination with efficacy outcomes delineate a nonsurgical and targeted stem cell-based approach to overcoming drug resistance in recurrent metastatic ovarian cancer.

Editor's Highlight: Farnesoid X Receptor Protects Against Low-Dose Carbon Tetrachloride-Induced Liver Injury Through the Taurocholate-JNK Pathway.

Hepatotoxicity is of major concern for humans exposed to industrial chemicals and drugs. Disruption of farnesoid X receptor (FXR), a master regulator of bile acid (BA) metabolism, enhanced the sensitivity to liver injury in mice after toxicant exposure, but the precise mechanism remains unclear. In this study, the interconnection between BA metabolism, FXR, and chemically induced hepatotoxicity was investigated using metabolomics, Fxr-null mice (Fxr-/-) and hepatocytes, and recombinant adenoviruses. A single low-dose intraperitoneal injection of carbon tetrachloride (CCl4), an inducer of acute hepatitis in mice, resulted in more severe hepatocyte damage and higher induction of pro-inflammatory mediators, such as chemokine (C-C motif) ligand 2 (Ccl2), in Fxr-/-. Serum metabolomics analysis revealed marked increases in circulating taurocholate (TCA) and tauro-ß-muricholate (T-ß-MCA) in these mice, and forced expression of bile salt export protein (BSEP) by recombinant adenovirus in Fxr-/- ameliorated CCl4-induced liver damage. Treatment of Fxr-null hepatocytes with TCA, but not T-ß-MCA, significantly increased c-Jun-N-terminal kinase (JNK) activation and Ccl2 mRNA levels, and up-regulation of Ccl2 mRNA was attenuated by co-treatment with a JNK inhibitor SP600125, indicating that TCA directly amplifies hepatocyte inflammatory signaling mainly mediated by JNK under FXR-deficiency. Additionally, pretreatment with SP600125 or restoration of FXR expression in liver by use of recombinant adenovirus, attenuated CCl4-induced liver injury. Collectively, these results suggest that the TCA-JNK axis is likely associated with increased susceptibility to CCl4-induced acute liver injury in Fxr-/-, and provide clues to the mechanism by which FXR and its downstream gene targets, such as BSEP, protects against chemically induced hepatotoxicity.

Novel cystine ester mimics for the treatment of cystinuria-induced urolithiasis in a knockout mouse model.

OBJECTIVE: To assess the effectiveness of l-cystine dimethyl ester (CDME), an inhibitor of cystine crystal growth, for the treatment of cystine urolithiasis in an Slc3a1 knockout mouse model of cystinuria. MATERIALS AND METHODS: CDME (200 µg per mouse) or water was delivered by gavage daily for 4 weeks. Higher doses by gavage or in the water supply were administered to assess organ toxicity. Urinary amino acids and cystine stones were analyzed to assess drug efficacy using several analytical methods. RESULTS: Treatment with CDME led to a significant decrease in stone size compared with that of the water group (P = .0002), but the number of stones was greater (P = .005). The change in stone size distribution between the 2 groups was evident by micro computed tomography. Overall, cystine excretion in urine was the same between the 2 groups (P = .23), indicating that CDME did not interfere with cystine metabolism. Scanning electron microscopy analysis of cystine stones from the CDME group demonstrated a change in crystal habit, with numerous small crystals. l-cysteine methyl ester was detected by ultra-performance liquid chromatography-mass spectrometer in stones from the CDME group only, indicating that a CDME metabolite was incorporated into the crystal structure. No pathologic changes were observed at the doses tested. CONCLUSION: These data demonstrate that CDME promotes formation of small stones but does not prevent stone formation, consistent with the hypothesis that CDME inhibits cystine crystal growth. Combined with the lack of observed adverse effects, our findings support the use of CDME as a viable treatment for cystine urolithiasis.

Role of oxidative stress and the mitochondrial permeability transition in methylmercury cytotoxicity.

Oxidative stress has been implicated in the pathogenesis of methylmercury (MeHg) neurotoxicity. Studies of mature neurons suggest that the mitochondrion may be a major source of MeHg-induced reactive oxygen species and a critical mediator of MeHg-induced neuronal death, likely by activation of apoptotic pathways. It is unclear, however, whether the mitochondria of developing and mature neurons are equally susceptible to MeHg. Murine embryonal carcinoma (EC) cells, which differentiate into neurons following exposure to retinoic acid, were used to compare the differentiation-dependent effects of MeHg on ROS production and mitochondrial depolarization. EC cells and their neuronal derivatives were pre-incubated with the ROS indicator 2',7'-dichlorofluoroscein diacetate or tetramethylrhodamine methyl ester, an indicator of mitochondrial membrane potential, with or without cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore opening, and examined by laser scanning confocal microscopy in the presence of 1.5 µM MeHg. To examine consequences of mitochondrial perturbation, immunohistochemical localization of cytochrome c (cyt c) was determined after incubation of cells in MeHg for 4 h. MeHg treatment induced earlier and significantly higher levels of ROS production and more extensive mitochondrial depolarization in neurons than in undifferentiated EC cells. CsA completely inhibited mitochondrial depolarization by MeHg in EC cells but only delayed this response in the neurons. In contrast, CsA significantly inhibited MeHg-induced neuronal ROS production. Cyt c release was also more extensive in neurons, with less protection afforded by CsA. These data indicate that neuronal differentiation state influences mitochondrial transition pore dynamics and MeHg-stimulated production of ROS.