Lung Sections Research Articles

Kiss1 receptor knockout exacerbates airway hyperresponsiveness and remodeling in a mouse model of allergic asthma

BackgroundIn asthma, sex-steroids signaling is recognized as a critical regulator of disease pathophysiology. However, the paradoxical role of sex-steroids, especially estrogen, suggests that an upstream mechanism or even independent of estrogen plays an important role in regulating asthma pathophysiology. In this context, in our previous studies, we explored kisspeptin (Kp) and its receptor Kiss1R’s signaling in regulating human airway smooth muscle cell remodeling in vitro and airway hyperresponsiveness (AHR) in vivo in a mouse (wild-type, WT) model of asthma. In this study, we evaluated the effect of endogenous Kp in regulating AHR and remodeling using Kiss1R knockout (Kiss1R−/−) mice.MethodsC57BL/6J WT (Kiss1R+/+) and Kiss1R−/− mice, both male and female, were intranasally challenged with mixed-allergen (MA) and/or phosphate-buffered saline (PBS). We used flexiVent analysis to assess airway resistance (Rrs), elastance (Ers), and compliance (Crs). Following this, broncho-alveolar lavage (BAL) was performed for differential leukocyte count (DLC) and cytokine analysis. Histology staining was performed using hematoxylin and eosin (H&E) for morphological analysis and Masson’s Trichrome (MT) for collagen deposition. Additionally, lung sections were processed for immunofluorescence (IF) of Ki-67, α-smooth muscle actin (α-SMA), and tenascin-c.ResultsInterestingly, the loss of Kiss1R exacerbated lung function and airway contractility in mice challenged with MA, with more profound effects in Kiss1R−/− female mice. MA-challenged Kiss1R−/− mice showed a significant increase in immune cell infiltration and proinflammatory cytokine levels. Importantly, the loss of Kiss1R aggravated Th2/Th17 biased cytokines in MA-challenged mice. Furthermore, histology of lung sections from Kiss1R−/− mice showed increased collagen deposition on airway walls and mucin production in airway cells compared to Kiss1R+/+ mice. In addition, immunofluorescence analysis showed loss of Kiss1R significantly aggravated airway remodeling and subsequently AHR.ConclusionsThese findings demonstrate the importance of inherent Kiss1R signaling in regulating airway inflammation, AHR, and remodeling in the pathophysiology of asthma.

Cardiac metastasis from carcinoma gall bladder - a rare case detected on autopsy

Metastasis to the heart is uncommon compared to other organs, posing notable diagnostic challenges. Incidence in autopsy studies typically ranges between 0.7% and 3.5%. Lung, breast, and hematologic cancers are the main sources of such metastases, leading to varied clinical presentations and severe complications. Postmortem viscera from a 47 year old male with a suspected history of jaundice. Only limited information was available. Gross examination showed a solid mass in the gall bladder with areas of hemorrhage and necrosis, while no other abnormalities were observed in other viscera received. Brain examination showed congested blood vessels, while heart sections exhibited atypical cell deposits in the pericardium and myocardium. Coronary arteries displayed pathological thickening. Lung and liver sections also displayed similar metastatic atypical cell deposits, infiltrating tissue and causing necrosis. Gall bladder examination revealed irregular glandular formations lined with polygonal tumor cells positive for cytokeratin. The final diagnosis based on histomorphological features and immunohistochemistry confirmed gall bladder adenocarcinoma metastasising to the heart, liver and lungs.

Nanoparticle delivery of VEGF and SDF-1α as an approach for treatment of pulmonary arterial hypertension.

Endothelial dysfunction is an underlying mechanism for the development of pulmonary arterial hypertension (PAH). Vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF) may help repair the dysfunctional endothelium and provide treatment for PAH. To examine this possibility, nanoparticles carrying human recombinant VEGF and SDF (VEGFNP and SDFNP) were aerosolized into the lungs of nude rats at Day 14 after monocrotaline (MCT) injection and analyses were performed at Day 28. The data show that the VEGFNP/SDFNP delivery led to a lower pulmonary arterial pressure and prevented right ventricular hypertrophy in the MCT rats: the right ventricular systolic pressure of the control, MCT, and MCT + VEGFNP/SDFNP treatment groups were 29±2, 70±9, and 44±5 (mean±SD) mmHg, respectively; the pulmonary vascular resistance indices of the groups were 0.6±0.3, 3.2±0.7, and 1.7±0.5, respectively; and the Fulton indices [-RV/(LV + Septum)] were 0.22±0.01, 0.44±0.07, and 0.23±0.02, respectively. The VEGFNP/SDFNP delivery delayed the thickening of distal pulmonary vessels: the number of nearly occluded vessels in a whole lung section from the MCT and MCT + VEGFNP/SDFNP groups were 46±12 and 2±3, respectively. Gene expression analysis of the endothelial cell markers, VE-cadherin, KDR, BMPR2, and eNOS, and smooth cell markers, SM-MHC and α-SMA, indicated significant loss of distal pulmonary vessels in the MCT- treated rats. VEGFNP/SDFNP delivery did not recover the loss, but significantly increased eNOS and decreased α-SMA expression in the MCT-treated lungs. Thus, the therapeutic effect of VEGFNP/SDFNP may be mediated by improving/repairing endothelial function in the PAH lungs.

Establishment of a stem cell administration imaging method in bleomycin-induced pulmonary fibrosis mouse models

Pulmonary fibrosis is a progressive disease caused by interstitial inflammation. Treatments are extremely scarce; therapeutic drugs and transplantation therapies are not widely available due to cost and a lack of donors, respectively. Recently, there has been a high interest in regenerative medicine and exponential advancements in stem cell-based therapies have occurred. However, a sensitive imaging technique for investigating the in vivo dynamics of transplanted stem cells has not yet been established and the mechanisms of stem cell-based therapy remain largely unexplored. In this study, we administered mouse adipose tissue-derived mesenchymal stem cells (mASCs) labeled with quantum dots (QDs; 8.0 nM) to a mouse model of bleomycin-induced pulmonary fibrosis in an effort to clarify the relationship between in vivo dynamics and therapeutic efficacy. These QD-labeled mASCs were injected into the trachea of C57BL/6 mice seven days after bleomycin administration to induce fibrosis in the lungs. The therapeutic effects and efficacy were evaluated via in vivo/ex vivo imaging, CT imaging, and H&E staining of lung sections. The QD-labeled mASCs remained in the lungs longer and suppressed fibrosis. The 3D imaging results showed that the transplanted cells accumulated in the peripheral and fibrotic regions of the lungs. These results indicate that mASCs may prevent fibrosis. Thus, QD labeling could be a suitable and sensitive imaging technique for evaluating in vivo kinetics in correlation with the efficacy of cell therapy.

Methods for Detecting Cough and Airway Inflammation in Mice.

Chronic cough, which lasts for more than 8 weeks, is one of the most common complaints requiring medical attention, and patients suffer from a huge socioeconomic burden and a marked decrement in quality of life. Animal models can mimic the complex pathophysiology of the cough and are important tools for cough research. The detection of cough sensitivity and airway inflammation is of great significance for studying the complex pathological mechanism of cough. This article describes the measurement of cough using a noninvasive and real-time whole-body plethysmography (WBP) system and the normative procedures for harvesting tissue samples (including blood, lung, spleen, and trachea) of mice. It introduces some methods to assess airway inflammation, including pathological changes in hematoxylin and eosin (HE)-stained lung and trachea sections, the total protein concentration, the uric acid concentration, and the lactate dehydrogenase (LDH) activity in the supernatant of bronchoalveolar lavage fluid (BALF), and the leukocytes and differential cell counts of BALF. These methods are reproducible and serve as valuable tools to study the complex pathophysiology of cough.

Fibroblast activation protein is a cellular marker of fibrotic activity in canine idiopathic pulmonary fibrosis.

Canine idiopathic pulmonary fibrosis (CIPF) is a progressive fibrotic interstitial lung disease of unknown etiology, afflicting aging West Highland white terriers (WHWTs) and leading to progressive respiratory failure. Fibroblast activation protein (FAP), a protease overexpressed in many cancers, is upregulated in idiopathic pulmonary fibrosis in humans. The aim of this study was to investigate FAP as a marker of active fibrosis in lung biopsies from WHWTs affected with CIPF, as well as the potential of plasmatic FAP as a biomarker. After establishing a scoring system to evaluate the severity and activity of fibrosis on histopathological lung sections, anti-FAP immunohistochemistry was performed on healthy and CIPF samples. FAP expression was characterized using both visual and digital quantitative pathology software analyses and then correlated to fibrosis severity and activity. Levels of plasmatic FAP in WHWTs affected with CIPF were measured by enzyme-linked immunosorbent assay and compared with healthy dogs. Lung samples from 22 WHWTs affected with CIPF were collected. According to the fibrosis scoring system, they were classified as cases of mild (5), moderate (9) and severe (8) fibrosis and were attributed scores of fibrosis activity. Fifteen healthy lung samples were classified as non-fibrotic. Healthy lung samples were FAP-negative, whereas fibroblasts were FAP-positive in 20 CIPF samples. FAP immunohistochemical expression correlated mildly with fibrosis severity (p < 0.05; R 2 = 0.22) but highly with fibrosis activity scores (p < 0.001; R 2 = 0.68). Digital image analysis detected a higher percentage of FAP-positive cells in areas of active fibrosis (p < 0.001) and FAP-positive cells were distributed outside mature fibrosis lesions, clustered in active fibrosis areas or scattered within alveolar septa. On the other hand, plasmatic FAP was significantly lower in dogs affected with CIPF compared with healthy dogs (p < 0.01). In conclusion, this study provides a valuable histological scoring system to assess the severity and activity of fibrosis in CIPF. It demonstrates that FAP is a good cellular marker of fibrotic activity in CIPF, and thus constitutes a promising target to be exploited for diagnostic and therapeutic applications. Additionally, it suggests that plasmatic FAP, although non-specific, could be altered in CIPF.

MMP-3 mediates copper oxide nanoparticle-induced pulmonary inflammation and fibrosis

BackgroundThe increasing production and usage of copper oxide nanoparticles (Nano-CuO) raise human health concerns. Previous studies have demonstrated that exposure to Nano-CuO could induce lung inflammation, injury, and fibrosis. However, the potential underlying mechanisms are still unclear. Here, we proposed that matrix metalloproteinase-3 (MMP-3) might play an important role in Nano-CuO-induced lung inflammation, injury, and fibrosis.ResultsExposure of mice to Nano-CuO caused acute lung inflammation and injury in a dose-dependent manner, which was reflected by increased total cell number, neutrophil count, macrophage count, lactate dehydrogenase (LDH) activity, and CXCL1/KC level in bronchoalveolar lavage fluid (BALF) obtained on day 3 post-exposure. The time-response study showed that Nano-CuO-induced acute lung inflammation and injury appeared as early as day 1 after exposure, peaked on day 3, and ameliorated over time. However, even on day 42 post-exposure, the LDH activity and macrophage count were still higher than those in the control group, suggesting that Nano-CuO caused chronic lung inflammation. The Nano-CuO-induced pulmonary inflammation was further confirmed by H&E staining of lung sections. Trichrome staining showed that Nano-CuO exposure caused pulmonary fibrosis from day 14 to day 42 post-exposure with an increasing tendency over time. Increased hydroxyproline content and expression levels of fibrosis-associated proteins in mouse lungs were also observed. In addition, Nano-CuO exposure induced MMP-3 overexpression and increased MMP-3 secretion in mouse lungs. Knocking down MMP-3 in mouse lungs significantly attenuated Nano-CuO-induced acute and chronic lung inflammation and fibrosis. Moreover, Nano-CuO exposure caused sustained production of cleaved osteopontin (OPN) in mouse lungs, which was also significantly decreased by knocking down MMP-3.ConclusionsOur results demonstrated that short-term Nano-CuO exposure caused acute lung inflammation and injury, while long-term exposure induced chronic pulmonary inflammation and fibrosis. Knocking down MMP-3 significantly ameliorated Nano-CuO-induced pulmonary inflammation, injury, and fibrosis, and also attenuated Nano-CuO-induced cleaved OPN level. Our study suggests that MMP-3 may play important roles in Nano-CuO-induced pulmonary inflammation and fibrosis via cleavage of OPN and may provide a further understanding of the mechanisms underlying Nano-CuO-induced pulmonary toxicity.Graphical

Effect of copper nanoparticles green-synthesized using Ocimum basilicum against Pseudomonas aeruginosa in mice lung infection model

Abstract The frequency of lung infection induced by multi-drug resistant strains of Pseudomonas aeruginosa has significantly risen, primarily due to the inadequate effectiveness of powerful chemotherapeutic methods. This study demonstrates that the Ocimum basilicum aqueous extract and copper nanoparticles (CuNPs) exhibited significant antioxidant and anti-infectious properties under in vivo conditions. To analyze the characteristics of the CuNPs synthesized from the reaction between copper nitrate solution and the aqueous O. basilicum extract, various techniques such as energy dispersive X-ray analysis, field emission scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction analysis, and transmission electron microscopy were employed. The in vivo study encompasses the assessment of P. aeruginosa lethal dose in mice and the disease manifestation analysis, which comprises reduction in body weight, hypothermia, bacteremia, and other parameters, over a 48 h infection period. The infected mice exhibited a notable decrease in body temperature, measuring at 25°C after 48 h, compared to the initial temperature of 39°C. Additionally, a 30% reduction in weight was seen at the conclusion of the study. To assess the effectiveness of CuNPs on lung infection caused by the calculated lethal dose and bacteremia, histopathology analysis was employed. The bacterial load in the CuNPs group was determined to be 0.5 Log10CFU/mL on Day 8, indicating a notable decrease from the initial measurement of 1.5 Log10CFU/mL on Day 1. The histopathological findings revealed a widespread and sporadic buildup of alveolar space inflammatory cells, with infiltrates observed throughout all lung sections in infected mice. Enhanced lung histology was observed in the group of animal treated with reduced exudates noted at 200 µg/kg. CuNPs demonstrated inhibitory effects on the growth of P. aeruginosa at 8 µg/mL, while at 16 µg/mL, they effectively eradicated P. aeruginosa. The research unequivocally demonstrates the efficacy of CuNPs extract in combating lung infections induced by P. aeruginosa at 200 µg/kg. The recent survey aims to further explore the biomedical characteristics of these CuNPs in order to develop a powerful treatment against this dangerous pathogen.

Histopathological and Immunohistochemical Studies of a Spontaneous Case of Pulmonary Adenomatosis in an Adult Ram: A Case Report

Aim: The present study aimed to describe histopathological findings and immunohistochemical expression of proliferating cell nuclear antigen (PCNA) in a spontaneous case of pulmonary adenomatosis in an adult ram. Case Presentation: A carcass of an adult ram was presented for necropsy in the Post-mortem Hall of the Department of Veterinary Pathology, College of Veterinary Sciences of the Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar with a history of respiratory distress. A detailed necropsy was performed. The tracheal mucosa showed mild congestion. The lungs were pale and firm in consistency. On histopathological examination, nasal turbinate and trachea both showed the vascular changes along with mild infiltration of mononuclear cells. The sections of lungs revealed pulmonary adenocarcinoma characterized by the presence of cuboidal to low columnar epithelial cells forming acinar or papillary-like structures in the parenchyma and surrounded by the normal alveoli. Areas of necrosis along with infiltration of mononuclear cells were also evident. An Immunohistochemical study of the lung sections revealed mild to moderate immunopositive reactivity for the PCNA in the nuclei of neoplastic cells of adenocarcinoma. It indicated the proliferative activity of neoplastic cells. Conclusion: Based on histopathological examination, the present case was diagnosed as pulmonary adenomatosis. PCNA expression indicated the presence of actively proliferating neoplastic cells.

ELUCIDATING THE EFFECTS OF FORMALIN AS FOOD PRESERVATIVE ON GROSS AND HISTOARCHITECTURE OF VITAL ORGANS OF SWISS ALBINO MICE

Formalin usage as food preservative in Bangladesh has dramatically increased recently, especially in fish, fruits, and many other food products, putting the vast majority of people in serious health risk. With this background, the study was aimed to detect the alterations of gross structure and histoarchitecture of vital organs in Swiss albino mice due to the consumption of formalin. Mice were divided into five groups at the age of four weeks. Normal drinking water was supplied to the control group of mice. Mice of group 1, 2, 3 and 4 were supplied normal drinking water mixed with formalin at 1%, 5%, 10% and 20% concentrations, respectively up to week 24. Mice treated with varying concentrations of formalin for 24 weeks was assessed for the weight change of their liver, heart, kidney and lung weights. Investigations were also conducted on histological alterations in the liver, heart, kidney and lung. After 24 weeks of treatment of mice with different concentrations of formalin, the weight of all four major organs (liver, heart, kidney and lung) was decreased in all formalin-treated mice in comparison to control mice. Petechial hemorrhages, congestion, necrosis, and parenchymatous cell degeneration have been found in liver. Kidney sections have shown decreased Bowman’s space, hypertrophied glomerulus and tubular dilatation. Leucocyte infiltration, necrosis, and rupture of connective tissue have been seen in the heart sections. Lung sections have shown pulmonary alterations, including hemorrhages, thicker alveolar walls, and inflammatory cells. It was, concluded that formalin has adverse effects on vital organs of Swiss Albino mice.

Repeated Silica exposures lead to Silicosis severity via PINK1/PARKIN mediated mitochondrial dysfunction in mice model

Background and objectivesSilicosis, one of the occupational health illnesses is caused by inhalation of crystalline silica. Deposition of extracellular matrix and fibroblast proliferation in lungs are linked to silicosis development. Mitochondrial dysfunction plays critical role in some diseases, but how these processes progress and regulated in silicosis, remains limited. Detailed study of silica induced pulmonary fibrosis in mouse model, its progression and severity may be helpful in designing future therapeutic strategies. MethodsIn present study, mice model of silicosis has been developed after repeated silica exposures which may closely resemble clinical symptoms of silicosis in human. In addition to efficiently mimicking the acute/chronic transformation processes of silicosis, this is practical and efficient in terms of time and output, which avoids mechanical injury to the upper respiratory tract due to surgical interventions. Sonicated sterile silica suspension (120 mg/kg) was administered through intranasal route thrice a week at regular intervals (21, 28 and 35 days). ResultsPresence of minute to larger silicotic nodules in H&E-stained lung sections were observed in all silica induced model groups. Enhanced ECM deposition was noted in MT stained lung sections of silica exposure groups as compared to control which were confirmed by significantly higher MMP9 expression levels and hydroxyproline content in silica 35 days group. Increase in Reactive oxygen species (ROS), inflammatory cell recruitment mainly, neutrophils and macrophage were observed in all three silica exposure groups. Transmission electron microscopic analysis has confirmed presence of many aberrant shaped mitochondria (swollen, round shape) in 35 days model where autophagosomes were minimum. Western blot analysis of mitophagy and autophagy markers such as Pink1, Parkin, Cytochrome c, SQSTM1/p62, the ratio of light chain LC3B II/LC3B I was found higher in 21 and 28 days which were significantly reduced in 35 days silica model. ConclusionsHigher MMP9 activity and MMP9 /TIMP1 ratio demonstrate excessive extracellular matrix damage and deposition in 35 days model. Significantly reduced expressions of autophagy and mitophagy markers have also confirmed progression in fibrosis severity and its association with repeated silica exposures in 35 days model group.

Imipenem reduces the efficacy of vancomycin against Elizabethkingia species.

Elizabethkingia spp. are emerging as nosocomial pathogens causing various infections. These pathogens express resistance to a broad range of antibiotics, thus requiring antimicrobial combinations for coverage. However, possible antagonistic interactions between antibiotics have not been thoroughly explored. This study aimed to evaluate the effectiveness of antimicrobial combinations against Elizabethkingia infections, focusing on their impact on pathogenicity, including biofilm production and cell adhesion. Double-disc diffusion, time-kill, and chequerboard assays were used for evaluating the combination effects of antibiotics against Elizabethkingia spp. We further examined the antagonistic effects of antibiotic combinations on biofilm formation and adherence to A549 human respiratory epithelial cells. Further validation of the antibiotic interactions and their implications was performed using ex vivo hamster precision-cut lung sections (PCLSs) to mimic in vivo conditions. Antagonistic effects were observed between cefoxitin, imipenem and amoxicillin/clavulanic acid in combination with vancomycin. The antagonism of imipenem toward vancomycin was specific to its effects on the genus Elizabethkingia. Imipenem further hampered the bactericidal effect of vancomycin and impaired its inhibition of biofilm formation and the adhesion of Elizabethkingia meningoseptica ATCC 13253 to human cells. In the ex vivo PCLS model, vancomycin exhibited dose-dependent bactericidal effects; however, the addition of imipenem also reduced the effect of vancomycin. Imipenem reduced the bactericidal efficacy of vancomycin against Elizabethkingia spp. and compromised its capacity to inhibit biofilm formation, thereby enhancing bacterial adhesion. Clinicians should be aware of the potential issues with the use of these antibiotic combinations when treating Elizabethkingia infections.

Experimental congenital diaphragmatic hernia features an alteration of DNA sensing targets cGAS and STING.

The pathogenesis of congenital diaphragmatic hernia (CDH) depends on multiple factors. Activation of the DNA-sensing cyclic-GMP-AMP-synthase (cGAS) and Stimulator-of-Interferon-Genes (STING) pathway by double-stranded DNA (dsDNA) links environmental stimuli and inflammation. We hypothesized that nitrofen exposure alters cGAS and STING in human bronchial epithelial cells and fetal rat lungs. We used the Quant-IT™-PicoGreen™ assay to assess dsDNA concentration in BEAS-2B cells after 24 h of nitrofen-exposure and performed immunofluorescence of cGAS/STING. We used nitrofen to induce CDH and harvested control and CDH lungs at embryonic day E15, E18 and E21 for cGAS/STING immunofluorescence, RT-qPCR and RNA-Scope™ in-situ-hybridization (E18, E21). We found a higher concentration of dsDNA following nitrofen treatment. Nitrofen-exposure to BEAS-2B cells increased cGAS and STING protein abundance. cGAS abundance was higher in nitrofen lungs at E15, E18 and E21. RNA-Scope in-situ-hybridization showed higher cGAS and STING expression in E18 and E21 lungs. RT-qPCR revealed higher mRNA expression levels of STING in E21 nitrofen-induced lungs. Our data suggest that nitrofen-exposure increases dsDNA content which leads to stimulation of the cGAS/STING pathway in human BEAS-2B cells and the nitrofen rat model of CDH. Consequently, DNA sensing and the cGAS-STING-pathway potentially contribute to abnormal lung development in CDH. We found an alteration of DNA sensing targets cGAS and STING in human BEAS-2B cells and experimental congenital diaphragmatic hernia with higher protein abundance and mRNA expression in cells and lung sections of nitrofen-treated rat pups. This is the first study to investigate DNA sensing, a potential link between environmental stimuli and inflammation, in experimental CDH. Our study extends the knowledge on the pathogenesis of experimental CDH.

Vinpocetine alleviated alveolar epithelial cells injury in experimental pulmonary fibrosis by targeting PPAR-γ/NLRP3/NF-κB and TGF-β1/Smad2/3 pathways

This study aimed to investigate the potential anti-fibrotic activity of vinpocetine in an experimental model of pulmonary fibrosis by bleomycin and in the MRC-5 cell line. Pulmonary fibrosis was induced in BALB/c mice by oropharyngeal aspiration of a single dose of bleomycin (5 mg/kg). The remaining induced animals received a daily dose of pirfenidone (as a standard anti-fibrotic drug) (300 mg/kg/PO) and vinpocetine (20 mg/kg/PO) on day 7 of the induction till the end of the experiment (day 21). The results of the experiment revealed that vinpocetine managed to alleviate the fibrotic endpoints by statistically improving (P ≤ 0.05) the weight index, histopathological score, reduced expression of fibrotic-related proteins in immune-stained lung sections, as well as fibrotic markers measured in serum samples. It also alleviated tissue levels of oxidative stress and inflammatory and pro-fibrotic mediators significantly elevated in bleomycin-only induced animals (P ≤ 0.05). Vinpocetine managed to express a remarkable attenuating effect in pulmonary fibrosis both in vivo and in vitro either directly by interfering with the classical TGF-β1/Smad2/3 signaling pathway or indirectly by upregulating the expression of Nrf2 enhancing the antioxidant system, activating PPAR-γ and downregulating the NLRP3/NF-κB pathway making it a candidate for further clinical investigation in cases of pulmonary fibrosis.

The mitigating effect of para-hydroxycinnamic acid in bleomycin-induced pulmonary fibrosis in mice through targeting oxidative, inflammatory and fibrotic pathways.

This study investigated the therapeutic benefits of para-hydroxycinnamic acid in mice with bleomycin-induced lung fibrosis. Forty male BALB/c mice were randomly assigned to four groups: normal, which received 0.9% normal saline; induced, which received a single dose of bleomycin (5mg/kg) by oropharyngeal challenge; pirfenidone-treated; and para-hydroxycinnamic acid-treated, which challenged with bleomycin and received a daily oral dose of 300 and 50 mg/kg, respectively, from day 7 to day 21. Tissue pro-fibrotic and inflammatory cytokines, oxidative indicators, pulmonary histopathology, immunohistochemistry of fibrotic proteins and the assessment of gene expression by RT-qPCR were evaluated on day 22 after euthanizing animals. Pirfenidone and para-hydroxycinnamic acid managed to alleviate the fibrotic endpoints by statistically improving the weight index, histopathological score and reduced expression of fibrotic-related proteins in immune-stained lung sections, as well as fibrotic markers measured in serum samples. They also managed to alleviate tissue levels of oxidative stress and inflammatory and pro-fibrotic mediators. para-Hydroxycinnamic acid enhanced the expression of crucial genes associated with oxidative stress, inflammation and fibrosis in vivo. para-Hydroxycinnamic acid has demonstrated similar effectiveness to pirfenidone, suggesting it could be a promising treatment for fibrotic lung conditions by inhibiting the TGF-β1/Smad3 pathway or through its anti-inflammatory and antioxidant properties.

A novel humanized mouse model for HIV and tuberculosis co-infection studies.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), continues to be a major public health problem worldwide. The human immunodeficiency virus (HIV) is another equally important life-threatening pathogen. HIV infection decreases CD4+ T cell levels markedly increasing Mtb co-infections. An appropriate animal model for HIV/Mtb co-infection that can recapitulate the diversity of the immune response in humans during co-infection would facilitate basic and translational research in HIV/Mtb infections. Herein, we describe a novel humanized mouse model. The irradiated NSG-SGM3 mice were transplanted with human CD34+ hematopoietic stem cells, and the humanization was monitored by staining various immune cell markers for flow cytometry. They were challenged with HIV and/or Mtb, and the CD4+ T cell depletion and HIV viral load were monitored over time. Before necropsy, the live mice were subjected to pulmonary function test and CT scan, and after sacrifice, the lung and spleen homogenates were used to determine Mtb load (CFU) and cytokine/chemokine levels by multiplex assay, and lung sections were analyzed for histopathology. The mouse sera were subjected to metabolomics analysis. Our humanized NSG-SGM3 mice were able to engraft human CD34+ stem cells, which then differentiated into a full-lineage of human immune cell subsets. After co-infection with HIV and Mtb, these mice showed decrease in CD4+ T cell counts overtime and elevated HIV load in the sera, similar to the infection pattern of humans. Additionally, Mtb caused infections in both lungs and spleen, and induced granulomatous lesions in the lungs. Distinct metabolomic profiles were also observed in the tissues from different mouse groups after co-infections. The humanized NSG-SGM3 mice are able to recapitulate the pathogenic effects of HIV and Mtb infections and co-infection at the pathological, immunological and metabolism levels and are therefore a reproducible small animal model for studying HIV/Mtb co-infection.

Abstract PO5-18-07: A Humanized Monoclonal Antibody to Secreted Frizzled-Related Protein 2 Inhibits TNBC Lung Metastases

Abstract Introduction: A promising novel target for breast cancer is secreted frizzled-related protein 2 (SFRP2). SFRP2 protects tumors against apoptosis, promotes T-cell exhaustion, and induces angiogenesis. SFRP2 is expressed in breast cancer but has not been specifically evaluated in triple negative breast cancer. We previously developed a humanized monoclonal antibody to SFRP2, (hSFRP2 mAb) that inhibits primary breast cancer growth in mice without toxicity. The purpose of this study is to establish the presence of SFRP2 in human TNBC, evaluate the efficacy of hSFRP2 mAb in metastatic TNBC in vivo, and in a cell line resistant to chemotherapy. Methods: Immunohistochemistry (IHC) with SFRP2 antibody on a human TNBC tissue microarray (TMA) containing 88 FFPE cores. After IHC with primary antibody to SFRP2, the TMA was scanned and staining intensity was quantified using spatial analysis. Results for SFRP2 staining were analyzed as previously described for analyses of estrogen and progesterone receptor positivity; categories are: 0-absence of staining; low positive (0-10%), and positive &amp;gt;10%. Development of a doxorubicin (dox)-resistant cell line and induction of apoptosis with hSFRP2 mAb. MDA-MB-231 cells were treated with 4nM dox and increased 2nM at a time until the cells were resistant to 10µM dox after 12 months. WT and dox-resistant cells were treated for 4 hours with either hSFRP2 mAb (10uM,) or IgG control (10uM) (n=6). Apoptosis was detected with Promokine Apoptosis/Necrosis/Healthy cells kit. Cells were imaged using the EVOS FLc and counted using Imagej software. Differences between treated and control were compared with two-tailed T-test. hSFRP2 mAb treatment of mice with metastatic TNBC. PY8119 and E0771 TNBC murine cells (5 × 105/100 μL) were injected iv into 6–8-week-old C57BL/6 female mice. The next day the mice were treated with IgG1 control 8 mg/kg iv or hSFRP2 mAb 8 mg/kg iv every 3 days. After four weeks, the lungs were resected, and surface metastases were counted. Differences between treated and control were compared with Poisson distributions. FFPE sections of lungs were stained with ApopTag kit. Three metastases per lung were counted at 20 X, and the difference between IgG1 and hSFRP2 mAb groups compared with two-tailed T-test. Results: SFRP2 protein is present in human TNBC. Out of 88 cores, 83 cores (94%) were positive, 4 cores (4.5%) were minimally positive, and 1 core (1.1%) was negative for SFRP2. Development of a doxorubicin (dox)-resistant cell line and induction of apoptosis with hSFRP2 mAb treatment in vitro. The number of apoptotic cells in IgG1 control-treated MDA-MB-231 cells was 11.5 ± 3.7, and 76.6 ± 5.0 for hSFRP2 mAb-treated tumors (p&amp;lt; 0.001, n=9). The mean number of apoptotic MDA-MB-231 dox-resistant cells in IgG1 control was 16.7 ± 6.0, and 68.4 ± 6.4 for hSFRP2 mAb-treated tumors, (p&amp;lt; 0.001, n=9). hSFRP2 mAb inhibits the growth of metastatic TNBC in vivo and increases apoptosis. For E0771, the number of surface metastases was 8.9 ±2.8 in the IgG1 control group and 4.9 ± 1.1 in the hSFRP2 mAb treated group (p&amp;lt; 0.05, n=15). The number of apoptotic cells/HPF was 9.8 ± 1.8 for the IgG1 control group and 21.0 ± 4.6 for the hSFRP2 mAb group (n=15, p&amp;lt; .01). For Py8119 tumors there were 6.4 ± 1.0 metastases in the IgG1 control group and 3.6 ± 0.9 in the hSFRP2 mAb group (p&amp;lt; 0.05, n=11). The number of apoptotic cells/HPF was 6 ± 1.8 for the IgG1 control group and 14 ±1.3 for the hSFRP2 mAb group (n=10, p&amp;lt; 0.001). Conclusions: SFRP2 protein is abundantly expressed in human TNBC. A humanized monoclonal antibody to SFRP2 reduces TNBC lung metastases growth in two in vivo models and increases tumor apoptosis. TNBC resistant to doxorubicin remains sensitive to apoptosis induced by the hSFRP2 mAb. Citation Format: Lillian Hsu, Patrick Nasarre, Julie Siegel, Eleanor Hilliard, Rupak Mukherjee, Nancy Klauber-Demore. A Humanized Monoclonal Antibody to Secreted Frizzled-Related Protein 2 Inhibits TNBC Lung Metastases [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-18-07.

L-citrulline Mitigates Systemic Inflammation and Associated Pulmonary Vascular Remodeling in a Neonatal Rat Model of Postnatal Undernutrition

Background &amp; Aim: Preterm infants with postnatal growth restriction and undernutrition are at increased risk of developing bronchopulmonary dysplasia (BPD), pulmonary hypertension (PH), metabolic syndrome, and sepsis. Preclinical and clinical studies report increased systemic inflammation, and lung and pancreatic histopathology in growth-restricted neonates. Safe therapies to mitigate these adverse outcomes associated with postnatal growth restriction and undernutrition in preterm infants are needed. L-citrulline is a non-essential amino acid, involved in the urea and nitric oxide cycles, that exhibits anti-inflammatory properties. This study aims to investigate whether L-citrulline may decrease systemic inflammation and improve histopathology in the lung, heart, and pancreas in a neonatal model of postnatal undernutrition. Methods: Neonatal Sprague Dawley rat pups were divided into 2 groups, normal nutrition (litter size n=12) and undernutrition (litter size n=18) by altering the litter size of the nursing dam. Body weight was measured daily and both groups received daily intraperitoneal injections of saline or L-citrulline (2.5g/kg) for 21 days. On postnatal day 21, heart, lung, and pancreatic tissue were collected for histology and mRNA analyses. Lung and pancreatic tissue were processed for qPCR to detect mRNA expression of pro-inflammatory mediators (MCP-1/CCL2, MIP-1α /CCL3, IL-6, and TNF-α). Right ventricular hypertrophy was calculated using Fulton’s index (weight of the right ventricle divided by the combined weight of the left ventricle and the intraventricular septum). Lung sections were stained with Movat Pentachrome and examined for the presence of immune cells and medial wall thickening of pulmonary arteries. In a separate group of animals, bronchoalveolar lavage fluid (BALF) and blood obtained by cardiac puncture were cytocentrifuged and stained with hematoxylin &amp; eosin for immune cell counts. Results: Undernourished rats had a lower body weight than normally nourished rats at postnatal day 21 (n=11-17; p&lt;0.0001). Undernourished animals had an increased number of immune cells in BALF and lung tissue and an elevated neutrophil count in the blood which were all decreased with concurrent L-citrulline treatment (n=3-4; p&lt;0.001). L-citrulline treatment also prevented the increased mRNA expression of pro-inflammatory mediators in the lungs [MCP-1/CCL2, TNF-α, and IL-1β] (n=8; p&lt;0.05) and pancreas [MCP-1/CCL2, MIP-1α /CCL3, IL-6, and TNF-α] (n=3; p&lt;0.05) of undernourished animals. These animals had a greater pulmonary artery medial wall area and Fulton’s index when compared to normally nourished animals treated with saline or L-citrulline (n=3-4; p&lt;0.001). Concurrent L-citrulline treatment in undernourished animals prevented pulmonary artery vascular remodeling and heart tissue histopathology such that the medial wall area and Fulton’s index did not differ from that of animals in control groups (n=4; p&lt;0.01). Conclusion: L-citrulline effectively mitigates the adverse effects of postnatal undernutrition, reducing lung, pancreatic, and systemic inflammation and histopathology in the lung and heart associated with PH. As a safe, well-tolerated, and inexpensive treatment, L-citrulline presents promising therapeutic potential that could prevent short and long-term morbidities associated with growth restriction and undernutrition in preterm infants. This work was funded by the Women's Auxiliary of The Hospital for Sick Children. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

CHD9NB Is a Novel Brush Border Marker in the Intestine and Beyond

The public availability of single cell RNA sequencing datasets has accelerated discovery of novel gene markers of specific cell types. A recently published dataset of human small intestine and colon (GSE185224) revealed 14 distinct epithelial clusters, including tuft cell, a rare sensory cell population. We probed this dataset for novel tuft cell-specific makers and investigated the localization of candidate marker proteins. Using dataset GSE185224, Leiden clustering and UMAP visualization were generated, and tuft cell clusters were identified by previously known markers, e.g. POU2F3. Genes were systematically parsed through and CHD9NB (Chromodomain Helicase DNA Binding Protein 9 neighbor) was identified as a candidate novel tuft cell marker gene. CHD9NB encodes a 52 amino acid protein with no published information for function. Antibodies were raised against recombinant human CHD9NB. Paraffn sections of tissues and enteroids from healthy individuals and mice were immunostained against CHD9NB and brush border or cell structural markers. CHD9NB was expressed along apical actin bundles of intestinal tuft cells. Contrary to transcriptional level, which was lower in enterocytes than tuft cells, intense CHD9NB immunostaining was observed at the tips of microvilli along mature enterocytes in human and mouse small intestine and colon. This microvillus tip localization was suggested by the presence of CHD9NB above the ACTG1 staining in brush border, similar to EPS8. Less intense CHD9NB was detected at both the tips and base of microvilli of the developing brush border in intestinal crypts, suggesting a differentiation stage-dependent localization of CHD9NB in the intestinal epithelial cells. Differentiated human enteroids exhibited either villus-like (microvillus tip-specific) or crypt-like (present at both the base and tips) CHD9NB staining, likely suggesting a range of enteroid differentiation states. To determine whether CHD9NB is specific to the intestinal microvilli or more generally expressed in brush borders of other organs, sections of kidney, inner ear, and lung were stained. CHD9NB staining was specific to brush border-containing, OAT1+ proximal tubules of the kidney, and absent in brush border-lacking distal tubules and glomeruli. In contrast to tips of microvilli in enterocytes, CHD9NB was localized to the base of the brush border of proximal tubules. CHD9NB staining in the murine cochlea was specific to the DCLK1+ cells of the stria vascularis, rather than hair cells. In the lung, CHD9NB was detected along the ciliated cells of the human bronchiole. We propose CHD9NB as a novel brush border and intestinal tuft cell marker, with organ-specific localization patterns beyond the intestine. Further studies will explore the function of CHD9NB in enteroids. NSF GRFP; NIH R01DK128190; RC2DK118640. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The pulmonary neuroepithelial body-vagal sensory nerve axis is altered in a mouse model of bronchopulmonary dysplasia (BPD)

Breathing control problems, like apnea of prematurity, are a common complication in preterm infants with bronchopulmonary dysplasia (BPD), but the underlying factors remain incompletely understood. A subpopulation of pulmonary vagal afferent nerve fibers (Calb1+) innervate CO2 and O2 sensitive clusters of lung neuroendocrine cells (neuroepithelial bodies (NEBs)) located along conducting airways. Calb1+ fibers provide direct sensory input to the brainstem that putatively limit tidal volume. NEBs are hyperplastic in BPD infants and may increase activity of inhibitory Calb1+ vagal fibers. Herein, we sought to determine if the NEB-Calb1 lung-brainstem axis is altered in association with breathing control in BPD. BPD in mice was established by exposure to chronic neonatal (postnatal day (P) 0-14) hyperoxia (Hx; 70% O2; n=20) or normoxia (Nx; n=17). Consistent with human BPD, Hx caused alveolar simplification supported by a reduction in alveolar density and alveoli (p&lt;0.05; separate t-tests) in P22 H&amp;E-stained lung sections (n=5-7/group). Like human infants at risk for BPD, Hx mice adapted to increased dead space (caused by reduction in alveoli) by hyperventilating (p&lt;0.05; generalized linear modeling accounting for fat free mass and heat production, measured by NMR and respirometry; n=10-24/group). This was facilitated by an increase in tidal volume that was significantly reduced at P14 but increased at P17 and P21 (n=19-28) (Tukey’s post-hoc: p&lt;0.05). NEBs and associated innervation (vagal and dorsal root ganglia) were next assessed in subsets of mice. First, IT instillation of WGA-594 lectin into subsets of mice (n=9 mice/18 ganglia per group) and subsequent quantification of 594+ neurons per vagal ganglia revealed fewer neurons (p=0.07; t-test) in Hx mice (110.8 ± 6.9 vs 95.2 ± 4.9). Second, CGRP+ innervation to NEBs is reduced in Hx mice (Quantification of CGRP+ nerve fibers only: 17740.4 +/- 4126 vs 10474.6 +/- 2140 +CGRP pixels; p=0.12 n=5-7/group). Third, there was greater frequency of NEBs per unit airway (p&lt;0.05; 0.306 ± 0.03 vs 0.401 ± 0.03) with a tendency for larger NEBs (p=0.06; 9.14 ± 0.7 vs 11.93 ± 1.3) indicating NEB hyperplasia. Together, these data indicate individual NEB innervation is reduced in BPD but that total NEB innervation may remain unchanged. If this is true for Calb1+ vagal fibers remain untested. To functionally assess Calb1-vagal fibers we activated, with DCZ, the excitatory synthetic Gq-DREAD receptor (DTR) expressed in Calb1cre mice via microinjection of AAV-Cre-Gq-mCherry into the left vagal ganglia. DCZ but not vehicle caused a major increase in peak inspiratory flow during acute hypoxic challenges compared to vehicle treated mice (Pre-DMSO, Post-DMSO, Pre-DCZ = ~1ml/sec vs 4ml/sec post-DCZ; p&lt;0.05, n=4/group). Collection of vagal ganglia confirms DTR expression but spillover of DTR is present. Repeating these studies with diluted AAV-Cre-Gq in control and BPD mice are ongoing. Together, our data verify that hyperoxia induced BPD in mice causes a similar reduction in alveoli and compensation to increased dead space by hyperventilation as observed in human BPD. Additionally, BPD structurally alters the NEB-vagal axis but whether these changes are functionally significant to nCOB is being addressed with ongoing studies. AHA 20CDA35310121, Advancing Healthier Wisconsin Endowment. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.