Increased Leukocyte Infiltration Research Articles

1, 6-dilauroyl-D-fructofuranose ameliorates lipopolysaccharide-induced septic acute kidney injury via inhibiting caspase 1 mediated pyroptosis formation in rat.

Sepsis is a systemic inflammatory state associated with acute kidney injury (AKI) and high mortality. However, sepsis-induced AKI cannot be effectively prevented or treated using current antimicrobial therapies and supportive measures. We explored the therapeutic effect of newly developed fructose esters on sepsis-induced AKI (S-AKI). We used the surface plasmon resonance technique and ultrasensitive chemiluminescence analyzer to characterize the lipopolysaccharide (LPS)/endotoxin binding activity and antioxidant capability of fructose esters. We assessed the extent of fructose ester gastrointestinal digestion using rat intestinal acetone powder. We examined the therapeutic effect of fructose esters on LPS-induced S-AKI by evaluating the blood and renal reactive oxygen species (ROS) amounts, caspase 1 mediated pyroptosis, inflammation, microcirculation, and renal dysfunction. Our data showed that the fructose esters are not easily hydrolyzed by the rat intestinal acetone powder, suggesting their high stability in the gastrointestinal tract. 1,6-dilauroyl-D-fructofuranose (FDL) dose-dependently scavenged H2O2 and displayed a higher binding affinity to LPS compared to sialic acid and fructose did. LPS significantly enhanced caspase 1 mediated pyroptosis and increased leukocyte infiltration, blood and renal ROS amount, and blood urea nitrogen (BUN) and creatinine level, whereas FDL significantly depressed these LPS-enhanced parameters. In addition, the increased plasma inflammatory cytokines levels using LPS could be reduced by intravenous fructose ester FDL treatment. Our data suggest that FDL, with its antioxidant activity against H2O2, can neutralize LPS toxicity using a high binding affinity, and attenuate S-AKI by inhibiting caspase 1 mediated pyroptosis, thereby ameliorating renal oxidative stress and dysfunction.

Central nervous system-associated macrophages modulate the immune response following stroke in aged mice.

Age is a major nonmodifiable risk factor for ischemic stroke. Central nervous system-associated macrophages (CAMs) are resident immune cells located along the brain vasculature at the interface between the blood circulation and the parenchyma. By using a clinically relevant thromboembolic stroke model in young and aged male mice and corresponding human tissue samples, we show that during aging, CAMs acquire a central role in orchestrating immune cell trafficking after stroke through the specific modulation of adhesion molecules by endothelial cells. The absence of CAMs provokes increased leukocyte infiltration (neutrophils and CD4+ and CD8+ T lymphocytes) and neurological dysfunction after stroke exclusively in aged mice. Major histocompatibility complex class II, overexpressed by CAMs during aging, plays a significant role in the modulation of immune responses to stroke. We demonstrate that during aging, CAMs become central coordinators of the neuroimmune response that ensure a long-term fine-tuning of the immune responses triggered by stroke.

Substance P Promotes Leukocyte Infiltration in the Liver and Lungs of Mice with Sepsis: A Key Role for Adhesion Molecules on Vascular Endothelial Cells.

Substance P (SP), encoded by the Tac1 gene, has been shown to promote leukocyte infiltration and organ impairment in mice with sepsis. Neurokinin-1 receptor (NK1R) is the major receptor that mediates the detrimental impact of SP on sepsis. This investigation studied whether SP affects the expression of adhesion molecules, including intercellular cell adhesion molecule-1 (ICAM1) and vascular cell adhesion molecule-1 (VCAM1) on vascular endothelial cells in the liver and lungs, contributing to leukocyte infiltration in these tissues of mice with sepsis. Sepsis was induced by caecal ligation and puncture (CLP) surgery in mice. The actions of SP were inhibited by deleting the Tac1 gene, blocking NK1R, or combining these two methods. The activity of myeloperoxidase and the concentrations of ICAM1 and VCAM1 in the liver and lungs, as well as the expression of ICAM1 and VCAM1 on vascular endothelial cells in these tissues, were measured. The activity of myeloperoxidase and the concentration of ICAM1 and VCAM1 in the liver and lungs, as well as the expression of ICAM1 and VCAM1 on vascular endothelial cells in these tissues, increased in mice with CLP surgery-induced sepsis. Suppressing the biosynthesis of SP and its interactions with NK1R attenuated CLP surgery-induced alterations in the liver and lungs of mice. Our findings indicate that SP upregulates the expression of ICAM1 and VCAM1 on vascular endothelial cells in the liver and lungs, thereby increasing leukocyte infiltration in these tissues of mice with CLP surgery-induced sepsis by activating NK1R.

Chronic endometritis. Local prooxidant, antioxidant and immunometabolic disorders. An open-label, prospective, randomized study

Aim. To determine local features of lipoperoxidation processes, antioxidant defense, state of matrix metalloproteinases in chronic endometritis. Materials and methods. Eighty women aged 29.33±6.71 (min-max [24–36]) with histologically and immunohistochemically confirmed chronic endometritis and a history of reproductive losses were examined (group 1). The control group consisted of 30 patients without chronic endometritis confirmed by the results of a histochemical study and no history of reproductive losses, suffering from infertility of tubal-peritoneal origin at the age of 30.11±6.9 years (min-max [24–37]; group 2). All 130 patients underwent transvaginal echography of the pelvic organs, and histological and immunohistochemical examination of endometrial samples was performed. The activity of lipid peroxidation factors was analyzed: primary, secondary and final products of lipid peroxidation in heptane and isopropanolol fractions of uterine cavity aspirate, enzymes of the antioxidant system: superoxide dismutase, catalase, glutathione peroxidase, deoxyribonuclease I (DNase), by spectrophotometric method, remodeling factors – matrix metalloproteinases MMP-2, -9 by flow fluorimetry. Results. In patients of group 1, according to transvaginal echography of the pelvic organs, M-echo was 6.29±1.22 mm, in group 2 – 9.10±1.15 mm. In chronic endometritis, disruption of pro-oxidant and activity of antioxidant factors in the endometrium in women with chronic endometritis and a history of reproductive losses. In the heptane fraction of the phenolic extract, the concentration of primary products of lipid peroxidation (diene conjugates) was 12.98% higher than in the control, the content of secondary products of lipid peroxidation (ketodienes) was higher by 15.45%, conjugated trienes by 18.67% higher than the values in the control group, which indicates increased lipid peroxidation of endometrial cell membranes during CE. In the isopropanol phase, the products of lipid peroxidation of phospholipid acyl residues are detected. In the isopropanol fraction of the lipid extract, the amount of diene conjugates was higher, ketodienes by 24.32% and conjugated trienes by 26.14%. An increase in the activity of the enzymes superoxide dismutase by 23.98%, catalase by 14.34%, glutathione peroxidase by 12.39%, and DNase by 2.9 times was recorded compared to the indicators of group 2. Conclusion. Chronic endometritis is accompanied by changes in the system of pro-oxidant and antioxidant factors, the content of tissue remodeling products. The pathogenetic changes underlying oxidative stress in the endometrium may be increased leukocyte infiltration, imbalance of primary, secondary and end products of lipid peroxidation, enzymes superoxide dismutase, catalase, glutathione peroxidase, deoxyribonuclease I (DNase), expression of matrix metalloproteinases-2, -9.

Intratumoral Fc-optimized agonistic CD40 antibody induces tumor rejection and systemic antitumor immunity in patients with metastatic cancer.

While CD40 agonism is an attractive approach for activating antigen-presenting cells and initiating antitumor responses, previous attempts have encountered limited clinical efficacy coupled with toxicity. We previously demonstrated that interactions between the antibody Fc domain and the inhibitory receptor FcγRIIB are critical for enhanced antitumor activity. Here, we present the results of a phase 1 study on intratumoral administration of an anti-CD40 agonistic antibody (2141-V11) Fc-engineered to enhance FcγRIIB binding. Primary endpoints included safety, maximum tolerated dose (MTD), and recommended phase 2 dose. Secondary objectives included preliminary clinical activity and correlative studies from biospecimens. 2141-V11 was well-tolerated without dose-limiting toxicities and MTD was not reached. In ten evaluable patients with metastatic cancer, the overall response rate was 20%, with complete responses in two patients (melanoma and breast carcinoma) and stable disease in six patients. 2141-V11 induced tumor regression in injected and non-injected lesions, with increased leukocyte infiltration and tertiary lymphoid structures (TLS) formation in post-treatment biopsies. In a humanized mouse model for CD40 and FcγRs, 2141-V11 induced TLS formation in mice bearing orthotopic breast carcinoma, correlating with local and abscopal antitumor effects, systemic immune activation, and immune memory. These findings support the safety and efficacy of 2141-V11, warranting phase 2 studies and suggesting a unique mechanism of action for this Fc-enhanced immunotherapy (NCT04059588).

Repeated doses of captopril induce airway hyperresponsiveness by modulating the TRPV1 receptor in rats

Although TRPV1 receptors play an essential role in the adverse effects on the airways following captopril treatment, there is no available evidence of their involvement in treatment regimens involving repeated doses of captopril. Comparing the difference in these two treatment regimens is essential since captopril is a continuous-use medication. Thus, this study explored the role of the transient receptor potential vanilloid 1 (TRPV1) in the effects of captopril on rat airways using two treatment regimens. Airway resistance, bronchoalveolar lavage (BAL), and histological and immunohistochemical analyses were conducted in rats administered with single or repeated doses of captopril. This study showed that the hyperresponsiveness to bradykinin and capsaicin in captopril-treated rats was acute. Treatment with the selective B2 antagonist, HOE140 reduced bradykinin hyperresponsiveness and abolished capsaicin exacerbation in single-dose captopril-treated rats. Likewise, degeneration of TRPV1-positive neurones also reduced hyperresponsiveness to bradykinin. Single-dose captopril treatment increased leukocyte infiltration in the BAL when compared with the vehicle and this increase was reduced by TRPV1-positive neurone degeneration. However, when compared with the vehicle treatment, animals treated with repeated doses of captopril showed an increase in leukocyte influx as early as 1 h after the last captopril treatment, but this effect disappeared after 24 h. Additionally, an increase in TRPV1 expression occurred only in animals who received repeated captopril doses and the degeneration of TRPV1-positive neurones attenuated TRPV1 upregulation. In conclusion, these data strongly indicate that a treatment regimen involving multiple doses of captopril not only enhances sensitisation but also upregulates TRPV1 expression. Consequently, targeting TRPV1 could serve as a promising strategy to reduce the negative impact of captopril on the airways.

17β-estradiol and methylprednisolone association as a therapeutic option to modulate lung inflammation in brain-dead female rats.

Brain death (BD) is known to compromise graft quality by causing hemodynamic, metabolic, and hormonal changes. The abrupt reduction of female sex hormones after BD was associated with increased lung inflammation. The use of both corticoids and estradiol independently has presented positive results in modulating BD-induced inflammatory response. However, studies have shown that for females the presence of both estrogen and corticoids is necessary to ensure adequate immune response. In that sense, this study aims to investigate how the association of methylprednisolone (MP) and estradiol (E2) could modulate the lung inflammation triggered by BD in female rats. Female Wistar rats (8 weeks) were divided into four groups: sham (animals submitted to the surgical process, without induction of BD), BD (animals submitted to BD), MP/E2 (animals submitted to BD that received MP and E2 treatment 3h after BD induction) and MP (animals submitted to BD that received MP treatment 3h after BD induction). Hemodynamics, systemic and local quantification of IL-6, IL-1β, VEGF, and TNF-α, leukocyte infiltration to the lung parenchyma and airways, and adhesion molecule expression were analyzed. After treatment, MP/E2 association was able to reinstate mean arterial pressure to levels close to Sham animals (p<0.05). BD increased leukocyte infiltration to the airways and MP/E2 was able to reduce the number of cells (p=0.0139). Also, the associated treatment modulated the vasculature by reducing the expression of VEGF (p=0.0616) and maintaining eNOS levels (p=0.004) in lung tissue. Data presented in this study show that the association between corticoids and estradiol could represent a better treatment strategy for lung inflammation in the female BD donor by presenting a positive effect in the hemodynamic management of the donor, as well as by reducing infiltrated leukocyte to the airways and release of inflammatory markers in the short and long term.

Utilization of a highly adaptable murine air pouch model for minimally invasive testing of the inflammatory potential of biomaterials.

Introduction: The biocompatibility of an implanted material strongly determines the subsequent host immune response. After insertion into the body, each medical device causes tissue reactions. How intense and long-lasting these are is defined by the material properties. The so-called foreign body reaction is a reaction leading to the inflammation and wound healing process after implantation. The constantly expanding field of implant technology and the growing areas of application make optimization and adaptation of the materials used inevitable. Methods: In this study, modified liquid silicone rubber (LSR) and two of the most commonly used thermoplastic polyurethanes (TPU) were compared in terms of induced inflammatory response in the body. We evaluated the production of inflammatory cytokines, infiltration of inflammatory cells and encapsulation of foreign bodies in a subcutaneous air-pouch model in mice. In this model, the material is applied in a minimally invasive procedure via a cannula and in one piece, which allows material testing without destroying or crushing the material and thus studying an intact implant surface. The study design includes short-term (6h) and long-term (10days) analysis of the host response to the implanted materials. Air-pouch-infiltrating cells were determined by flow cytometry after 6h and 10days. Inflammation, fibrosis and angiogenesis markers were analyzed in the capsular tissue by qPCR after 10days. Results: The foreign body reaction was investigated by macroscopic evaluation and scanning electron microscopy (SEM). Increased leukocyte infiltration was observed in the air-pouch after 6h, but it markedly diminished after 10days. After 10days, capsule formations were observed around the materials without visible inflammatory cells. Discussion: For biocompatibility testing materials are often implanted in muscle tissue. These test methods are not sufficiently conclusive, especially for materials that are intended to come into contact with blood. Our study primarily shows that the presented model is a highly adaptable and minimally invasive test system to test the inflammatory potential of and foreign body reaction to candidate materials and offers more precise analysis options by means of flow cytometry.

Therapy-Induced Senescence Contributes to the Efficacy of Abemaciclib in Patients with Dedifferentiated Liposarcoma.

We conducted research on CDK4/6 inhibitors (CDK4/6i) simultaneously in the preclinical and clinical spaces to gain a deeper understanding of how senescence influences tumor growth in humans. We coordinated a first-in-kind phase II clinical trial of the CDK4/6i abemaciclib for patients with progressive dedifferentiated liposarcoma (DDLS) with cellular studies interrogating the molecular basis of geroconversion. Thirty patients with progressing DDLS enrolled and were treated with 200 mg of abemaciclib twice daily. The median progression-free survival was 33 weeks at the time of the data lock, with 23 of 30 progression-free at 12 weeks (76.7%, two-sided 95% CI, 57.7%-90.1%). No new safety signals were identified. Concurrent preclinical work in liposarcoma cell lines identified ANGPTL4 as a necessary late regulator of geroconversion, the pathway from reversible cell-cycle exit to a stably arrested inflammation-provoking senescent cell. Using this insight, we were able to identify patients in which abemaciclib induced tumor cell senescence. Senescence correlated with increased leukocyte infiltration, primarily CD4-positive cells, within a month of therapy. However, those individuals with both senescence and increased TILs were also more likely to acquire resistance later in therapy. These suggest that combining senolytics with abemaciclib in a subset of patients may improve the duration of response. Abemaciclib was well tolerated and showed promising activity in DDLS. The discovery of ANGPTL4 as a late regulator of geroconversion helped to define how CDK4/6i-induced cellular senescence modulates the immune tumor microenvironment and contributes to both positive and negative clinical outcomes. See related commentary by Weiss et al., p. 649.

Topical formulations of Aprepitant are safe and effective in relieving pain and inflammation, and drive neural regeneration

PurposeTo test long-term ocular toxicity and analgesic/anti-inflammatory efficacy of two novel ocular formulations of neurokinin 1 receptor (NK1R) antagonist Aprepitant. Methodsfor toxicity studies, two Aprepitant formulations (X and Y) were tested on C57BL/6 N mice. Gold standards were 0.4% Oxybuprocaine, 0.1% Diclofenac, or saline. For efficacy studies, C57BL/6 N mice underwent corneal alkali burn, and then received Aprepitant formulation X, Dexamethasone or saline. Eye-drops were applied 3 times/day for 90 days (toxicity) and 14 days (efficacy). Stromal opacity, corneal epithelial damage, nociception and sensitivity were assessed in vivo. The eye-wiping test and corneal sensitivity were assessed to evaluate analgesic efficacy and nerve function. At the end of the experiments mice were euthanized, and corneas were dissected for immunohistochemistry and RT-PCR analyses. ResultsIn normal mice, formulation X was not toxic when topically administered for 90 days. Formulation Y was associated with increased leukocyte infiltration in the cornea (p < 0.001). X1 and X2 formulations significantly reduced corneal pain, as Diclofenac and Oxybuprocaine, but did not reduce corneal sensitivity. Formulation Y, instead, was not analgesic at any time point. In the alkali burn model, X1 and X2 formulation enhanced epithelial damage recovery, and reduced inflammation both at day 7 and 14. Moreover, formulation X showed a stronger analgesic effect when compared to the saline and Dexamethasone groups (p < 0.01). Finally, formulation X1 and X2 restored corneal sensitivity by promoting corneal nerve regeneration. ConclusionsAprepitant X formulation is a promising candidate for the treatment of pain associated with inflammation of the ocular surface.

Ethanol exacerbates pulmonary complications after burn injury in mice, regardless of frequency of ethanol exposures

Burn injuries are associated with significant morbidity and mortality, and lungs are the most common organ to fail. Interestingly, patients with alcohol intoxication at the time of burn have worse clinical outcomes, including pulmonary complications. Using a clinically relevant murine model, we have previously reported that episodic ethanol exposure before burn exacerbated lung inflammation. Specifically, intoxicated burned mice had worsened pulmonary responses, including increased leukocyte infiltration and heightened levels of CXCL1 and IL-6. Herein, we examined whether a single binge ethanol exposure before scald burn injury yields similar pulmonary responses. C57BL/6 male mice were given ethanol (1.2 g/kg) 30 min before a 15 % total body surface area burn. These mice were compared to a second cohort given episodic ethanol binge for a total of 6 days (3 days ethanol, 4 days rest, 3 days ethanol) prior to burn injury. 24 h after burn, histopathological examination of lungs were performed. In addition, survival, and levels of infiltrating leukocytes, CXCL1, and IL-6 were quantified. Episodic and single ethanol exposure before burn decreased survival compared to burn only mice and sham vehicle mice, respectively (p < 0.05). However, no difference in survival was observed between burned mice with single and episodic ethanol binge. Examination of H&E-stained lung sections revealed that regardless of ethanol binge frequency, intoxication prior to burn worsened pulmonary inflammation, evidenced by elevated granulocyte accumulation and congestion, relative to burned mice without any ethanol exposure. Levels of infiltrating granulocyte in the lungs were significantly higher in burned mice with both episodic and single ethanol intoxication, compared to burn injury only (p < 0.05). In addition, there was no difference in the granulocyte count between single and ethanol binge mice with burn injury. Neutrophil chemoattractant CXCL1 levels in the lung were similarly increased following single and episodic ethanol exposure prior to burn compared to burn alone (22-fold and 26-fold respectively, p < 0.05). Lastly, we assessed pulmonary IL-6, which revealed that irrespective of frequency, ethanol exposure combined with burn injury raised pro-inflammatory cytokine IL-6 in the lungs relative to burn mice. Again, we did not find any difference in the amount of IL-6 in lungs of burned mice with single and episodic ethanol intoxication. Taken altogether, these data demonstrate that both single and episodic exposure to ethanol prior to burn injury similarly worsens pulmonary inflammation. These results suggest that ethanol-induced exacerbation of the pulmonary responses to burn injury is due to presence of ethanol at the time of injury rather than longer-term effects of ethanol exposure.

Prostaglandin F2α requires activation of calcium-dependent signalling to trigger inflammation in human myometrium.

Preterm birth is one of the major causes of neonatal morbidity and mortality across the world. Both term and preterm labour are preceded by inflammatory activation in uterine tissues. This includes increased leukocyte infiltration, and subsequent increase in chemokine and cytokine levels, activation of pro-inflammatory transcription factors as NF-κB and increased prostaglandin synthesis. Prostaglandin F2α (PGF2α) is one of the myometrial activators and stimulators. Here we investigated the role of PGF2α in pro-inflammatory signalling pathways in human myometrial cells isolated from term non-labouring uterine tissue. Primary myometrial cells were treated with G protein inhibitors, calcium chelators and/or PGF2α. Nuclear extracts were analysed by TranSignal cAMP/Calcium Protein/DNA Array. Whole cell protein lysates were analysed by Western blotting. mRNA levels of target genes were analysed by RT-PCR. The results show that PGF2α increases inflammation in myometrial cells through increased activation of NF-κB and MAP kinases and increased expression of COX-2. PGF2α was found to activate several calcium/cAMP-dependent transcription factors, such as CREB and C/EBP-β. mRNA levels of NF-κB-regulated cytokines and chemokines were also elevated with PGF2α stimulation. We have shown that the increase in PGF2α-mediated COX-2 expression in myometrial cells requires coupling of the FP receptor to both Gαq and Gαi proteins. Additionally, PGF2α-induced calcium response was also mediated through Gαq and Gαi coupling. In summary, our findings suggest that PGF2α-induced inflammation in myometrial cells involves activation of several transcription factors - NF-κB, MAP kinases, CREB and C/EBP-β. Our results indicate that the FP receptor signals via Gαq and Gαi coupling in myometrium. This work provides insight into PGF2α pro-inflammatory signalling in term myometrium prior to the onset of labour and suggests that PGF2α signalling pathways could be a potential target for management of preterm labour.

The MIF promoter SNP rs755622 is associated with immune activation in glioblastoma.

Intratumoral heterogeneity is a defining hallmark of glioblastoma, driving drug resistance and ultimately recurrence. Many somatic drivers of microenvironmental change have been shown to affect this heterogeneity and, ultimately, the treatment response. However, little is known about how germline mutations affect the tumoral microenvironment. Here, we find that the single-nucleotide polymorphism (SNP) rs755622 in the promoter of the cytokine macrophage migration inhibitory factor (MIF) is associated with increased leukocyte infiltration in glioblastoma. Furthermore, we identified an association between rs755622 and lactotransferrin expression, which could also be used as a biomarker for immune-infiltrated tumors. These findings demonstrate that a germline SNP in the promoter region of MIF may affect the immune microenvironment and further reveal a link between lactotransferrin and immune activation.

A micro-sized vaccine based on recombinant Lactiplantibacillus plantarum fights against SARS-CoV-2 infection via intranasal immunization.

COVID-19 has globally spread to burden the medical system. Even with a massive vaccination, a mucosal vaccine offering more comprehensive and convenient protection is imminent. Here, a micro-sized vaccine based on recombinant Lactiplantibacillus plantarum (rLP) displaying spike or receptor-binding domain (RBD) was characterized as microparticles, and its safety and protective effects against SARS-CoV-2 were evaluated. We found a 66.7% mortality reduction and 100% protection with rLP against SARS-CoV-2 in a mouse model. The histological analysis showed decreased hemorrhage symptoms and increased leukocyte infiltration in the lung. Especially, rLP:RBD significantly decreased pulmonary viral loads. For the first time, our study provides a Lactiplantibacillus plantarum-vectored vaccine to prevent COVID-19 progress and transmission via intranasal vaccination.

Increased plasma renin by vasodilators promotes the progression of abdominal aortic aneurysm.

Background: It is well-accepted that antihypertensive therapy is the cornerstone of treatment for abdominal aortic aneurysm (AAA) patients with hypertension. Direct-acting vasodilators were used in the treatment of hypertension by directly relaxing vascular smooth muscle but may have destructive effects on the aortic wall by activating the renin-angiotensin system axis. Their roles in AAA disease remain to be elucidated. In this study, we used hydralazine and minoxidil, two classical direct-acting vasodilators, to investigate their influence and potential mechanisms on AAA disease. Methods and results: In this study, we investigated the plasma renin level and plasma renin activity in AAA patients. Simultaneously, age and gender ratio-matched patients diagnosed with peripheral artery disease and varicose veins were selected as the control group using a ratio of 1:1:1. Our regression analysis suggested both the plasma renin level and plasma renin activity are positively associated with AAA development. In view of the well-established relationship between direct-acting vasodilators and increased plasma renin concentration, we established a porcine pancreatic elastase-infused AAA mouse model, followed by oral administration of hydralazine (250mg/L) and minoxidil (120mg/L) to investigate effects of direct-acting vasodilators on AAA disease. Our results suggested both hydralazine and minoxidil promoted the progression of AAA with increased aortic degeneration. Mechanistically, the vasodilators aggravated aortic inflammation by increased leukocyte infiltration and inflammatory cytokine secretion. Conclusion and relevance: The plasma renin level and plasma renin activity are positively associated with AAA development. Direct vasodilators aggravated experimental AAA progression, which raised cautionary concerns about their applications in AAA disease.

Neurosensory abnormalities and stability of a mouse model of dry eye disease

This study aimed to use a mouse model of dry eye disease (DED) induced by topical administration of benzalkonium chloride (BAK) and assess its stability and the presence of neurosensory abnormalities, including ocular pain. Eight-week-old C57BL6/6 N male mice were used in this study. Mice were treated with 10 μL of 0.2% BAK dissolved in artificial tears (AT), administered twice daily for 7 days. After one week, animals were randomized into two groups: one was administered with 0.2% BAK in AT once per day for 7 days, while the other was not further treated. Corneal epitheliopathy was quantified at days 0, 3, 7, 12, and 14. Moreover, tear secretions, corneal nociception, and corneal nerve integrity were measured after BAK treatment. After sacrifice, corneas were dissected to assess nerve density and leukocyte infiltration by immunofluorescence. Topical BAK instillation for 14 days significantly increased corneal fluorescein staining (p < 0.0001) compared to day 0. On the other hand, interruption of BAK instillation was associated with improvement of corneal epitheliopathy (day 12, p < 0.0001; day 14, p < 0.001). BAK treatment increased ocular pain (p < 0.0001) and resulted in a significant increase in leukocyte infiltration in the cornea (p < 0.01). Moreover, corneal sensitivity was reduced (p < 0.0001), together with corneal nerve density (p < 0.0001) and tear secretion (p < 0.0001). One week twice a day, followed by one additional week once a day, of 0.2% BAK topical administration induces stable clinical and histological signs of DED, which is associated with neurosensory abnormalities, including pain.

CTNNAL1 enhances glucocorticoid sensitivity in HDM-induced asthma mouse model through deactivating hsp90 signaling pathway

AimsAdhesion molecules play vital roles in the induction of airway hyperresponsiveness (AHR) or airway inflammation. The down-regulation of catenin alpha-like 1 (CTNNAL1) in the bronchial epithelial cells of asthma patients and mice models has been noted in our previous study. In this work, we further explore the underlying mechanism of CTNNAL1 in asthma. Main methodsWe constructed a house dust mite (HDM)-induced asthma animal model on control mice and applied CTNNAL1-siRNA transfection to create CTNNAL1-deficient mice. Key findingsWe documented much more severe airway inflammation and increased leukocyte infiltration in the lungs of the CTNNAL1-deficient mice comparing to control mice, along with elevated expression of inflammatory cytokines. Dexamethasone (DEX) treatment led to less reduced inflammation in CTNNAL1-deficient mice compared with control mice. Immunoprecipitation confirmed the interaction between heat shock protein90 (hsp90) and CTNNAL1. The expression of hsp90 was upregulated after CTNNAL1 silencing. Meanwhile, the use of hsp90 inhibitor geldanamycin significantly decreased the expression of NR3C1, ICAM-1 and the ratio of p-p65/p65 in CTNNAL1-silenced 16HBE14o- cells. Both geldanamycin and DEX could function to suppress the expression of ICAM-1 and the phosphorylation level of p65. Nevertheless, the anti-inflammatory effect of DEX proved less potent than geldanamycin in the CTNNAL1-silenced group. The combined therapy of geldanamycin and DEX significantly decreased the inflammatory responses in CTNNAL1-deficient HBE cells than DEX monotherapy. SignificanceOur study corroborates that CTNNAL1 deficiency induced aggravated airway inflammation and rendered insensitivity to glucocorticoids via triggering hsp90 signaling pathway.

Negative air ions through the action of antioxidation, anti-inflammation, anti-apoptosis and angiogenesis ameliorate lipopolysaccharide induced acute lung injury and promote diabetic wound healing in rat.

Negative air ions (NAIs) being bioactive and negative charged molecules may confer antioxidant and anti-inflammatory activity. We assessed the effect of NAIs on two inflammatory diseases in animal models including lipopolysaccharide (LPS) induced acute lung injury (ALI) and wound healing in diabetic rats. We used intra-tracheal infusion of LPS to induce ALI and made a full-thickness cutaneous wound in streptozotocin-induced diabetic female Wistar rats. We evaluated NAIs effects on reactive oxygen species amount, leukocyte infiltration, wound healing rate, western blot, and immunohistochemistry in the lungs of ALI and skin sections of wounds. Our data found NAIs exposed saline displayed higher antioxidant activity vs. non-exposed saline. NAIs exposure did not significantly affect arterial blood pressure and respiratory frequency in control and LPS treated groups. LPS increased leukocyte infiltration, caspase 3/Poly-ADP-ribose-polymerase-mediated apoptosis formation and decreased Beclin-1/LC3-II-mediated autophagy in lungs. NAIs exposure conferred pulmonary protection by depressed leukocyte infiltration and caspase 3/Poly-ADP-ribose-polymerase mediated apoptosis and enhanced LC3-II-mediated autophagy in LPS induced ALI. NAIs treatment resulted in a significantly accelerated wound closure rate, decreased erythrocyte accumulation and leukocyte infiltration mediated oxidative stress and inflammation, and upregulated expression of skin collagen, vascular endothelial growth factor receptor-2 (VEGFR-2) and factor transforming growth factor-beta 1 (TGF-β1) vs non-treated group. Based on these results, it is suggested that NAIs conferred a protection through the upregulating LC3-II-dependent autophagy mechanism and downregulating leukocyte infiltration mediated inflammation and caspase 3/Poly-ADP-ribose-polymerase signaling in the LPS-treated ALI and promoted diabetic wound healing through the enhancing skin collagen synthesis, VEGFR-2 and TGF-β1 pathways.

Immunological profiles of the breast cancer microenvironment represented by tumor-infiltrating lymphocytes and PD-L1 expression

Tumor-infiltrating lymphocytes (TILs) and programmed cell death 1 ligand 1 (PD-L1) are established prognostic and predictive biomarkers for certain breast cancer subsets. However, their association with the immune response complexity is not fully understood. Therefore, we analyzed the association between the immune cell fractions in breast cancer tissues and histologically assessed TIL (hTIL) and PD-L1 (hPD-L1). Forty-five tumor and eighteen blood samples were collected from patients with breast cancer. Total leukocyte counts, frequency of 11 immune cell populations, and PD-L1 expression in each cell fraction were evaluated by flow cytometry. TILs and PD-L1 were assessed by hematoxylin and eosin staining and immunohistochemistry, respectively. A higher hTIL score showed association with increased leukocyte infiltration, higher CD4+ and CD8+ T cell proportions, and lower natural killer and natural killer T cell proportions. PD-L1 was highly expressed in nonclassical monocytes, monocyte/macrophages, myeloid-derived suppressor cells, myeloid dendritic cells, dendritic cells, and other lineages in tumors. hPD-L1 positivity reflected PD-L1 expression accurately in these fractions, as well as increased leukocyte infiltration in tumors. These results indicate that hTILs reflect differences in the immune responses in the tumor microenvironment, and certain immune cell fractions are favorably expressed in the PD-L1 pathway in breast cancer microenvironments.

Liver pathological alterations in fetal rabbit model of congenital diaphragmatic hernia.

To date, fetal liver implication is not a well-understood phenomenon in congenital diaphragmatic hernia (CDH). We evaluated the fetal morphologic changes on liver growth after surgical procedure in CDH experimental model. A diaphragmatic defect at gestational day E25 and tracheal occlusion (TO) at E27 were surgically created in rabbit fetuses. Five experimental groups were assessed: control group, left CDH, right CDH, CDH + TO, and TO alone. Body and organ growth were measured. For histological evaluation of the CDH effect, liver sections were collected. Left-CDH group had livers with increased leukocyte infiltration in comparison with controls (p= 0.02). Increased capillary sinusoid congestion and hepatocyte vacuolation were greater in left-CDH compared with the right-CDH group (p= 0.05). Capillary sinusoid congestion and interstitial edema were more evident in the left-CDH compared with CDH + TO group (p= 0.05). Increases in sinusoid congestion, hepatocyte vacuolation, and interstitial edema were also greater in the CDH + TO compared with controls (p≤ 0.02). Intrathoracic liver weight was higher in right-CDH compared with left-CDH group (p< 0.001). Total lung weights (TLW) were significantly lower in both left-CDH compared with controls (p< 0.001), CDH + TO (p= 0.01), and TO (p< 0.01) and in right-CDH compared with CDH + TO (p< 0.01) and TO (p< 0.01). Decreased kidney and heart weights were also recorded. Hemodynamics and structural fetal liver changes in laterality were noted in CDH model. Regulation of intrathoracic liver weights seems to be disturbed by the absence of diaphragmic contact. Pulmonary injury is supported by the effect of a first hit, while the growth of internal organs suggests a multisystemic remodeling related to the fetal adaptation.