Intrapleural Injection Research Articles

A2A and 5‐HT Receptors are Differentially Required for Respiratory Plasticity Over the Course of Motor Neuron Loss in Intrapleurally CTB‐SAP Treated Rats

Loss of respiratory (e.g., phrenic) motor neurons leads to ventilatory failure in patients with neuromuscular diseases, but there are currently no treatments to maintain ventilation or to significantly and consistently preserve/restore ventilatory capacity in these patients. Genetic models have been used to study the impact of motor neuron loss; however, the rate and amount of motor neuron death cannot be controlled in these models, and degeneration is not limited to only respiratory motor neurons making it difficult to study treatment modalities to improve ventilation. Thus, we have developed a novel model of controlled respiratory motor neuron death using intrapleural injection of cholera toxin B conjugated to saporin (CTB‐SAP), in which we find that eupneic ventilation is maintained despite deficits in maximal ventilatory capacity. Our previous results demonstrated that respiratory plasticity is not only observed 7 days (d) after CTB‐SAP delivery, but that it is actually enhanced compared to control treatments. At 28d, however, respiratory plasticity is comparable to control levels, suggesting that the underlying mechanisms of plasticity at both time points may differ. Further, we suggest that neuroplasticity mechanisms contribute to maintain ventilation in the face of respiratory motor neuron loss. Although multiple cellular pathways give rise to respiratory plasticity, this study will focus on serotonin (5‐HT) and adenosine 2A (A2A) receptors based on our preliminary receptor expression data. Our preliminary data indicate that A2A receptor expression is increased in 7d, not 28d, CTB‐SAP treated rats, whereas 5‐HT receptor expression decreases at 7d and recovers to control levels in 28d CTB‐SAP treated rats. Thus, we hypothesize that distinct mechanisms (A2A receptor‐dependent at 7d, 5‐HT receptor‐dependent at 28d) enable respiratory plasticity and maintain eupneic ventilation in CTB‐SAP‐induced neuropathology. Using anesthetized, paralyzed and ventilated CTB‐SAP treated adult Sprague Dawley rats, we studied acute intermittent hypoxia (AIH; 3, 5 min bouts of 10.5% O2) induced phrenic long‐term facilitation (pLTF) following intrathecal C4 delivery of either the A2A receptor antagonist (10 μM MSX‐3) or the 5‐HT receptor antagonist (20 mM methysergide) to rats that received bilateral, intrapleural injections of: 1) CTB‐SAP (25μg), or 2) un‐conjugated CTB and SAP (control) in rats treated 7d or 28d prior. Our preliminary results indicate that pLTF is abolished after MSX‐3 at 7d and methysergide at 28d vs. controls and vehicle treated CTB‐SAP rats. These observations suggest that A2A receptors are necessary for respiratory plasticity early (7d), but 5‐HT receptors are required late (28d). Studies are underway to assess whether these receptors are required to maintain eupneic ventilation using whole‐body plethysmography. Results from this project will increase our understanding of how respiratory plasticity is initiated and how eupneic ventilation is maintained over the course of neuropathology, and will have broad implications for potential therapies to preserve/restore ventilatory function in disorders in which respiratory motor neuron death is associated.Support or Funding InformationNIH R00 HL119606 and SCIDRPThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Balancing Neuroprotection with Functional Recovery: The Potential Role of the PNN in Preventing Excitotoxicity after SCI

In spinal cord injury (SCI), initial mechanical trauma causes debilitating primary damage to neural cells and blood vessels. Following this, secondary cascades of downstream events occur, including inflammation, ischemia, and excitotoxicity — an increase in intracellular Ca2+ concentration from overactive glutamate (Glu) receptors leading to cell death. Additionally, there is an upregulation of the perineuronal net (PNN), a lattice‐like structure of the extracellular matrix (ECM) which modulates neural communication and homeostasis. The PNN is partially composed of negatively charged chondroitin sulfate proteoglycans (CSPGs). While CSPGs can stabilize plasticity and neuronal growth during development, these molecules become inhibitory to regeneration, sprouting and plasticity after injury, as well as contribute greatly to the glial scar. However, administration of the bacterial enzyme chondroitinase ABC (ChABC) can digest the PNN and CSPGs, ultimately promoting functional recovery. What remains unknown are the other impacts of removing the PNN at very acute stages of injury. We hypothesize that the PNN and its negatively charged CSPGs are upregulated after SCI as a neuroprotective response that attenuates excitotoxicity by acting as a buffer for Ca2+. To test our hypothesis, we induced excitotoxicity by injecting rats with a threshold dose of NMDA with or without ChABC utilizing the well defined spinal respiratory motor system. 59% of SCI occurs at the cervical level, and mechanical ventilation is the leading cause of death and restriction of independence in these cases. Therefore, we administered the dose instraspinally at the C4 level and paired treatment with intrapleural injection of cholera toxin‐B to retrogradely label the phrenic motor neuron pool, which innervates the diaphragm. Preliminary findings suggest that animals lacking the PNN have exacerbated cell death versus control. This implies that, following SCI, the body's main focus is to survive and not necessarily to preserve function. PNN upregulation after injury could be a conserved mechanism which promotes survival and CNS tissue preservation at the expense of plasticity and functional regeneration. The next steps are to detail the spread of ChABC and how long it takes for the PNN to regenerate. Future directions will investigate the optimal timing of ChABC administration to balance preserving cell survival with promoting functional recovery.Support or Funding InformationNINDS R01NS101105 (WJA)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

IL-10 promotes malignant pleural effusion in mice by regulating TH 1- and TH 17-cell differentiation and migration.

The role of IL-10 in malignant pleural effusion (MPE) remains unknown. By using murine MPE models, we observed that an increase in pleural IL-10 was a significant predictor of increased risk of death. We noted that TH 1- and TH 17-cell content in MPE was higher in IL-10-/- mice than in WT mice, and IL-10 deficiency promoted differentiation into TH 1 but not into TH 17 cells. A higher fraction of TH 1 and TH 17 cells in the MPE of IL-10-/- mice expressed CXCR3 compared with WT mice. We also demonstrated that Lewis lung cancer and colon adenocarcinoma cells secreted large amounts of CXCL10, a ligand of CXCR3, which induced the migration of TH 1 and TH 17 cells into the MPE, and IFN-γ could promote this signaling cascade. Furthermore, intrapleural injection of mice with CXCL10-deficient tumor cells led to decreased TH 1- and TH 17-cell content in MPE, increased MPE volume, and reduced survival of MPE-bearing mice. Taken together, we demonstrated that IL-10 deficiency promoted T-cell differentiation into TH 1 cells and upregulated the CXCR3-CXCL10 signaling pathway that recruits TH 1 and TH 17 cells into MPE, ultimately resulting in decreased MPE formation and longer survival time of mice-bearing MPE.

Abstract A165: Depletion of macrophages in the tumor-draining lymph node enhances dendritic cell-induced antitumor immunity and survival

Abstract Effective antitumor immunity involves successful priming of tumor-specific T-cells by dendritic cells (DC) in the lymph node (LN), followed by trafficking, infiltration and sustained elimination of tumor cells by T-cells. Immunotherapies aim to facilitate or further invigorate these processes, but at which anatomic sites these therapies act and in which specific patients remains unknown. We and others have identified macrophages as key mediators of immune suppression in the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) are capable of negating the effectiveness of multiple conventional- and immune-targeted anticancer therapies. Several current therapeutic strategies aim to deplete or reprogram macrophages; however, as these drugs act systemically, their precise mechanism and site of action remains unclear. Besides TAMs, macrophages in the LN have been identified as potent immune modulatory cells in diverse settings. Using a novel method that allows for the specific interrogation of LN-macrophages, we aimed to investigate the role of LN macrophages in regulating DC-induced anti-tumor immunity. To determine the immune modulatory functions of the tumor on the LN, we extensively characterized the immune contexture and phenotype of the tumor-draining lymph node (TDLN) compared to a distant non-tumor draining lymph node (non-TDLN) using multicolor flow- and histo-cytometry, in an orthotopic mouse model of peritoneal mesothelioma. In addition, the effects of systemic macrophage depletion using an CSF1R-kinase inhibitor or clodronate encapsulated liposomes (CEL) were evaluated. We developed a method to specifically deplete TDLN-macrophages while leaving TAMs and other tissue macrophages intact, by intrapleural (i.pl) injection of low-dose CEL. Using this model, we investigated the immune-regulatory properties of LN-macrophages following adoptive transfer of activated, tumor-loaded DCs in the TDLN by both flow- and histo-cytometry. Comparison of the TDLN and non-TDLN immune contexture revealed prominent shifts in immune cell frequencies and phenotypes, including a decrease in T-cell frequencies and a marked increase in CD169+ LN-macrophages in the TDLN. Systemic macrophage targeting using CSF1R-kinase inhibition or CEL effectively minimized LN-macrophage subsets as well as TAMs, therefore preventing the specific interrogation of LN-macrophage biology during tumor growth and in the context of immune activation. Conversely, titrating CEL doses down to 5% of the total dosing volume injected i.pl. allowed for the specific depletion of LN-macrophages while leaving systemic macrophages undisturbed. LN-macrophages limited the presence of migratory ex vivo activated and tumor-loaded DCs, as indicated by increased counts of CFSE+ DCs in the in the TDLN following adoptive transfer in CEL-pretreated mice. Depletion of TDLN-macrophages increased LN- and blood frequencies and activation status of DC-induced CD8+ T-cells and CD4+ T-helper cells, indicative of enhanced antitumor immunity. Furthermore, whereas DC-induced immune activation alone was insufficient to impede tumor growth and prolong survival, elimination of LN-macrophages prior to DC-administration enabled the successful impediment of tumor growth. Interestingly, interrogation of tumor-infiltrating T-cell (TIL) phenotype and distribution showed a robust increase in CD4+ T-helper TILs and a concurrent decrease in CD8+ TIL- exhaustion phenotype, as evidenced by diminished PD-1 and LAG-3 expression. Altogether, these results indicate an important immune inhibitory role for LN-macrophages in settings of immune activation in the solid tumor setting. These findings further increase our knowledge of where and how macrophages exert their immune-inhibitory functions and allow for better insight into the underpinnings of immunotherapy resistance. Citation Format: Floris F. Dammeijer, Mandy van Gulijk, Melanie M. Lukkes, Menno van Nimwegen, Rudi W. Hendriks, Thorbald T. van Hall, Heleen H. Vroman, Joachim J.G.J.V. Aerts. Depletion of macrophages in the tumor-draining lymph node enhances dendritic cell-induced antitumor immunity and survival [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A165.

ADAM10 mediates malignant pleural mesothelioma invasiveness

Malignant pleural mesothelioma (MPM) is an aggressive cancer with limited therapeutic options and treatment efficiency. Even if the latency period between asbestos exposure, the main risk factor, and mesothelioma development is very long, the local invasion of mesothelioma is very rapid leading to a mean survival of one year after diagnosis. ADAM10 (A Disintegrin And Metalloprotease) sheddase targets membrane-bound substrates and its overexpression is associated with progression in several cancers. However, nothing is known about ADAM10 implication in MPM. In this study, we demonstrated higher ADAM10 expression levels in human MPM as compared to control pleural samples and in human MPM cell line. This ADAM10 overexpression was also observed in murine MPM samples. Two mouse mesothelioma cell lines were used in this study including one primary cell line obtained by repeated asbestos fibre injections. We show, in vitro, that ADAM10 targeting through shRNA and pharmacological (GI254023X) approaches reduced drastically mesothelioma cell migration and invasion, as well as for human mesothelioma cells treated with siRNA targeting ADAM10. Moreover, ADAM10 downregulation in murine mesothelioma cells significantly impairs MPM progression in vivo after intrapleural cell injection. We also demonstrate that ADAM10 sheddase downregulation decreases the production of a soluble N-cadherin fragment through membrane N-cadherin, which stimulated mesothelioma cell migration. Taken together, we demonstrate that ADAM10 is overexpressed in MPM and takes part to MPM progression through the generation of N-cadherin fragment that stimulates mesothelioma cell migration. ADAM10 inhibition is worth considering as a therapeutic perspective in mesothelioma context.

Pleural fluid penetration of moxifloxacin and doripenem: An experimental model of empyema.

OBJECTIVE:This study aimed to evaluate the penetration of moxifloxacin and doripenem into the pleural fluid (PF) using a rabbit model of empyema.METHODS:An empyema was induced using the intrapleural injection of turpentine (1 mL), followed 24 h later by instillation of 5 mL Klebsiella Pneumoniae (ATCC 33495), Fusobacterium nucleatum (ATCC 25586) and Streptokok Pneumoniae (ATCC 6305) into the pleural space. After an empyema was corroborated, Moxifloxacin (25 mg/kg-1) and Doripenem (20 mg/kg-1) were administered intraperitoneally. To determine the levels of antibiotics measured by High-Performance Liquid Chromatography in pleural and blood samples were obtained serially at 8, 24, 48 and 72nd hour.RESULTS:The penetration of both antibiotics into the PF was very good. The penetration rate of doripenem (area under the curve (AUC) for PF/blood (AUCPF/AUCblood) ratio=1.68) was better than moxifloxacin (ratio=0.78). Equalization time between the PF and blood concentration of doripenem was more quickly than moxifloxacin. Peak PF concentration of moxifloxacin was 0,81 μg/mL-1 and occurred 8 h after infusion and then gradually decreased; at the beginning of the blood and pleural fluid concentrations of doripenem were equal. While the pleura concentration was increasing, blood concentration was almost the same. Doripenem reached a peak concentration (0.54 μg/ml) 24 h post-administration.CONCLUSION:Differences were found in the penetration of the two antibiotics. Doripenem had convenient penetration PF compared to moxifloxacin. Due to the differences between human and rabbit pleural thickness, doripenem’s pleural penetration should be examined in infection models in animals with equal pleura thickness and clinical trials.

Anti-EGFR reduces malignant pleural effusion and morbidity in experimental model of adenocarcinoma

Introduction: A lot patients with lung cancer develop pleural effusion in an advanced stage of the disease, with high morbidity and mortality. The pathogenesis of the malignant pleural effusion is not well known and the therapeutic options are limited. Currently target therapy, like the antibody anti-EGFR, is been used more often in the treatment of the lung cancer. The EGFR is a tyrosine-kinase receptor considered oncogenic and responsible for the growing, the survival, the proliferation and the differentiation of loads of cell types. The use of antibodies anti-EGFR may entail positive effects in the fight against the tumor and the effects of the malignant pleural effusion. Goals: To evaluate the effects of intrapleural therapy with anti-EGFR in experimental model of malignant pleural effusion. Material e Methods: This study received the approval of the Comitê de Ética no Uso de Animais (CEUA) from HCFMUSP. Sixty C57BL/6 mice received intrapleural injection of 0,5x105 Lewis Lung Carcinoma (LLC) cells. The animals were divided in two groups that received the intrapleural injection with anti-EGFR or PBS (No treatment/Control) after 3, 7, 10 and 14 days from the induction of the pleural neoplasia. Ten animals from each group were observed until their death to evaluate a survival graphic curve. Forty animals were submitted to euthanasia after 7, 10, 14 or 21 days. We evaluated the weight gain or loss in grams (g), the mobility (score 0-3), the volume of recovered pleural effusion in milliliters (mL), and the presence of tumor in in the pleura, the pericardium, the inflammatory cells of the lungs and the histological changes in the kidneys, liver and spleen. Tumor apoptosis (TUNEL) and proliferation (PCNA) were evaluated by scores (0-4). Statistical analysis: One Way ANOVA, Kaplan-Meier, p <0,05. Results: On the survival analysis we did not observe any statistical difference between the groups. The mice weight loss was observed in all the groups after 21 days. It was observed a better mobility in the groups that receives the anti-EGFR treatment. The pleural effusion volume was higher in the control group during all the study. The presence of pleural tumor implants was higher in the control group in comparison to the group that received treatment after 14 days. Pulmonary inflammation was discreet in all groups. The histological evaluation of the pericardium and the cardiac muscle showed tumor implants in the control group after 21 days. Hepatic and renal steatosis were more evident after 14 days in the control group. White flesh hyperplasia of the spleen was observed in all the periods of the study, especially in the 21st day in the control group. Higher levels of apoptosis and lower levels of tumor proliferation were observed in the groups that received treatment with anti-EGFR. Conclusions: In this experimental model, although it did not showed any benefic change in the animals survival, the target therapy with anti-EGFR reduced the pleural effusion volume, the morbidity and showed good histological parameters.

Spinal protein phosphatase 1 constrains respiratory plasticity after sustained hypoxia

Plasticity is an important aspect of the neural control of breathing. One well-studied form of respiratory plasticity is phrenic long-term facilitation (pLTF) induced by acute intermittent but not sustained hypoxia. Okadaic acid-sensitive protein phosphatases (PPs) differentially regulate phrenic nerve activity with intermittent vs. sustained hypoxia, at least partially accounting for pLTF pattern sensitivity. However, okadaic acid inhibits multiple serine/threonine phosphatases, and the relevant phosphatase (PP1, PP2A, PP5) for pLTF pattern sensitivity has not been identified. Here, we demonstrate that sustained hypoxia (25 min, 9-10.5% O2) elicits phrenic motor facilitation in rats pretreated with bilateral intrapleural injections of small interfering RNAs (siRNAs; Accell-modified to preferentially transfect neurons, 3.33 μM, 3 days) targeting PP1 mRNA (48 ± 14% change from baseline, n = 6) but not PP2A (14 ± 9% baseline, n = 6) or nontargeting siRNAs (4 ± 10% baseline, n = 7). In time control rats (no hypoxia) treated with siRNAs ( n = 6), no facilitation was evident (-9 ± 9% baseline). siRNAs had no effect on the hypoxic phrenic response. Immunohistochemistry revealed PP1 and PP2A protein in identified phrenic motoneurons. Although PP1 and PP2A siRNAs significantly decreased PP1 and PP2A mRNA in PC12 cell cultures, we were not able to verify "knockdown" in vivo after siRNA treatment. On the other hand, PP1 and PP2A siRNAs significantly decreased PP1 and PP2A mRNA in PC12 cell cultures, verifying the intended siRNA effects. In conclusion, PP1 (not PP2A) is the relevant okadaic acid-sensitive phosphatase constraining phrenic motor facilitation after sustained hypoxia and likely contributing to pLTF pattern sensitivity. NEW & NOTEWORTHY This study demonstrates that the relevant okadaic acid-sensitive Ser/Thr protein phosphatase (PP) constraining facilitation after sustained hypoxia is PP1 and not PP2A. It suggests that PP1 may be critical in the pattern sensitivity of hypoxia-induced phrenic motor plasticity.

Severe Adverse Reactions Induced by the Chest Injection of Elemene: An Analysis of 7 Cases

背景与目的恶性胸腔积液（malignant pleural effusion, MPE）指胸膜原发恶性肿瘤或其他部位恶性肿瘤转移至胸膜引起的胸腔积液。胸腔内注射榄香烯对MPE有良好的治疗效果，在临床中应用较普遍，同时存在一些不良反应，但文献缺乏对严重不良反应的报道及处理。本研究旨在观察榄香烯注射液治疗恶性胸腔积液导致不良反应发生情况，探讨严重不良反应发生原因和应对方案。方法回顾性分析14例胸腔注射榄香烯病例，对其中7例发生严重不良反应病例进行详细报道。结果榄香烯注射液胸腔注射所致严重不良反应主要表现为剧烈胸痛、呼吸困难、喘憋、意识模糊和凝血功能障碍，经临床对症治疗后均可缓解。结论胸腔注射榄香烯榄香烯对MPE有一定治疗价值，使用时应严格把握适应证、筛选患者、严密监测。

OK-432 pleurodesis for the treatment of pneumothorax in patients with interstitial pneumonia.

Pneumothorax occasionally develops in patients with interstitial pneumonia (IP) and is often intractable. As there exists no well-established treatment for pneumothorax with IP, we evaluated the efficacy and safety of pleurodesis with OK-432, a lyophilized preparation of Streptococcus pyogenes Su strain that has been inactivated by benzylpenicillin. We retrospectively evaluated the efficacy and safety of pleurodesis using OK-432 in 39 patients treated for IP-related pneumothorax between January 2006 and May 2017. Five to 10 Klinische Einheit (KE) of OK-432 was injected through the chest tube of each patient. Pleurodesis was considered successful if 1) the chest tube was removed without air leaks and 2) there was no recurrence of pneumothorax within 4 weeks after tube removal, and no additional treatment was required. OK-432 pleurodesis was performed 46 times in 39 patients. The median number of OK-432 intrapleural injections received was 1 (range, 1-6), and median dose was 10 KE (range, 5-55 KE). The success rate was 63% (29/46) and recurrence rate was 17.4% (8/46). Grade 5 adverse events were observed in eight patients, including two patients who developed acute exacerbation of IP. Patients in whom the first OK-432 pleurodesis was successful had a significantly longer median survival time than patients in whom it was unsuccessful (322 days vs. 70 days, p = 0.036). Our results show that OK-432 pleurodesis is an effective treatment for pneumothorax associated with IP; however, clinicians should be aware of the possibility of adverse events, especially in patients who are critically ill.

Analysis of the etiology and treatment of chylothorax in 119 pediatric patients in a single clinical center

PurposeTo summarize the etiology and treatment of 119 patients with chylothorax in our hospital. MethodsA total of 119 patients with chylothorax, divided into a neonate group, an infant group, and an older-than-1-year group, were included in our study and analyzed from January 2000 to July 2017 in the Children’s Hospital of Chongqing Medical University. ResultsA total of 90 males and 29 females were included in our study. In the neonate group, 21 chylothorax cases were due to idiopathic factors, constituting 72.4% of the cases; 8 were related to cardiothoracic surgery, constituting 27.6%. In the infant group, 15 chylothorax cases were due to cardiothoracic surgery, constituting 55.6% of the cases; 11 were related to idiopathic factors, constituting 40.7%; and 1 was related to lymphoma, constituting 3.8%. In the older-than-1-year group, 33 chylothorax cases were due to idiopathic factors, constituting 52.4% of the cases; 25 were cardiothoracic surgery, constituting 39.7%; 2 were related to another internal medicine disease, constituting 3.2%; 2 were due to injury, constituting 3.2%; and 1 was related to lymphoma, constituting 1.6%. All the patients sequentially underwent thoracic drainage therapy, followed by fasting, thoracic injection of an adhesion-promoting agent, and thoracic duct ligation surgery. Among the neonates, 23 patients (79%) with fasting therapy improved, and 5 patients with fasting + intrapleural injection improved (17%). In the infant group, fasting promoted recovery in 14 patients, accounting for 51%, and fasting + thoracic injection improved the conditions of 10 patients, accounting for 37%. In the older-than-1-year group, fasting was effective in 35 patients, accounting for 55%; fasting + thoracic injection was effective in 22 patients, accounting for 34%; and fasting + thoracic injection + thoracic duct ligation surgery enabled the recovery of 2 patients, accounting for 3.2%. ConclusionIn our center, the main causes of chylothorax in the neonates group are idiopathic factors but may also include a history of unspecified birth trauma; the proportions of idiopathic factors in the infant and older-than-1-year groups are also higher, but the iatrogenic factors are significantly increased in the latter 2 groups. All the patients underwent thoracic drainage therapy, and fasting promoted the recovery of most children. When fasting was ineffective, subsequent thoracic injections were effective. If the above two methods failed, surgery was a method of choice, but it was not always effective. Level of EvidenceLevel IV. Type of StudyRetrospective study.

Inflammation in the pleural cavity following injection of multi-walled carbon nanotubes is dependent on their characteristics and the presence of IL-1 genes

Upon inhalation, multi-walled carbon nanotubes (MWCNTs) may reach the subpleura and pleural spaces, and induce pleural inflammation and/or mesothelioma in humans. However, the mechanisms of MWCNT-induced pathology after direct intrapleural injections are still only partly elucidated. In particular, a role of the proinflammatory interleukin-1 (IL-1) cytokines in pleural inflammation has so far not been published. We examined the MWCNT-induced pleural inflammation, gene expression abnormalities, and the modifying role of IL-1α and β cytokines following intrapleural injection of two types of MWCNTs (CNT-1 and CNT-2) compared with crocidolite asbestos in IL-1 wild-type (WT) and IL-1α/β KO (IL1-KO) mice. Histopathological examination of the pleura 28 days post-exposure revealed mesothelial cell hyperplasia, leukocyte infiltration, and fibrosis occurring in the CNT-1 (Mitsui-7)-exposed group. The pleura of these mice also showed the greatest changes in mRNA and miRNA expression levels, closely followed by CNT-2. In addition, the CNT-1-exposed group also presented the greatest infiltrations of leukocytes and proliferation of fibrous tissue. WT mice were more prone to development of sustained inflammation and fibrosis than IL1-KO mice. Prominent differences in genetic and epigenetic changes were also observed between the two genotypes. In conclusion, the fibrotic response to MWCNTs in the pleura depends on the particles’ physico-chemical properties and on the presence or absence of the IL-1 genes. Furthermore, we found that CNT-1 was the most potent inducer of inflammatory responses, followed by CNT-2 and crocidolite asbestos.

Evaluation of efficacy and safety for Brucea javanica oil emulsion in the control of the malignant pleural effusions via thoracic perfusion

BackgroundBrucea javanica oil emulsion (BJOE) is traditional Chinese medicine with implicated anti-tumor activity, which has been used for treating lung cancer in China. The aim of this investigation was to evaluate the effects and safety of intrapleural injection of BJOE in treating malignant pleural effusion (MPE).MethodsThe randomised controlled trials (RCTs) on the effects and safety of BJOE in treating MPE were searched from electronic medical database including MEDLINE, SCI, EMBASE, Cochrance Library and CNKI. A total of 14 RCTs with 1085 patients were involved in this meta-analysis.ResultsThe overall response rate (ORR) of traditional chemotherapy drugs plus BJOE was higher than that of traditional chemotherapy drugs alone (p = 0.001; odds ratio = 1.39). Meanwhile, the combination of BJOE and traditional chemotherapy drugs improved the quality of life (QOL) of patients with MPE (p < 0.001; odds ratio = 1.56) compared with traditional chemotherapy drugs alone. Moreover, the participation of BJOE reduced the myelotoxicity and digestive reactions caused by traditional chemotherapy drugs (p < 0.05).ConclusionsThe efficacy and safety of traditional chemotherapy drugs plus BJOE was superior to traditional chemotherapy drugs alone via intrapleural injection in controlling MPE, which suggested that BJOE can be used to treat MPE.

Plasminogen and the Plasminogen receptor, Plg‐RKT, regulate efferocytosis and macrophage reprogramming

Inflammation resolution is an active process that functions to restore tissue homeostasis. Phagocytic clearance of apoptotic leukocytes (efferocytosis) at sites of inflammation plays an important role in inflammation resolution by inducing remarkable macrophage phenotypic plasticity. The M1/M2 nomenclature provides a basic classification system to distinguish proinflammatory (M1) from anti‐inflammatory/resolving macrophages (M2/Mres). Plasminogen (Plg) is synthesized by the liver and is the proenzyme of the broad‐spectrum serine protease, plasmin (Pla), which is the major enzyme responsible for fibrinolysis. The participation of the Plg system in the productive phase of inflammation is well known, but the involvement of the Plg system in the resolving phase of inflammation is just emerging. Here we investigated the effects of deletion of Plg and the Plg receptor, Plg‐RKT, separately, in the context of resolution of inflammation in vivo. Plg−/− and Plg‐RKT −/− mice and their respective wild type littermates were challenged by intrapleural injection of LPS (250ng/mouse, i.pl.) or PBS and cells present in the pleural cavity were harvested at 4, 8, 24 and 48 hr and processed for total and differential leukocyte counts and flow cytometric analyses. In this model of pleurisy there is a time‐dependent influx of leukocytes into the pleural cavity. The number of neutrophils and M1 macrophages peaks at 8 hr, and decreases at 48 hr. There is a significant increase in mononuclear cell recruitment into the pleural cavity that coincides with the resolution phase of inflammation and with prevalence of M2 and Mres macrophages at 48hr. In the current study, in the setting of LPS‐induced inflammation, increased numbers of M1 macrophages were present in Plg−/− mice 8h after LPS challenge without changes in number of M2 or Mres macrophages, as compared to Plg+/+ littermates [Plg+/+ = 155 ± 39 and Plg−/− = 301 ± 57 × 103 M1 macrophages/cavity (P &lt; 0.05, n=6)]. There were no genotype‐dependent effects on the number of neutrophils recruited to the pleural cavity at all time points evaluated. At 48 hr the total number of mononuclear cells recruited to the pleural cavity was significantly decreased in both Plg−/− and Plg‐RKT −/− mice, relative to their respective wild type littermate controls [Plg+/+ = 3,432 ± 335 and Plg−/− = 2,377 ± 242 (P &lt; 0.01, n=5); Plg‐RKT +/+ = 2,780 ± 341 and Plg‐RKT −/− = 1,725 ± 95 × 103 mononuclear cells/cavity (P &lt; 0.05, n=6)]. Additionally, the frequency of the M2 marker, CD206, and Annexin A1 (both important for clearance of apoptotic cells) was decreased in Plg−/− macrophages 48 hr after LPS injection. To explore the roles of Plg and Plg‐RKT in efferocytosis, mice were injected with zymosan intraperitoneally, followed by an intraperitoneal injection of apoptotic human neutrophils labeled with carboxyfluorescein succinimidyl ester (CFSE). Significantly decreased efferocytosis of apoptotic neutrophils by Plg−/− macrophages and by Plg‐RKT−/− macrophages, compared with their wild type littermate controls was observed [Plg+/+ = 20.16 ± 3.57 and Plg−/− = 9.04 ± 3.50 (P &lt; 0.05, n=5); Plg‐RKT +/+ = 18.11 ± 4.57 and Plg‐RKT−/− = 5.80 ± 2.90 frequency of total cells/cavity (P &lt; 0.05, n=6)]. These results suggest that Plg regulates macrophage polarization and efferocytosis of apoptotic cells, thereby contributing to the resolution of inflammation and that these effects are mediated by the Plg receptor, Plg‐RKT.Support or Funding InformationCAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil), National Institutes of Health (grant HL 081046 to L.A.M.) and by Merit Review Award #5I01BX002026 from the U.S. Department of Veterans Affairs (to R.J.P.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

5‐HT2B Receptor Expression in the Phrenic Motor Nucleus of Rats with Intrapleural CTB‐Saporin‐Induced Phrenic Motor Neuron Death

Intrapleural injection of cholera toxin B fragment conjugated to saporin (CTB‐SAP) selectively kills respiratory motor neurons, and mimics aspects of neuromuscular disorders and neurodegenerative diseases including phrenic motor neuron death and decreased phrenic motor output. In this model, respiratory plasticity (acute intermittent hypoxia induced phrenic long‐term facilitation; pLTF) is enhanced following 7 days (d) of CTB‐SAP compared to control treatment. In contrast, pLTF in 28d CTB‐SAP treated rats is comparable to pLTF in controls, suggesting that the underlying mechanisms of plasticity at both time points may differ. pLTF in naïve rats predominately requires 5‐HT2 receptors; however, there is an alternative mechanism for pLTF that requires A2A receptors. Our preliminary data suggest that A2A receptors are increased in the phrenic motor nucleus of 7d, not 28d, CTB‐SAP treated rats (Craig et al., 2017), suggesting that 7d CTB‐SAP rats utilize the alternative mechanism for enhanced pLTF. However, it remains unknown how CTB‐SAP induced phrenic motor neuron death affects 5‐HT2A/B receptor expression, and if 5‐HT2A/B receptors are utilized for pLTF in CTB‐SAP treated rats. Here, we tested the hypothesis that 5‐HT2B receptor expression in the phrenic motor nucleus is decreased in 7d CTB‐SAP treated rats, but is not different in 28d CTB‐SAP treated rats when compared to controls using immunohistochemistry. 5‐HT2B receptor expression was studied in Sprague Dawley rats 7 and 28 days following bilateral intrapleural injections of: 1) CTB‐SAP (25μg), or 2) un‐conjugated CTB and SAP (control). Our preliminary data indicate that following CTB‐SAP injection, total 5‐HT2B receptor positive pixels and C4 fractional area are decreased in the phrenic motor nucleus and non‐phrenic ventral horn at 7d, not 28d, vs. controls (n=3 for each group; p&lt;0.05). This suggests 5‐HT2B receptors recover to control levels and are potentially utilized for pLTF in 28d CTB‐SAP treated rats. Future studies will be focused on whether 5‐HT2A receptor expression is similarly affected, whether 5‐HT2A/B receptor expression is impacted on other cell types (i.e., glial cells), and which 5‐HT receptor isoform is required for pLTF following CTB‐SAP‐induced phrenic motor neuron death. This project increases our understanding of the expression of receptors associated with respiratory plasticity and its implications for breathing following motor neuron death.Support or Funding InformationSupported by NIH K99/R00 HL119606This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inflammation Differentially Impacts Phrenic Long‐term Facilitation (pLTF) in Rats with Motor Neuron Death Induced by Intrapleural CTB‐saporin Injections

Intrapleural injection of cholera toxin B fragment conjugated to saporin (CTB‐SAP) selectively kills respiratory motor neurons, and mimics aspects of neuromuscular disorders and neurodegenerative diseases including phrenic motor neuron death, decreased phrenic motor output, and increased microglial cell number. Our previous results demonstrated unexpectedly that respiratory plasticity is not only observed 7 days (d) after CTB‐SAP delivery, but that it is actually enhanced compared to control treatments. At 28d, however, respiratory plasticity is comparable to control levels, suggesting that the underlying mechanisms of plasticity at both time points may differ. Because inflammation is a hallmark of all neurodegenerative diseases, and it is known to prevent the development of acute intermittent hypoxia‐induced phrenic long‐term facilitation (pLTF), we predicted that inflammation would impair pLTF in CTB‐SAP‐treated rats and thus, anti‐inflammatory drugs would enhance pLTF at both time points. Using anesthetized, paralyzed and ventilated CTB‐SAP‐treated adult Sprague Dawley rats, we studied acute intermittent hypoxia (AIH; 3, 5 min bouts of 10.5% O2) induced pLTF following intrathecal C4 delivery of the nonsteroidal anti‐inflammatory drug ketoprofen to rats that received bilateral, intrapleural injections of: 1) CTB‐SAP (25μg), or 2) un‐conjugated CTB and SAP (control) in rats treated 7d or 28d prior. Surprisingly, preliminary results indicate that ketoprofen attenuates pLTF in 7d CTB‐SAP‐treated rats, but enhances it in the 28d CTB‐SAP‐treated rats. These observations suggest that inflammation potentially contributes to pLTF in 7d CTB‐SAP‐treated rats, whereas it may undermine pLTF in 28d CTB‐SAP‐treated rats. Studies are underway to assess cervical spinal levels of both pro‐inflammatory and anti‐inflammatory and neurotrophic molecules at both time points, and their cellular source. Future studies will investigate the potential contributions of microglia to the mechanisms whereby inflammation differentially impacts pLTF in CTB‐SAP‐treated rats. Results from this project will increase our understanding of how inflammation and microglial activities influence respiratory plasticity following motor neuron death, and will have broad implications for therapies restoring ventilatory function in many disorders in which motor neuron death is associated.Support or Funding InformationNIH K99/R00 HL119606This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phrenic Motor Neuron Survival Below a Cervical Spinal Cord Injury

Cervical spinal injury (cSCI) severs descending brainstem projections to spinal respiratory motor neurons, paralyzing respiratory muscles caudal to injury. Spinal hemisection at C2 (C2Hx) has frequently been used as an experimental model of cSCI to study both spontaneous and induced functional recovery of respiratory and non‐respiratory somatic motor function. One assumption implicit in these studies is that C2Hx dennervates motor neurons below the injury, but does not cause significant motor neuron pathology or cell death. However, one recent study reported substantial, bilateral motor neuron death caudal to C2Hx (Satkunendrarajah et al, 2016); in this study, motor neurons were identified by ChAT immunoreactivity in the spinal ventral horn. We have also observed that retrograde labeling with Cholera toxin B fragment (CtB) is diminished in phrenic motor neurons following C2Hx (unpublished data) when the tracer was administered post‐C2Hx. However, these observations are potentially misleading since: 1) C2Hx may diminish ChAT immunoreactivity without motor neuron cell death; and 2) retrograde CtB transport may be diminished following injury due to motor neuron inactivity below the injury, leading to the false impression that phrenic motor neurons had died. Because phrenic motor neuron cell death following cSCI would have profound implications for studies of spontaneous and/or induced phrenic motor plasticity after C2Hx, we tested the hypothesis that C2Hx has minimal impact on phrenic motor neuron cell counts when phrenic motor neurons are retrogradely labeled with CtB prior to cSCI. Phrenic motor neurons were retrogradely labeled in intact adult Sprague Dawley rats via intrapleural injections of CtB. Rats were randomly assigned to one of four groups: 1) 2 week post‐C2Hx, 2) 8 week post‐C2Hx, or 3 &amp; 4) age matched uninjured controls. Two weeks after CtB injection, rats in the C2Hx groups underwent left lateral C2Hx injury under isoflurane anesthesia. Rats were perfused at 2 or 8 wks post‐injury, and longitudinal sections (40 μm) from C3 to C8 were processed to reveal CtB and ChAT. CtB labeled phrenic motor neurons were quantified in serial sections; no loss of phrenic motor neurons was observed at either time post‐C2Hx versus spinally intact rats (p&gt;0.05). Analysis of ChAT immunoreactivity is ongoing; however preliminary data suggest minimal changes in phrenic motor neuron ChAT expression following injury. Although we found little evidence for phrenic motor neuron cell death caudal to C2Hx, we cannot rule out greater cell loss in other motor neuron pools. Consequently, ChAT immunoreactivity is currently underway in non‐phrenic cervical motoneurons caudal to C2Hx. These findings have important implications for studies concerning plasticity in cervical motor systems following cervical spinal injury.Support or Funding InformationSupported by: K12HD055929 (EGR), RO1HL69064 (GSM), T32HD043730 (Allen), and grants from the DoD (GSM), the American Physiological Society (AH, JVS), the McNair Scholars Foundation (JVS), and the McKnight Brain Institute (GSM, MCC).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phrenic motor neuron adenosine 2A receptors elicit phrenic motor facilitation.

Although adenosine 2A (A2A ) receptor activation triggers specific cell signalling cascades, the ensuing physiological outcomes depend on the specific cell type expressing these receptors. Cervical spinal adenosine 2A (A2A ) receptor activation elicits a prolonged facilitation in phrenic nerve activity, which was nearly abolished following intrapleural A2A receptor siRNA injections. A2A receptor siRNA injections selectively knocked down A2A receptors in cholera toxin B-subunit-identified phrenic motor neurons, sparing cervical non-phrenic motor neurons. Collectively, our results support the hypothesis that phrenic motor neurons express the A2A receptors relevant to A2A receptor-induced phrenic motor facilitation. Upregulation of A2A receptor expression in the phrenic motor neurons per se may potentially be a useful approach to increase phrenic motor neuron excitability in conditions such as spinal cord injury. Cervical spinal adenosine 2A (A2A ) receptor activation elicits a prolonged increase in phrenic nerve activity, an effect known as phrenic motor facilitation (pMF). The specific cervical spinal cells expressing the relevant A2A receptors for pMF are unknown. This is an important question since the physiological outcome of A2A receptor activation is highly cell type specific. Thus, we tested the hypothesis that the relevant A2A receptors for pMF are expressed in phrenic motor neurons per se versus non-phrenic neurons of the cervical spinal cord. A2A receptor immunostaining significantly colocalized with NeuN-positive neurons (89±2%). Intrapleural siRNA injections were used to selectively knock down A2A receptors in cholera toxin B-subunit-labelled phrenic motor neurons. A2A receptor knock-down was verified by a ∼45% decrease in A2A receptor immunoreactivity within phrenic motor neurons versus non-targeting siRNAs (siNT; P<0.05). There was no evidence for knock-down in cervical non-phrenic motor neurons. In rats that were anaesthetized, subjected to neuromuscular blockade and ventilated, pMF induced by cervical (C3-4) intrathecal injections of the A2A receptor agonist CGS21680 was greatly attenuated in siA2A (21%) versus siNT treated rats (147%; P<0.01). There were no significant effects of siA2A on phrenic burst frequency. Collectively, our results support the hypothesis that phrenic motor neurons express the A2A receptors relevant to A2A receptor-induced pMF.

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice.

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Efficacy and safety of intrapleural cisplatin versus silver nitrate in treatment of malignant pleural effusion

IntroductionMalignant pleural effusion is a frequent problem. Pleurodesis is performed to prevent its recurrence. New, effective, and safe sclerosing agents are needed.AimThe aim of this was to compare efficacy and safety of silver nitrate solution 0.5% versus cisplatin in achievement of pleurodesis in malignant pleural effusion.Patients and methodsProspective randomized single-blinded clinical trial performed at Chest, Clinical Oncology and Nuclear Medicine and Pathology Departments, Mansoura University, from February 2016 to March 2017. A total of 60 patients (26 male and 34 female) with malignant pleural effusion were divided into two groups: first group included 30 patients who were managed with silver nitrate pleurodesis, and second group included 30 patients who were managed by intrapleural cisplatin injection. The success rate of pleurodesis was considered if there was no clinical or radiological recurrence of effusion for 1 month after intervention.ResultsThere were significant improvements in cough, chest pain, and dyspnea in the two groups after 1 month versus that before pleurodesis. The success rate of pleurodesis in silver nitrate group was 90 versus 76.7% in cisplatin group, without significant difference (P=0.166). Chest pain was reported in 26.7% in silver nitrate group and 13.3% in cisplatin group, and fever was reported in 33.3% in silver nitrate group and 20.0% in cisplatin group. Recurrence was reported in 10% in silver nitrate group and in 23.3% in cisplatin group.ConclusionSilver nitrate and cisplatin were nearly equally effective, safe, and less expensive agents in achievement of pleurodesis in patients with malignant effusion with high success rate and low complications.