Objective— Antibodies against human neutrophil antigen-3a (HNA-3a) located on choline transporter-like protein 2 induce severe transfusion-related acute lung injury (TRALI). This study aims to identify the mechanism implicated in anti–HNA-3a-mediated TRALI. Approach and Results— Our analysis shows that anti–HNA-3a recognizes 2 choline transporter-like protein 2 isoforms (P1 and P2) on human microvascular endothelial cells from lung blood vessels but reacts only with the P1 isoform on neutrophils. Direct treatment of HNA-3a–positive endothelial cells with anti–HNA-3a, but not with anti–HNA-3b, leads to reactive oxygen species production, increased albumin influx, and decreased endothelial resistance associated with the formation of actin stress filaments and loosening of junctional vascular endothelium-cadherin. In a novel in vivo mouse model, TRALI was documented by significant increase in lung water content, albumin concentration, and neutrophil numbers in the bronchoalveolar lavage on injection of human anti–HNA-3a in lipopolysaccharides-treated, as well as nontreated mice. Interestingly, although neutrophil depletion alleviated severity of lung injury, it failed to prevent TRALI in this model. Infusion of anti–HNA-3a F(ab′) 2 fragments caused moderate TRALI. Finally, mice lacking nicotinamide adenine dinucleotide phosphate oxidase (NOX2 y/ - ) were protected from anti–HNA-3a-mediated TRALI. Conclusions— These data demonstrate the initiation of endothelial barrier dysfunction in vitro and in vivo by direct binding of anti–HNA-3a on endothelial cells. It seems, however, that the presence of neutrophils aggravates barrier dysfunction. This novel mechanism of TRALI primarily mediated by endothelial cell dysfunction via choline transporter-like protein 2 may help to define new treatment strategies to decrease TRALI-related mortality.

This study demonstrated that HEK293T cells expressing stable recombinant HNA-3 are suitable for the detection of HNA-3 alloantibodies allowing reliable screening of blood products.

These results suggested that rapid ELISA using recombinant neutrophil antigens may provide a valuable method for rapid screening of human alloantibodies against HNA-1a, -1b, and -1c in patients with neutropenia and in blood donors.

Ocimum sanctum (OS) is tropical herbal plant which is easy to find and widely used as a vegetable food in Indonesia. In last decade, lung adenocarcinoma was in top position as male cancer disease in Indonesia. Recently, emerging data showing the extracts of different species of Ocimum exhibiting the anti-tumor properties. Further studies on lung lewis carcinoma demonstrated pro-apoptosis effects after the treatment with Ocimum extracts. However, the effect of OS of Indonesian origin in human alveolar pulmonary adenocarcinoma A549 cells remain unclear. Therefore, we aimed to investigate effects of ethanolic extract OS (EEOS) in A549 cell culture systems. Cell adhesion and viability assays revealed that EEOS significantly decreased the attachment into extracellular matrix of A549 cells. Morphological examination AO/EB and DAPI staining indicated that EEOS induced the cells shrinkage, DNA fragmentation and condensation of A549 cells. Further, EEOS treatment induced the apoptosis rate followed by up-regulation of reactive oxygen species (ROS), caspase-3 expression and decreased anti-apoptotic protein Bcl-2. This condition also suppressed the expression of SOD2 as well as the GPx. In conclusion, our findings indicate that EEOS suppressed the viability of A549 cells, which may result from the activation of ROS promoting the apoptosis signaling via mitochondrial intrinsic pathway. Taken together, EEOS might be a good therapeutic potential to further understand its properties in the treatment of lung carcinoma.

Acetylsalicylic acid is used as a non-steroidal anti-inflammatory drugs (NSAID) and antiplatelet agents by inhibiting cyclooxygenases. However, therapy using acetylsalicylic acid could induce gastric bleeding and cause other gastrointestinal toxicity. The aim of this study was to demonstrate the synthesis of a new compound bearing salicylic acid residue namely 2-((3-(chloromethyl)benzoyl)oxy)benzoic acid, to analyze its potential as a ligand for human cyclooxygenase-2 (COX-2) receptor, to evaluate its toxicity level and its effectiveness for analgesic and antiplatelet agent compared with acetylsalicylic acid.Synthesis of 2-((3-(chloromethyl)benzoyl)oxy)benzoic acid was conducted by microwave irradiation. The purity of this compound was evaluated with TLC, IR, NMR, and EDS spectroscopy. The chemical characterization and docking studies against human COX-2 (PDB:5F1A) was performed in-silico. The acute oral toxicity assay was performed under OECD guidelines. The analgesic activity study was performed by plantar and writhing test on animal model. For anti-platelet activity study, we performed tail-bleeding assay and flow cytometry based platelet aggregation assay. We could successfully synthesize a pure white crystalline 2-((3-(chloromethyl)benzoyl)oxy)benzoic acid. In-Silico G-Score result of those compounds gives us preliminary hint of the potential affinity of this compound as a ligand for COX-2 receptor (PDB: 5F1A). Acute toxicity and microscopic gastrointestinal assessments indicated non-observable harmful toxicity parameters. The plantar response time of 2-((3-(chloromethyl)benzoyl)oxy)benzoic acid treated groups showed a significant increment (P < 0.01), and the nociceptive response in writhing test demonstrated a significant dose-dependent decrement. This indicated that its analgesic activity was better than acetylsalicylic acid. The platelet aggregation of 2-((3-(chloromethyl)benzoyl)oxy)benzoic acid was lower than its controls, indicating an aggregation inhibition pattern. The animals treated with 2-((3-(chloromethyl)benzoyl)oxy)benzoic acid gave a longer bleeding time. Overall, this study demonstrated a successful synthesis of pure 2-((3-(chloromethyl)benzoyl)oxy) benzoic acid. We postulated that this compound was better than acetylsalicylic acid, exhibiting excellent analgesic and antiplatelet activity with no toxicity impact.

Introduction: Salicylic acid derivate is very popular for its activity to suppress pain, fever, and inflam ma tion. One of its derivatives is acetylsalicylic acid (ASA) wh ich has be en reported repeatedly that, as a nonsteroidal anti-inflam ma tory drug (NSAID), it has a cardioprotective effect. Although ASA has various advantages, several stud ies have reported that it ma y indu ce severe peptic ulcer disease. We recently synthesized a new compoun d derived from salicylic acid, name ly 2-((3-(chloromethyl)be nz oyl)oxy)be nz oic acid (3 -CH 2 Cl) wh ich still has the be nefit of acetylsalicylic acid as an analgesic and antiplatelet, bu t lacks its harmful side effects (Caroline et al., 20 19 ). In addition, in silico stud ies of 3-CH 2 Cl showed a higher affinity towards protein receptor cyclooxygenase-2 (COX-2; PDB: 5F1A ) than ASA. We hypothesized that 3-CH 2 Cl inhibits the COX-2 activity wh ich could presumably decrease the inflam ma tory responses. However, no knowledge is available on the anti-inflam ma tory response and molecular signaling of this new compoun d. Henc e, in this stud y, we investigated the potential func tional relevanc e of 3-CH 2 Cl in regulating the inflamma tory response in lipopolysacch aride (LPS)-indu ced rats. The results of this stud y show that this compoun d could significantly redu ce the inflam ma tory parame ter in LPS-indu ced rats. Material and methods: Rats were indu ced with LPS of 0.5 mg/kg bw intravenously, prior oral administration with vehicle (3 % Pulvi s Gumm i Arabicum / PGA), 50 0 mg/60 kg body weight (b w; rat dosage converted to hu ma n) of 3-CH 2 Cl and ASA. The inflam ma tory parame ters such as ch anges in the temperature of septic shock, cardiac blood plasma concentrations of IL-1β and TNF-α (ELISA), blood inflam ma tion parame ters, wh ite blood cell concentrations, and lung histopathology were observed. Meanwh ile, the stability of 3-CH 2 Cl powder was evaluated. Result: After the administration of 50 0 mg/60 kg bw of 3-CH 2 Cl (rat dosage converted to hu ma n) to LPSindu ced rats, we observed a significant redu ction of both TNF-α (5 .70+ /-1.04 × 10 3 pg/mL, p= <0 .001 ) and IL-1β (2 .32+ /-0.28 × 10 3 pg/mL, p= <0 .001 ) cardiac blood plasma concentrations. Besides, we foun d a redu ction of wh ite blood cell concentration and the severity of lung injury in the 3-CH 2 Cl group compared to the LPS-indu ced rat group. Ad ditionally, this compoun d ma intained the rat body temperature within norma l limits du ring inflam ma tion, preventing the rats to un dergo septic shock, ch aracterized by hypothermic (t = 12 0 min.) or hyperthermic (t = 36 0 min) conditions. Furthermore, 3-CH 2 Cl was foun d to be stable un til 3 years at 25°C with a relative hu midity of 75 ± 5% . Conclusion: 3-CH 2 Cl compoun d inhibited inflam ma tion in the LPS-indu ced inflam ma tion response model in rats, hypothetically through bind ing to COX-2, and presumably inhibited LPS-indu ced NF-κβ signaling pathways. This stud y could be used as a preliminary hint to investigate the t...

Objective— In contrast to other antibodies involved in transfusion-related acute lung injury, anti–HNA-3a antibodies are incapable of inducing direct neutrophil activation and seem to interact with endothelial cells (ECs) primarily. In animal studies, anti–HNA-3a-mediated transfusion-related acute lung injury could be precipitated in the absence of neutrophils, but was stronger when neutrophils were present. In a different context the target protein of these antibodies, choline transporter-like protein-2 (CTL-2), was reported to interact with a protein of the inner ear carrying 2 von Willebrand factor (VWF) A-domains. These observations prompted us to investigate whether VWF might be involved in anti–HNA-3a-mediated neutrophil activation, and whether signaling via CD11b/CD18 is involved, as in various other experimental settings. Approach and Results— Cell adhesion demonstrated specific binding of CTL-2 to VWF. Immunoprecipitation analysis of CTL-2/CD11b/CD18 coexpressing cells indicated that anti–HNA-3a colocalizes CTL-2 and CD11b/CD18 when VWF is present. Functional studies revealed that anti–HNA-3a-mediated neutrophil agglutination is an active, protein kinase C-dependent and partially Fc-dependent process. Agglutination and the production of reactive oxygen species seem to require the formation of a trimolecular complex between the target antigen (CTL-2), CD11b/CD18 and VWF. In line with these observations, anti–HNA-3a induced less severe transfusion-related acute lung injury and less neutrophil recruitment to the alveolar space in VWF knockout mice. Conclusions— We introduce CTL-2 as a new binding partner for VWF. Interaction of neutrophils with VWF via CTL-2 allows anti–HNA-3a to induce signal transduction via CD11b/CD18, which leads to neutrophil activation and agglutination. In transfusion-related acute lung injury, this mechanism may further aggravate endothelial leakage.

Prostate carcinoma and benign prostate hyperplasia (BPH) with associated lower urinary tract symptoms (LUTS) are among the most prevalent and clinically relevant diseases in men. BPH is characterized by an enlargement of prostate tissue associated with increased tone of smooth muscle cells (SMCs) which surround the single glands composing the prostate. Secretions of the glands leave the prostate through local excretory ducts during the emission phase of ejaculation. Pharmacological treatment of BPH suggests different local drug targets based on reduction of prostate smooth muscle tone as the main effect and disturbed ejaculation as a common side effect. This highlights the need for detailed investigation of single prostate glands and ducts. We combined structural and functional imaging techniques-notably, clear lipid-exchanged, acrylamide-hybridized rigid imaging/immunostaining/ in situ hybridization-compatible tissue-hydrogel (CLARITY) and time-lapse imaging-and defined glands and ducts as distinct SMC compartments in human and rat prostate tissue. The single glands of the prostate (comprising the secretory part) are characterized by spontaneous contractions mediated by the surrounding SMCs, whereas the ducts (excretory part) are quiescent. In both SMC compartments, phosphodiesterase (PDE)-5 is expressed. PDE5 inhibitors have recently emerged as alternative treatment options for BPH. We directly visualized that the PDE5 inhibitors sildenafil and tadalafil act by reducing spontaneous contractility of the glands, thereby reducing the muscle tone of the organ. In contrast, the ductal (excretory) system and thus the prostate's contribution to ejaculation is unaffected by PDE5 inhibitors. Our differentiated imaging approach reveals new details about prostate function and local drug actions and thus may support clinical management of BPH.-Kügler, R., Mietens, A., Seidensticker, M., Tasch, S., Wagenlehner, F. M., Kaschtanow, A., Tjahjono, Y., Tomczyk, C. U., Beyer, D., Risbridger, G. P., Exintaris, B., Ellem, S. J., Middendorff, R. Novel imaging of the prostate reveals spontaneous gland contraction and excretory duct quiescence together with different drug effects.