Late-Onset Exudative Pleural Effusions Without Concomitant Airway Obstruction or Lung Parenchymal Abnormalities: A Novel Presentation of Chronic Lung Allograft Dysfunction.

Restrictive allograft syndrome (RAS) is an aggressive variant of CLAD characterized by progressive restrictive ventilatory decline and persistent pleuro-parenchymal changes that can be seen on chest CT. We identified four lung transplant recipients with a progressive restrictive ventilatory defect due to lymphocyte-predominant exudative pleural effusions, but no pleuro-parenchymal abnormalities typical of RAS. Using molecular analysis, we also found increased levels of previously described immune markers of RAS, including NFkB, 20S proteasome, lipocalin, TNFα, and TGFß, within the circulating small extracellular vesicles of the remaining living lung transplant recipient. Despite the absence of lung parenchymal changes, these patients had a poor prognosis with rapid deterioration in allograft function and no response to pleural-based interventions such as thoracentesis, decortication, and pleurodesis. We hypothesize that these cases represent a distinct CLAD phenotype characterized by progressive restriction due to pleural inflammation, lymphocyte-predominant pleural effusion, resultant compressive atelectasis, and eventual respiratory failure in the absence of lung parenchymal involvement.

Downregulation of a tumor suppressor gene LKB1 in lung transplantation as a biomarker for chronic murine lung allograft rejection.

BACKGROUND: We recently demonstrated decreased tumor suppressor gene liver kinase B1 (LKB1) level in lung transplant recipients diagnosed with bronchiolitis obliterans syndrome. STE20-related adaptor alpha (STRADα) functions as a pseudokinase that binds and regulates LKB1 activity. METHODS: A murine model of chronic lung allograft rejection in which a single lung from a B6D2F1 mouse was orthotopically transplanted into a DBA/2J mouse was employed. We examined the effect of LKB1 knockdown using CRISPR-CAS9 in vitro culture system. RESULTS: Significant downregulation of LKB1 and STRADα expression was found in donor lung compared to recipient lung. STRADα knockdown significantly inhibited LKB1, pAMPK expression but induced phosphorylated mammalian target of rapamycin (mTOR), fibronectin, and Collagen-I, expression in BEAS-2B cells. LKB1 overexpression decreased fibronectin, Collagen-I, and phosphorylated mTOR expression in A549 cells. CONCLUSIONS: We demonstrated that downregulation of LKB1-STRADα pathway accompanied with increased fibrosis, results in development of chronic rejection following murine lung transplantation.

Moonlighting proteins: beacon of hope in era of drug resistance in bacteria.

Moonlighting proteins (MLPs) are ubiquitous and provide a unique advantage to bacteria performing multiple functions using the same genomic content. Targeting MLPs can be considered as a futuristic approach in fighting drug resistance problem. This review follows the MLP trail from its inception to the present-day state, describing a few bacterial MLPs, viz., glyceraldehyde 3'-phosphate dehydrogenase, phosphoglucose isomerase glutamate racemase (GR), and DNA gyrase. Here, we carve out that targeting MLPs are the beacon of hope in an era of increasing drug resistance in bacteria. Evolutionary stability, structure-functional relationships, protein diversity, possible drug targets, and identification of new drugs against bacterial MLP are given due consideration. Before the final curtain calls, we provide a comprehensive list of small molecules that inhibit the biochemical activity of MLPs, which can aid the development of novel molecules to target MLPs for therapeutic applications.

Cutting Edge: Circulating Exosomes with COVID Spike Protein Are Induced by BNT162b2 (Pfizer-BioNTech) Vaccination prior to Development of Antibodies: A Novel Mechanism for Immune Activation by mRNA Vaccines.

Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) causes severe acute respiratory syndrome. mRNA vaccines directed at the SARS-CoV-2 spike protein resulted in development of Abs and protective immunity. To determine the mechanism, we analyzed the kinetics of induction of circulating exosomes with SARS-CoV-2 spike protein and Ab following vaccination of healthy individuals. Results demonstrated induction of circulating exosomes expressing spike protein on day 14 after vaccination followed by Abs 14 d after the second dose. Exosomes with spike protein, Abs to SARS-CoV-2 spike, and T cells secreting IFN-γ and TNF-α increased following the booster dose. Transmission electron microscopy of exosomes also demonstrated spike protein Ags on their surface. Exosomes with spike protein and Abs decreased in parallel after four months. These results demonstrate an important role of circulating exosomes with spike protein for effective immunization following mRNA-based vaccination. This is further documented by induction of humoral and cellular immune responses in mice immunized with exosomes carrying spike protein.

Low-dose IL-2 prevents murine chronic cardiac allograft rejection: Role for IL-2-induced T regulatory cells and exosomes with PD-L1 and CD73.

To determine the effects and immunological mechanisms of low-dose interleukin-2 (IL-2) in a murine model of chronic cardiac allograft rejection (BALB/c to C57BL/6) after costimulatory blockade consisting of MR1 (250 µg/ip day 0) and CTLA4-Ig (200 µg/ip day 2), we administered low-dose IL-2 (2000 IU/day) starting on posttransplant day 14 for 3 weeks. T regulatory (Treg) cell infiltration of the grafts was determined by immunohistochemistry; circulating exosomes by western blot and aldehyde bead flow cytometry; antibodies to donor MHC by immunofluorescent staining of donor cells; and antibodies to cardiac self-antigens (myosin, vimentin) by ELISA. We demonstrated that costimulation blockade after allogeneic heart transplantation induced circulating exosomes containing cardiac self-antigens and antibodies to both donor MHC and self-antigens, leading to chronic rejection by day 45. Treatment with low-dose IL-2 prolonged allograft survival (>100 days), prevented chronic rejection, and induced splenic and graft-infiltrating CD4+ CD25+ Foxp3 Treg cells by day 45 and circulating exosomes (Foxp3+) with PD-L1 and CD73. MicroRNA 142, associated with the TGFß pathway, was significantly downregulated in exosomes from IL-2-treated mice. In conclusion, low-dose IL-2 delays rejection in a murine model of chronic cardiac allograft rejection and also induces graft-infiltrating Tregs and circulating exosomes with immunoregulatory molecules.

Novel role for tumor suppressor gene, liver kinase B1, in epithelial-mesenchymal transition leading to chronic lung allograft dysfunction.

Epithelial-mesenchymal transition (EMT) has been implicated to play a role in chronic lung allograft dysfunction (CLAD). Liver kinase B1 (LKB1), a tumor suppressor gene, can regulate EMT. However, its role in CLAD development following lung transplantation remains unknown. Using qRT-PCR, biopsies from lung transplant recipients with bronchiolitis obliterans syndrome (BOS) demonstrated significant downregulation of LKB1 (p = .0001), compared to stable biopsies. To determine the role of LKB1 in EMT development, we analyzed EMT in human bronchial epithelial cell line BEAS-2B. Knockdown of LKB1 by siRNA significantly dysregulated mesenchymal markers expression in BEAS-2B cells. Following incubation of human primary bronchial epithelial cell or BEAS-2B cells with exosomes isolated from BOS or stable lung transplant recipients, LKB1 expression was inhibited when incubated with BOS-exosome. Incubation with BOS-exosomes also decreased LKB1 expression and induced EMT markers in air-liquid interface culture method. Our results provide novel evidence that exosomes released from transplanted lungs undergoing chronic rejection are associated with inactivated tumor suppressor gene LKB1 and this loss induces EMT leading to the pathogenesis of CLAD following human lung transplantation.

Pre-existing Ab against vimentin leads to false-positive HLA Ab results in two pediatric heart transplant candidates.

BACKGROUND: HLA Ab analysis is carried out as a routine assay both pre- and post-heart transplantation to identify Abs directed against HLA with a focus post-transplant on those Abs that are donor-specific. While virtual crossmatching has decreased the requirement for prospective crossmatching in many cases, the management of highly sensitized children on the heart transplant waitlist remains challenging and can delay the ability to successfully identify a suitable organ. METHODS: This report describes the histocompatibility assessment and management of identical twin boys with familial restrictive cardiomyopathy serially listed for transplant. The boys presented with HLA Ab testing that demonstrated broad pan-DR reactivity which included Abs directed against SAgs. RESULTS: Our team began investigating the initial Ab results soon after listing the first child; the brother was listed 8 days later and had the same broad Ab profile. The clinical lab ran multiple investigative crossmatches using donor samples with known antigen typing and continued to see broad reactivity. We then partnered with an affiliated research lab where we identified high-level Abs directed against vimentin along with vimentin-positive exosomes in both boys. CONCLUSIONS: While Abs directed against the self-antigen vimentin has been described to cause false-positive tissue crossmatches, this is the first report of these Abs being associated with false-positive Ab screens using solid-phase assays. This finding informed our management and surveillance in these two vulnerable pediatric heart transplant candidates.

COVID-19 in a lung transplant recipient: Exploring the diagnostic role of circulating exosomes and the clinical impact of advanced immunosuppression.

Exosomes isolated from plasma of lung transplant recipients with allograft injury contain donor-derived lung self-antigens (collagen V and Kα1 tubulin) and human leukocyte antigen (HLA) molecules. We present a case of a 76-year-old, female lung transplant recipient treated for acute cellular rejection with methylprednisolone and anti-thymocyte globulin, who subsequently contracted SARS-CoV-2 and developed a sharp increase in the mean fluorescent intensity of anti-HLA antibodies. Analysis of circulating exosomes during rejection, but before SARS-CoV-2 infection, revealed the presence of lung self-antigens and HLA class II molecules. After the patient contracted SARS-CoV-2, exosomes with the SARS-CoV-2 spike protein were also found. After resolution of infectious symptoms, exosomes with SARS-CoV-2 spike protein were no longer detected; however, exosomes with lung self-antigens and HLA class II molecules persisted, which coincided with a progressive decline in spirometric flows, suggesting chronic lung allograft dysfunction. We propose that the analysis of circulating exosomes may be used to detect allograft injury mediated by both rejection and infection. Furthermore, the detection of exosomes containing viral proteins may be helpful in identifying allograft injury driven by viral pathogens.

The role of miRNA-155 in the immunopathogenesis of obliterative airway disease in mice induced by circulating exosomes from human lung transplant recipients with chronic lung allograft dysfunction.

Human lung transplant recipients undergoing rejection induce circulatory exosomes with lung self-antigens (SAgs), K-alpha 1 Tubulin and Collagen V, and immunization of C57BL/6 mice with exosomes induced obliterative airway disease (HEI-OAD). We analyzed whether exosomes with SAgs induced immunity in microRNA-155 knockout mice (miR-155KO), as microRNA-155 is an immune regulator. C57BL/6 and miR-155KO were immunized with exosomes from stable or chronic rejection (bronchiolitis obliterans syndrome (BOS) and on day 30, induction of exosomes, antibodies (Abs) to SAgs and cellular immunity were determined. C57BL/6 immunized with exosomes from BOS developed OAD. These immunized animals also developed Abs to SAgs and increased frequency of SAg-specific IFNγ and IL17- producing cells. In contrast, Abs to SAgs did not develop in miR-155KO and there was reduction in frequency of cells producing IL10. Upregulation of suppressor of cytokine signaling for lung inflammation was also noted resulting in abrogation of induction of exosomes with SAgs OAD.

Benzimidazoles: Selective Inhibitors of Topoisomerase I with Differential Modes of Action.

DNA topoisomerases are unique enzymes that alter the topology of DNA by cleavage and religation mechanisms. Small molecules such as camptothecins and noncamptothecins are reported to inhibit different classes of topoisomerases. Benzimidazole, 2-(3,4-dimethoxyphenyl)-5-[5-(4-methylpiperazin-1-yl)-1 H-benzimidazol-2-yl]-1 H benzimidazole (DMA), a new analogue of Hoechst 33342, was observed as a selective and differential inhibitor of human and Escherichia coli DNA topoisomerase I. In this study, we have concluded that DMA and Hoechst 33342 have differential binding toward human and E. coli topoisomerase I. We also dissected the mechanism of differential binding, as DMA and Hoechst 33342 bind to human topoisomerase I with linear kinetics with reversible binding, whereas the same molecules bind to E. coli topoisomerase I in a nonlinear and irreversible manner, which contributes to higher affinity and comparatively good IC50 values toward E. coli topoisomerase I. Interestingly, DMA and Hoechst 33342 showed inhibition of mutant human topoisomerases I, i.e., A653P, N722S, and T729P, whereas these clinically relevant mutants are resistant to camptothecins.

Bisphosphonate-Generated ATP-Analogs Inhibit Cell Signaling Pathways.

Bisphosphonates are a major class of drugs used to treat osteoporosis, Paget's disease, and cancer. They have been proposed to act by inhibiting one or more targets including protein prenylation, the epidermal growth factor receptor, or the adenine nucleotide translocase. Inhibition of the latter is due to formation in cells of analogs of ATP: the isopentenyl ester of ATP (ApppI) or an AppXp-type analog of ATP, such as AMP-clodronate (AppCCl2p). We screened both ApppI as well as AppCCl2p against a panel of 369 kinases finding potent inhibition of some tyrosine kinases by AppCCl2p, attributable to formation of a strong hydrogen bond between tyrosine and the terminal phosphonate. We then synthesized bisphosphonate preprodrugs that are converted in cells to other ATP-analogs, finding low nM kinase inhibitors that inhibited cell signaling pathways. These results help clarify our understanding of the mechanisms of action of bisphosphonates, potentially opening up new routes to the development of bone resorption, anticancer, and anti-inflammatory drug leads.

The role of exosomes in allograft immunity.

Extracellular vesicles are emerging as potent vehicles of intercellular communication. In this review, we focus on a subclass of extracellular vesicles called exosomes. Previously considered an unimportant catch-all, exosomes have recently been recognized for their role in various diseases and their potential for therapeutic use. We have examined the role of exosomes after human lung transplantation and have delineated the composition of circulating exosomes isolated from lung transplant recipients diagnosed with acute and chronic rejection, primary graft dysfunction, and respiratory viral infection. The presence of lung-associated self-antigens (K-alpha 1 Tubulin and collagen V) and mismatched donor HLA in exosomes isolated from lung transplant recipients signifies that these exosomes originated in the transplanted lungs, and therefore dramatically affect transplant biology and immune pathways. Exosomes released from transplanted organs also carry other proteins, costimulatory molecules, and nucleic acids. Therefore, these molecules may be used as biomarkers for allograft rejection and immunity.

Topoisomerases: Resistance versus Sensitivity, How Far We Can Go?

DNA topoisomerases are ubiquitously present remarkable molecular machines that help in altering topology of DNA in living cells. The crucial role played by these nucleases during DNA replication, transcription, and recombination vis-à-vis less sequence similarity among different species makes topoisomerases unique and attractive targets for different anticancer and antibacterial drugs. However, druggability of topoisomerases by the existing class of molecules is increasingly becoming questationable due to resistance development predominated by mutations in the corresponding genes. The current scenario facing a decline in the development of new molecules further comprises an important factor that may challenge topoisomerase-targeting therapy. Thus, it is imperative to wisely use the existing inhibitors lest with this rapid rate of losing grip over the target we may not go too far. Furthermore, it is important not only to design new molecules but also to develop new approaches that may avoid obstacles in therapies due to multiple resistance mechanisms. This review provides a succinct account of different classes of topoisomerase inhibitors, focuses on resistance acquired by mutations in topoisomerases, and discusses the various approaches to increase the efficacy of topoisomerase inhibitors. In a later section, we also suggest the possibility of using bisbenzimidazoles along with efflux pump inhibitors for synergistic bactericidal effects.

Pulsed Electron Paramagnetic Resonance Insights into the Ligand Environment of Copper in Drosophila Lysyl Oxidase.

Lysyl oxidase (LOX) is a copper amine oxidase that cross-links collagens and elastin in connective tissue and plays an important role in fibrosis, cancer development, and formation of the "metastatic niche". Despite its important biological functions, the structure of human LOX remains unknown (unlike that of an unrelated LOX, from Pichia pastoris). Here, we expressed active LOX from Drosophila melanogaster, DmLOXL1, a close homologue of human LOX, and characterized it by MS, UV-vis, activity, and inhibition assays. We then used bioinformatics, electron paramagnetic resonance, electron spin-echo envelope modulation, and hyperfine sublevel-correlation (HYSCORE) spectroscopies to probe Cu-ligand bonding finding direct evidence for pH-dependent Cu-His interactions. At pH = 9.3, the spectroscopic data indicated primarily a single His bound to Cu, but at pH = 7.5, there was evidence for a ∼ 1:1 mixture of species containing 1 and 3 His ligands. We then used HYSCORE to probe possible interactions between the LOX inhibitor BAPN (ß-aminopropionitrile; 1-[13C15N]cyano-2-aminoethane) and the copper center-finding none. Overall, the results are of interest since they provide new spectroscopic information about the nature of the catalytic site in LOX, an important anticancer drug target.

Cell penetrating synthetic antimicrobial peptides (SAMPs) exhibiting potent and selective killing of mycobacterium by targeting its DNA.

Naturally occurring antimicrobial peptides (AMPs) are powerful defence tools to tackle pathogenic microbes. However, limited natural production and high synthetic costs in addition to poor selectivity limit large-scale use of AMPs in clinical settings. Here, we present a series of synthetic AMPs (SAMPs) that exhibit highly selective and potent killing of Mycobacterium (minimum inhibitory concentration <20 µg mL(-1)) over E. coli or mammalian cells. These SAMPs are active against rapidly multiplying as well as growth saturated Mycobacterium cultures. These SAMPs are not membrane-lytic in nature, and are readily internalized by Mycobacterium and mammalian cells; whereas in E. coli, the lipopolysaccharide layer inhibits their cellular uptake, and hence, their antibacterial action. Upon internalization, these SAMPs interact with the unprotected genomic DNA of mycobacteria, and impede DNA-dependent processes, leading to bacterial cell death.

Deciphering the role of charge, hydration, and hydrophobicity for cytotoxic activities and membrane interactions of bile acid based facial amphiphiles.

We synthesized four cationic bile acid based facial amphiphiles featuring trimethyl ammonium head groups. We evaluated the role of these amphiphiles for cytotoxic activities against colon cancer cells and their membrane interactions by varying charge, hydration and hydrophobicity. The singly charged cationic Lithocholic acid based amphiphile (LCA-TMA1) is most cytotoxic, whereas the triply charged cationic Cholic acid based amphiphile (CA-TMA3) is least cytotoxic. Light microscopy and Annexin-FITC assay revealed that these facial amphiphiles caused late apoptosis. In addition, we studied the interactions of these amphiphiles with model membrane systems by Prodan-based hydration, DPH-based anisotropy, and differential scanning calorimetry. LCA-TMA1 is most hydrophobic with a hard charge causing efficient dehydration and maximum perturbations of membranes thereby facilitating translocation and high cytotoxicity against colon cancer cells. In contrast, the highly hydrated and multiple charged CA-TMA3 caused least membrane perturbations leading to low translocation and less cytotoxicity. As expected, Chenodeoxycholic acid and Deoxycholic acid based amphiphiles (CDCA-TMA2, DCA-TMA2) featuring two charged head groups showed intermediate behavior. Thus, we deciphered that charge, hydration, and hydrophobicity of these amphiphiles govern membrane interactions, translocation, and resulting cytoxicity against colon cancer cells.

Extracellular Vesicles in Transplantation: Friend or Foe.

The long-term function of transplanted organs, even under immunosuppression, is hindered by rejection, especially chronic rejection. Chronic rejection occurs more frequently after lung transplantation, termed chronic lung allograft dysfunction (CLAD), than after transplantation of other solid organs. Pulmonary infection is a known risk factor for CLAD, as transplanted lungs are constantly exposed to the external environment; however, the mechanisms by which respiratory infections lead to CLAD are poorly understood. The role of extracellular vesicles (EVs) in transplantation remains largely unknown. Current evidence suggests that EVs released from transplanted organs can serve as friend and foe. EVs carry not only major histocompatibility complex antigens but also tissue-restricted self-antigens and various transcription factors, costimulatory molecules, and microRNAs capable of regulating alloimmune responses. EVs play an important role in antigen presentation by direct, indirect, and semidirect pathways in which CD8 and CD4 cells can be activated. During viral infections, exosomes (small EVs <200 nm in diameter) can express viral antigens and regulate immune responses. Circulating exosomes may also be a viable biomarker for other diseases and rejection after organ transplantation. Bioengineering the surface of exosomes has been proposed as a tool for targeted delivery of drugs and personalized medicine. This review focuses on recent studies demonstrating the role of EVs with a focus on exosomes and their dual role (immune activation or tolerance induction) after organ transplantation, more specifically, lung transplantation.

Design, synthesis, and mechanistic investigations of bile acid-tamoxifen conjugates for breast cancer therapy.

We have synthesized two series of bile acid tamoxifen conjugates using three bile acids lithocholic acid (LCA), deoxycholic acid (DCA), and cholic acid (CA). These bile acid-tamoxifen conjugates possess 1, 2, and 3 tamoxifen molecules attached to hydroxyl groups of bile acids having free acid and amine functionalities at the tail region of bile acids. The in vitro anticancer activities of these bile acid-tamoxifen conjugates show that the free amine headgroup based cholic acid-tamoxifen conjugate (CA-Tam3-Am) is the most potent anticancer conjugate as compared to the parent drug tamoxifen and other acid and amine headgroup based bile acid-tamoxifen conjugates. The cholic acid-tamoxifen conjugate (CA-Tam3-Am) bearing three tamoxifen molecules shows enhanced anticancer activities in both estrogen receptor +ve and estrogen receptor -ve breast cancer cell lines. The enhanced anticancer activity of CA-Tam3-Am is due to more favorable irreversible electrostatic interactions followed by intercalation of these conjugates in hydrophobic core of membrane lipids causing increase in membrane fluidity. Annexin-FITC based FACS analysis showed that cells undergo apoptosis, and cell cycle analysis showed the arrest of cells in sub G0 phase. ROS assays showed a high amount of generation of ROS independent of ER status of the cell line indicating changes in mitochondrial membrane fluidity upon the uptake of the conjugate that further leads to the release of cytochrome c, a direct and indirect regulator of ROS. The mechanistic studies for apoptosis using PCR and western analysis showed apoptotsis by intrinsic and extrinsic pathways in ER +ve MCF-7 cells and by only an intrinsic pathway in ER -ve cells. In vivo studies in the 4T1 tumor model showed that CA-Tam3-Am is more potent than tamoxifen. These studies showed that bile acids provide a new scaffold for high drug loading and that their anticancer activities strongly depend on charge and hydrophobicity of lipid-drug conjugates.

Association Between Antibodies Against Lung Self-Antigens and Gastroesophageal Reflux in Lung Transplant Candidates.

Gastroesophageal reflux (GER) and pretransplant antibodies against lung self-antigens (SAbs) collagen-V and/or k-alpha 1 tubulin are both independently associated with allograft dysfunction after lung transplantation (LTx). The role of GER in inducing lung injury and SAbs is unknown. We aimed to study the association between pre-LTx GER and SAbs. After IRB approval, we retrieved SAb assays conducted between 2015 and 2019 and collected 24 hour GER data for these patients. Patients were divided into 2 groups: no reflux (GER-) and pathologic reflux (GER+) to compare the prevalence of SAbs. Multivariate analysis was used to study the association between GER and SAbs in the whole cohort and in restrictive lung disease (RLD) and obstructive lung disease (OLD) subsets. Proximal esophageal reflux (PER) events ≥5 was considered abnormal. Patients (n = 134; 73 men) were divided into groups: GER- (54.5%, n = 73) and GER+ (45.5%, n = 61). The prevalence of GER was higher in the RLD than in the OLD subset (p < 0.001). The overall prevalence of SAbs was 53.7% (n = 72), higher in the GER+ than the GER- group (65.6% vs 43.8%, p = 0.012), but comparable between RLD and OLD subsets. Overall, SAbs were associated with GER (p = 0.012) and abnormal PER (p = 0.017). GER and abnormal PER increased the odds of SAbs in the RLD subset (OR [95% CI]: 2.825 [1.033-7.725], p = 0.040 and OR [95% CI]: 3.551 [1.271-9.925], p = 0.014, respectively) but not in the OLD subset. LTx candidates have a high prevalence of SAbs, which are significantly associated with GER and abnormal PER in patients with RLD.