An Inhaled Galectin-3 Inhibitor in COVID-19 Pneumonitis: A Phase Ib/IIa Randomized Controlled Clinical Trial (DEFINE).

Rationale: High circulating galectin-3 is associated with poor outcomes in patients with coronavirus disease (COVID-19). We hypothesized that GB0139, a potent inhaled thiodigalactoside galectin-3 inhibitor with antiinflammatory and antifibrotic actions, would be safely and effectively delivered in COVID-19 pneumonitis. Objectives: Primary outcomes were safety and tolerability of inhaled GB0139 as an add-on therapy for patients hospitalized with COVID-19 pneumonitis. Methods: We present the findings of two arms of a phase Ib/IIa randomized controlled platform trial in hospitalized patients with confirmed COVID-19 pneumonitis. Patients received standard of care (SoC) or SoC plus 10 mg inhaled GB0139 twice daily for 48 hours, then once daily for up to 14 days or discharge. Measurements and Main Results: Data are reported from 41 patients, 20 of which were assigned randomly to receive GB0139. Primary outcomes: the GB0139 group experienced no treatment-related serious adverse events. Incidences of adverse events were similar between treatment arms (40 with GB0139 + SoC vs. 35 with SoC). Secondary outcomes: plasma GB0139 was measurable in all patients after inhaled exposure and demonstrated target engagement with decreased circulating galectin (overall treatment effect post-hoc analysis of covariance [ANCOVA] over days 2-7; P = 0.0099 vs. SoC). Plasma biomarkers associated with inflammation, fibrosis, coagulopathy, and major organ function were evaluated. Conclusions: In COVID-19 pneumonitis, inhaled GB0139 was well-tolerated and achieved clinically relevant plasma concentrations with target engagement. The data support larger clinical trials to determine clinical efficacy. Clinical trial registered with ClinicalTrials.gov (NCT04473053) and EudraCT (2020-002230-32).

Tissue Proteomic Analysis Identifies Mechanisms and Stages of Immunopathology in Fatal COVID-19.

Immunopathology occurs in the lung and spleen in fatal coronavirus disease (COVID-19), involving monocytes/macrophages and plasma cells. Antiinflammatory therapy reduces mortality, but additional therapeutic targets are required. We aimed to gain mechanistic insight into COVID-19 immunopathology by targeted proteomic analysis of pulmonary and splenic tissues. Lung parenchymal and splenic tissue was obtained from 13 postmortem examinations of patients with fatal COVID-19. Control tissue was obtained from cancer resection samples (lung) and deceased organ donors (spleen). Protein was extracted from tissue by phenol extraction. Olink multiplex immunoassay panels were used for protein detection and quantification. Proteins with increased abundance in the lung included MCP-3, antiviral TRIM21, and prothrombotic TYMP. OSM and EN-RAGE/S100A12 abundance was correlated and associated with inflammation severity. Unsupervised clustering identified "early viral" and "late inflammatory" clusters with distinct protein abundance profiles, and differences in illness duration before death and presence of viral RNA. In the spleen, lymphocyte chemotactic factors and CD8A were decreased in abundance, and proapoptotic factors were increased. B-cell receptor signaling pathway components and macrophage colony stimulating factor (CSF-1) were also increased. Additional evidence for a subset of host factors (including DDX58, OSM, TYMP, IL-18, MCP-3, and CSF-1) was provided by overlap between 1) differential abundance in spleen and lung tissue; 2) meta-analysis of existing datasets; and 3) plasma proteomic data. This proteomic analysis of lung parenchymal and splenic tissue from fatal COVID-19 provides mechanistic insight into tissue antiviral responses, inflammation and disease stages, macrophage involvement, pulmonary thrombosis, splenic B-cell activation, and lymphocyte depletion.

Aspirin inhibits mTOR signaling, activates AMP-activated protein kinase, and induces autophagy in colorectal cancer cells.

BACKGROUND & AIMS: Aspirin reduces the incidence of and mortality from colorectal cancer (CRC) by unknown mechanisms. Cancer cells have defects in signaling via the mechanistic target of rapamycin (mTOR), which regulates proliferation. We investigated whether aspirin affects adenosine monophosphate-activated protein kinase (AMPK) and mTOR signaling in CRC cells. METHODS: The effects of aspirin on mTOR signaling, the ribosomal protein S6, S6 kinase 1 (S6K1), and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) were examined in CRC cells by immunoblotting. Phosphorylation of AMPK was measured; the effects of loss of AMPKα on the aspirin-induced effects of mTOR were determined using small interfering RNA (siRNA) in CRC cells and in AMPK(α1/α2-/-) mouse embryonic fibroblasts. LC3 and ULK1 were used as markers of autophagy. We analyzed rectal mucosa samples from patients given 600 mg aspirin, once daily for 1 week. RESULTS: Aspirin reduced mTOR signaling in CRC cells by inhibiting the mTOR effectors S6K1 and 4E-BP1. Aspirin changed nucleotide ratios and activated AMPK in CRC cells. mTOR was still inhibited by aspirin in CRC cells after siRNA knockdown of AMPKα, indicating AMPK-dependent and AMPK-independent mechanisms of aspirin-induced inhibition of mTOR. Aspirin induced autophagy, a feature of mTOR inhibition. Aspirin and metformin (an activator of AMPK) increased inhibition of mTOR and Akt, as well as autophagy in CRC cells. Rectal mucosal samples from patients given aspirin had reduced phosphorylation of S6K1 and S6. CONCLUSIONS: Aspirin is an inhibitor of mTOR and an activator of AMPK, targeting regulators of intracellular energy homeostasis and metabolism. These could contribute to its protective effects against development of CRC.

Phototherapeutic Induction of Immunogenic Cell Death and CD8+ T Cell-Granzyme B Mediated Cytolysis in Human Lung Cancer Cells and Organoids.

Augmenting T cell mediated tumor killing via immunogenic cancer cell death (ICD) is the cornerstone of emerging immunotherapeutic approaches. We investigated the potential of methylene blue photodynamic therapy (MB-PDT) to induce ICD in human lung cancer. Non-Small Cell Lung Cancer (NSCLC) cell lines and primary human lung cancer organoids were evaluated in co-culture killing assays with MB-PDT and light emitting diodes (LEDs). ICD was characterised using immunoblotting, immunofluorescence, flow cytometry and confocal microscopy. Phototherapy with MB treatment and low energy LEDs decreased the proliferation of NSCLC cell lines inducing early necrosis associated with reduced expression of the anti-apoptotic protein, Bcl2 and increased expression of ICD markers, calreticulin (CRT), intercellular cell-adhesion molecule-1 (ICAM-1) and major histocompatibility complex I (MHC-I) in NSCLC cells. MB-PDT also potentiated CD8+ T cell-mediated cytolysis of lung cancer via granzyme B in lung cancer cells and primary human lung cancer organoids.

Randomised controlled trial of intravenous nafamostat mesylate in COVID pneumonitis: Phase 1b/2a experimental study to investigate safety, Pharmacokinetics and Pharmacodynamics.

BACKGROUND: Many repurposed drugs have progressed rapidly to Phase 2 and 3 trials in COVID19 without characterisation of Pharmacokinetics /Pharmacodynamics including safety data. One such drug is nafamostat mesylate. METHODS: We present the findings of a phase Ib/IIa open label, platform randomised controlled trial of intravenous nafamostat in hospitalised patients with confirmed COVID-19 pneumonitis. Patients were assigned randomly to standard of care (SoC), nafamostat or an alternative therapy. Nafamostat was administered as an intravenous infusion at a dose of 0.2 mg/kg/h for a maximum of seven days. The analysis population included those who received any dose of the trial drug and all patients randomised to SoC. The primary outcomes of our trial were the safety and tolerability of intravenous nafamostat as an add on therapy for patients hospitalised with COVID-19 pneumonitis. FINDINGS: Data is reported from 42 patients, 21 of which were randomly assigned to receive intravenous nafamostat. 86% of nafamostat-treated patients experienced at least one AE compared to 57% of the SoC group. The nafamostat group were significantly more likely to experience at least one AE (posterior mean odds ratio 5.17, 95% credible interval (CI) 1.10 - 26.05) and developed significantly higher plasma creatinine levels (posterior mean difference 10.57 micromol/L, 95% CI 2.43-18.92). An average longer hospital stay was observed in nafamostat patients, alongside a lower rate of oxygen free days (rate ratio 0.55-95% CI 0.31-0.99, respectively). There were no other statistically significant differences in endpoints between nafamostat and SoC. PK data demonstrated that intravenous nafamostat was rapidly broken down to inactive metabolites. We observed no significant anticoagulant effects in thromboelastometry. INTERPRETATION: In hospitalised patients with COVID-19, we did not observe evidence of anti-inflammatory, anticoagulant or antiviral activity with intravenous nafamostat, and there were additional adverse events. FUNDING: DEFINE was funded by LifeArc (an independent medical research charity) under the STOPCOVID award to the University of Edinburgh. We also thank the Oxford University COVID-19 Research Response Fund (BRD00230).

Aspirin Rescues Wnt-Driven Stem-like Phenotype in Human Intestinal Organoids and Increases the Wnt Antagonist Dickkopf-1.

BACKGROUND & AIMS: Aspirin reduces colorectal cancer (CRC) incidence and mortality. Understanding the biology responsible for this protective effect is key to developing biomarker-led approaches for rational clinical use. Wnt signaling drives CRC development from initiation to progression through regulation of epithelial-mesenchymal transition (EMT) and cancer stem cell populations. Here, we investigated whether aspirin can rescue these proinvasive phenotypes associated with CRC progression in Wnt-driven human and mouse intestinal organoids. METHODS: We evaluated aspirin-mediated effects on phenotype and stem cell markers in intestinal organoids derived from mouse (ApcMin/+ and Apcflox/flox) and human familial adenomatous polyposis patients. CRC cell lines (HCT116 and Colo205) were used to study effects on motility, invasion, Wnt signaling, and EMT. RESULTS: Aspirin rescues the Wnt-driven cystic organoid phenotype by promoting budding in mouse and human Apc deficient organoids, which is paralleled by decreased stem cell marker expression. Aspirin-mediated Wnt inhibition in ApcMin/+ mice is associated with EMT inhibition and decreased cell migration, invasion, and motility in CRC cell lines. Chemical Wnt activation induces EMT and stem-like alterations in CRC cells, which are rescued by aspirin. Aspirin increases expression of the Wnt antagonist Dickkopf-1 in CRC cells and organoids derived from familial adenomatous polyposis patients, which contributes to EMT and cancer stem cell inhibition. CONCLUSIONS: We provide evidence of phenotypic biomarkers of response to aspirin with an increased epithelial and reduced stem-like state mediated by an increase in Dickkopf-1. This highlights a novel mechanism of aspirin-mediated Wnt inhibition and potential phenotypic and molecular biomarkers for trials.

Truncation of C-mip (Tc-mip), a new proximal signaling protein, induces c-maf Th2 transcription factor and cytoskeleton reorganization.

Several arguments suggest that minimal change nephrotic syndrome (MCNS) results from yet unknown systemic disorder of T cell function. By screening a cDNA library from T cell relapse, we identified a new pleckstrin homology (PH) domain-containing protein encoded by a gene located on chromosome 16q24. Two alternative transcripts were identified. The first species (c-mip) was expressed in fetal liver, kidney, and peripheral blood mononuclear cells (PBMCs), but weakly detected in PBMCs from MCNS patients. The second form (Tc-mip, standing for truncated c-maf inducing protein), corresponds to subtracted transcript and lacks the NH2-terminal PH domain. The expression of Tc-mip was restricted to fetal liver, thymus, and MCNS PBMCs where it was specifically recruited in CD4+ T cells subset. Overexpression of Tc-mip in T cell Jurkat induced c-maf, transactivated the interleukin 4 gene and down-regulated the interferon gamma expression, characteristic of a Th2 commitment. Moreover, the overexpression of Tc-mip induced Src phosphorylation, T cell clustering, and a cellular redistribution of the cytoskeleton-associated L-plastin, by a PI3 kinase independent pathway. Tc-mip represents therefore the first identified protein, which links proximal signaling to c-maf induction.

C-mip interacts physically with RelA and inhibits nuclear factor kappa B activity.

The fine regulation of NF-kappaB activity is crucial for both resting and stimulated cells and relies on complex balance between multiple activators and inhibitors. We report here that c-mip, a recently identified pleckstrin homology (PH) and leucine-rich repeat (LRR)-domain-containing protein, inactivates GSKbeta and interacts with RelA, a key member of the NF-kappaB family. We show that c-mip inhibits the degradation of I-kappaBalpha and impedes the dissociation of the NF-kappaB/I-kappaBalpha complexes. C-mip acts downstream signaling of classical NF-kappaB pathway and may represent one of the missing links in the control of NF-kappaB activity.

Potential role of soluble ST2 protein in idiopathic nephrotic syndrome recurrence following kidney transplantation.

BACKGROUND: Corticosteroid-resistant idiopathic nephrotic syndrome (INS) recurs rapidly after transplantation in 30% to 50% of transplant recipients, suggesting the presence of 1 or more circulating factors that alter the glomerular filtration barrier. We investigated the possible role in INS recurrence of soluble ST2 (sST2) protein, a marker of T helper type 2 (T(H)2) cells and a factor predicted to be regulated by the transcription factor c-Maf; involvement of sST2 protein would be consistent with the observation that both T(H)2 cells and c-Maf appear to be activated during INS relapse. STUDY DESIGN: Retrospective observational study. SETTING & PARTICIPANTS: Patients with biopsy-proven corticosteroid-resistant INS who had undergone kidney transplantation between September 1983 and April 2007 (n = 71). A control group consisting of proteinuric transplant recipients with kidney failure unrelated to INS (n = 34). PREDICTOR: Patients who developed INS recurrence after transplantation (n = 31) were compared with those in whom INS did not recur (n = 40) and the control group. Recurrence of INS was defined as urine protein excretion greater than 2 g/d immediately after transplantation that persisted at greater than 1 g/d despite treatment or a kidney graft biopsy showing minimal change glomerulonephritis or focal segmental glomerulosclerosis. OUTCOMES & MEASUREMENTS: Urine protein excretion in the 3 groups was 5.0 g/d (range, 1.3 to 10.5), 0.14 g/d (range, 0 to 0.46), and 4.3 g/d (range, 3 to 6.2). The sST2 protein was analyzed both quantitatively and qualitatively in patient sera, and its activity was tested in vitro on a mouse podocyte cell line and in vivo in rats. RESULTS: sST2 protein levels were significantly increased after transplantation in patients with INS recurrence compared with the 2 other groups (617.5 versus 23 pg/mL; P < 0.001 and 158.5 pg/mL; P < 0.01 respectively). However, patients with recurrence expressed a normal sST2 isoform, and the sST2 protein was unable to induce podocyte injury in vitro or trigger proteinuria in rats. LIMITATIONS: Pretransplantation and posttransplantation sera do not always represent paired samples. CONCLUSIONS: These data suggest that sST2 protein is a marker of INS recurrence that does not seem to be involved in the development of INS.

The Filamin-A is a partner of Tc-mip, a new adapter protein involved in c-maf-dependent Th2 signaling pathway.

Using a yeast two-hybrid screen, we identified Filamin-A as a binding partner of the new adapter protein c-mip (c-maf inducing protein) and it's splice variant Tc-mip (truncated c-maf inducing protein). We have previously shown that Tc-mip is involved in Th2 signaling pathway and cytoskeletal reorganization in patients with minimal change nephrotic syndrome (MCNS), the most frequent glomerular disease in children. We showed that Filamin-A and c-mip or Tc-mip co-immunoprecipitate from c-mip or Tc-mip Jurkat transfected cells using antibodies directed against both types of proteins. In co-immunoprecipitate Jurkat cells, Filamin-A and c-mip were distributed evenly in the cytoplasm, whereas in Tc-mip-transfected Jurkat cells, Filamin-A was expressed in zones facing the cell contact. Moreover, we found that Filamin-A was upregulated in T lymphocytes of MCNS patients, as compared to normal subjects. These findings suggest that Filamin-A interacts with c-mip/Tc-mip in this new T-cell signaling pathway.

C-mip interacts with the p85 subunit of PI3 kinase and exerts a dual effect on ERK signaling via the recruitment of Dip1 and DAP kinase.

In naive T cells, Lck exerts a negative control on the ERK/MAPK pathway. We show that c-mip (c-maf inducing protein) interacts with the p85 subunit of PI3 kinase and inactivates Lck, which results in Erk1/2 and p38 MAPK activation. This effect is not enough to activate AP1 given the inability of ERK to migrate into the nucleus and to transactivate its target genes. We demonstrate that c-mip interacts with Dip1 and upregulates DAPK, which blocks the nuclear translocation of ERK1/2. This dual effect of c-mip is unique and might represent a potential mechanism to prevent the development of an immune response.

Abnormal RNA processing and altered expression of serin-rich proteins in minimal-change nephrotic syndrome.

Mechanisms underlying the pathophysiology of minimal-change nephrotic syndrome (MCNS), the most frequent glomerular disease in children, remain elusive, but recent findings argue for a T cell dysfunction. Starting from a differential cDNA library from T cells of a patient under relapse and remission, we identified 16 transcripts specific for MCNS. All of these transcripts that were selectively up-regulated during the relapse phase of the disease were generated by alternative splicing of known genes. This abnormal RNA expression was associated with a down-regulation of serin-rich protein 75 and serin-rich protein 40, two proteins involved in mRNA splicing. Taken together, these data suggest that T cell dysfunction in MCNS is associated with abnormal mRNA splicing.

A novel approach to investigation of the pathogenesis of active minimal-change nephrotic syndrome using subtracted cDNA library screening.

Clinical and experimental observations suggest that minimal-change nephrotic syndrome (MCNS) results from T cell dysfunction, via unknown mechanisms. For the identification of genes that are potentially involved in MCNS, a subtractive cDNA library was constructed from cDNA from T cell-enriched peripheral blood mononuclear cells obtained from the same patient during relapse versus remission ("relapse minus remission"). This library was screened by differential hybridization with forward ("relapse minus remission") and reverse ("remission minus relapse") subtractive cDNAs probes, as well as unsubtracted probes from relapse and remission, and irrelevant nephrotic syndrome (membranous nephropathy). A total of 84 transcripts were isolated, of which 12 matched proteins of unknown function and 30 were unknown clones. Among the 42 known transcripts, at least 18 are closely involved in the T cell receptor-mediated complex signaling cascade, including genes encoding components of the T cell receptor and proteins associated with the cytoskeletal scaffold, as well as transcription factors. In particular, it was demonstrated that the expression levels of Fyb/Slap, L-plastin, and grancalcin were increased during relapse, suggesting that the integration of proximal signaling after T cell engagement involves the preferential recruitment of these cytoskeleton-associated proteins in MCNS. Because very low levels of interleukin-12 receptor beta2 mRNA were detected in relapse samples, the interleukin-12 signaling pathway might be defective, suggesting that, in MCNS, T cell activation evolves toward a T helper 2 phenotype. Therefore, the combination of subtractive cloning and differential screening constitutes an efficient approach to the identification of genes that are likely to be involved in the pathophysiologic processes of MCNS.

Transcriptional and post-transcriptional alterations of IkappaBalpha in active minimal-change nephrotic syndrome.

Minimal-change nephrotic syndrome (MCNS) is a renal disease characterized by heavy glomerular proteinuria and increased production of cytokines by immune cells. Because of the central role of nuclear factor-kappaB (NF-kappaB) in the regulation of cytokine expression, its activity during the relapse and remission phases of steroid-sensitive MCNS was analyzed. During relapse, nuclear extracts from peripheral blood mononuclear cells displayed high levels of NF-kappaB DNA-binding activity, consisting primarily of p50/RelA (p65) complexes. NF-kappaB p65 and IkappaBalpha proteins were barely detected or not detected in cytosolic fractions during relapse, in contrast to remission. The lack of expression of IkappaBalpha protein was associated with downregulation of IkappaBalpha mRNA and increases in the levels of the mRNA encoding the proteasome alpha2 subunit proteolytic pathway. In addition, inhibition of proteasome activity induced cytosolic accumulation of phosphorylated IkappaBalpha and significant reductions in the NF-kappaB binding activity in nuclear extracts from peripheral blood mononuclear cells from patients experiencing relapses. These results suggest that alterations in the NF-kappaB/IkappaBalpha regulatory feedback loop may contribute to the immunologic abnormalities that occur in steroidsensitive MCNS.

NF-kappa B p65 antagonizes IL-4 induction by c-maf in minimal change nephrotic syndrome.

Mechanisms underlying the pathophysiology of minimal change nephrotic syndrome (MCNS), the most frequent of glomerular diseases in children, remain elusive, although recent arguments suggest that T cell dysfunction may be involved in the pathogenesis of this disease. Recently, we reported that activated T cells of these patients display a down-regulation of IL-12R beta2 chain, suggesting an early commitment toward Th2 phenotype. In this study, we show that the short form of the proto-oncogene c-maf, a known activator of the IL-4 gene, is highly induced in MCNS T cells during relapse, where it translocates to the nuclear compartment and binds to the DNA responsive element. Unexpectedly, the nuclear localization of c-maf did not promote the IL-4 gene transcription in relapse. Using several approaches, we show in this study that RelA blunts IL-4 induction in T cells during the relapse in these patients. We demonstrate that the ex vivo inhibition of proteasome activity in T cells from relapse, which blocks NF-kappaB activity, strongly increases the IL-4 mRNA levels. Overexpression of c-maf in T cells induces a high level of IL-4 promoter-driven luciferase activity. In contrast, coexpression of c-maf with NF-kappaB RelA/p50, or RelA, but not p50, inhibits the c-maf-dependent IL-4 promoter activity. Finally, we demonstrated that, in T cell overexpressing RelA and c-maf, RelA expelled c-maf from its DNA binding site on IL-4 gene promoter, which results in active inhibition of IL-4 gene transcription. Altogether, these results suggest that the involvement of c-maf in Th2 commitment in MCNS operates through IL-4-independent mechanisms.