Wnt proteins synergize to activate ß-catenin signaling.

Wnt ligands are involved in diverse signaling pathways that are active during development, maintenance of tissue homeostasis and in various disease states. While signaling regulated by individual Wnts has been extensively studied, Wnts are rarely expressed alone, and the consequences of Wnt gene co-expression are not well understood. Here, we studied the effect of co-expression of Wnts on the ß-catenin signaling pathway. While some Wnts are deemed 'non-canonical' due to their limited ability to activate ß-catenin when expressed alone, unexpectedly, we find that multiple Wnt combinations can synergistically activate ß-catenin signaling in multiple cell types. WNT1- and WNT7B-mediated synergistic Wnt signaling requires FZD5, FZD8 and LRP6, as well as the WNT7B co-receptors GPR124 (also known as ADGRA2) and RECK. Unexpectedly, this synergistic signaling occurs downstream of ß-catenin stabilization, and is correlated with increased lysine acetylation of ß-catenin. Wnt synergy provides a general mechanism to confer increased combinatorial control over this important regulatory pathway.

First-in-Human Study to Evaluate Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of a Rapidly Developed SARS-CoV-2 Therapeutic Antibody, AOD01, in Healthy Adults.

INTRODUCTION: AOD01 is a novel, fully human immunoglobulin (Ig) G1 neutralizing monoclonal antibody that was developed as a therapeutic against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). This first-in-human study assessed safety, tolerability, pharmacokinetics (PK), and pharmacodynamics of AOD01 in healthy volunteers. METHODS: Intravenous doses of AOD01 were evaluated in escalating cohorts [four single-dose cohorts (2, 5, 10, and 20 mg/kg) and one two-dose cohort (two doses of 20 mg/kg, 24 h apart)]. RESULTS: Twenty-three subjects were randomized to receive AOD01 or a placebo in blinded fashion. A total of 34 treatment-emergent adverse events (TEAEs) were reported; all were mild in severity. Related events (headache and diarrhea) were reported in one subject each. No event of infusion reactions, serious adverse event (SAE), or discontinuation due to AE were reported. The changes in laboratory parameters, vital signs, and electrocardiograms were minimal. Dose-related exposure was seen from doses 2 to 20 mg/kg as confirmed by Cmax and AUC0-tlast. The median Tmax was 1.5-3 h. Clearance was dose independent. Study results revealed long half-lives (163-465 h). Antidrug antibodies (ADA) to AOD01 were not detected among subjects, except in one subject of the two-dose cohort on day 92. Sustained ex vivo neutralization of SARS-CoV-2 was recorded until day 29 with single doses from 2 to 20 mg/kg and until day 43 with two doses of 20 mg/kg. CONCLUSIONS: AOD01 was safe and well tolerated, demonstrated dose-related PK, non-immunogenic status, and sustained ex vivo neutralization of SARS-CoV-2 after single intravenous dose ranging from 2 to 20 mg/kg and two doses of 20 mg/kg and show good potential for treatment of SARS-CoV-2 infection. (Health Sciences Authority identifier number CTA2000119).

Mechanism of the immunomodulatory activity of glycodelin.

Glycodelin, a progesterone regulated protein synthesized by the endometrium (GdA) has been well documented to inhibit the proliferation of activated T-cells and is an indispensable molecule in the maternal system for the establishment, maintenance and progression of pregnancy. Data from our laboratory have unequivocally shown that the immunosuppression by GdA is via induction of apoptosis in activated T cells. Another isoform of glycodein, GdS, from the male reproductive system, in spite of sharing an identical amino acid sequence as that of GdA has been shown not to harbour the immunosuppressive activity of GdA. As the only difference between the two proteins is glycosylation, we proposed to study the role of the sugars in imparting apoptotic activity to Gd. Using the recombinant baculovirus system, Gd lacking glycosylation was expressed and from the experimental observations we could conclude that the activity of Gd lies in the protein backbone. Recombinant Gd expressed in P. pastoris, and Chinese hamster ovary cells, like the GdS did not exhibit apoptotic activity. A close analyses of the glycans associated with the Gd molecules from various sources suggested that though the apoptogenic activity of Gd lies in the protein backbone, the glycans modulate the activity by masking (as in case of GdS and most recombinant Gd expressed in our laboratory) or unmasking (as in case of GdA and baculovirus expressed Gd), the functional region of the molecule.

The role of glycodelin as an immune-modulating agent at the feto-maternal interface.

Glycodelin A is a progesterone-induced endometrial glycoprotein which has been amply documented to play a role in down-modulation of the maternal immune response to fetal allo-antigens and to be indispensable for the maintenance and progression of pregnancy. Earlier studies from our laboratory have focused on the effect of glycodelin on T cells, key regulators of both the antibody and cell-mediated arms of the acquired immune system. Glycodelin-induced apoptosis in activated T cells occurs through a caspase-dependent intrinsic mitochondrial pathway. Interestingly, glycodelin inhibited the proliferation of B cells but did not induce apoptosis. More recently, we have studied the effect of glycodelin on the cells of the innate immune system, namely monocytes and NK cells. We have found that glycodelin induced apoptosis in monocytic cells before their differentiation to macrophages, via the mitochondrial pathway, but did not affect their phagocytic capacity after differentiation. Glycodelin induced apoptosis in NK cells but this activity was independent of caspases. In conclusion, glycodelin is observed to affect many cells of the immune system, although the nature of the effect and signaling mechanisms involved in each cell type may be distinct.

Glycodelin A, an immunomodulatory protein in the endometrium, inhibits proliferation and induces apoptosis in monocytic cells.

Glycodelin A (GdA), is a lipocalin with an immunomodulatory role, secreted by the endometrium under progesterone regulation and proposed to play a role in protecting the fetus from maternal immune attack. Glycodelin A has an inhibitory effect on the proliferation of T cells and B cells and also on the activity of natural killer cells. We have earlier demonstrated that the inhibitory effect of glycodelin A on T cell proliferation is due to apoptosis induced in these cells through the caspase-dependent intrinsic mitochondrial pathway. Studies reported until now have shown that glycodelin modulates the adaptive immune responses. We, therefore, decided to look at its effect, if any, on the innate immune system. The effect of glycodelin on monocytes was studied using human monocytic cell lines, THP1 and U937, and primary human monocytes as model systems. We demonstrated that glycodelin inhibited the proliferation of THP1 and U937 and induced apoptosis in these cells as well as in primary monocytes. We found that this signaling was caspase-independent but followed the intrinsic mitochondrial pathway of apoptosis. No effect of glycodelin was seen on the phagocytic ability of monocytes post-differentiation into macrophages. These observations suggest that, at the fetomaternal interface, glycodelin plays a protective role by deleting the monocytes that could become pro-inflammatory. Importantly, leaving the macrophages untouched to carry on with efficient clearance of the apoptotic cells.

Glycodelin A, not glycodelin S, is apoptotically active. Relevance of sialic acid modification.

Glycodelin, previously known as PP14 (placental protein-14), is a kernel lipocalin secreted by the glandular epithelium of the endometrium upon progesterone stimulation and by the seminal vesicles. The isoform of the protein present in female reproductive tissue, glycodelin A (GdA), and the male counterpart, glycodelin S (GdS), have identical amino acid sequences, but strikingly different N-linked glycans. It is well documented in literature that GdA is an immunosuppressive protein, and we have shown that this activity is due to its ability to induce apoptosis in activated T cells. The precise role of GdS in seminal plasma is not known. In this study, we report that GdS is not apoptotically active. We observe that the apoptotic activity requires the presence of sialic acid residues on the complex glycans, as in the case of GdA; however, complex glycans of GdS are non-sialylated. We have expressed the wild-type protein in Pichia pastoris, which does not add sialic acid to the secreted proteins, and confirmed our observations that the protein is apoptotically inactive in the non-sialylated form. Our results indicate that differential glycosylation modulates the function of the different glycodelin isoforms.