Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein.

SARS-CoV-2 enters host cells after binding through its spike glycoprotein to the angiotensin-converting enzyme 2 (ACE2) receptor. Soluble ACE2 ectodomains bind and neutralize the virus, yet their short in vivo half-live limits their therapeutic use. This limitation can be overcome by fusing the fragment crystallizable (Fc) part of human immunoglobulin G (IgG) to the ACE2 ectodomain, but this bears the risk of Fc-receptor activation and antibody-dependent cellular cytotoxicity. Here, we describe optimized ACE2-IgG4-Fc fusion constructs that avoid Fc-receptor activation, preserve the desired ACE2 enzymatic activity and show promising pharmaceutical properties. The engineered ACE2-IgG4-Fc fusion proteins neutralize the original SARS-CoV, pandemic SARS-CoV-2 as well as the rapidly spreading SARS-CoV-2 alpha, beta and delta variants of concern. Importantly, these variants of concern are inhibited at picomolar concentrations proving that ACE2-IgG4 maintains - in contrast to therapeutic antibodies - its full antiviral potential. Thus, ACE2-IgG4-Fc fusion proteins are promising candidate anti-antivirals to combat the current and future pandemics.

Smad7 regulates the adult neural stem/progenitor cell pool in a transforming growth factor beta- and bone morphogenetic protein-independent manner.

Members of the transforming growth factor beta (TGF-beta) family of proteins modulate the proliferation, differentiation, and survival of many different cell types. Neural stem and progenitor cells (NPCs) in the adult brain are inhibited in their proliferation by TGF-beta and by bone morphogenetic proteins (BMPs). Here, we investigated neurogenesis in a hypomorphic mouse model for the TGF-beta and BMP inhibitor Smad7, with the hypothesis that NPC proliferation might be reduced due to increased TGF-beta and BMP signaling. Unexpectedly, we found enhanced NPC proliferation as well as an increased number of label-retaining cells in vivo. The enhanced proliferation potential of mutant cells was retained in vitro in neurosphere cultures. We observed a higher sphere-forming capacity as well as faster growth and cell cycle progression. Use of specific inhibitors revealed that these effects were independent of TGF-beta and BMP signaling. The enhanced proliferation might be at least partially mediated by elevated signaling via epidermal growth factor (EGF) receptor, as mutant cells showed higher expression and activation levels of the EGF receptor. Conversely, an EGF receptor inhibitor reduced the proliferation of these cells. Our data indicate that endogenous Smad7 regulates neural stem/progenitor cell proliferation in a TGF-beta- and BMP-independent manner.

Evaluation of vascular endothelial growth factor addition in vitro on mouse inner ear progenitor cell cultures.

We analyzed progenitor cell cultures of inner ear tissue from newborn mice, and found proliferating cells, morphologically differentiating cells and subpopulations of cells expressing either neuronal or glial markers. In addition, we observed the expression of fetal liver kinase-1, a receptor for the vascular endothelial growth factor in a subpopulation of the cultured cells. Consistent with the expression of fetal liver kinase-1, addition of vascular endothelial growth factor at a dose of 10 ng/ml increased the expansion rate of inner ear-derived progenitor cells. Together with other published data, these results suggest that the vascular endothelial growth factor might be involved in inducing or supporting cochlear repair processes.

Transforming growth factor-beta1 is a negative modulator of adult neurogenesis.

Transforming growth factor (TGF)-beta1 has multiple functions in the adult central nervous system (CNS). It modulates inflammatory responses in the CNS and controls proliferation of microglia and astrocytes. In the diseased brain, TGF-beta1 expression is upregulated and, depending on the cellular context, its activity can be beneficial or detrimental regarding regeneration. We focus on the role of TGF-beta1 in adult neural stem cell biology and neurogenesis. In adult neural stem and progenitor cell cultures and after intracerebroventricular infusion, TGF-beta1 induced a long-lasting inhibition of neural stem and progenitor cell proliferation and a reduction in neurogenesis. In vitro, although TGF-beta1 specifically arrested neural stem and progenitor cells in the G0/1 phase of the cell cycle, it did not affect the self-renewal capacity and the differentiation fate of these cells. Also, in vivo, TGF-beta1 did not influence the differentiation fate of newly generated cells as shown by bromo-deoxyuridine incorporation experiments. Based on these data, we suggest that TGF-beta1 is an important signaling molecule involved in the control of neural stem and progenitor cell proliferation in the CNS. This might have potential implications for neurogenesis in a variety of TGF-beta1-associated CNS diseases and pathologic conditions.

Bile salt-induced apoptosis in human colon cancer cell lines involves the mitochondrial transmembrane potential but not the CD95 (Fas/Apo-1) receptor.

BACKGROUND AND AIMS: Depending on their physico-chemical characteristics, bile acids can be potent inducers of apoptosis in colon cancer cells. This observation contrasts with bile acids being promoters of colorectal cancer carcinogenesis. Our recent observation of caspase activation in deoxycholate (DC)-treated colon cancer cell lines prompted us to analyze the mechanisms of bile acid-induced colon cancer cell death. METHODS: CD95 expression was correlated to DC-induced cell death in four colon cancer cell lines. Mitochondrial transmembrane potential (MTP) was determined in whole cells as well as in isolated mitochondria. RESULTS: On 2 of the 4 human colon cancer cell lines investigated, no CD95 was detected. These data were supported by a lack of CD95 mRNA in those cell lines that did not express CD95 on their surface. The apoptotic response to bile acids did not correlate with CD95 receptor expression on the respective cell lines. Therefore, we analyzed the MTP after the addition of toxic bile acids. MTP was destabilized early after the addition of deoxycholate to SW480 cells. These data were confirmed in isolated mitochondria, which showed strong swelling after the addition of DC. Accordingly, release of cytochrome-c from the mitochondrial intermembrane space into the cytosol, indicating dissipation of the MTP, and subsequent caspase-3 cleavage were detectable as early as 3 min after the addition of DC. CONCLUSION: In contrast to hepatocytes and hepatic carcinoma cell lines, DC induces apoptosis in colon cancer cell lines via a CD95 receptor-independent mechanism. Direct induction of the mitochondrial permeability transition by toxic bile acids is suggested as the apoptosis-inducing mechanism in colon cancer cells.

Direct stimulation of adult neural stem cells in vitro and neurogenesis in vivo by vascular endothelial growth factor.

Hypoxia as well as global and focal ischemia are strong activators of neurogenesis in the adult mammalian central nervous system. Here we show that the hypoxia-inducible vascular endothelial growth factor (VEGF) and its receptor VEGFR-2/Flk-1 are expressed in clonally-derived adult rat neural stem cells in vitro. VEGF stimulated the expansion of neural stem cells whereas blockade of VEGFR-2/Flk-1-kinase activity reduced neural stem cell expansion. VEGF was also infused into the lateral ventricle to study changes in neurogenesis in the ventricle wall, olfactory bulb and hippocampus. Using a low dose (2.4 ng/d) to avoid endothelial proliferation and changes in vascular permeability, VEGF stimulated adult neurogenesis in vivo. After VEGF infusion, we observed reduced apoptosis but unaltered proliferation suggesting a survival promoting effect of VEGF in neural progenitor cells. Strong expression of VEGFR-2/Flk-1 was detected in the ventricle wall adjacent to the choroid plexus, a site of significant VEGF production, which suggests a paracrine function of endogenous VEGF on neural stem cells in vivo. We propose that VEGF acts as a trophic factor for neural stem cells in vitro and for sustained neurogenesis in the adult nervous system. These findings may have implications for the pathogenesis and therapy of neurodegenerative diseases.

The neurogenic competence of progenitors from the postnatal rat retina in vitro.

The mammalian retina develops from stem or progenitor cells that are of neuroectodermal origin and derive from bilateral invaginations of the neuroepithelium, the optic vesicles. Shortly after birth, around 12 days postnatal in rats, the retina is fully developed in its cellular parts. Even though different cell types in the adult might be potential sources for retinal stem cells or progenitor cells, the retina is a non-neurogenic region and the diseased retina is devoid of any spontaneous regeneration. In an attempt to link late developmental processes to the adult situation, we analyzed the presence and the neurogenic potential of retinal progenitors during the postnatal period and compared it to adult ciliary body (CB) derived retinal progenitors and subventricular zone (SVZ) derived neural stem cells. Retinal progenitor properties were identified by the capacity to proliferate and by the expression of the progenitor markers Nestin, Flk-1, Chx10, Pax6 and the radial glia marker BLBP. The neurogenic potential was assayed by the expression of the neuronal markers doublecortin, betaIII Tubulin, Map2 and NSE, the glial makers A2B5, NG2, GalC and GFAP, and by incorporation of BrdU. The number of Flk-1 positive cells and concomitantly the number of newly born betaIII Tubulin-positive cells decreased within the first postnatal week in retinal progenitor cultures and no newly generated betaIII Tubulin, but GFAP positive cells were detected thereafter. In contrast to neural stem cells derived from the adult SVZ, postnatal and adult CB derived progenitors had a lower and a restricted proliferation potential and did not generate oligodendrocytes. The work demonstrates, however, that the existence of retinal progenitor cells is not restricted to embryonic development. In the sensory retina the differentiation potential of late retinal progenitors becomes restricted to the glial lineage, whereas neurogenic progenitor cells are still present in the CB. In addition, major differences in growth and differentiation potential of adult neural stem cells and postnatal and adult retinal progenitors are presented.

High efficacy of clonal growth and expansion of adult neural stem cells.

Neural stem cells (NSCs) from the adult central nervous system are currently being investigated for their potential use in autologous cell replacement strategies. High expansion rates of NSCs in culture are crucial for the generation of a sufficient amount of cells needed for transplantation. Here, we describe efficient growth of adult NSCs in Neurobasal medium containing B27 supplement under clonal and low-density conditions in the absence of serum or conditioned medium. Expansion of up to 15-fold within 1 week was achieved on low-density NSC cultures derived from the lateral ventricle wall, the hippocampal formation, and the spinal cord of adult rats. A 27% single-cell cloning efficiency in Neurobasal/B27 combination further demonstrates its growth-promoting ability. Multipotency and nontumorgenicity of NSCs were retained despite the high rate of culture expansion. In addition, increased cell survival was obtained when Accutase, instead of trypsin, was used for enzymatic dissociation of NSC cultures. This work provides an important step toward the development of standardized protocols for highly efficient in vitro expansion of NSCs from the adult central nervous system to move more closely to the clinical use of NSCs.