The administration of amnion-derived multipotent cell secretome ST266 protects against necrotizing enterocolitis in mice and piglets.

Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in premature infants and is steadily rising in frequency. Patients who develop NEC have a very high mortality, illustrating the importance of developing novel prevention or treatment approaches. We and others have shown that NEC arises in part from exaggerated signaling via the bacterial receptor, Toll-like receptor 4 (TLR4) on the intestinal epithelium, leading to widespread intestinal inflammation and intestinal ischemia. Strategies that limit the extent of TLR4 signaling, including the administration of amniotic fluid, can reduce NEC development in mouse and piglet models. We now seek to test the hypothesis that a secretome derived from amnion-derived cells can prevent or treat NEC in preclinical models of this disease via a process involving TLR4 inhibition. In support of this hypothesis, we show that the administration of this secretome, named ST266, to mice or piglets can prevent and treat experimental NEC. The protective effects of ST266 occurred in the presence of marked TLR4 inhibition in the intestinal epithelium of cultured epithelial cells, intestinal organoids, and human intestinal samples ex vivo, independent of epidermal growth factor. Strikingly, RNA-seq analysis of the intestinal epithelium in mice reveals that the ST266 upregulates critical genes associated with gut remodeling, intestinal immunity, gut differentiation. and energy metabolism. These findings show that the amnion-derived secretome ST266 can prevent and treat NEC, suggesting the possibility of novel therapeutic approaches for patients with this devastating disease.NEW & NOTEWORTHY This work provides hope for children who develop NEC, a devastating disease of premature infants that is often fatal, by revealing that the secreted product of amniotic progenitor cells (called ST266) can prevent or treat NEC in mice, piglet, and "NEC-in-a-dish" models of this disease. Mechanistically, ST266 prevented bacterial signaling, and a detailed transcriptomic analysis revealed effects on gut differentiation, immunity, and metabolism. Thus, an amniotic secretome may offer novel approaches for NEC.

Exploratory Phase II Multicenter, Open-Label, Clinical Trial of ST266, a Novel Secretome for Treatment of Persistent Corneal Epithelial Defects.

Objective: An exploratory phase II, multicenter, open-label, clinical trial (NCT03687632) was conducted to evaluate the safety and effectiveness in treating persistent corneal epithelial defects (PEDs) with ST266, a proprietary novel multi-cytokine platform biologic solution secreted by cultured Amnion-derived Multipotent Progenitor (AMP) cells. Methods: Subjects with a PED were treated with ST266 eye drops 4 times daily for 28 days, then followed for 1 week. Safety was assessed by monitoring of adverse events (AEs) and serious adverse events (SAEs). Efficacy was assessed by measuring the area of the PED by slit lamp biomicroscopy. Tolerability of ST266, percentage of eyes with complete healing, reduction in area of the epithelial defect, and maintenance of a reduction in the area of the epithelial defect 7 days after treatment were recorded. Results: Thirteen patients were enrolled into the trial at one of eight sites. The first patient withdrew after 5 days. The remaining 12 patients with PEDs with median duration of 39 days (range = 12 to 393 days) completed treatment. Ten of the 12 eyes had been refractory to treatment with various conventional therapies prior to enrollment. After 28 days of treatment, there was a significant decrease in mean PED area compared with baseline (66.4% ± 35.3%, P = 0.001). At follow-up, 1 week after completion of treatment, on day 35, the PED area was further reduced by 78.8% ± 37.5% (P = 0.01) compared with baseline. During 28 days of treatment, 5 eyes (41.7%) had complete wound closure. There were no AEs of concern thought to be related to the drug, and no SAEs were noted. Conclusions: In this trial, we found ST266 eye drops might promote corneal epithelization, thereby reducing the PED area, including in refractory cases in a wide range of etiologies. ST266 was well-tolerated by most patients.

Age Impacts the Burden That Reference Memory Imparts on an Increasing Working Memory Load and Modifies Relationships With Cholinergic Activity.

Rodent aging research often utilizes spatial mazes, such as the water radial-arm-maze (WRAM), to evaluate cognition. The WRAM can simultaneously measure spatial working and reference memory, wherein these two memory types are often represented as orthogonal. There is evidence, however, that these two memory forms yield interference at a high working memory load. The current study systematically evaluated whether the presence of a reference memory component impacts handling of an increasing working memory load. Young and aged female rats were tested to assess whether aging impacts this relationship. Cholinergic projections from the basal forebrain to the hippocampus and cortex can affect cognitive outcomes, and are negatively impacted by aging. To evaluate whether age-related changes in working and reference memory profiles are associated with cholinergic functioning, we assessed choline acetyltransferase activity in these behaviorally-tested rats. Results showed that young rats outperformed aged rats on a task testing solely working memory. The addition of a reference memory component deteriorated the ability to handle an increasing working memory load, such that young rats performed similar to their aged counterparts. Aged rats also had challenges when reference memory was present, but in a different context. Specifically, aged rats had difficulty remembering which reference memory arms they had entered within a session, compared to young rats. Further, aged rats that excelled in reference memory also excelled in working memory when working memory demand was high, a relationship not seen in young rats. Relationships between cholinergic activity and maze performance differed by age in direction and brain region, reflecting the complex role that the cholinergic system plays in memory and attentional processes across the female lifespan. Overall, the addition of a reference memory requirement detrimentally impacted the ability to handle working memory information across young and aged timepoints, especially when the working memory challenge was high; these age-related deficits manifested differently with the addition of a reference memory component. This interplay between working and reference memory provides insight into the multiple domains necessary to solve complex cognitive tasks, potentially improving the understanding of complexities of age- and disease- related memory failures and optimizing their respective treatments.

Neuroprotection mediated by ST266 requires full complement of proteins secreted by amnion-derived multipotent progenitor cells.

ST266 is the biological secretome of cultured Amnion-derived Multipotent Progenitor cells containing multiple growth factors and cytokines. While intranasally-administered ST266 improves the phenotype in experimental optic neuritis, specific ST266 components mediating these effects are not known. We compared the effects of ST266 with and without removal of large molecular weight proteins both in vitro and in the multiple sclerosis model experimental autoimmune encephalomyelitis (EAE) in C57BL/6J mice. Mice were treated daily with intranasal vehicle, ST266 or lower molecular weight fraction of ST266. Retinal ganglion cells were counted in isolated retinas, and optic nerves were assessed for inflammation and demyelination. ST266 treatment significantly improved retinal ganglion cell survival and reduced optic nerve demyelination in EAE mice. The lower molecular weight ST266 fraction significantly improved optic nerve demyelination, but only showed a trend towards improved retinal ganglion cell survival. ST266 fractions below 50kDa increased Schwann cell proliferation in vitro, but were less effective than non-fractionated ST266. Demyelination attenuation was partially associated with the lower molecular weight ST266 fraction, but removal of higher molecular weight biomolecules from ST266 diminishes its neuroprotective effects, suggesting at least some high molecular weight proteins play a role in ST266-mediated neuroprotection.

Estradiol and selective estrogen receptor agonists differentially affect brain monoamines and amino acids levels in transitional and surgical menopausal rat models.

Estrogens have many beneficial effects in the brain. Previously, we evaluated the effects of two models of menopause (surgical vs. transitional) on multiple monoaminergic endpoints in different regions of the adult rat brain in comparison with levels in gonadally intact rats. Here we evaluated the effects of estrogen receptor (ER) agonist treatments in these same two models of menopause. Neurochemical endpoints were evaluated in the hippocampus (HPC), frontal cortex (FCX), and striatum (STR) of adult ovariectomized (OVX) rats and in rats that underwent selective and gradual ovarian follicle depletion by daily injection of 4-vinylcyclohexene-diepoxide (VCD), after 1- and 6-weeks treatment with 17ß-estradiol (E2), or with selective ERα (PPT), ERß (DPN), or GPR30 (G-1) agonists. Endpoints included serotonin (5-HT) and 5-Hydroxyindoleacetic acid, dopamine (DA), 3,4-Dihydroxyphenylacetic acid and homovanillic acid, norepinephrine (NE) and epinephrine, as well as the amino acids tryptophan (TRP) and tyrosine (TYR). Significant differences between the models were detected. OVX rats were much more sensitive to ER agonist treatments than VCD-treated rats. Significant differences between brain regions also were detected. Within OVX rats, more agonist effects were detected in the HPC than in any other region. One interesting finding was the substantial decrease in TRP and TYR detected in the HPC and FCX in response to agonist treatments, particularly in OVX rats. This is on top of the substantial decreases in TRP and TYR previously reported one week after OVX or VCD-treatments in comparison with gonadally intact controls. Other interesting findings included increases in the levels of 5-HT, DA, and NE in the HPC of OVX rats treated with DPN, increases in DA detected in the FCX of OVX rats treated with any of the ER agonists, and increases in 5-HT and DA detected in the STR of OVX rats treated with E2. Many effects that were observed after 1-week of treatment were no longer observed after 6-weeks of treatment, demonstrating that effects were temporary despite continued agonist treatment. Collectively, the results demonstrate significant differences in the effects of ER agonists on monoaminergic endpoints in OVX vs. VCD-treated rats that also were brain region-specific and time dependent. The fact that agonist treatments had lesser effects in VCD treated rats than in OVX rats may help to explain reports of lesser effects of estrogen replacement on cognitive performance in women that have undergone transitional vs. surgical menopause.

Comparison of transitional vs surgical menopause on monoamine and amino acid levels in the rat brain.

Loss of ovarian function has important effects on neurotransmitter production and release with corresponding effects on cognitive performance. To date, there has been little direct comparison of the effects of surgical and transitional menopause on neurotransmitter pathways in the brain. In this study, effects on monoamines, monoamine metabolites, and the amino acids tryptophan (TRP) and tyrosine (TYR) were evaluated in adult ovariectomized (OVX) rats and in rats that underwent selective and gradual ovarian follicle depletion by daily injection of 4-vinylcyclohexene-diepoxide (VCD). Tissues from the hippocampus (HPC), frontal cortex (FCX), and striatum (STR) were dissected and analyzed at 1- and 6-weeks following OVX or VCD treatments. Tissues from gonadally intact rats were collected at proestrus and diestrus to represent neurochemical levels during natural states of high and low estrogens. In gonadally intact rats, higher levels of serotonin (5-HT) were detected at proestrus than at diestrus in the FCX. In addition, the ratio of 5-hydroxyindoleacetic acid (5-HIAA)/5HT in the FCX and HPC was lower at proestrus than at diestrus, suggesting an effect on 5-HT turnover in these regions. No other significant differences between proestrus and diestrus were observed. In OVX- and VCD-treated rats, changes were observed which were both brain region- and time point-dependent. In the HPC levels of norepinephrine, 5-HIAA, TRP and TYR were significantly reduced at 1 week, but not 6 weeks, in both OVX and VCD-treated rats relative to proestrus and diestrus. In the FCX, dopamine levels were elevated at 6 weeks after OVX relative to diestrus. A similar trend was observed at 1 week (but not 6 weeks) following VCD treatment. In the STR, norepinephrine levels were elevated at 1 week following OVX, and HVA levels were elevated at 1 week, but not 6 weeks, following VCD treatment, relative to proestrus and diestrus. Collectively, these data provide the first comprehensive analysis comparing the effects of two models of menopause on multiple neuroendocrine endpoints in the brain. These effects likely contribute to effects of surgical and transitional menopause on brain function and cognitive performance that have been reported.