The Multi-Component Causes of Late Neonatal Sepsis-Can We Regulate Them?

Elucidating the mechanisms of bacterial translocation is crucial for the prevention and treatment of neonatal sepsis. In the present study, we aimed to evaluate the potential of lactoferrin to inhibit the development of late-onset blood infection in neonates. Our investigation evaluates the role of key stress factors leading to the translocation of intestinal bacteria into the bloodstream and, consequently, the development of life-threatening sepsis. Three stress factors, namely weaning, intraperitoneal administration of Gram-positive cocci and oral intake of Gram-negative rods, were found to act synergistically. We developed a novel model of rat pups sepsis induced by bacterial translocation and observed the inhibition of this process by supplementation of various forms of lactoferrin: iron-depleted (apolactoferrin), iron-saturated (hololactoferrin) and manganese-saturated lactoferrin. Additionally, lactoferrin saturated with manganese significantly increases the Lactobacillus bacterial population, which contributes to the fortification of the intestinal barrier and inhibits the translocation phenomenon. The acquired knowledge can be used to limit the development of sepsis in newborns in hospital neonatal intensive care units.

Extracellular vesicles containing the transferrin receptor as nanocarriers of apotransferrin.

Previous work by our group has shown the pro-differentiating effects of apotransferrin (aTf) on oligodendroglial cells in vivo and in vitro. Further studies showed the remyelinating effect of aTf in animal demyelination models such as hypoxia/ischemia, where the intranasal administration of human aTf provided brain neuroprotection and reduced white matter damage, neuronal loss, and astrogliosis in different brain regions. These data led us to search for a less invasive and controlled technique to deliver aTf to the CNS. To such end, we isolated extracellular vesicles (EVs) from human and mouse plasma and different neuron and glia conditioned media and characterized them based on their quality, quantity, identity, and structural integrity by western blot, dynamic light scattering, and scanning electron microscopy. All sources yielded highly pure vesicles whose size and structures were in keeping with previous literary evidence. Given that, remarkably, EVs from all sources analyzed contained Tf receptor 1 (TfR1) in their composition, we employed two passive cargo-loading strategies which rendered successful EV loading with aTf, specifically through binding to TfR1. These results unveil EVs as potential nanovehicles of aTf to be delivered into the CNS parenchyma, and pave the way for further studies into their possible clinical application in the treatment of demyelinating diseases.

A new proprietary gel containing glabridin, andrographolide, and apolactoferrin improves the appearance of epidermal melasma in adult women: A 6-month pilot, uncontrolled open-label study.

BACKGROUND: Melasma-a localized chronic acquired hypermelanosis-is common in adult women and it is difficult to treat. AIMS: We designed a pilot, uncontrolled open-label study to evaluate the appearance of epidermal melasma after 6 months of twice-daily application of a nonprescription proprietary gel formulation containing glabridin, andrographolide, and apolactoferrin. PATIENTS/METHODS: A total of 40 Caucasian women with epidermal melasma (Fitzpatrick skin types II-VI) were enrolled. The study endpoints included standardized clinical photography, determination of Melasma Area and Severity Index (MASI) scores, spectrocolorimeter X-Rite analysis, in vivo reflectance confocal microscopy (RCM), and self-assessment of cosmetic acceptability. RESULTS: All endpoints showed a statistically significant improvement of epidermal melasma from baseline to the end of the study. There were no dropouts and cosmetic acceptability was rated as excellent by all of the study patients. The only observed adverse event was a mild, transient xerosis (n = 3). CONCLUSIONS: Favorable outcomes, as demonstrated by investigator and instrumental assessments, were demonstrated using a proprietary gel for the treatment of epidermal melasma in adult women. Our results need to be confirmed in independent placebo-controlled studies.

Lactoferrin-loaded contact lenses counteract cytotoxicity caused in vitro by keratoconic tears.

Lactoferrin (LF), an iron-binding protein with antioxidant activity, is significantly reduced in the lacrimal film of patients affected by keratoconus (KC) compared to healthy subjects, and this is supposed to be the cause for tear iron increase and consequent iron deposition and oxygen free radical accumulation in the cornea. We decided to study if LF-loaded contact lenses (LF-CLs) could exert antioxidant activity on epithelial cells incubated in tears collected from two patients affected by keratoconus (KC1 and KC2). Moreover through this model we indirectly estimated iron concentration in the tears of healthy and KC subjects. Reflex tears were collected during the first 2 min of light or onion-induced lacrimation and stored at -20 °C. After incubation in tears for 18 h, mortality of epithelial cells was investigated by trypan blue exclusion test. Successively, LF-CLs were deposed on cells incubated in KC1 tears. For the indirect determination of iron content, cells were incubated with LF 1.2 mg/mL and different FeSO4 concentrations, and for the estimation of iron from the patient's tears, cells were incubated with free serum medium and healthy tears (1:1) and different FeSO4 concentrations. Epithelial cells incubated with reflex tears of KC patients showed increased mortality (27.7 ± 3.9%, p = 0.0003, for KC1 and 17.6 ± 0.95%, p = 0.014, for KC2) compared to epithelial cells maintained in control healthy tears (8.6 ± 1.2%). This difference in mortality was correlated with tear iron concentration, which was estimated at 4.58 µg/mL for the healthy subjects, at 56.28 µg/mL for KC1, and at 8.7 µg/mL for KC2 patient. Application of LF-CLs counteracted KC tear cytotoxicity restoring viability obtained in the presence of control tears. Therapeutic contact lenses obtained by LF loading can reduce oxidative stress induced by patients' tears and might represent an efficient device to arrest the progression of keratoconus.

Iron-loaded transferrin (Tf) is detrimental whereas iron-free Tf confers protection against brain ischemia by modifying blood Tf saturation and subsequent neuronal damage.

Despite transferrin being the main circulating carrier of iron in body fluids, and iron overload conditions being known to worsen stroke outcome through reactive oxygen species (ROS)-induced damage, the contribution of blood transferrin saturation (TSAT) to stroke brain damage is unknown. The objective of this study was to obtain evidence on whether TSAT determines the impact of experimental ischemic stroke on brain damage and whether iron-free transferrin (apotransferrin, ATf)-induced reduction of TSAT is neuroprotective. We found that experimental ischemic stroke promoted an early extravasation of circulating iron-loaded transferrin (holotransferrin, HTf) to the ischemic brain parenchyma. In vitro, HTf was found to boost ROS production and to be harmful to primary neuronal cultures exposed to oxygen and glucose deprivation. In stroked rats, whereas increasing TSAT with exogenous HTf was detrimental, administration of exogenous ATf and the subsequent reduction of TSAT was neuroprotective. Mechanistically, ATf did not prevent extravasation of HTf to the brain parenchyma in rats exposed to ischemic stroke. However, ATf in vitro reduced NMDA-induced neuronal uptake of HTf and also both the NMDA-mediated lipid peroxidation derived 4-HNE and the resulting neuronal death without altering Ca2+-calcineurin signaling downstream the NMDA receptor. Removal of transferrin from the culture media or blockade of transferrin receptors reduced neuronal death. Together, our data establish that blood TSAT exerts a critical role in experimental stroke-induced brain damage. In addition, our findings suggest that the protective effect of ATf at the neuronal level resides in preventing NMDA-induced HTf uptake and ROS production, which in turn reduces neuronal damage.

A Brief Review of Three Common Supplements Used in Alzheimer's Disease.

Individuals with Alzheimer's disease (AD) and their caregivers are using supplements in an effort to halt the progression of the disease. Individuals at risk for or fearing Alzheimer's may use these supplements to try to prevent the disease. Senior care pharmacists are accessible and uniquely qualified to answer questions, make recommendations, and attempt to make drug therapy safe and effective for these individuals. With this in mind, it is important to know the data supporting (or not supporting) common supplements marketed toward those with AD. A review of efficacy and safety data, drug interactions, as well as the mechanism of action believed to benefit those with AD of three common supplements (Prevagen, Cerefolin NAC, and the omega-3 polyunsaturated fatty acid DHA), are highlighted.

Decreasing TfR1 expression reverses anemia and hepcidin suppression in ß-thalassemic mice.

Iron availability for erythropoiesis and its dysregulation in ß-thalassemia are incompletely understood. We previously demonstrated that exogenous apotransferrin leads to more effective erythropoiesis, decreasing erythroferrone (ERFE) and derepressing hepcidin in ß-thalassemic mice. Transferrin-bound iron binding to transferrin receptor 1 (TfR1) is essential for cellular iron delivery during erythropoiesis. We hypothesize that apotransferrin's effect is mediated via decreased TfR1 expression and evaluate TfR1 expression in ß-thalassemic mice in vivo and in vitro with and without added apotransferrin. Our findings demonstrate that ß-thalassemic erythroid precursors overexpress TfR1, an effect that can be reversed by the administration of exogenous apotransferrin. In vitro experiments demonstrate that apotransferrin inhibits TfR1 expression independent of erythropoietin- and iron-related signaling, decreases TfR1 partitioning to reticulocytes during enucleation, and enhances enucleation of defective ß-thalassemic erythroid precursors. These findings strongly suggest that overexpressed TfR1 may play a regulatory role contributing to iron overload and anemia in ß-thalassemic mice. To evaluate further, we crossed TfR1+/- mice, themselves exhibiting iron-restricted erythropoiesis with increased hepcidin, with ß-thalassemic mice. Resultant double-heterozygote mice demonstrate long-term improvement in ineffective erythropoiesis, hepcidin derepression, and increased erythroid enucleation in relation to ß-thalassemic mice. Our data demonstrate for the first time that TfR1+/- haploinsufficiency reverses iron overload specifically in ß-thalassemic erythroid precursors. Taken together, decreasing TfR1 expression during ß-thalassemic erythropoiesis, either directly via induced haploinsufficiency or via exogenous apotransferrin, decreases ineffective erythropoiesis and provides an endogenous mechanism to upregulate hepcidin, leading to sustained iron-restricted erythropoiesis and preventing systemic iron overload in ß-thalassemic mice.

Investigation of the enhanced antimicrobial activity of combination dry powder inhaler formulations of lactoferrin.

The airways of most people with cystic fibrosis are colonized with biofilms of the Gram-negative, opportunistic pathogen Pseudomonas aeruginosa. Delivery of antibiotics directly to the lung in the form of dry powder aerosols offers the potential to achieve high local concentrations directly to the biofilms. Unfortunately, current aerosolised antibiotic regimes are unable to efficiently eradicate these biofilms from the airways. We investigated the ability of the innate antimicrobial, lactoferrin, to enhance the activity of two aminoglycoside antibiotics (tobramycin and gentamicin) against biofilms of P. aeruginosa strain PAO1. Biofilms were prepared in 96 well polystyrene plates. Combinations of the antibiotics and various lactoferrin preparations were spray dried. The bacterial cell viability of the various spray dried combinations was determined. Iron-free lactoferrin (apo lactoferrin) induced a 3-log reduction in the killing of planktonic cell by the aminoglycoside antibiotics (p<0.01) and also reduced both the formation and persistence of P. aeruginosa biofilms (p<0.01). Combinations of lactoferrin and an aminoglycoside displays potential as an effective new therapeutic strategy in the treatment of P. aeruginosa biofilms infections such as those typical of the CF lungs.

Effects of a Supplement Containing Apoaequorin on Verbal Learning in Older Adults in the Community.

CONTEXT: The changes in verbal learning and working memory that often occur with aging may result in reduced social and intellectual interactions. These changes significantly affect an individual's quality of life. As humans age, the body's ability to regulate and maintain calcium levels is diminished. Pharmacological manipulation of the entry of free calcium (Ca2+) has been shown to be effective in increasing some aspects of cognitive function in the aged brain. Apoaequorin has been shown in laboratory studies to regulate levels of intracellular calcium in neuronal cells and to provide protection against ischemic cell death. OBJECTIVE: The study was designed to assess the effects of a supplement of apoaequorin on verbal learning and working memory. DESIGN: The current study, the Madison Memory Study, was a randomized, double-blind, placebo-controlled trial. SETTING: The study occurred in Madison, WI, USA. PARTICIPANTS: Participants were 218 community-dwelling adults, aged 40-91 y, with self-reported memory concerns. INTERVENTION: Participants were randomly assigned to receive either apoaequorin (apoaequorin group) or a matched placebo (control group) for 90 d. OUTCOME MEASURES: The study used quantitative, computerized tools for cognitive assessment the CogState International Shopping List (ISL) and the CogState ISL-Delayed Recall (ISL-DR). Scores from computerized cognitive tasks were measured at baseline and at several points during the 90-d study. RESULTS: No significant differences existed between the intervention and control groups in any parameter at baseline. The intervention group (apoaequorin group) showed a statistically significant improvement in verbal learning and recall on the ISL and the ISL-DR, respectively, during the 90-d study. Apoaequorin was tolerated very well in the study. CONCLUSIONS: The results indicated a strong relationship between apoaequorin and improvements on a quantitative measure of cognitive function, specifically verbal learning. The study found that apoaequorin is a well-tolerated supplement that improved cognitive function in aging adults. The results suggest potential utility for apoaequorin in addressing the declines in cognitive function associated with aging.

Biodegradable Eri silk nanoparticles as a delivery vehicle for bovine lactoferrin against MDA-MB-231 and MCF-7 breast cancer cells.

This study used the Eri silk nanoparticles (NPs) for delivering apo-bovine lactoferrin (Apo-bLf) (~2% iron saturated) and Fe-bLf (100% iron saturated) in MDA-MB-231 and MCF-7 breast cancer cell lines. Apo-bLf and Fe-bLf-loaded Eri silk NPs with sizes between 200 and 300 nm (±10 nm) showed a significant internalization within 4 hours in MDA-MB-231 cells when compared to MCF-7 cells. The ex vivo loop assay with chitosan-coated Fe-bLf-loaded silk NPs was able to substantiate its future use in oral administration and showed the maximum absorption within 24 hours by ileum. Both Apo-bLf and Fe-bLf induced increase in expression of low-density lipoprotein receptor-related protein 1 and lactoferrin receptor in epidermal growth factor (EGFR)-positive MDA-MB-231 cells, while transferrin receptor (TfR) and TfR2 in MCF-7 cells facilitated the receptor-mediated endocytosis of NPs. Controlled and sustained release of both bLf from silk NPs was shown to induce more cancer-specific cytotoxicity in MDA-MB-231 and MCF-7 cells compared to normal MCF-10A cells. Due to higher degree of internalization, the extent of cytotoxicity and apoptosis was significantly higher in MDA-MB-231 (EGFR+) cells when compared to MCF-7 (EGFR-) cells. The expression of a prominent anticancer target, survivin, was found to be downregulated at both gene and protein levels. Taken together, all the observations suggest the potential use of Eri silk NPs as a delivery vehicle for an anti-cancer milk protein, and indicate bLf for the treatment of breast cancer.

Lactoferrin-loaded contact lenses: eye protection against oxidative stress.

PURPOSE: Tear fluid contains antioxidative compounds, vitamin C, glutathione, superoxide dismutase, and lactoferrin (LF), which protect the corneal epithelium from the effects of ultraviolet irradiation, direct airflow, and chemical agents. However, these natural defenses against oxidative stress can decrease, favoring the development of anterior eye disorders, such as keratoconus, dry eye, and Sjögren syndrome. LF is an iron-binding glycoprotein, present in mammalian secretions such as tears and milk, endowed with different physiological functions such as antimicrobial, antiviral, and antioxidant activities. In this work, we studied the capability of different soft contact lenses to adsorb and release LF to restore cellular viability in oxidative stress conditions. METHODS: Three types of contact lenses (filcon V, galyfilcon A, and filcon IB) were loaded with LF and then incubated with TsA or human corneal epithelial primary cells. After oxidative stress induction with 250 µM or 125 µM H2O2, cell viability was evaluated. RESULTS: Data showed that the highest quantity of LF loaded in contact lenses was between 61 µg (for filcon V) and 39 µg (for filcon IB); the release was between 49% and 100% of protein adsorbed. LF released from contact lenses maintained its antioxidant activity at least for 24 hours and was able to protect human epithelial cells from the detrimental effects of oxidative stress. CONCLUSIONS: These results demonstrate that LF-loaded contact lenses could represent a new therapeutic approach to treat ocular surface pathologies characterized by high levels of oxidative stress.

Reversal of hemochromatosis by apotransferrin in non-transfused and transfused Hbbth3/+ (heterozygous B1/B2 globin gene deletion) mice.

Intermediate beta-thalassemia has a broad spectrum of sequelae and affected subjects may require occasional blood transfusions over their lifetime to correct anemia. Iron overload in intermediate beta-thalassemia results from a paradoxical intestinal absorption, iron release from macrophages and hepatocytes, and sporadic transfusions. Pathological iron accumulation in parenchyma is caused by chronic exposure to non-transferrin bound iron in plasma. The iron scavenger and transport protein transferrin is a potential treatment being studied for correction of anemia. However, transferrin may also function to prevent or reduce iron loading of tissues when exposure to non-transferrin bound iron increases. Here we evaluate the effects of apotransferrin administration on tissue iron loading and early tissue pathology in non-transfused and transfused Hbb(th3/+) mice. Mice with the Hbb(th3/+) phenotype have mild to moderate anemia and consistent tissue iron accumulation in the spleen, liver, kidneys and myocardium. Chronic apotransferrin administration resulted in normalization of the anemia. Furthermore, it normalized tissue iron content in the liver, kidney and heart and attenuated early tissue changes in non-transfused Hbb(th3/+) mice. Apotransferrin treatment was also found to attenuate transfusion-mediated increases in plasma non-transferrin bound iron and associated excess tissue iron loading. These therapeutic effects were associated with normalization of transferrin saturation and suppressed plasma non-transferrin bound iron. Apotransferrin treatment modulated a fundamental iron regulatory pathway, as evidenced by decreased erythroid Fam132b gene (erythroferrone) expression, increased liver hepcidin gene expression and plasma hepcidin-25 levels and consequently reduced intestinal ferroportin-1 in apotransferrin-treated thalassemic mice.

A Gelatinases-targeting scFv-based Fusion Protein Shows Enhanced Antitumour Activity with Endostar against Hepatoma.

Gelatinases play important roles in tumour invasion and metastasis and are thus considered promising targets for cancer therapy. In this study, a new single-chain variable fragment (scFv)-based fusion protein Fv-LDP, composed of the anti-gelatinases scFv and lidamycin apoprotein (LDP), was prepared, and its combination with angiogenesis inhibitor Endostar was then investigated. The fusion protein Fv-LDP specifically bound to various tumour cells, and its binding capability to human pulmonary giant cell carcinoma (PG) cells was higher than that of LDP. Fv-LDP inhibited the expression and secretion of gelatinases and could be internalized into tumour cells via endocytosis. Fv-LDP also suppressed the growth of human hepatoma cells and murine hepatoma 22 transplanted in Kunming mice in various degrees. In addition, Endostar could enhance the synergistic or additive inhibition of Fv-LDP on the growth, migration or invasion of human hepatoma cells shown by a colony formation assay and a transwell-based migration or invasion assay, respectively. In vivo, Fv-LDP/Endostar combination showed a significantly synergistic effect on the growth of a human hepatoma xenograft, with an inhibition rate of 80.8% compared with the Fv-LDP (44.1%) or Endostar (8.9%)-treated group. The above-mentioned results indicate that the fusion protein Fv-LDP is effective against transplantable hepatoma in mice and human hepatoma xenografts in athymic mice. Moreover, Endostar can potentiate the inhibition effect of Fv-LDP on the growth of human hepatoma cells and xenografts. These data will provide a new combined strategy for improving the therapeutic efficacy of treatments for hepatoma or other gelatinase-overexpressing tumours.

Effect of bovine apo-lactoferrin on the growth and virulence of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae (App) is a Gram-negative bacterium that causes porcine pleuropneumonia, leading to economic losses in the swine industry. Due to bacterial resistance to antibiotics, new treatments for this disease are currently being sought. Lactoferrin (Lf) is an innate immune system glycoprotein of mammals that is microbiostatic and microbicidal and affects several bacterial virulence factors. The aim of this study was to investigate whether bovine iron-free Lf (BapoLf) has an effect on the growth and virulence of App. Two serotype 1 strains (reference strain S4074 and the isolate BC52) and a serotype 7 reference strain (WF83) were analyzed. First, the ability of App to grow in iron-charged BLf was discarded because in vivo, BapoLf sequesters iron and could be a potential source of this element favoring the infection. The minimum inhibitory concentration of BapoLf was 14.62, 11.78 and 10.56 µM for the strain BC52, S4074 and WF83, respectively. A subinhibitory concentration (0.8 µM) was tested by assessing App adhesion to porcine buccal epithelial cells, biofilm production, and the secretion and function of toxins and proteases. Decrease in adhesion (24-42 %) was found in the serotype 1 strains. Biofilm production decreased (27 %) for only the strain 4074 of serotype 1. Interestingly, biofilm was decreased (60-70 %) in the three strains by BholoLf. Hemolysis of erythrocytes and toxicity towards HeLa cells were not affected by BapoLf. In contrast, proteolytic activity in all strains was suppressed in the presence of BapoLf. Finally, oxytetracycline produced synergistic effect with BapoLf against App. Our results suggest that BapoLf affects the growth and several of the virulence factors in App.

Safety assessment of the calcium-binding protein, apoaequorin, expressed by Escherichia coli.

Calcium-binding proteins are ubiquitous modulators of cellular activity and function. Cells possess numerous calcium-binding proteins that regulate calcium concentration in the cytosol by buffering excess free calcium ion. Disturbances in intracellular calcium homeostasis are at the heart of many age-related conditions making these proteins targets for therapeutic intervention. A calcium-binding protein, apoaequorin, has shown potential utility in a broad spectrum of applications for human health and well-being. Large-scale recombinant production of the protein has been successful; enabling further research and development and commercialization efforts. Previous work reported a 90-day subchronic toxicity test that demonstrated this protein has no toxicity by oral exposure in Sprague-Dawley rodents. The current study assesses the allergenic potential of the purified protein using bioinformatic analysis and simulated gastric digestion. The results from the bioinformatics searches with the apoaequorin sequence show the protein is not a known allergen and not likely to cross-react with known allergens. Apoaequorin is easily digested by pepsin, a characteristic commonly exhibited by many non-allergenic dietary proteins. From these data, there is no added concern of safety due to unusual stability of the protein by ingestion.

Apotransferrin inhibits interleukin-2 expression and protects mice from experimental autoimmune encephalomyelitis.

Transferrin (Tf) has a major role in T cell activation and proliferation. Here, we investigated whether Tf exerts immunomodulatory effects on T cells and in development of T-cell driven experimental autoimmune encephalomyelitis (EAE). While treatment of concanavalin A-stimulated splenocytes with apotransferrin (ApoTf) did not affect release of IL-1ß, TNF, IFN-γ, IL-17, IL-4, and IL-10, it markedly and dose-dependently down-regulated synthesis of IL-2 in these cells. ApoTf also inhibited IL-2 generation in purified CD3+ T cells and the effect was accompanied with down-regulation of MAPK p44/42 and NFκB signaling. Despite impeded IL-2 release, proliferation of splenocytes was not inhibited by ApoTf. Importantly, ApoTf ameliorated EAE in mice and significantly reduced ex vivo IL-2 production in proteolipid protein-specific lymphocytes. Thus ApoTf may be a promising beneficial agent for multiple sclerosis.

Biocompatibility, absorption and safety of protein nanoparticle-based delivery of doxorubicin through oral administration in rats.

Doxorubicin, a potent anticancer drug associated with cardiotoxicity and low oral bioavailability, was loaded into apotransferrin nanoparticles to improve its pharmacological performance. Here, doxorubicin (doxo)-loaded apotransferrin nanoparticles were termed as Apodoxonano, and they were prepared by sol-oil chemistry. The pH-dependent stability of nanoparticles in simulated fluids was evaluated, and the in vitro release was investigated in phosphate-buffered saline. The pharmacokinetic and toxicity studies were conducted in Wistar rats. Nanoparticles have an average size of 75 nm, with 63% entrapment efficiency, at 10 mg w/w of apotransferrin. The particles displayed good pH-dependent stability in the pH range 1.1-7.4, but sensitive at endosomal pH of 5.5, thus facilitating intracellular drug release in endosomes. Multiplex assay showed high transport ability of nano form across epithelial cells (caco-2) when compared to doxo. Moreover, during oral administration, Apodoxonano localizes significantly in esophagus, stomach and small intestine, suggesting that it was absorbed in GI tract through epithelial lining. The drug localization was shown to be significantly lower in the heart reflecting its decreased cardiotoxic nature. The Apodoxonano with a longer bioavailability and a negligible cardiotoxicity can serve as an effective and safe vehicle of drug delivery.

Safety assessment of Apoaequorin, a protein preparation: subchronic toxicity study in rats.

Apoaequorin, a calcium-binding protein originally isolated from jellyfish is available commercially as a dietary supplement. The objective of the present study was to investigate potential adverse effects, if any, of Apoaequorin, a recombinant protein preparation, in rats following subchronic administration. For this study, Sprague-Dawley (Hsd:SD) rats (10/sex/group) were administered via oral gavage 0 (control), 92.6, 462.9, and 926.0mg/kg body weight (bw)/day of Apoaequorin preparation, for 90 days. The corresponding amount of Apoaequorin protein was 0, 66.7, 333.3 and 666.7 mg/kg bw/day, respectively. Administration of the Apoaequorin preparation did not result in any mortality. There were no clinical or ophthalmological signs, body weight, body weight gain, food consumption, food efficiency, clinical pathology or histopathological changes attributable to administration of Apoaequorin. Any changes noted were incidental and in agreement with those historically observed in the age and strain of rats used in this study. Based on the results of this study, the No Observed-Adverse-Effect Level (NOAEL) for Apoaequorin was determined as 666.7 mg/kg bw/day, the highest dose tested.

Intranasal administration of aTf protects and repairs the neonatal white matter after a cerebral hypoxic-ischemic event.

Our previous studies showed that the intracerebral injection of apotransferrin (aTf) attenuates white matter damage and accelerates the remyelination process in a neonatal rat model of cerebral hypoxia-ischemia (HI) injury. However, the intracerebral injection of aTf might not be practical for clinical treatments. Therefore, the development of less invasive techniques capable of delivering aTf to the central nervous system would clearly aid in its effective clinical use. In this work, we have determined whether intranasal (iN) administration of human aTf provides neuroprotection to the neonatal mouse brain following a cerebral hypoxic-ischemic event. Apotransferrin was infused into the naris of neonatal mice and the HI insult was induced by right common carotid artery ligation followed by exposure to low oxygen concentration. Our results showed that aTf was successfully delivered into the neonatal HI brain and detected in the olfactory bulb, forebrain and posterior brain 30 min after inhalation. This treatment successfully reduced white matter damage, neuronal loss and astrogliosis in different brain regions and enhanced the proliferation and survival of oligodendroglial progenitor cells (OPCs) in the subventricular zone and corpus callosum (CC). Additionally, using an in vitro hypoxic model, we demonstrated that aTf prevents oligodendrocyte progenitor cell death by promoting their differentiation. In summary, these data suggest that iN administration of aTf has the potential to be used for clinical treatment to protect myelin and to induce remyelination in demyelinating hypoxic-ischemic events in the neonatal brain.