New Anti-RSV Nucleoprotein Monoclonal Antibody Pairs Discovered Using Rabbit Phage Display Technology.

Human respiratory syncytial virus (hRSV) is one of the major contagious viruses and causes complicated respiratory issues, especially in young children. The sensitive and fast detection of hRSV is critical for taking the most effective actions. In the present study, rabbit antibodies against the hRSV nucleoprotein (NP) were developed using phage display technology. A female rabbit was immunized with an hRSV strain A2 recombinant NP. A Fab library was built and sorted during two successive panning rounds for strain B and the A2 NP (recombinant preparations), respectively. The choice of candidates was performed using ELISA on the two NP strains. The obtained library was 3 × 106 cfu/mL, with an insertion rate of >95%. The two panning rounds permitted an enrichment factor of 100. ELISA screening allowed us to obtain 28 NP-specific Fab candidates. Among them, 10 retained candidates were reformatted into rabbit full IgG; thereafter, pairing tests on the recombinant strains and native lysate samples were performed. After the pairing tests on the recombinant strains, 53 pairs were identified. Eleven pairs were identified as being able to detect RSVs from native lysates. This work presents new high-potential monoclonal antibodies mAbs (mAbs), which would benefit from lateral flow testing data with patient materials.

CDNF rescues motor neurons in models of amyotrophic lateral sclerosis by targeting endoplasmic reticulum stress.

Amyotrophic lateral sclerosis is a progressive neurodegenerative disease that affects motor neurons in the spinal cord, brainstem and motor cortex, leading to paralysis and eventually to death within 3-5 years of symptom onset. To date, no cure or effective therapy is available. The role of chronic endoplasmic reticulum stress in the pathophysiology of amyotrophic lateral sclerosis, as well as a potential drug target, has received increasing attention. Here, we investigated the mode of action and therapeutic effect of the endoplasmic reticulum-resident protein cerebral dopamine neurotrophic factor in three preclinical models of amyotrophic lateral sclerosis, exhibiting different disease development and aetiology: (i) the conditional choline acetyltransferase-tTA/TRE-hTDP43-M337V rat model previously described; (ii) the widely used SOD1-G93A mouse model; and (iii) a novel slow-progressive TDP43-M337V mouse model. To specifically analyse the endoplasmic reticulum stress response in motor neurons, we used three main methods: (i) primary cultures of motor neurons derived from embryonic Day 13 embryos; (ii) immunohistochemical analyses of spinal cord sections with choline acetyltransferase as spinal motor neuron marker; and (iii) quantitative polymerase chain reaction analyses of lumbar motor neurons isolated via laser microdissection. We show that intracerebroventricular administration of cerebral dopamine neurotrophic factor significantly halts the progression of the disease and improves motor behaviour in TDP43-M337V and SOD1-G93A rodent models of amyotrophic lateral sclerosis. Cerebral dopamine neurotrophic factor rescues motor neurons in vitro and in vivo from endoplasmic reticulum stress-associated cell death and its beneficial effect is independent of genetic disease aetiology. Notably, cerebral dopamine neurotrophic factor regulates the unfolded protein response initiated by transducers IRE1α, PERK and ATF6, thereby enhancing motor neuron survival. Thus, cerebral dopamine neurotrophic factor holds great promise for the design of new rational treatments for amyotrophic lateral sclerosis.

Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) Is Highly Expressed in Mouse Tissues With Metabolic Function.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) form a family of atypical growth factors discovered for their neuroprotective properties in the central nervous system (CNS) in animal models of neurodegenerative diseases. Although their mechanism of protective action still remains unclear, it has been suggested that both MANF and CDNF promote cell survival through regulating the unfolded protein response (UPR), thereby relieving endoplasmic reticulum (ER) stress. Recent studies identified MANF for its emerging roles in metabolic function, inflammation and pancreatic ß-cells. We have found that MANF deletion from the pancreas and ß-cells leads to postnatal depletion of ß-cells and diabetes. Moreover, global MANF-deficiency in mice results in severe diabetes-independent growth retardation. As the expression pattern of MANF in mouse tissues has not been extensively studied, we set out to thoroughly investigate MANF expression in embryonic and adult mice using immunohistochemistry, histochemical X-gal staining, enzyme-linked immunosorbent assay (ELISA), and quantitative reverse transcription PCR (RT-qPCR). We found that MANF is highly expressed in brain neurons regulating energy homeostasis and appetite, as well as in hypothalamic nuclei producing hormones and neuropeptides important for different body functions. Strong expression of MANF was also observed in peripheral mouse tissues and cells with high secretory and metabolic function. These include pituitary gland and interestingly we found that the anterior pituitary gland is smaller in MANF-deficient mice compared to wild-type mice. Consequently, we found reduction in the number of growth hormone- and prolactin-producing cells. This combined with increased expression of UPR genes, reduced number of proliferating cells in the anterior pituitary and dysregulated expression of pituitary hormones might contribute to the severe growth defect seen in the MANF knockout mice. Moreover, in this study we compared MANF and CDNF levels in mouse tissues. Unlike MANF, CDNF protein levels are generally lower in mouse tissues, and the highest levels of CDNF was observed in the tissues with high-energy demands and oxidative roles, including heart, muscle, testis, and brown adipose tissue.

Mesencephalic Astrocyte-Derived Neurotrophic Factor Is Upregulated with Therapeutic Fasting in Humans and Diet Fat Withdrawal in Obese Mice.

Dietary restriction induces beneficial metabolic changes and prevents age-related deterioration. Mesencephalic astrocyte-derived neurotrophic factor (MANF) shows protective effects on cells in various models of degenerative diseases. Here we studied whether circulating concentrations of MANF are associated with fasting-induced positive effects. We quantified the levels of circulating MANF from 40 human subjects before and after therapeutic fasting. As measured by an enzyme-linked immunosorbent assay (ELISA), the mean concentration of plasma MANF increased after an average fasting of 15 days. Plasma MANF levels correlated inversely with adiponectin, a hormone that regulates metabolism, thus suggesting that MANF levels are related to metabolic homeostasis. To study the effects of dietary intervention on MANF concentrations in mice, we developed an ELISA for mouse MANF and verified its specificity using MANF knock-out (KO) tissue. A switch from high-fat to normal diet increased MANF levels and downregulated the expression of unfolded protein response (UPR) genes in the liver, indicating decreased endoplasmic reticulum (ER) stress. Liver MANF and serum adiponectin concentrations correlated inversely in mice. Our findings demonstrate that MANF expression and secretion increases with dietary intervention. The MANF correlation to adiponectin and its possible involvement in metabolic regulation and overall health warrants further studies.

Increased Serum Levels of Mesencephalic Astrocyte-Derived Neurotrophic Factor in Subjects With Parkinson's Disease.

BACKGROUND: Mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) promote the survival of midbrain dopamine neurons in animal models of Parkinson's disease (PD). However, little is known about endogenous concentrations of MANF and CDNF in human PD patients, and their relation to PD pathogenesis. Our main objective was to study whether circulating concentrations of MANF and CDNF differ between PD patients and controls, and if they correlate with clinical parameters. Levels of circulating CDNF were studied for the first time. METHODS: MANF and CDNF levels were measured from serum samples of 34 PD patients and 35 controls using validated in-lab-designed enzyme-linked immunosorbent assay (ELISAs). MANF and CDNF mRNA levels in whole blood samples of 60 PD patients and 30 controls were measured by quantitative real time polymerase chain reaction (qRT-PCR). MANF concentrations in different blood cell types were measured by ELISA. RESULTS: Circulating MANF concentrations were significantly higher in PD patients compared to controls (P < 0.001) and were positively correlated with Beck Depression Inventory (BDI) depression rating. MANF protein was present in blood cells, however, MANF mRNA levels in the blood did not differ between PD patients and controls (P = 0.44). The mean concentration of serum CDNF was 33 pg/ml in the controls. CDNF levels were not altered in PD patients (P = 0.25). CONCLUSION: MANF but not CDNF level was increased in the blood of PD patients. It would be interesting to examine the blood level of MANF from early stage PD patients in future studies to test whether MANF can be used as a clinical marker of PD.

Levels of glial cell line-derived neurotrophic factor are decreased, but fibroblast growth factor 2 and cerebral dopamine neurotrophic factor are increased in the hippocampus in Parkinson's disease.

Growth factors can facilitate hippocampus-based learning and memory and are potential targets for treatment of cognitive dysfunction via their neuroprotective and neurorestorative effects. Dementia is common in Parkinson's disease (PD), but treatment options are limited. We aimed to determine if levels of growth factors are altered in the hippocampus of patients with PD, and if such alterations are associated with PD pathology. Enzyme-linked immunosorbent assays were used to quantify seven growth factors in fresh frozen hippocampus from 10 PD and nine age-matched control brains. Western blotting and immunohistochemistry were used to explore cellular and inflammatory changes that may be associated with growth factor alterations. In the PD hippocampus, protein levels of glial cell line-derived neurotrophic factor were significantly decreased, despite no evidence of neuronal loss. In contrast, protein levels of fibroblast growth factor 2 and cerebral dopamine neurotrophic factor were significantly increased in PD compared to controls. Levels of the growth factors epidermal growth factor, heparin-binding epidermal growth factor, brain-derived neurotrophic factor and mesencephalic astrocyte-derived neurotrophic factor did not differ between groups. Our data demonstrate changes in specific growth factors in the hippocampus of the PD brain, which potentially represent targets for modification to help attenuate cognitive decline in PD. These data also suggest that multiple growth factors and direction of change needs to be considered when approaching growth factors as a potential treatment for cognitive decline.

Characterization of CDNF-Secreting ARPE-19 Cell Clones for Encapsulated Cell Therapy.

Cerebral dopamine neurotrophic factor (CDNF) shows beneficial effects in rodent models of Parkinson's and Alzheimer's disease. The brain is a challenging target for protein therapy due to its exclusive blood-brain barrier. Hence, the therapeutic protein should be delivered directly to the brain parenchyma. Implantation of encapsulated mammalian cells that constantly secrete CDNF is a potential approach for targeted and long-term protein delivery to the brain. In this study, we generated several CDNF-secreting cell clones derived from human retinal pigment epithelial cell line ARPE-19, and studied CDNF secretion from the clones maintained as monolayers and in polymeric microcapsules. The secretion of wild type (wt) CDNF transgene was low and the majority of the produced protein remained intracellular, locating mainly to the endoplasmic reticulum (ER). The secretion of wtCDNF decreased to even lower levels when the clones were in a non-dividing state, as in the microcapsules. Both codon optimization and deletion of the putative ER-retrieval signal (four last amino acids: KTEL) improved CDNF secretion. More importantly, the secretion of KTEL-deleted CDNF remained constant in the non-dividing clones. Thus, cells expressing KTEL-deleted CDNF, in contrast to wtCDNF, can be considered for cell encapsulation applications if the KTEL-deleted CDNF is proven to be biologically active in vivo.

Insulin mutations impair beta-cell development in a patient-derived iPSC model of neonatal diabetes.

Insulin gene mutations are a leading cause of neonatal diabetes. They can lead to proinsulin misfolding and its retention in endoplasmic reticulum (ER). This results in increased ER-stress suggested to trigger beta-cell apoptosis. In humans, the mechanisms underlying beta-cell failure remain unclear. Here we show that misfolded proinsulin impairs developing beta-cell proliferation without increasing apoptosis. We generated induced pluripotent stem cells (iPSCs) from people carrying insulin (INS) mutations, engineered isogenic CRISPR-Cas9 mutation-corrected lines and differentiated them to beta-like cells. Single-cell RNA-sequencing analysis showed increased ER-stress and reduced proliferation in INS-mutant beta-like cells compared with corrected controls. Upon transplantation into mice, INS-mutant grafts presented reduced insulin secretion and aggravated ER-stress. Cell size, mTORC1 signaling, and respiratory chain subunits expression were all reduced in INS-mutant beta-like cells, yet apoptosis was not increased at any stage. Our results demonstrate that neonatal diabetes-associated INS-mutations lead to defective beta-cell mass expansion, contributing to diabetes development.

MANF protects human pancreatic beta cells against stress-induced cell death.

AIMS/HYPOTHESIS: There is a great need to identify factors that could protect pancreatic beta cells against apoptosis or stimulate their replication and thus prevent or reverse the development of diabetes. One potential candidate is mesencephalic astrocyte-derived neurotrophic factor (MANF), an endoplasmic reticulum (ER) stress inducible protein. Manf knockout mice used as a model of diabetes develop the condition because of increased apoptosis and reduced proliferation of beta cells, apparently related to ER stress. Given this novel association between MANF and beta cell death, we studied the potential of MANF to protect human beta cells against experimentally induced ER stress. METHODS: Primary human islets were challenged with proinflammatory cytokines, with or without MANF. Cell viability was analysed and global transcriptomic analysis performed. Results were further validated using the human beta cell line EndoC-ßH1. RESULTS: There was increased expression and secretion of MANF in human beta cells in response to cytokines. Addition of recombinant human MANF reduced cytokine-induced cell death by 38% in human islets (p < 0.05). MANF knockdown in EndoC-ßH1 cells led to increased ER stress after cytokine challenge. Mechanistic studies showed that the protective effect of MANF was associated with repression of the NF-κB signalling pathway and amelioration of ER stress. MANF also increased the proliferation of primary human beta cells twofold when TGF-ß signalling was inhibited (p < 0.01). CONCLUSIONS/INTERPRETATION: Our studies show that exogenous MANF protein can provide protection to human beta cells against death induced by inflammatory stress. The antiapoptotic and mitogenic properties of MANF make it a potential therapeutic agent for beta cell protection.

Increased circulating concentrations of mesencephalic astrocyte-derived neurotrophic factor in children with type 1 diabetes.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) was recently shown to be essential for the survival and proliferation of pancreatic ß-cells in mice, where deletion of MANF resulted in diabetes. The current study aimed at determining whether the concentration of circulating MANF is associated with the clinical manifestation of human type 1 diabetes (T1D). MANF expression in T1D or MANF levels in serum have not been previously studied. We developed an enzyme-linked immunosorbent assay (ELISA) for MANF and measured serum MANF concentrations from 186 newly diagnosed children and adolescents and 20 adults with longer-term T1D alongside with age-matched controls. In healthy controls the mean serum MANF concentration was 7.0 ng/ml. High MANF concentrations were found in children 1-9 years of age close to the diagnosis of T1D. The increased MANF concentrations were not associated with diabetes-predictive autoantibodies and autoantibodies against MANF were extremely rare. Patients with conspicuously high MANF serum concentrations had lower C-peptide levels compared to patients with moderate MANF concentrations. Our data indicate that increased MANF concentrations in serum are associated with the clinical manifestation of T1D in children, but the exact mechanism behind the increase remains elusive.

Cerebral dopamine neurotrophic factor improves long-term memory in APP/PS1 transgenic mice modeling Alzheimer's disease as well as in wild-type mice.

Cerebral dopamine neurotrophic factor (CDNF) protects and repairs dopamine neurons in animal models of Parkinson's disease, which motivated us to investigate its therapeutic effect in an animal model of Alzheimer's disease (AD). We employed an established APP/PS1 mouse model of AD and gave intrahippocampal injections of CDNF protein or CDNF transgene in an AAV2 viral vector to 1-year-old animals. We performed a behavioral test battery 2 weeks after the injections and collected tissue samples after the 3-week test period. Intrahippocampal CDNF-therapy improved long-term memory in both APP/PS1 mice and wild-type controls, but did not affect spontaneous exploration, object neophobia or early stages of spatial learning. The memory improvement was not associated with decreased brain amyloid load or enhanced hippocampal neurogenesis. Intracranial CDNF treatment has beneficial effects on long-term memory and is well tolerated. The CDNF molecular mechanisms of action on memory await further studies.

Gene therapy with AAV2-CDNF provides functional benefits in a rat model of Parkinson's disease.

Cerebral dopamine neurotrophic factor (CDNF) protein has been shown to protect the nigrostriatal dopaminergic pathway when given as intrastriatal infusions in rat and mouse models of Parkinson's disease (PD). In this study, we assessed the neuroprotective effect of CDNF delivered with a recombinant adeno-associated viral (AAV) serotype 2 vector in a rat 6-hydroxydopamine (6-OHDA) model of PD. AAV2 vectors encoding CDNF, glial cell line-derived neurotrophic factor (GDNF), or green fluorescent protein were injected into the rat striatum. Protein expression analysis showed that our AAV2 vector efficiently delivered the neurotrophic factor genes into the brain and gave rise to a long-lasting expression of the proteins. Two weeks after AAV2 vector injection, 6-OHDA was injected into the rat striatum, creating a progressive degeneration of the nigrostriatal dopaminergic system. Treatment with AAV2-CDNF resulted in a marked decrease in amphetamine-induced ipsilateral rotations while it provided only partial protection of tyrosine hydroxylase (TH)-immunoreactive cells in the rat substantia nigra pars compacta and TH-reactive fibers in the striatum. Results from this study provide additional evidence that CDNF can be considered a potential treatment of Parkinson's disease.

Mechanisms of polyethylenimine-mediated DNA delivery: free carrier helps to overcome the barrier of cell-surface glycosaminoglycans.

BACKGROUND: Polyethylenimine (PEI) polyplexes mediate efficient gene transfer only at high +/- charge ratios at which free noncomplexed PEI is present. The excess of PEI gives polyplexes a positive surface charge that plays a role in polyplex binding on the cell membrane. Although positively charged PEI polyplexes are known to interact with anionic cell-surface glycosaminoglycans (GAGs), the exact role of free PEI in such interactions is unclear. METHODS: Chinese hamster ovary wild-type cells and mutants lacking cell-surface GAGs were transfected with marker genes using PEI polyplexes with and without free PEI. The total amount of cell-associated plasmid DNA (pDNA) delivered by polyplexes was determined by quantitative real-time PCR and transgene expression was determined using ß-galactosidase and luciferase assays. RESULTS: Transfection activity of polyplexes without free PEI in cells expressing cell-surface GAGs was low even though pDNA was delivered to cells. In the absence of cell-surface GAGs, polyplexes without free PEI had high transfection efficacy. This indicates that the cell-surface GAGs inhibit transfection by purified polyplexes. PEI polyplexes with free carrier mediated transfection in both normal and GAG-deficient cells because free PEI overcomes the inhibitory effect of cell-surface GAGs on transfection. The intracellular elimination of pDNA was faster in the presence of GAGs and, despite improved transfection, free PEI reduced pDNA association with the cells. CONCLUSIONS: Free PEI is essential for minimizing the undesirable binding of polyplexes to cell-surface GAGs that have a negative impact on transfection. The same mechanism may be important in transfections with other polyplexes that require high charge ratios for transfection.