Search Results (27)

Background: The Nerve Growth Factor (NGF) is essential for neuronal survival and function and represents a key therapeutic target for pain and inflammation-related disorders, as well as for neurodegenerative diseases. Small-molecule antagonists of human NGF (hNGF) offer advantages over monoclonal antibodies, including oral availability and reduced immunogenicity. However, their development is often hindered by solubility challenges, necessitating the use of solvents like dimethyl sulfoxide (DMSO). This study investigates whether DMSO directly interacts with hNGF and affects its receptor-binding properties. Methods: Integrative/hybrid computational and experimental biophysical approaches were used to assess DMSO-NGF interaction by combining machine-learning tools and Nuclear Magnetic Resonance (NMR), Fourier Transform Infrared (FT-IR) spectroscopy, Differential Scanning Fluorimetry (DSF) and Grating-Coupled Interferometry (GCI). These techniques evaluated binding affinity, conformational stability, and receptor-binding dynamics. Results: Our findings demonstrate that DMSO binds hNGF with low affinity in a specific yet non-disruptive manner. Importantly, DMSO does not induce significant conformational changes in hNGF nor affect its interactions with its receptors. Conclusions: These results highlight the importance of considering solvent–protein interactions in drug discovery, as these low-affinity yet specific interactions can affect experimental outcomes and potentially alter the small molecules binding to the target proteins. By characterizing DMSO-NGF interactions, this study provides valuable insights for the development of NGF-targeting small molecules, supporting their potential as effective alternatives to monoclonal antibodies for treating pain, inflammation, and neurodegenerative diseases. Full article

Background:Mesenchymal stem/stromal cells (MSCs) are non-hematopoietic, plastic-adherent, and self-renewing cells capable of in vitro trilineage differentiation into fat, bone, and cartilage tissue. Suggestively, MSCs have additional plasticity, as demonstrated by their ability to differentiate in vitro into myocytes, neuron-like cells, and hepatocytes. MSCs are ideal for therapeutic application owing to their numerous advantages; they exhibit limited growth and differentiation abilities, leading to heterogeneous cell populations with inconsistent functions. However, highly purified MSCs, namely, rapidly expanding clones (RECs) that are isolated by single-cell sorting, display uniform functionality. RECs have the potential to offer many benefits, such as transplantable cells for treating several disorders of bone, heart, peripheral nerves, brain, and other organs. This study aimed to assess the effects of RECs on the pheochromocytoma (PC12) cell line, a well-known neuronal cell model.Methods: PC12 cells were cultured under the following conditions: co-culture with RECs, treatment with REC-derived conditioned medium (CM), or co-culture with RECs using Transwell inserts for 7 days. The cells were stained with anti-βIII-tubulin antibody; the lengths of neurites were measured by image analysis. Results: Regarding the co-culture with RECs, PC12’s outgrowth was significantly increased. The RECs expressed nerve growth factor (NGF), a neurotrophic factor that could act on PC12 cells to trigger cellular differentiation.Conclusions: Our findings suggest that RECs via direct culture, intercellular communication in Transwell culture, and RECs CM promoted PC12 cell survival and outgrowth via NGF signaling. Full article

Monoclonal antibody therapy has been a cornerstone of human healthcare for nearly four decades, effectively treating a wide range of diseases including cancers, autoimmune disorders, and inflammatory conditions. However, its application in veterinary medicine is a relatively recent development, offering a promising therapeutic approach for managing chronic diseases in small animals. Dogs and cats, like humans, suffer from chronic conditions such as cancer, arthritis, allergies, and chronic pain, which mAb therapy could potentially address. This review aims to explore the therapeutic potential of mAb therapy in small animal medicine, focusing on currently authorized products, including their mechanisms of action, clinical efficacy, and safety concerns. A comprehensive review of the literature was conducted to evaluate the use of mAbs in veterinary medicine, specifically in the treatment of chronic disorders. While mAb therapy has shown significant benefits in human healthcare, challenges remain in its application to veterinary practice, including safety concerns and the limited availability of approved products. Despite these challenges, mAb therapy holds great promise for improving the management of chronic diseases in animals, with future research and development potentially expanding its clinical use. Full article

Nerve growth factor (NGF) is one of the essential components that have been implicated in the pathophysiology of neuropathic pain, a condition that develops following nerve injury or dysfunction. This neurotrophin is critical for the survival and maintenance of sensory neurons, and its dysregulation has been implicated in the sensitization of pain pathways. NGF interacts with its receptor TrkA and p75^NTR to activate intracellular signaling pathways associated with nociception and the emergence of allodynia and hyperalgesia. Therapeutic approaches employing neutralizing antibodies and molecule inhibitors have been highly effective at both preclinical and clinical levels, hence giving hope again for the use of NGF as an important biomarker and therapeutic target in the management of neuropathic pain. By exploiting the unique properties of NGF and its interactions within the nervous system, new therapeutic modalities could be designed to enhance efficacy while minimizing side effects. In conclusion, taking advantage of the multifaceted dynamics of NGF could provide effective pain management therapies to finally respond to the unmet needs of patients experiencing neuropathic pain. Full article

Biological drugs, including monoclonal antibodies, represent a revolutionary strategy in all fields of medicine, offering promising results even in the treatment of osteoarthritis (OA). However, their safety and efficacy have not been fully validated, highlighting the need for in-depth studies. Therefore, we provided a comprehensive systematic review of the intra-articular use of monoclonal antibodies for the treatment of OA in animal models, reflecting ongoing efforts to advance therapeutic strategies and improve patient outcomes. A systematic literature search was conducted in December 2023 following the PRISMA guidelines, using the Web of Science, Google Scholar, and PUBMED databases. Out of a total of 456, 10 articles were included in the study analyzing intra-articular antibodies and focusing on various targets, including vascular endothelial growth factor (VEGF), nerve growth factor (NGF), interleukin 4-10 (IL4-10), tumor necrosis factor α (TNF-α), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and matrix metalloproteinase 13 (MMP-13). Most studies administered the antibodies weekly, ranging from 1 to 10 injections. Animal models varied, with mean follow-up periods of 8.9 ± 4.1 weeks. The methods of assessing outcomes, including pain and morpho-functional changes, varied. Some studies reported only morphological and immunohistochemical data, while others included a quantitative analysis of protein expression. In conclusion, monoclonal antibodies represent a promising avenue in the treatment of OA, offering targeted approaches to modulate disease pathways. Further research and clinical trials are needed to validate their safety and efficacy, with the potential to revolutionize the management of OA and reduce reliance on prosthetic interventions. Full article

Peripheral nerve regeneration depends on close interaction between neurons and Schwann cells (SCs). After nerve injury, SCs produce growth factors and cytokines that are crucial for axon re-growth. Previous studies revealed the supernatant of SCs exposed to nuclear magnetic resonance therapy (NMRT) treatment to increase survival and neurite formation of rat dorsal root ganglion (DRG) neurons in vitro. The aim of this study was to identify factors involved in transferring the observed NMRT-induced effects to SCs and consequently to DRG neurons. Conditioned media of NMRT-treated (CM NMRT) and untreated SCs (CM CTRL) were tested by beta-nerve growth factor (ßNGF) ELISA and multiplex cytokine panels to profile secreted factors. The expression of nociceptive transient receptor potential vanilloid 1 (TRPV1) channels was assessed and the intracellular calcium response in DRG neurons to high-potassium solution, capsaicin or adenosine triphosphate was measured mimicking noxious stimuli. NMRT induced the secretion of ßNGF and pro-regenerative-signaling factors. Blocking antibody experiments confirmed ßNGF as the main factor responsible for neurotrophic/neuritogenic effects of CM NMRT. The TRPV1 expression or sensitivity to specific stimuli was not altered, whereas the viability of cultured DRG neurons was increased. Positive effects of CM NMRT supernatant on DRG neurons are primarily mediated by increased ßNGF levels. Full article

β-Nerve growth factor (β-NGF) is a protein produced in the reproductive tract of camelids (camels, llamas, and alpacas) that has been identified as the ovulation inducing factor in seminal plasma. β-NGF from seminal plasma deposited into the reproductive tract of the female camelid acts systemically to stimulate the secretion of luteinizing hormone (LH) from the anterior pituitary, which in turn induces follicle maturation and ovulation. The objectives of the present study were to determine if β-NGF is present in the reproductive tract of the stallion and identify the specific site(s) of production. The hypotheses were that β-NGF would be present in the stallion reproductive tract and would primarily be localized in Sertoli cells of the testes and the prostate gland. Immunohistochemistry on paraffin-embedded paraformaldehyde-fixed tissues was performed using a rabbit polyclonal anti-β-NGF antibody on a total of six male equine reproductive tracts, including a one-day old colt, a one-year-old colt, and four adult stallion tracts. Strong immunostaining was observed in the efferent ducts of the testes and the epithelial cells of the prostate, seminal vesicles, bulbourethral glands, and ampullae. Weaker β-NGF staining was noted in Leydig cells, Sertoli cells, and spermatogonia within the testes and in epithelial cells of the epididymis. In conclusion, immunohistochemistry revealed that β-NGF is present in the stallion reproductive tract, and the protein is primarily present in the efferent ducts of the testes and in all accessory sex glands. Full article

Stress exposure worsens allergic inflammatory diseases substantially. Mast cells (MCs) play a key role in peripheral immune responses to neuroendocrine stress mediators such as nerve growth factor (NGF) and substance P (SP). Mast cell proteases (MCPs) and cholinergic factors (Chrna7, SLURP1) were recently described to modulate MC stress response. We studied MCPs and Chrna7/SLURP1 and their interplay in a mouse model for noise induced stress (NiS) and atopic dermatitis-like allergic inflammation (AlD) and in cultured MC lacking Chrna7. We found that the cholinergic stress axis interacts with neuroendocrine stress mediators and stress-mediator cleaving enzymes in AlD. SP-cleaving mMCP4+ MC were upregulated in AlD and further upregulated by stress in NiS+AlD. Anti-NGF neutralizing antibody treatment blocked the stress-induced upregulation in vivo, and mMCP4+ MCs correlated with measures of AlD disease activity. Finally, high mMCP4 production in response to SP depended on Chrna7/SLURP1 in cultured MCs. In conclusion, mMCP4 and its upstream regulation by Chrna7/SLURP1 are interesting novel targets for the treatment of allergic inflammation and its aggravation by stress. Full article

Spermatogenesis is the process of proliferation and differentiation of spermatogonial cells to meiotic and post-meiotic stages and sperm generation. Normal spermatogenesis occurs in vivo at 34 °C to 35 °C, and high temperatures are known to cause male infertility. The aim of the present study was to examine the effect of temperature (35 °C compared to 37 °C) on the viability/apoptosis of developed cells, on the development of different stages of spermatogenesis in 3D in vitro culture conditions, and the functionality of Sertoli cells under these conditions. We used isolated cells from seminiferous tubules of sexually immature mice. The cells were cultured in methylcellulose (as a three-dimensional (3D) in vitro culture system) and incubated in a CO₂ incubator at 35 °C or 37 °C. After two to six weeks, the developed cells and organoids were collected and examined for cell viability and apoptosis markers. The development of different stages of spermatogenesis was evaluated by immunofluorescence staining or qPCR analysis using specific antibodies or primers, respectively, for cells at each stage. Factors that indicate the functionality of Sertoli cells were assessed by qPCR analysis. The developed organoids were examined by a confocal microscope. Our results show that the percentages and/or the expression levels of the developed pre-meiotic, meiotic, and post-meiotic cells were significantly higher at 35 °C compared to those at 37 °C, including the expression levels of the androgen receptor, the FSH receptor, transferrin, the androgen-binding protein (ABP), and the glial-derived nerve growth factor (GDNF) which were similarly significantly higher at 35 °C than at 37 °C. The percentages of apoptotic cells (according to acridine orange staining) and the expression levels of BAX, FAS, and CASPAS 3 were significantly higher in cultures incubated at 37 °C compared to those incubated at 35 °C. These findings support the in vivo results regarding the negative effect of high temperatures on the process of spermatogenesis and suggest a possible effect of high temperatures on the viability/apoptosis of spermatogenic cells. In addition, increasing the temperature in vitro also impaired the functionality of Sertoli cells. These findings may deepen our understanding of the mechanisms behind optimal conditions for normal spermatogenesis in vivo and in vitro. Full article

Leukocyte- and Platelet-Rich Fibrin (L-PRF) is a second-generation platelet concentrate that is prepared directly from the patient’s own blood. It is widely used in the field of regenerative medicine, and to better understand its clinical applicability we aimed to further explore the biological properties and effects of L-PRF on cells from the central and peripheral nervous system. To this end, L-PRF was prepared from healthy human donors, and confocal, transmission, and scanning electron microscopy as well as secretome analysis were performed on these clots. In addition, functional assays were completed to determine the effect of L-PRF on neural stem cells (NSCs), primary cortical neurons (pCNs), and peripheral dorsal root ganglion (DRG) neurons. We observed that L-PRF consists of a dense but porous fibrin network, containing leukocytes and aggregates of activated platelets that are distributed throughout the clot. Antibody array and ELISA confirmed that it is a reservoir for a plethora of growth factors. Key molecules that are known to have an effect on neuronal cell functions such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) were slowly released over time from the clots. Next, we found that the L-PRF secretome had no significant effect on the proliferative and metabolic activity of NSCs, but it did act as a chemoattractant and improved the migration of these CNS-derived stem cells. More importantly, L-PRF growth factors had a detrimental effect on the survival of pCNs, and consequently, also interfered with their neurite outgrowth. In contrast, we found a positive effect on peripheral DRG neurons, and L-PRF growth factors improved their survival and significantly stimulated the outgrowth and branching of their neurites. Taken together, our study demonstrates the positive effects of the L-PRF secretome on peripheral neurons and supports its use in regenerative medicine but care should be taken when using it for CNS applications. Full article

The matricellular protein cell communication factor 2/connective tissue growth factor (CCN2/CTGF) is critical to development of neuromuscular fibrosis. Here, we tested whether anti-CCN2 antibody treatment will reduce established forepaw fibro-degenerative changes and improve function in a rat model of overuse injury. Adult female rats performed a high repetition high force (HRHF) task for 18 weeks. Tissues were collected from one subset after 18 wks (HRHF-Untreated). Two subsets were provided 6 wks of rest with concurrent treatment with anti-CCN2 (HRHF-Rest/anti-CCN2) or IgG (HRHF-Rest/IgG). Results were compared to IgG-treated Controls. Forepaw muscle fibrosis, neural fibrosis and entheseal damage were increased in HRHF-Untreated rats, compared to Controls, and changes were ameliorated in HRHF-Rest/anti-CCN2 rats. Anti-CCN2 treatment also reduced phosphorylated-β-catenin (pro-fibrotic protein) in muscles and distal bone/entheses complex, and increased CCN3 (anti-fibrotic) in the same tissues, compared to HRHF-Untreated rats. Grip strength declines and mechanical sensitivity observed in HRHF-Untreated improved with rest; grip strength improved further in HRHF-Rest/anti-CCN2. Grip strength declines correlated with muscle fibrosis, entheseal damage, extraneural fibrosis, and decreased nerve conduction velocity, while enhanced mechanical sensitivity (a pain-related behavior) correlated with extraneural fibrosis. These studies demonstrate that blocking CCN2 signaling reduces established forepaw neuromuscular fibrosis and entheseal damage, which improves forepaw function, following overuse injury. Full article

Efficient induction of target-specific antibodies can be elicited upon immunization with highly immunogenic virus-like particles (VLPs) decorated with desired membrane-anchored target antigens (Ags). However, for example, for diagnostic purposes, monoclonal antibodies (mAbs) are required to enable the histological examination of formaldehyde-fixed paraffin-embedded (FFPE) biopsy tissue samples. Aiming at the generation of FFPE-antigen-specific mAbs and as a proof of concept (POC), we first established a simplified protocol using only formaldehyde and 90 °C heat fixation (FF90) of cells expressing the target Ag nerve growth factor receptor (NGFR). The FF90 procedure was validated using flow cytometric analysis and two mAbs recognizing either the native and FFPE-Ag or exclusively the native Ag. C-terminally truncated NGFR (trNGFR)-displaying native and FF90-treated VLPs derived from HIV-1 did not reveal distinctive changes in particle morphology using transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis. Mice were subsequently repetitively immunized with trNGFR-decorated FF90-VLPs and hybridoma technology was used to establish mAb-producing cell clones. In multiple screening rounds, nine cell clones were identified producing mAbs distinctively recognizing epitopes in FF90- and FFPE-NGFR. This POC of a new methodology should foster the future generation of mAbs selectively targeting FFPE-fixed cell-surface Ags. Full article

Background and Objectives: Diabetic gastroenteropathy (DG) is a common complication of diabetes mellitus type 2. Interstitial cells are non-neural cells of mesenchymal origin inserted between nerve elements and smooth muscle cells, necessary for normal function and peristaltic contractions in the gastrointestinal (GI) tract. There are at least two types of interstitial cells within the GI muscle layer—interstitial cells of Cajal (ICC) and interstitial platelet-derived growth factor receptor α-positive cells (IPC). The mechanism of diabetic gastroenteropathy is unclear, and interstitial cells disorders caused by metabolic changes in diabetes mellitus (DM) could explain the symptoms of DG (slow intestinal transit, constipation, fecal incontinence). The aim of this study was to identify PDGFRα and c-kit immunoreactive cells in the colon of rats with streptozotocin–nicotinamide-induced diabetes mellitus type 2, as well as to determine their distribution in relation to smooth muscle cells and enteric nerve structures. Materials and Methods: Male Wistar rats were used, and diabetes type 2 was induced by an intraperitoneal injection of streptozotocin, immediately after intraperitoneal application of nicotinamide. The colon specimens were exposed to PDGFRα and anti-c-kit antibodies to investigate interstitial cells; enteric neurons and smooth muscle cells were immunohistochemically labeled with NF-M and desmin antibodies. Results: Significant loss of the intramuscular ICC, myenteric ICC, and loss of their connection in intramuscular linear arrays and around the ganglion of the myenteric plexus were observed with no changes in nerve fiber distribution in the colon of rats with diabetes mellitus type 2. IPC were rarely present within the colon muscle layer with densely distributed PDGFRα+ cells in the colon mucosa and submucosa of both experimental groups. In summary, a decrease in intramuscular ICC, discontinuities and breakdown of contacts between myenteric ICC without changes in IPC and nerve fibers distribution were observed in the colon of streptozotocin/nicotinamide-induced diabetes type 2 rats. Full article

Glioblastoma multiforme (GBM) is the most lethal adult brain cancer. Temozolomide (TMZ), the standard chemotherapeutic drug used in GBM, has limited benefit and alternate therapies are needed to improve GBM treatment. Nerve growth factor (NGF) and its precursor proNGF are increasingly recognized as stimulators of human tumor progression. The expression and stimulatory effect of NGF on GBM cell growth has previously been reported, but the status of proNGF in GBM is unreported. In this study, we have investigated proNGF expression and biological activity in GBM. A clinical cohort of GBM (n = 72) and low-grade glioma (n = 20) was analyzed by immunohistochemistry for proNGF and digital quantification. ProNGF expression was significantly increased in GBM compared to low grade gliomas and proNGF was also detected in patient plasma samples. ProNGF was also detected in most GBM cell lines by Western blotting. Although anti-proNGF blocking antibodies inhibited cell growth in GBM cells with methylated MGMT gene promoter, targeting proNGF could not potentiate the efficacy of TMZ. In subcutaneous xenograft of human GBM cells, anti-proNGF antibodies slightly reduced tumor volume but had no impact on TMZ efficacy. In conclusion, this data reveals that proNGF is overexpressed in GBM and can stimulate cancer cell growth. The potential of proNGF as a clinical biomarker and therapeutic target warrants further investigations. Full article

Expression of the nerve growth factor NGF is increased in pulmonary hypertension (PH). We have here studied whether oxidative stress and inflammation, two pathological conditions associated with transforming growth factor-β1 (TGF-β1) in PH, may trigger NGF secretion by pulmonary arterial (PA) cells. Effects of hydrogen peroxide (H₂O₂) and interleukin-1β (IL-1β) were investigated ex vivo on rat pulmonary arteries, as well as in vitro on human PA smooth muscle (hPASMC) or endothelial cells (hPAEC). TβRI expression was assessed by Western blotting. NGF PA secretion was assessed by ELISA after TGF-β1 blockade (anti-TGF-β1 siRNA, TGF-β1 blocking antibodies, TβRI kinase, p38 or Smad3 inhibitors). TβRI PA expression was evidenced by Western blotting both ex vivo and in vitro. H₂O₂ or IL-1β significantly increased NGF secretion by hPASMC and hPAEC, and this effect was significantly reduced when blocking TGF-β1 expression, binding to TβRI, TβRI activity, or signaling pathways. In conclusion, oxidative stress and inflammation may trigger TGF-β1 secretion by hPASMC and hPAEC. TGF-β1 may then act as an autocrine factor on these cells, increasing NGF secretion via TβRI activation. Since NGF and TGF-β1 are relevant growth factors involved in PA remodeling, such mechanisms may therefore be relevant to PH pathophysiology. Full article