Primary Cultures Research Articles

Efficient Protocol for Isolating Human Fibroblast from Primary Skin Cell Cultures; Application to Keloid, Hypertrophic Scar and Normal Skin Biopsies

Abstract This protocol introduces a streamlined and efficient method for isolating human fibroblast from skin primary cell culture with a specific focus on its application to keloid, hypertrophic scar, and normal skin biopsies. Additionally, the absence of suitable animal models for keloid and hypertrophic scar has led preclinical research to rely on in vitro studies using primary cell cultures. This approach addresses the challenges of existing protocols in terms of time, cost, equipment, and technical expertise required. The method involves derivation, culture, and characterization analysis including cell proliferation, migration, and fibroblastic marker (Vimentin, CD90, CD73, and CD105) expression. Our study yielded high amounts of fibroblast from tested skin explants while maintaining their in vivo-like characteristics and behaviour. Immunostaining assay confirmed that the cultivated fibroblast was positively expressed Vimentin. Flowcytometry results showed high expression of CD90 and CD73 while relatively showing lower expression of CD105. Fibroblast derived from keloid tissue showed the highest rate of proliferation and migration ability compared to the other samples. These findings suggest an efficient and reproducible technique to cultivate high qualified fibroblast from human skin in normal or pathological condition, particularly for keloid and hypertrophic scar. The application of this protocol provides a foundation for further studies to investigate the progression and potential intervention of aberrant fibrotic dermatological disorder, in vitro.

Shortcomings, limitations and gaps in physiological roles of extracellular vesicles in obesity.

Extracellular vesicles (EVs) play a crucial role in mediating communication between cells across species and kingdoms. The intercellular communication facilitated by EVs through autocrine and paracrine signalling mechanisms is essential for cell survival, maintaining normal metabolic functions and ensuring overall bodily homeostasis and health. Extracellular vesicles are present in various bodily fluids, such as pleural effusions, plasma, breast milk, amniotic fluid, semen and saliva. Additionally, the generation and release of EVs contribute to the removal of cellular waste. Patients with obesity exhibit a higher release and amount of circulating EVs than individuals with normal weight. This increased EV release in obesity might contribute to the inflammatory state characteristic of this metabolic condition, because higher levels of pro-inflammatory molecules are found within their cargo. However, interpreting results related to EV abundance, cargo and biological actions can be complicated by several factors; these include variations in cell sources, a wide age range (from children to the elderly), a mix of females and males, medication use and health status, a range of body weights (from normal weight to morbid obesity) and differences between in vitro assays using cell lines versus primary cultures. This article addresses the shortcomings, limitations and gaps in knowledge, providing a framework for enhancing our understanding of the physiological effects of EVs on obesity.

Epigenetic modifications in transdifferentiation of adipose tissue-derived stromal cells into spontaneously beating cardiomyocyte-like cells

Abstract Background Adipose tissue-derived stromal vascular fraction (adipose SVF) contains pluripotent mesenchymal stem cells and rarely transdifferentiate into beating cardiomyocytes. We developed a simple culture protocol under which the adult murine inguinal adipose SVF reproductively transdifferentiates into beating cardiomyocyte-like cells (beating CM) without inductions in the primary culture. We also reported that Mef2c is a crucial factor in this conversion. However, the characteristics of beating CM and the factors that regulate the differentiation of adipose SVF toward the cardiac lineage are unknown. Purpose To investigate sequential changes in global gene expression profiles of adipose SVF in primary culture and determine the mechanism of differentiation into beating CM. Methods Adipose SVF cells were isolated from adult mice's inguinal subcutaneous fat pad and cultured using the previously established beating CM induction method (Sci Rep. 2021). At 6 time points (days 0, 3, 7, 14, 21, and 28) in primary culture, total RNA was extracted, and RNA-sequencing was performed (n = 3). The data was analyzed using Subio Platform and MetaCore software. To assess the candidate gene and proteins, adipose SVF cells were transduced with the gene using a lentivirus vector and supplemented with the specific inhibitors. The cardiac differentiation was evaluated by quantitative PCR on day 28. Results The beating CM was confirmed on days 14, 21, and 28 in the primary culture. Among prefiltered 14,574 genes, the expression level of 749 genes were significantly changed (153 genes upregulated vs. 596 genes downregulated) between days 7 and 14 (2-fold, P < 0.05), in which beating CM appeared. The GO molecular functions analysis showed that O-GluNAC transferase and histone deacetylase (HDAC)-associated genes were expressed dominantly before day 7, and KLF4-associated genes were expressed prominently after day 14 in primary culture. The expression of cardiac troponin T (Tnnt2) and myosin light chain ventricular type (Myl2) increased on day 3 and day 14, respectively. The HDAC7, an epigenetic regulator, decreased the expression on day 14 or later. Treatment with the HDAC inhibitor (150 nmol/L) increased the expression of Tnnt2 (5-fold vs. control) on day 28 in adipose SVF. Furthermore, treatment of Mef2c-transduced adipose SVFs with HDAC inhibitors increased expression of cardiac troponin T (29-fold vs. control, P < 0.05). Conclusion The time course analysis demonstrated that a remarkable change occurred in the regulation of transcription on day 14 in adipose SVF primary culture. Alterations in epigenetic modifications increased beating CM in adipose SVFs. Adipose SVF could be applied to new cardiac regeneration therapies by increasing the efficiency of introduction into the beating CM.

Itga9 protects against thoracic aortic aneurysm or dissection

Abstract Background The thoracic aortic aneurysm or dissection (TAAD) is a fatal cardiovascular disease with unclear pathogenic mechanisms. Integrins are important in coordinating the transmembrane connections between the extracellular matrix (ECM) and cytoskeleton, contributing to pathological processes of various diseases. Integrin α9 (Itga9), one of the integrin family subunits, has emerged as critical for vascular homeostasis. However, the contribution of Itga9 to TAAD remains elusive. Purpose The present study aimed to determine the role of Itga9 in the development of TAAD and its underlying mechanisms. Methods Western blotting and immunofluorescence staining were used to evaluate ITGA9 expression in the human Standford-A AAD tissues and rodent model of TAAD. Beta-aminopropionitrile monofumarate (BAPN) was used to induce TAAD mouse model. Vascular smooth muscle cell (VSMC)-specific Itga9 knockout mice or adeno-associated virus (AAV)-treated Itga9 overexpressed mice were generated to detect the role of Itga9 in the progression of TAAD. RNA-sequencing analysis was performed to discern possible transcriptome changes in the aorta tissues of VSMC-specific Itga9 knockout mice after BAPN treatment. Primary mouse aortic VSMC culture, flow cytometry analysis, EdU incorporation assay, transmission electron microscopy, and autophagy flux experiments were performed to evaluate the function of Itga9 on VSMCs and uncover its downstream target. Results ITGA9 expression levels were reduced in the aorta tissues of Standford-A AAD patients and BAPN-induced TAAD mice. VSMC-specific deletion of Itga9 exacerbated the incidence of BAPN-induced TAAD in mice. Itga9 deficiency downregulated Valosin-containing protein (Vcp) expression in mice aorta and caused reduced autophagy biomarkers of LC3 after BAPN treatment. In the primary aortic VSMCs, Itga9 deficiency resulted in repressed autophagy, accelerated apoptosis, and reduced proliferation ability. Mechanistically, Itga9 recruited the transcription factor Pre-B-cell leukemia transcription factor 1 (Pbx1) to activate the Vcp and promote VSMC autophagy. Consistently, overexpression of Itga9 in vitro and in vivo rescued the autophagic change and protected against the TAAD progression by upregulating the expression of Pbx1 and Vcp. Conclusions Our findings indicate a critical role of Itga9 in the regulation of VSMC autophagy by controlling Pbx1-Vcp. The Itga9-Pbx1-Vcp axis may represent a novel therapeutic target against TAAD.

3D-printed microfluidic and millifluidic devices for cell culture and mechanotransduction studies: A review

The practice of conventional cell culture techniques affects the quality of biological research in mechanobiology by not replicating the cell’s microenvironment adequately. Microfluidic devices resembling 3D in vivo cell culture offer viable alternatives for mechanobiology studies and other applications including cell screening, cell separation, and point-of-care diagnostics. This review highlights recent advances in additive manufacturing (AM) to fabricate microfluidic and millifluidic devices through fast design iterations and cost reduction compared with conventional device manufacturing. Key advances in AM technologies such as fused deposition modeling (FDM), stereolithography apparatus (SLA), and digital light processing (DLP) have allowed the direct manufacture of complex microfluidic devices with master molds and sacrificial structures using multiple material components. Currently, available 3D printed devices for the precise fabrication of milli- and microfluidic devices, essential to mimicking the cellular microenvironment, while economically reducing production costs by eliminating expensive clean-room environments and reducing material waste are highlighted.

Non-genetic differences underlie variability in proliferation among esophageal epithelial clones.

Individual cells grown in culture exhibit remarkable differences in their growth, with some cells capable of forming large clusters, while others are limited or fail to grow at all. While these differences have been observed across cell lines and human samples, the growth dynamics and associated cell states remain poorly understood. In this study, we performed clonal tracing through imaging and cellular barcoding of an in vitro model of esophageal epithelial cells (EPC2-hTERT). We found that about 10% of clones grow exponentially, while the remaining have cells that become non-proliferative leading to a halt in the growth rate. Using mathematical models, we demonstrate two distinct growth behaviors: exponential and logistic. Further, we discovered that the propensity to grow exponentially is largely heritable through four doublings and that the less proliferative clones can become highly proliferative through increasing plating density. Combining barcoding with single-cell RNA-sequencing (scRNA-seq), we identified the cellular states associated with the highly proliferative clones, which include genes in the WNT and PI3K pathways. Finally, we identified an enrichment of cells resembling the highly proliferative cell state in the proliferating healthy human esophageal epithelium.

Toxicity and Dermatokinetic Analysis of Ibrutinib in Human Skin Models

Background/Objectives: Ibrutinib (IBR) is a tyrosine kinase inhibitor under investigation in preclinical and clinical settings as an alternative treatment for melanoma. Nevertheless, the limited oral bioavailability of IBR and the need for high doses of the drug to kill melanoma cells are major drawbacks for this purpose. Considering that melanoma is restricted to the skin at early stages, the topical application of IBR might constitute an effective and safer administration route. In this study, we determined IBR’s toxicity and dermatokinetics using human primary cells and human organotypic skin explant cultures (hOSECs). Methods: After demonstrating that human primary fibroblasts and keratinocytes present IBR target genes, the cytotoxicity of the drug was determined using the MTT and annexin V/PI staining assays. IBR toxicity in the skin was assessed using the TTC assay, and the irritation potential was established using histological assessment. Finally, IBR cutaneous permeation was assessed ex vivo to determine the drug dermatokinetics. Results: Our findings reveal that IBR exerts dose-dependent toxicity towards skin cells, presenting an IC50 in the same range as melanoma cells. The topical application of the drug successfully reduced irritation and toxicity in the skin, and the drug was shown to successfully permeate the stratum corneum and reach the viable skin layers in therapeutic concentrations. Conclusions: Overall, our data encourage the topical application of IBR to treat melanoma, paving the way for future studies in this theme.

Hepatic Stellate Cell-mediated Increase in CCL5 Chemokine Expression after X-ray Irradiation Determined In Vitro and In Vivo.

Radiation exposure causes hepatitis which induces hepatic steatosis and fibrosis. Although hepatic stellate cells (HSCs) have been considered potential pathological modulators for the development of hepatitis due to viral and microbial infections, their involvement in radiation-induced hepatitis is yet to be determined. This study aimed to clarify the relationship between radiation exposure and expressions of inflammatory cytokines and chemokines in HSCs in vitro and invivo. HSCs were obtained from 1-week-old mice, known to be highly sensitive to radiation-induced hepatocellular carcinoma, using a newly established method combining liver perfusion, cell dissociation, and density gradient centrifugation, followed by magnetic negative selection of hematopoietic and endothelial cells with anti-CD45.2 and CD146 antibodies. The isolated HSCs were confirmed by the expression of desmin and glial fibrillary acidic protein (GFAP). We demonstrated that primary cultured HSCs, exposed to X-ray irradiation (0, 1.9, and 3.8 Gy) and cultured for 3 and 7 days, produced elevated levels of C-C motif chemokine ligand 5 (CCL5, also known as RANTES) inflammatory chemokine in a dose-dependent manner. An invivo immunofluorescence method confirmed that increased CCL5 signals were observed in GFAP-positive HSCs in mouse livers 7 days after whole-body X-ray irradiation (1.9 and 3.8 Gy). Adequate expression of C-C motif chemokine receptor 5 (Ccr5), a receptor for CCL5, was also detected using real-time PCR in the liver of both irradiated and non-irradiated mice. Taken together, our data suggest that HSCs may drive hepatitis via CCL5/CCR5 axis in the liver under radiation-induced stress. Furthermore, this newly established experimental protocol can help evaluate the expression of other inflammatory cytokines in primary cultures of HSCs isolated from infant mice.

Features of proinflammatory activation of macrophages in patients with rheumatoid arthritis and systemic lupus erythematosus

Autoimmune rheumatic diseases (ARDs) are chronic pathological conditions that arise from an abnormal immune response and are accompanied by systemic inflammation. The most common ARDs include rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and systemic sclerosis (SSc). The exact pathogenesis of ARDs remains unclear, but the complex influence of genetic, immunological and external environmental factors leads to the occurrence and further progression of ARDs. It has been shown that the cause of chronic inflammation may be proinflammatory activation of macrophages, in which an increase in the secretion of cytokines is observed. The aim of this study was to evaluate the inflammatory response of macrophages in patients with RA, SLE and SSc. Materials and methods. The study included 143 participants: 47 patients with RA, 45 patients with SLE, 34 patients with SSc, and 17 people without ARDs and other chronic diseases. Isolation of a primary culture of monocytes was carried out by centrifugation in a ficoll gradient using magnetic separation from the whole blood of study participants. Lipopolysaccharide (LPS) was added to stimulate cells along the proinflammatory pathway. Cell cultivation was carried out for 24 hours. Determination of basal and LPS-stimulated secretion of IL-8 by macrophages was carried out in the culture fluid using an enzyme-linked immunosorbent assay (ELISA). Proinflammatory activation of macrophages was calculated as the ratio of LPS-stimulated and basal IL-8 secretion. Research results. Basal secretion of IL-8 by macrophages was statistically significantly higher in the groups of patients with RA and SSc compared with the SLE and control groups. LPS-stimulated secretion of IL-8 by macrophages in the SSc group had statistically higher values compared to the RA and SLE groups. Proinflammatory activation of macrophages was reduced in the group of patients with RA compared to patients with SLE and the control group, and was also statistically significantly lower in patients with SSc compared to the control group.

Targeted NAD+ repletion via biomimetic nanoparticle enables simultaneous management of intimal hyperplasia and accelerated re-endothelialization: A proof-of-concept study toward next-generation of endothelium-protective, anti-restenotic therapy

Endovascular interventions often fail due to restenosis, primarily caused by smooth muscle cell (SMC) proliferation, leading to intimal hyperplasia (IH). Current strategies to prevent restenosis are far from perfect and impose significant collateral damage on the fragile endothelial cell (EC), causing profound thrombotic risks. Nicotinamide adenine dinucleotide (NAD+) is a co-enzyme and signaling substrate implicated in redox and metabolic homeostasis, with a pleiotropic role in protecting against cardiovascular diseases. However, a functional link between NAD+ repletion and the delicate duo of IH and EC regeneration has yet to be established. NAD+ repletion has been historically challenging due to its poor cellular uptake and low bioavailability. We have recently invented the first nanocarrier that enables direct intracellular delivery of NAD+ in vivo. Combining the merits of this prototypic NAD + -loaded calcium phosphate (CaP) nanoparticle (NP) and biomimetic surface functionalization, we created a biomimetic P-NAD + -NP with platelet membrane coating, which enabled an injectable modality that targets IH with excellent biocompatibility. Using human cell primary culture, we demonstrated the benefits of NP-assisted NAD+ repletion in selectively inhibiting the excessive proliferation of aortic SMC, while differentially protecting aortic EC from apoptosis. Moreover, in a rat balloon angioplasty model, a single-dose treatment with intravenously injected P-NAD + -NP immediately post angioplasty not only mitigated IH, but also accelerated the regeneration of EC (re-endothelialization) in vivo in comparison to control groups (i.e., saline, free NAD+ solution, empty CaP-NP). Collectively, our current study provides proof-of-concept evidence supporting the role of targeted NAD+ repletion nanotherapy in managing restenosis and improving reendothelialization.

Interspecific hybridization and cell engineering of lettuce (Lactuca L.)

Lactuca sativa L. is a leafy vegetable crop of the Asteraceae family, widely cultivated throughout the world. The main breeding trends for lettuce include higher yields, better taste quality, earliness, and resistance to abiotic and biotic stressors. Some wild Lactuca spp. have actively been employed by lettuce breeders as donors of resistance to various diseases. Conventional and biotechnological breeding methods are both currently used to develop new lettuce cultivars. This is an overview of the main advances in the production of interspecific Lactuca hybrids, including the use of cell and tissue culture techniques, and genetic engineering. Studying artificial hybridization and natural populations makes it possible to identify evolutionary relationships among various Lactuca spp. Somatic hybridization is an overlooked but promising technology in Lactuca breeding: it allows a breeder to obtain a wider range of variations, and is beyond the strict control by GMO laws. This technique faces problems associated with complicated protoplast regeneration and the loss of reproductive ability in hybrids. Genome-editing methods are more effective and better controllable, but society is still wary of any interference with the plant genome and legally regulates the sale of GM products as food. Thus, researchers are challenged with the task to improve these techniques.

Evaluation of Cross-Linked Actin Networks (CLANs) in Human Trabecular Meshwork Cells and Tissues.

Elevated intraocular pressure (IOP) is a major risk factor for the development and progression of glaucoma, the leading cause of irreversible vision loss and blindness. An overall increase in resistance to aqueous humor outflow causes sustained elevation in IOP. Glaucomatous insults in the aqueous humor outflow pathway, including the trabecular meshwork (TM), precede such chronic physiological changes in IOP. These insults include ultrastructural changes with excessive extracellular matrix deposition and actin cytoskeletal reorganization that leads to pathological stiffening of the ocular tissues. One of the most common cytoskeletal changes associated with TM tissue stiffness in glaucoma is the increased prevalence of cross-linked actin networks (CLANs) in cells of the trabecular meshwork (TM) and lamina cribrosa (LC). In glaucomatous cells, rearrangement of linear actin stress fibers leads to formation of polygonal arrays within the cytoplasm, resembling a geodesic dome-like structure, that we identified as CLANs. In addition to increased amounts of CLANs in POAG TM cells and tissues, we also discovered that glucocorticoid (GC) and TGFβ2 signaling pathways associated with the development of ocular hypertension (OHT) and glaucoma also induced CLANs in the TM. Despite a clear association, we are yet to completely understand the mechanisms involved in CLAN formation and their direct relevance to disease pathology. In this chapter, we will describe methods to identify and characterize CLANs using fluorescent microscopy in primary TM cell cultures, ex vivo perfusion cultured human anterior segments, and in situ in human donor eyes.

Unveiling the Human Brain on a Chip: An Odyssey to Reconstitute Neuronal Ensembles and Explore Plausible Applications in Neuroscience.

The brain is an incredibly complex structure that consists of millions of neural networks. In developmental and cellular neuroscience, probing the highly complex dynamics of the brain remains a challenge. Furthermore, deciphering how several cues can influence neuronal growth and its interactions with different brain cell types (such as astrocytes and microglia) is also a formidable task. Traditional in vitro macroscopic cell culture techniques offer simple and straightforward methods. However, they often fall short of providing insights into the complex phenomena of neuronal network formation and the relevant microenvironments. To circumvent the drawbacks of conventional cell culture methods, recent advancements in the development of microfluidic device-based microplatforms have emerged as promising alternatives. Microfluidic devices enable precise spatiotemporal control over compartmentalized cell cultures. This feature facilitates researchers in reconstituting the intricacies of the neuronal cytoarchitecture within a regulated environment. Therefore, in this review, we focus primarily on modeling neuronal development in a microfluidic device and the various strategies that researchers have adopted to mimic neurogenesis on a chip. Additionally, we have presented an overview of the application of brain-on-chip models for the recapitulation of the blood-brain barrier and neurodegenerative diseases, followed by subsequent high-throughput drug screening. These lab-on-a-chip technologies have tremendous potential to mimic the brain on a chip, providing valuable insights into fundamental brain processes. The brain-on-chip models will also serve as innovative platforms for developing novel neurotherapeutics to address several neurological disorders.

Artemisiae Iwayomogii Herba mitigates excessive neuroinflammation and Aβ accumulation by regulating the pro-inflammatory response and autophagy-lysosomal pathway in microglia in 5xFAD mouse model of Alzheimer's disease.

Alzheimer's disease (AD) presents a growing societal challenge, driven by an aging population. It is characterized by neurodegeneration linked to β-amyloid (Aβ) and tau protein aggregation. Reactive glial cell-mediated neuroinflammation exacerbates disease progression by facilitating the accumulation of Aβ and impairing its clearance, thus highlighting potential therapeutic targets. Aerial parts of Artemisia iwayomogi (AIH), a kind of mugwort, has been consumed as a medicinal herb in East Asia for relieving inflammation-related diseases. Previously, AIH was found to exert potent inhibitory effects on neuroinflammation. This study aimed to examine whether AIH mitigates AD pathogenesis by regulating neuroinflammation and reducing Aβ deposition. AIH treatment to primary mixed glial cultures attenuated the pro-inflammatory responses evoked by Aβ stimulation. When treated to 5 × familial AD (5xFAD) mice, AIH improved learning and cognitive ability and reduced Aβ burden in the brain. AIH suppressed glial overactivation, as well as inhibited the expressions of pro-inflammatory mediators in the brain. Moreover, AIH regulated AKT signaling and elevated the expression of autophagy-lysosomal mediators in vitro. It was confirmed that lysosome-associated membrane protein 1 (LAMP1) was increased in the Aβ-associated microglia in the mouse hippocampus. Finally, it was observed that tau phosphorylation was alleviated, and synaptic protein expression was increased in AIH-treated 5xFAD mice. Overall, this study demonstrated that AIH ameliorated excessive neuroinflammation and Aβ accumulation by regulating microglial activation and autophagy-lysosomal pathway, thereby suggesting AIH as a promising therapeutic candidate for AD treatment.

Metabolic Response to Small Molecule Therapy in Colorectal Cancer Tracked with Raman Spectroscopy and Metabolomics.

Despite numerous screening tools for colorectal cancer (CRC), 25 % of patients are diagnosed with advanced disease. Novel diagnostic technologies that are early, accurate, and rapid are imperative to assess the therapeutic efficacy of clinical drugs and identify new biomarkers of treatment response. Here Raman spectroscopy (RS) was used to track metabolic reprogramming in KRAS-mutant HCT116 and SW837 cells, and KRAS wild-type CC cells. RS combined with multivariate analysis methods distinguished nonresponsive, partially responsive, and responsive cells treated with cetuximab, a monoclonal antibody for EGFR inhibition, sotorasib, a clinically approved KRAS inhibitor, and various doses of trametinib, an inhibitor of the MAPK pathway. Cells treated with a combination of subtoxic doses of trametinib and BKM120, an inhibitor of the PI3K pathway, showed a synergistic response between the two pathways. Using a supervised machine learning regression model, we established a scoring methodology trained to a priori predict therapeutic response to new treatment combinations. RS metabolites were verified with mass spectrometry, and enrichment pathways were identified, including amino acid, purine, and nicotinate and nicotinamide metabolism that differentiated monotherapy from combination therapy. Our approach may ultimately be applicable to patient-derived primary cells and cultures of patient tumors to predict effective drugs for individualized care.

Association between premature vascular smooth muscle cells senescence and vascular inflammation in Takayasu’s arteritis

ObjectivesArterial wall inflammation and remodelling are the characteristic features of Takayasu’s arteritis (TAK). It has been proposed that vascular smooth muscle cells (VSMCs) are the main targeted cells of inflammatory...

Early life cisplatin exposure induces nerve growth factor mediated neuroinflammation and chemotherapy induced neuropathic pain.

Chemotherapy-induced neuropathic pain (CINP) is a common adverse health related comorbidity that manifests later in life in paediatric cancer patients. Current analgesia is ineffective, aligning closely with our lack of understanding of CINP. The aim of this study was to investigate how cisplatin induces nerve growth factor mediated neuroinflammation and nociceptor sensitisation. In a rodent model of cisplatin induced survivorship pain, cisplatin induced a neuroinflammatory environment in the dorsal root ganglia (DRG) demonstrated by nerve growth factor (NGF) positive macrophages infiltrating into the DRG. Cisplatin treated CD11b/F480 positive macrophages expressed more NGF compared to vehicle treated. Primary DRG sensory neuronal cultures demonstrated enhanced NGF-dependent TRPV1 mediated nociceptor activity after cisplatin treatment. Increased nociceptor activity was also observed when cultured DRG neurons were treated with conditioned media from cisplatin activated macrophages. Elevated nociceptor activity was dose-dependently inhibited by a neutralising NGF antibody. Intraperitoneal administration of a NGF neutralising antibody reduced cisplatin-induced mechanical hypersensitivity and aberrant nociceptor intraepidermal nerve fibre density. These findings identify that a monocyte/macrophage driven NGF/TrkA pathway is a novel analgesic target for adult survivors of childhood cancer.

Strain-specific differences in reovirus infection of murine macrophages segregate with polymorphisms in viral outer-capsid protein σ3.

Mammalian orthoreovirus (reovirus) strains type 1 Lang (T1L) and type 3 Dearing-RV (T3D-RV) infect the intestine in mice but differ in the induction of inflammatory responses. T1L infection is associated with the blockade of oral immunological tolerance to newly introduced dietary antigens, whereas T3D-RV is not. T1L infection leads to an increase in infiltrating phagocytes, including macrophages, in gut-associated lymphoid tissues that are not observed in T3D-RV infection. However, the function of macrophages in reovirus intestinal infection is unknown. Using cells sorted from infected intestinal tissue and primary cultures of bone-marrow-derived macrophages (BMDMs), we discovered that T1L infects macrophages more efficiently than T3D-RV. Analysis of T1L × T3D-RV reassortant viruses revealed that the viral S4 gene segment, which encodes outer-capsid protein σ3, is responsible for strain-specific differences in infection of BMDMs. Differences in the binding of T1L and T3D-RV to BMDMs also segregated with the σ3-encoding S4 gene. Paired immunoglobulin-like receptor B (PirB), which serves as a receptor for reovirus, is expressed on macrophages and engages σ3. We found that PirB-specific antibody blocks T1L binding to BMDMs and that T1L binding to PirB-/- BMDMs is significantly diminished. Collectively, our data suggest that reovirus T1L infection of macrophages is dependent on engagement of PirB by viral outer-capsid protein σ3. These findings raise the possibility that macrophages function in the innate immune response to reovirus infection that blocks immunological tolerance to new food antigens.IMPORTANCEMammalian orthoreovirus (reovirus) infects humans throughout their lifespan and has been linked to celiac disease (CeD). CeD is caused by a loss of oral immunological tolerance (LOT) to dietary gluten and leads to intestinal inflammation following gluten ingestion, which worsens with prolonged exposure and can cause malnutrition. There are limited treatment options for CeD. While there are genetic risk factors associated with the illness, triggers for disease onset are not completely understood. Enteric viruses, including reovirus, have been linked to CeD induction. We found that a reovirus strain associated with oral immunological tolerance blockade infects macrophages by virtue of its capacity to bind macrophage receptor PirB. These data contribute to an understanding of the innate immune response elicited by reovirus, which may shed light on how viruses trigger LOT and inform the development of CeD vaccines and therapeutic agents.

Complement C5a Implication in Axonal Growth After Injury.

Complement C5a protein has been shown to play a major role in tissue regeneration through interaction with its receptor (C5aR) on target cells. Expression of this receptor has been reported in the nervous system which, upon injury, has no treatment to restore the lost functions. This work aimed at investigating the Complement C5a effect on axonal growth after axotomy in vitro. Primary hippocampal neurons were isolated from embryonic Wistar rats. Cell expression of C5aR mRNA was verified by RT-PCR while its membrane expression, localization, and phosphorylation were investigated by immunofluorescence. Then, the effects of C5a on injured axonal growth were investigated using a 3D-printed microfluidic device. Immunofluorescence demonstrated that the primary cultures contained only mature neurons (93%) and astrocytes (7%), but no oligodendrocytes or immature neurons. Immunofluorescence revealed a co-localization of NF-L and C5aR only in the mature neurons where C5a induced the phosphorylation of its receptor. C5a application on injured axons in the microfluidic devices significantly increased both the axonal growth speed and length. Our findings highlight a new role of C5a in regeneration demonstrating an enhancement of axonal growth after axotomy. This may provide a future therapeutic tool in the treatment of central nervous system injury.

Nucleophosmin 1 overexpression enhances neuroprotection by attenuating cellular stress in traumatic brain injury

BackgroundTraumatic Brain Injury (TBI) is a multifaceted injury that can cause a wide range of symptoms and impairments, leading to significant effects on brain function. Nucleophosmin 1 (NPM1), a versatile phosphoprotein located in the nucleolus, is being recognized as a possible controller of cellular stress reactions and could be important in reducing neuro dysfunction caused by TBI. However the critical roles of NPM1 in cellular stress in TBI remains unclear. MethodsWe employed a control cortical impact mouse model and a scratch-induced primary neuronal culture model. Hematoxylin and eosin staining was used to evaluate tissue damage and cellular changes, with NPM1 expression in the cortical area assessed through immunofluorescence staining and Western blot analysis. Neuronal morphology was assessed using Nissl staining. Behavioral assessments were performed to evaluate the impact of NPM1 overexpression on neurobehavioral results in TBI mice. Mitochondrial function was assessed using an Extracellular Flux Analyzer. ResultsFollowing TBI, an increase in NPM1 expression was observed, with a peak at 72 h post-injury. Increased levels of NPM1 resulted in decreased neuronal cell death, as shown by Nissl staining, and lower levels of Caspase 8, APE1, H2AX, and 8-OHDG expression, indicating a reduction in DNA damage. NPM1 overexpression also resulted in improved neurobehavioral outcomes, characterized by decreased neurological deficits and enhanced motor function post-TBI. Additionally, in vitro, scratch-induction experiments revealed that NPM1 overexpression mitigated mitochondrial damage, as evidenced by the downregulation of P53, BCL2, and Cyto C expression levels and improvements in mitochondrial respiratory function. ConclusionThese findings suggest NPM1 as a promising target for developing interventions to alleviate TBI-related cellular stress and promote neuronal survival.