The gastrointestinal tract has extensive, surgically accessible nerve connections with the central nervous system. This provides the opportunity to exploit rapidly advancing methods of nerve ...stimulation to treat gastrointestinal disorders. Bioelectric neuromodulation technology has considerably advanced in the past decade, but sacral nerve stimulation for faecal incontinence currently remains the only neuromodulation protocol in general use for a gastrointestinal disorder. Treatment of other conditions, such as IBD, obesity, nausea and gastroparesis, has had variable success. That nerves modulate inflammation in the intestine is well established, but the anti-inflammatory effects of vagal nerve stimulation have only recently been discovered, and positive effects of this approach were seen in only some patients with Crohn's disease in a single trial. Pulses of high-frequency current applied to the vagus nerve have been used to block signalling from the stomach to the brain to reduce appetite with variable outcomes. Bioelectric neuromodulation has also been investigated for postoperative ileus, gastroparesis symptoms and constipation in animal models and some clinical trials. The clinical success of this bioelectric neuromodulation therapy might be enhanced through better knowledge of the targeted nerve pathways and their physiological and pathophysiological roles, optimizing stimulation protocols and determining which patients benefit most from this therapy.
The emergence of COVID-19 as a global pandemic had sharply illustrated the limitations of research and development pipelines and scaled manufacturing. Although existing vaccines were created in ...record time, global deployment remains limited by regional production scales. Similarly, the most effective treatments for infected COVID-19 patients are also constrained by production scales as well as by the cost of production and thus expense per treatment. The need to produce these interventions more cost-effectively, at larger scales, in less time while retaining high quality is paramount. The ConamaxTM platform is based on a Thraustochytrid-an order of microorganisms well established in industry for world-scale production of omega-3 fatty acids by fermentation. Thraustochytrids, and the species Aurantiochytrium acetophilum in particular, possess a number of innate qualities which make it ideal for production of monoclonal antibodies and other biotherapeutic proteins. In this study, the Conamax system was used to produce several targets which may be relevant as interventions in the fight against COVID-19; an anti-SARS-CoV-2 antibody (CR3022), tocilizumab, and the ACE2 receptor. Our system was capable of producing all of these targets and each was assayed in vitro for an activity which confirmed proper structural folding. Purified CR3022 antibody produced from Conamax was capable of reducing the cytopathic effect of SARS-CoV-2. Conamax-derived tocilizumab was shown to bind to its target IL6R. Both the full-length and soluble versions of ACE2 protein produced in the Conamax platform exhibited ACE2-specific proteolytic activity. These data indicate that the Conamax platform has great potential in the production of therapeutic agents.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A fast-growing field of neuroscience and medicine is the treatment of disease via electrical stimulation of the peripheral nervous system. Peripheral nerve stimulation delivers stimulation to nerves ...of the periphery where the target nerve can and is often located deep within the abdomen. Long-term preclinical animal models that demonstrate the safety and/or efficacy of electrical stimulation have predominantly used a skull mount to connect to neural interfaces. When targeting nerves of the extremities and abdomen, this mount location is less favourable due to its distance to the implant causing complications in surgery and to the longevity of the device in vivo . Objective: Here we aimed to develop and validate a chronic magnetic percutaneous connector designed for placement on the dorsal-lumbar aspect of the spine of awake, freely moving rats. Methods: A pedestal and external connector was developed, bench tested to assess for continuity, durability and disconnection forces, and validated in awake rats chronically implanted with an abdominal vagus nerve electrode array. The implanted pedestal and external connector were designed with custom PCBs, spring-loaded pins, magnets and biocompatible 3D printed housing. Results: The magnetic coupling mechanism allowed disconnection with minimal force, was highly reliable in maintaining electrical connection in awake rats and allowed recording of electrically evoked compound action potentials after chronic implantation. Conclusion: In conclusion, this percutaneous connector is a useful research tool for peripheral nerve stimulation studies. Significance: The connector described will allow investigation into the safety and efficacy of emerging neuromodulation therapies for the treatment of disease.
Abdominal vagus nerve stimulation (VNS) can be applied to the subdiaphragmatic branch of the vagus nerve of rats. Due to its anatomical location, it does not have any respiratory and cardiac ...off-target effects commonly associated with cervical VNS. The lack of respiratory and cardiac off-target effects means that the intensity of stimulation does not need to be lowered to reduce side effects commonly experienced during cervical VNS. Few recent studies demonstrate the anti-inflammatory effects of abdominal VNS in rat models of inflammatory bowel disease, rheumatoid arthritis, and glycemia reduction in a rat model of type 2 diabetes. Rat is a great model to explore the potential of this technology because of the well-established anatomy of the vagus nerve, the large size of the nerve that allows easy handling, and the availability of many disease models. Here, we describe the methods for cleaning and sterilizing the abdominal VNS electrode array and surgical protocol in rats. We also describe the technology required for confirmation of suprathreshold stimulation by recording evoked compound action potentials. Abdominal VNS has the potential to offer selective, effective treatment for a variety of conditions, including inflammatory diseases, and the application is expected to expand similarly to cervical VNS.
Electrical stimulation of the cervical vagus nerve is an emerging treatment for inflammatory bowel disease (IBD). However, side effects from cervical vagal nerve stimulation (VNS) are often reported ...by patients. Here we hypothesized that stimulating the vagus nerve closer to the end organ will have fewer off-target effects and will effectively reduce intestinal inflammation. Specifically, we aimed to: (i) compare off-target effects during abdominal and cervical VNS; (ii) verify that VNS levels were suprathreshold; and (iii) determine whether abdominal VNS reduces chemically-induced intestinal inflammation in rats. An electrode array was developed in-house to stimulate and record vagal neural responses. In a non-recovery experiment, stimulation-induced off-target effects were measured by implanting the cervical and abdominal vagus nerves of anaesthetized rats (
= 5) and recording changes to heart rate, respiration and blood pressure during stimulation (10 Hz; symmetric biphasic current pulse; 320 nC per phase). In a chronic experiment, the efficacy of VNS treatment was assessed by implanting an electrode array onto the abdominal vagus nerve and recording
electrically-evoked neural responses during the implantation period. After 14 days, the intestine was inflamed with TNBS (2.5% 2,4,6-trinitrobenzene sulphonic acid) and rats received therapeutic VNS (
= 7; 10 Hz; 320 nC per phase; 3 h/day) or no stimulation (
= 8) for 4.5 days. Stool quality, plasma C-reactive protein and histology of the inflamed intestine were assessed. Data show that abdominal VNS had no effect (two-way RM-ANOVA:
≥ 0.05) on cardiac, respiratory and blood pressure parameters. However, during cervical VNS heart rate decreased by 31 ± 9 beats/minute (
≥ 0.05), respiration was inhibited and blood pressure decreased. Data addressing efficacy of VNS treatment show that electrically-evoked neural response thresholds remained stable (one-way RM ANOVA:
≥ 0.05) and therapeutic stimulation remained above threshold. Chronically stimulated rats, compared to unstimulated rats, had improved stool quality (two-way RM ANOVA:
< 0.0001), no blood in feces (
< 0.0001), reduced plasma C-reactive protein (two-way RM ANOVA:
< 0.05) and a reduction in resident inflammatory cell populations within the intestine (Kruskal-Wallis:
< 0.05). In conclusion, abdominal VNS did not evoke off-target effects, is an effective treatment of TNBS-induced inflammation, and may be an effective treatment of IBD in humans.
Rheumatoid arthritis (RA) is a chronic, autoimmune inflammatory disease. Despite therapeutic advances, a significant proportion of RA patients are resistant to pharmacological treatment. Stimulation ...of the cervical vagus nerve is a promising alternative bioelectric neuromodulation therapeutic approach. However, recent clinical trials show cervical vagus nerve stimulation (VNS) was not effective in a significant proportion of drug resistant RA patients. Here we aim to assess if abdominal vagus nerve stimulation reduces disease severity in a collagen-induced arthritis (CIA) rat model. The abdominal vagus nerve of female Dark Agouti rats was implanted and CIA induced using collagen type II injection. VNS (1.6 mA, 200 μs pulse width, 50 μs interphase gap, 27 Hz frequency) was applied to awake freely moving rats for 3 h/day (days 11-17). At 17 days following the collagen injection, unstimulated CIA rats (
= 8) had significantly worse disease activity index, tumor necrosis factor-alpha (TNF-α) and receptor activator of NFκB ligand (RANKL) levels, synovitis and cartilage damage than normal rats (
= 8, Kruskal-Wallis:
< 0.05). However, stimulated CIA rats (
= 5-6) had significantly decreased inflammatory scores and ankle swelling (Kruskal-Wallis:
< 0.05) compared to unstimulated CIA rats (
= 8). Levels of tumor necrosis factor-alpha (TNF-α) remained at undetectable levels in stimulated CIA rats while levels of receptor activator of NFκB ligand (RANKL) were significantly less in stimulated CIA rats compared to unstimulated CIA rats (
< 0.05). Histopathological score of inflammation and cartilage loss in stimulated CIA rats were no different from that of normal (
> 0.05). In conclusion, abdominal VNS alleviates CIA and could be a promising therapy for patients with RA.
Partial injury to the central nervous system (CNS) is exacerbated by additional loss of neurons and glia via toxic events known as secondary degeneration. Using partial transection of the rat optic ...nerve (ON) as a model, we have previously shown that myelin decompaction persists during secondary degeneration. Failure to repair myelin abnormalities during secondary degeneration may be attributed to insufficient OPC proliferation and/or differentiation to compensate for loss of oligodendrocyte lineage cells (oligodendroglia). Following partial ON transection, we found that sub-populations of oligodendroglia and other olig2+ glia were differentially influenced by injury. A high proportion of NG2+/olig2-, NG2+/olig2+ and CC1-/olig2+ cells proliferated (Ki67+) at 3 days, prior to the onset of death (TUNEL+) at 7 days, suggesting injury-related cues triggered proliferation rather than early loss of oligodendroglia. Despite this, a high proportion (20%) of the NG2+/olig2+ OPCs were TUNEL+ at 3 months, and numbers remained chronically lower, indicating that proliferation of these cells was insufficient to maintain population numbers. There was significant death of NG2+/olig2- and NG2-/olig2+ cells at 7 days, however population densities remained stable, suggesting proliferation was sufficient to sustain cell numbers. Relatively few TUNEL+/CC1+ cells were detected at 7 days, and no change in density indicated that mature CC1+ oligodendrocytes were resistant to secondary degeneration in vivo. Mature CC1+/olig2- oligodendrocyte density increased at 3 days, reflecting early oligogenesis, while the appearance of shortened myelin internodes at 3 months suggested remyelination. Taken together, chronic OPC decreases may contribute to the persistent myelin abnormalities and functional loss seen in ON during secondary degeneration.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Throughout pregnancy, some degree of insulin resistance is necessary to divert glucose towards the developing foetus. In gestational diabetes mellitus (GDM), insulin resistance is exacerbated in ...combination with insulin deficiency, causing new-onset maternal hyperglycaemia. The rapid reversal of insulin resistance following delivery strongly implicates the placenta in GDM pathogenesis. In this case-control study, we investigated the proteomic cargo of human syncytiotrophoblast-derived extracellular vesicles (STBEVs), which facilitate maternal-fetal signalling during pregnancy, in a UK-based cohort comprising patients with a gestational age of 38-40 weeks. Medium/large (m/l) and small (s) STBEVs were isolated from GDM (n = 4) and normal (n = 5) placentae using ex vivo dual-lobe perfusion and subjected to mass spectrometry. Bioinformatics were used to identify differentially carried proteins and mechanistic pathways. In m/lSTBEVs, 56 proteins were differently expressed while in sSTBEVs, no proteins reached statistical difference. Differences were also observed in the proteomic cargo between m/lSTBEVs and sSTBEVs, indicating that the two subtypes of STBEVs may have divergent modes of action and downstream effects. In silico functional enrichment analysis of differentially expressed proteins in m/lSTBEVs from GDM and normal pregnancy found positive regulation of cytoskeleton organisation as the most significantly enriched biological process. This work presents the first comparison of two populations of STBEVs' protein cargos (m/l and sSTBEVs) from GDM and normal pregnancy isolated using placenta perfusion. Further investigation of differentially expressed proteins may contribute to an understanding of GDM pathogenesis and the development of novel diagnostic and therapeutic tools.
Venous endothelial cells are molecularly and functionally distinct from their arterial counterparts. Although veins are often considered the default endothelial state, genetic manipulations can ...modulate both acquisition and loss of venous fate, suggesting that venous identity is the result of active transcriptional regulation. However, little is known about this process. Here we show that BMP signalling controls venous identity via the ALK3/BMPR1A receptor and SMAD1/SMAD5. Perturbations to TGF-β and BMP signalling in mice and zebrafish result in aberrant vein formation and loss of expression of the venous-specific gene Ephb4, with no effect on arterial identity. Analysis of a venous endothelium-specific enhancer for Ephb4 shows enriched binding of SMAD1/5 and a requirement for SMAD binding motifs. Further, our results demonstrate that BMP/SMAD-mediated Ephb4 expression requires the venous-enriched BMP type I receptor ALK3/BMPR1A. Together, our analysis demonstrates a requirement for BMP signalling in the establishment of Ephb4 expression and the venous vasculature.
Despite advancements in pharmacotherapies, glycemia is poorly controlled in type 2 diabetic patients. As the vagus nerve regulates energy metabolism, here we evaluated the effect various electrical ...vagus nerve stimulation strategies have on glycemia and glucose‐regulating hormones, as a first step to developing a novel therapy of type 2 diabetes. Sprague–Dawley rats were anesthetized, the abdominal (anterior) vagus nerve implanted, and various stimulation strategies applied to the nerve: (a) 15 Hz; (b) 4 kHz, or 40 kHz and; (c) a combination of 15 Hz and 40 kHz to directionally activate afferent or efferent vagal fibers. Following a glucose bolus (500 mg/kg, I.V.), stimulation strategies were applied (60 min) and serial blood samples taken. No stimulation was used as a crossover control sequence. Applying 15 Hz stimulation significantly increased glucose (+2.9 ± 0.2 mM·hr, p = .015) and glucagon (+17.1 ± 8.0 pg·hr/ml, p = .022), compared to no stimulation. Application of 4 kHz stimulation also significantly increased glucose levels (+1.5 ± 0.5 mM·hr, p = .049), while 40 kHz frequency stimulation resulted in no changes to glucose levels but did significantly lower glucagon (−12.3 ± 1.1 pg·hr/ml, p = .0009). Directional afferent stimulation increased glucose (+2.4 ± 1.5 mM·hr) and glucagon levels (+39.5 ± 15.0 pg·hr/ml). Despite hyperglycemia resulting when VNS, aVNS, and 4 kHz stimulation strategies were applied, the changes in insulin levels were not significant (p ≥ .05). In summary, vagus nerve stimulation modulates glycemia by effecting glucagon and insulin secretions, and high‐frequency 40 kHz stimulation may have potential application for the treatment of type 2 diabetes.
Blood glucose can be controlled by electrically stimulating the vagus nerve. This approach could be a promising therapy of type 2 diabetes.
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