Assessment of recombinant protein production in E. coli with Time-Gated Surface Enhanced Raman Spectroscopy (TG-SERS).

Time-Gated Surface-Enhanced Raman spectroscopy (TG-SERS) was utilized to assess recombinant protein production in Escherichia coli. TG-SERS suppressed the fluorescence signal from the biomolecules in the bacteria and the culture media. Characteristic protein signatures at different time points of the cell cultivation were observed and compared to conventional continuous wave (CW)-Raman with SERS. TG-SERS can distinguish discrete features of proteins such as the secondary structures and is therefore indicative of folding or unfolding of the protein. A novel method utilizing nanofibrillar cellulose as a stabilizing agent for nanoparticles and bacterial cells was used for the first time in order to boost the Raman signal, while simultaneously suppressing background signals. We evaluated the expression of hCNTF, hHspA1, and hHsp27 in complex media using the batch fermentation mode. HCNTF was also cultivated using EnBase in a fed-batch like mode. HspA1 expressed poorly due to aggregation problems within the cell, while hCNTF expressed in batch mode was correctly folded and protein instabilities were identified in the EnBase cultivation. Time-gated Raman spectroscopy showed to be a powerful tool to evaluate protein production and correct folding within living E. coli cells during the cultivation.

NGF and CNTF expression and regulation mechanism by miRNA in acute paralytic strabismus.

BACKGROUND: Nerve growth factor (NGF) and ciliary neurotrophic factor (CNTF) are well-known neurotrophic factors and widely used in the clinical treatment for its promotion effect on peripheral nerve regeneration. And they were also recommended for the acute paralytic strabismus treatment. However, whether the NGF and CNTF have protective effect for the extraocular muscles of acute paralytic strabismus patients is still poorly understood. PURPOSE: In this study, we want to evaluate the biological function of NGF and CNTF on the extraocular muscle cells and reveale the regulation mechanism behind it. METHODS: Firstly, the relative expression of ngf and cntf was assessed by quantitative real-time RT-PCR. Then, the influence of NGF and CNTF on the extraocular muscle cell proliferation was determined by CCK-8. The inflammatory response in muscle cells after NGF and CNTF treatment was evaluated by ELISA and ROS detection. In addition to this, the up-stream regulation of the ngf and cntf expression was also studied. The TargetScan was used for the predication of potential miRNAs targeting with ngf and cntf 30-UTR, which is soon confirmed by luciferase activity assay. RESULTS: all the results in this research indicated that NGF and CNTF could promote the muscle cell proliferation and inhibit the inflammatory levels, then exert protective effect on the muscle cell function. RESULTS: All the results in this research indicated that NGF and CNTF could promote the muscle cell proliferation and inhibit the inflammatory levels, then exert protective effect on the muscle cell function. CONCLUSION: It was conceivable that let 7-5p was the up-stream regulator of ngf and cntf, and let 7-5p might serve as a potential molecular target for acute paralytic strabismus treatment.

The Combination of Adipose-derived Schwann-like Cells and Acellular Nerve Allografts Promotes Sciatic Nerve Regeneration and Repair through the JAK2/STAT3 Signaling Pathway in Rats.

Schwann cells (SCs) combined with acellular nerve allografts (ANAs) effectively promote the regeneration and repair of peripheral nerves, but the exact mechanism has not been fully elucidated. However, the disadvantages of SCs include their limited source and slow rate of expansion in vitro. Previous studies have found that adipose-derived stem cells have the ability to differentiate into Schwann-like cells. Therefore, we speculated that Schwann-like cells combined with ANAs could profoundly facilitate nerve regeneration and repair. The aim of the present study was to investigate the cellular and molecular mechanisms of regeneration and repair. In this study, tissue-engineered nerves were first constructed by adipose-derived Schwann-like cells and ANAs to bridge missing sciatic nerves. Then, the rats were randomly divided into five groups (n = 12 per group): a Control group; a Model group; an ADSC group; an SC-L group; and a DMEM group. Twelve weeks postsurgery, behavioral function tests and molecular biological techniques were used to evaluate the function of regenerated nerves and the relevant molecular mechanisms after sciatic nerve injury (SNI). The results showed that adipose-derived Schwann-like cells combined with ANAs markedly promoted sciatic nerve regeneration and repair. These findings also demonstrated that the expression of neurotrophic factors (NFs) was increased, and the expression of Janus activated kinase2 (JAK2)/P-JAK2, signal transducer and activator of transcription-3 (STAT3)/P-STAT3 was decreased in the spinal cord after SNI. Therefore, these results suggested that highly expressed NFs in the spinal cord could promote nerve regeneration and repair by inhibiting activation of the JAK2/STAT3 signaling pathway.

Vitronectin from brain pericytes promotes adult forebrain neurogenesis by stimulating CNTF.

Vitronectin (VTN) is a glycoprotein in the blood and affects hemostasis. VTN is also present in the extracellular matrix of various organs but little is known about its function in healthy adult tissues. We show, in adult mice, that VTN is uniquely expressed by approximately half of the pericytes of subventricular zone (SVZ) where neurogenesis continues throughout life. Intracerebral VTN antibody injection or VTN knockout reduced neurogenesis as well as expression of pro-neurogenic CNTF, and anti-neurogenic LIF and IL-6. Conversely, injections of VTN, or plasma from VTN+/+, but not VTN-/- mice, increased these cytokines. VTN promoted SVZ neurogenesis when LIF and IL-6 were suppressed by co-administration of a gp130 inhibitor. Unexpectedly, VTN inhibited FAK signaling and VTN-/- mice had increased FAK signaling in the SVZ. Further, an FAK inhibitor or VTN increased CNTF expression, but not in conditional astrocytic FAK knockout mice, suggesting that VTN increases CNTF through FAK inhibition in astrocytes. These results identify a novel role of pericyte-derived VTN in the brain, where it regulates SVZ neurogenesis through co-expression of CNTF, LIF and IL-6. VTN-integrin-FAK and gp130 signaling may provide novel targets to induce neurogenesis for cell replacement therapies.

All-trans retinoic acid upregulates the expression of ciliary neurotrophic factor in retinal pigment epithelial cells.

Retinopathy of prematurity, a leading cause of visual impairment in low birth-weight infants, remains a crucial therapeutic challenge. Ciliary neurotrophic factor (CNTF) is a promyelinating trophic factor that promotes rod and cone photoreceptor survival and cone outer segment regeneration in the degenerating retina. Ciliary neurotrophic factor expression is regulated by many factors such as all-trans retinoic acid (ATRA). In this study, we found that ATRA increased CNTF expression in mouse retinal pigment epithelial (RPE) cells in a dose- and time-dependent manner, and PKA signaling pathway is necessary for ATRA-induced CNTF upregulation. Furthermore, we showed that ATRA promoted CNTF expression through CREB binding to its promoter region. In addition, CNTF levels were decreased in serum of retinopathy of prematurity children and in retinal tissue of oxygen-induced retinopathy mice. In mouse RPE cells cultured with high oxygen, CNTF expression and secretion were decreased, but could be recovered after treatment with ATRA. In conclusion, our data suggest that ATRA administration upregulates CNTF expression in RPE cells.

High hydrostatic pressure enables almost 100% refolding of recombinant human ciliary neurotrophic factor from inclusion bodies at high concentration.

Protein refolding from inclusion bodies (IBs) often encounters a problem of low recovery at high protein concentration. In this study, we demonstrated that high hydrostatic pressure (HHP) could simultaneously achieve high refolding concentration and high refolding yield for IBs of recombinant human ciliary neurotrophic factor (rhCNTF), a potential therapeutic for neurodegenerative diseases. The use of dilution refolding obtained 18% recovery at 3 mg/mL, even in the presence of 4 M urea. In contrast, HHP refolding could efficiently increase the recovery up to almost 100% even at 4 mg/mL. It was found that in the dilution, hydrophobic aggregates were the off-path products and their amount increased with the protein concentration. However, HHP could effectively minimize the formation of hydrophobic aggregates, leading to almost complete conversion of the rhCNTF IBs to the correct configuration. The stable operation range of concentration is 0.5-4.0 mg/mL, in which the refolding yield was almost 100%. Compared with the literatures where HHP failed to increase the refolding yield beyond 90%, the reason could be attributed to the structural difference that rhCNTF has no disulfide bond and is a monomeric protein. After purification by one-step of anionic chromatography, the purity of rhCNTF reached 95% with total process recovery of 54.1%. The purified rhCNTF showed similar structure and in vitro bioactivity to the native species. The whole process featured integration of solubilization/refolding, a high refolding yield of 100%, a high concentration of 4 mg/mL, and a simple chromatography to ensure a high productivity.

Protective role of selenium-enriched supplement on spinal cord injury through the up-regulation of CNTF and CNTF-Ralpha.

OBJECTIVE: Spinal cord injury (SCI), as one of the common serious spine disorders, often leads to severe neurological dysfunction and even permanent disability, which will cause heavy economical burden for family and society. Currently, selenium-enriched products have an obvious role in the protection and recovery of SCI; however, its protective mechanism is still unclear. MATERIALS AND METHODS: In order to explore the protective effect of selenium-enriched supplement (SES) on SCI, the adult rats were randomly divided into sham operation control (SC) group, ischemia-reperfusion model (IM) group and SES pretreatment (ST) group to investigate the change of ciliary neurotrophic factor (CNTF) and its receptor-alpha (CNTF-Ralpha) during SCI in the presence of SES. The rats in IM and ST groups were subjected to the blockage of their abdominal aorta to establish the model of SCI; but the rats in SC group were subjected to sham operation without the blockage of abdominal aorta. The rats in ST group were fed with foods containing SES at the dose of equivalent 5 mg/L selenium in water before blocking their abdominal aorta. After 7 days, the rats were sacrificed to observe the structure of nerve cells through HE staining and the expression of CNTF and CNTF-Ralpha by Western blot, immunohistochemical, and RT-PCR methods, respectively. RESULTS: Both protein and mRNA of CNTF and CNTF-Ralpha were positively expressed in rats from SC group. The mRNA expression levels of CNTF and CNTF-Ralpha in ST group were much higher than SCI model group. CONCLUSIONS: SES can execute a protective role in SCI through up-regulating the expression of CNTF and CNTF-Ralpha.

Sodium Benzoate, a Metabolite of Cinnamon and a Food Additive, Upregulates Ciliary Neurotrophic Factor in Astrocytes and Oligodendrocytes.

Ciliary neurotrophic factor (CNTF) is a promyelinating trophic factor that plays an important role in multiple sclerosis (MS). However, mechanisms by which CNTF expression could be increased in the brain are poorly understood. Recently we have discovered anti-inflammatory and immunomodulatory activities of sodium benzoate (NaB), a metabolite of cinnamon and a widely-used food additive. Here, we delineate that NaB is also capable of increasing the mRNA and protein expression of CNTF in primary mouse astrocytes and oligodendrocytes and primary human astrocytes. Accordingly, oral administration of NaB and cinnamon led to the upregulation of astroglial and oligodendroglial CNTF in vivo in mouse brain. Induction of experimental allergic encephalomyelitis, an animal model of MS, reduced the level of CNTF in the brain, which was restored by oral administration of cinnamon. While investigating underlying mechanisms, we observed that NaB induced the activation of protein kinase A (PKA) and H-89, an inhibitor of PKA, abrogated NaB-induced expression of CNTF. The activation of cAMP response element binding (CREB) protein by NaB, the recruitment of CREB and CREB-binding protein to the CNTF promoter by NaB and the abrogation of NaB-induced expression of CNTF in astrocytes by siRNA knockdown of CREB suggest that NaB increases the expression of CNTF via the activation of CREB. These results highlight a novel myelinogenic property of NaB and cinnamon, which may be of benefit for MS and other demyelinating disorders.

Human ciliary neurotrophic factor-overexpressing stable bone marrow stromal cells in the treatment of a rat model of traumatic spinal cord injury.

BACKGROUND AIMS: Traumatic injury to the central nervous system (CNS) often causes motor dysfunctions. However, because of the CNS complexity and variability in the clinical presentations, efforts to repair damaged CNS tissue and restoring its functions are particularly demanding. On the other hand, recent progress in the regenerative therapy field have led to novel approaches for the treatment of traumatic CNS injury and renewed hopes to overcome the obstacles. It appears that the balance between neurite re-growth-inhibiting and neurite re-growth-inducing molecules determines the axonal re-growth fate. Neurotrophic factors can tilt this balance and indeed promote cell survival and axonal re-growth over neurodegeneration. One of the promising neurotrophic factors in this field is ciliary neurotrophic factor (CNTF). METHODS: We transfected rat bone marrow stromal cells with a mammalian expression vector-inserted human CNTF gene through the use of a non-viral method to prepare human CNTF-overexpressing stem cells under ex vivo conditions. We transplanted these modified cells to the rat model of spinal cord traumatic injury to explore functional recovery after contusion induction. RESULTS: Our data from immunocytochemistry and behavioral tests showed that such cells can act as a powerful potential approach to treat traumatic CNS injuries because these modified cells improved the behavioral test scores in the rat model of spinal cord injury. CONCLUSIONS: CNTF-overexpressing bone marrow stromal cells can ameliorate spinal cord traumatic injury and can be used in the treatment of traumatic CNS injuries in the near future.

1,25-Dihydroxyvitamin D3 enhances neural stem cell proliferation and oligodendrocyte differentiation.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) has recently been found to suppress experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Although its effect was attributed to an anti-inflammatory mechanism, it is not clear whether this treatment can also directly act on neural cells to promote CNS recovery. The present study investigates the effect of various concentrations of 1,25(OH)2D3 on neural stem cell (NSC) proliferation and their differentiation to oligodendrocytes, the myelinating cells. We have, for the first time, shown that NSCs constitutively express vitamin D receptor (VDR), which can be upregulated by 1,25(OH)2D3. This vitamin significantly enhanced proliferation of NSCs, and enhanced their differentiation into neurons and oligodendrocytes, but not astrocytes. NSCs treated with 1,25(OH)2D3 showed increased expression of NT-3, BDNF, GDNF and CNTF, important neurotrophic factors for neural cell survival and differentiation. Overall, we demonstrated that 1,25(OH)2D3 has a direct effect on NSC proliferation, survival, and neuron/oligodendrocyte differentiation, thus representing a novel mechanism underlying its remyelinating and neuroprotective effect in MS/EAE therapy.

High level soluble expression, purification, and characterization of human ciliary neuronotrophic factor in Escherichia coli by single protein production system.

Ciliary neurotrophic factor (CNTF) is characterized as a neuropoietic cytokine for a broad spectrum of neurons, leading to its evaluation in humans suffering from neurodegenerative diseases. Due to its wide range of biological applications, high yield production of soluble biologically active recombinant human CNTF (rhCNTF) in heterologous expression system is demanded. Many attempts had been undertaken to product rhCNTF in Escherichia coli (E. coli), however, the expression level of rhCNTF was low and most of which formed insoluble inclusion bodies. In this study, we described a new and efficient method to express rhCNTF. The human CNTF gene was codon optimized and then expressed by the single protein production (SPP) expression system in E. coli. The results showed that rhCNTF was expressed as a soluble biologically active protein, and upon purification, the final yield was about 250 mg/L in shake flask with a specific neuroprotective activity in Aß-induced SH-SY5Y cell injury model. Our study might open up a new strategy for large-scale production of functional rhCNTF for clinical applications as well as basic research.

P2X7 receptor inhibition increases CNTF in the subventricular zone, but not neurogenesis or neuroprotection after stroke in adult mice.

Increasing endogenous ciliary neurotrophic factor (CNTF) expression with a pharmacological agent might be beneficial after stroke as CNTF both promotes neurogenesis and, separately, is neuroprotective. P2X7 purinergic receptor inhibition is neuroprotective in rats and increases CNTF release in rat CMT1A Schwann cells. We, first, investigated the role of P2X7 in regulating CNTF and neurogenesis in adult mouse subventricular zone (SVZ). CNTF expression was increased by daily intravenous injections of the P2X7 antagonist Brilliant Blue G (BBG) in naïve C57BL/6 or Balb/c mice over 3 days. Despite the ∼40-60 % increase or decrease in CNTF with BBG or the agonist BzATP, respectively, the number of proliferated BrdU+SVZ nuclei did not change. BBG failed to increase FGF2, which is involved in CNTF-regulated neurogenesis, but induced IL-6, LIF, and EGF, which are known to reduce SVZ proliferation. Injections of IL-6 next to the SVZ induced CNTF and FGF2, but not proliferation, suggesting that IL-6 counteracts their neurogenesis-inducing effects. Following ischemic injury of the striatum by middle cerebral artery occlusion (MCAO), a 3-day BBG treatment increased CNTF in the medial penumbra containing the SVZ. BBG also induced CNTF and LIF, which are known to be protective following stroke, in the whole striatum after MCAO, but not GDNF or BDNF. However, BBG treatment did not reduce the lesion area or apoptosis in the penumbra. Even so, this study shows that P2X7 can be targeted with systemic drug treatments to differentially regulate neurotrophic factors in the brain following stroke.

Minocycline reduces remyelination by suppressing ciliary neurotrophic factor expression after cuprizone-induced demyelination.

Remyelination is disrupted in demyelinating diseases such as multiple sclerosis, but the underlying pathogenetic mechanisms are unclear. In this study, we employed the murine cuprizone model of demyelination, in which remyelination occurs after removal of the toxin from the diet, to examine the cellular and molecular changes during demyelination and remyelination. Microglia accumulated in the corpus callosum during weeks 2-4 of the cuprizone diet, and these cells remained activated 2 weeks after the change to the normal diet. To examine the role of microglia in remyelination, mice were treated with minocycline to inactivate these cells after cuprizone-induced demyelination. Minocycline treatment reduced the number of CC1-positive oligodendrocytes, as well as levels of myelin basic protein (MBP) and CNPase in the remyelination phase. The expression of CNTF mRNA in the corpus callosum increased after 4 weeks on the cuprizone diet and remained high 2 weeks after the change to the normal diet. Minocycline suppressed CNTF expression during the remyelination phase on the normal diet. Primary culture experiments showed that CNTF was produced by microglia in addition to astrocytes. In vitro, CNTF directly affected the differentiation of oligodendrocytic cells. These findings suggest that minocycline reduces remyelination by suppressing CNTF expression by microglia after cuprizone-induced demyelination.

The effect of methylprednisolone intravenous infusion on the expression of ciliary neurotrophic factor in a rat spinal cord injury model.

BACKGROUND CONTEXT: Methylprednisolone (MP) infusion after acute spinal cord injury (SCI) remains controversial despite large randomized studies, including the National Acute Spinal Cord Injury Studies (NASCIS). PURPOSE: To determine the effect of NASCIS protocol MP infusion on the expression of ciliary neurotrophic factor (CNTF), a neuroprotective cytokine, in a rat model after SCI. STUDY DESIGN: Animal laboratory study. METHODS: Thirty rats were randomized into an MP infusion group (intravenous [IV]-MP) versus normal saline (NS) control group (IV-NS) after a standardized SCI. Ciliary neurotrophic factor expression was measured by reverse transcription-polymerase chain reaction at 6, 12, 24, 48, and 72 hours post-SCI. RESULTS: Mean CNTF expression was diminished in the MP group at 12 (p=.006) and 24 (p=.008) hours postinjury compared with the control group. Expression of CNTF was not significantly different between the groups at 6, 48, and 72 hours post-SCI. CONCLUSIONS: Standardized MP infusion post-SCI reduces CNTF activation in a rat SCI model. Further study is needed to determine if this effect is seen in human SCIs.

S-nitrosoglutathione induces ciliary neurotrophic factor expression in astrocytes, which has implications to protect the central nervous system under pathological conditions.

Accumulating evidence suggests that reactive astrogliosis has beneficial and detrimental outcomes in various CNS disorders, but the mechanism behind this dichotomy is unclear. Recent advances in this direction suggested that NO signaling is critical to regulate the outcomes of reactive astrogliosis in vivo. Using biochemical and genetic approaches, we here investigated the effect of S-nitrosoglutathione (GSNO; a physiological NO donor) in astrocytes in vitro settings. GSNO enhanced the expressions of glial fibrillary acidic protein and neurotrophic factors including ciliary neurotrophic factor (CNTF) in astrocytes in a dose-dependent manner. The enhanced CNTF expression in GSNO-treated astrocytes was ascribed to NO-mediated sGC/cGMP/PKG signaling. It was associated with p38 MAPK-dependent increased peroxisome proliferator-activated receptor-γ transactivation. In addition, the chromatin accessibility of peroxisome proliferator-activated receptor-γ accompanied with ATF2 and CREB (cAMP-response element-binding protein) was enhanced across the CNTF gene promoter in GSNO treated astrocytes. Interestingly, secreted CNTF was responsible for increased expression of glial fibrillary acidic protein in GSNO-treated astrocytes in an autocrine manner via a JAK2- and STAT3-dependent mechanism. In addition, CNTF secreted by GSNO-treated astrocytes enhanced the differentiation of immature oligodendrocytes in vitro. These effects of GSNO were consistent with an endogenously produced NO in astrocytes stimulated with proinflammatory cytokines in vitro. We conclude that NO signaling induces CNTF expression in astrocytes that favors the beneficial outcomes of reactive astrogliosis in vivo. Our data suggest that the endogenously produced NO or its exogenous source has potential to modulate the outcomes of reactive astrogliosis to protect CNS under pathological conditions.

Exogenous ciliary neurotrophic factor (CNTF) reduces synaptic depression during repetitive stimulation.

It has been shown that ciliary neurotrophic factor (CNTF) has trophic and maintenance effects on several types of peripheral and central neurons, glia, and cells outside the nervous system. Both CNTF and its receptor, CNTF-Rα, are expressed in the muscle. We use confocal immunocytochemistry to show that the trophic cytokine and its receptor are present in the pre- and post-synaptic sites of the neuromuscular junctions (NMJs). Applied CNTF (7.5-200 ng/ml, 60 min-3 h) does not acutely affect spontaneous potentials (size or frequency) or quantal content of the evoked acetylcholine release from post-natal (in weak or strong axonal inputs on dually innervated end plates or in the most mature singly innervated synapses at P6) or adult (P30) NMJ of Levator auris longus muscle of the mice. However, CNTF reduces roughly 50% the depression produced by repetitive stimulation (40 Hz, 2 min) on the adult NMJs. Our findings indicate that, unlike neurotrophins, exogenous CNTF does not acutely modulate transmitter release locally at the mammalian neuromuscular synapse but can protect mature end plates from activity-induced synaptic depression.

Loss of neuron-astroglial interaction rapidly induces protective CNTF expression after stroke in mice.

Ciliary neurotrophic factor (CNTF) is a potent neural cytokine with very low expression in the CNS, predominantly by astrocytes. CNTF increases rapidly and greatly following traumatic or ischemic injury. Understanding the underlying mechanisms would help to design pharmacological treatments to increase endogenous CNTF levels for neuroprotection. Here, we show that astroglial CNTF expression in the adult mouse striatum is increased twofold within 1 h and increases up to >30-fold over 2 weeks following a focal stroke caused by a transient middle cerebral artery occlusion (MCAO). Selective neuronal loss caused by intrastriatal injection of quinolinic acid resulted in a comparable increase. Cocultured neurons reduced CNTF expression in astrocytes, which was prevented by light trypsinization. RGD (arginine-glycine-aspartic acid) blocking peptides induced CNTF expression, which was dependent on transcription. Astroglial CNTF expression was not affected by diffusible neuronal molecules or by neurotransmitters. The transient ischemia does not seem to directly increase CNTF, as intrastriatal injection of an ischemic solution or exposure of naive mice or cultured cells to severe hypoxia had minimal effects. Inflammatory mechanisms were probably also not involved, as intrastriatal injection of proinflammatory cytokines (IFNγ, IL6) in naive mice had no or small effects, and anti-inflammatory treatments did not diminish the increase in CNTF after MCAO. CNTF-/- mice had more extensive tissue loss and similar astrocyte activation after MCAO than their wild-type littermates. These data suggest that contact-mediated integrin signaling between neurons and astrocytes normally represses CNTF expression and that neuronal dysfunction causes a rapid protective response by the CNS.

Adult rat mesenchymal stem cells delay denervated muscle atrophy.

To evaluate the function of rat mesenchymal stem cells (rMSCs) on denervated gastrocnemius muscles and to address the role of ciliary neurotrophic factor (CNTF) in rMSCs, denervated Wistar rats were separately injected with culture media (sham control), CNTF protein, 2.5 × 10(5) siCNTF-treated rMSCs, 2.5 × 10(5) GFP-transfected rMSCs, or 2.5 × 10(5) untreated rMSCs. Muscle function was assessed at different time points post-surgery. Tibial nerve and gastrocnemius muscle samples were taken at 4, 8, and 12 weeks for histochemistry, and neuromuscular junction repair was also examined by electron microscopy. Fluorescence immunocytochemistry on tissue sections confirmed neurotrophin expression in rMSCs but with little evidence of neuronal differentiation. The engraftment of rMSCs significantly preserved the function of denervated gastrocnemius muscle based both on evaluation of muscle function and direct examination of muscle tissue. Further, the density and depth of the junctional folds were visibly reduced 12 weeks after surgery and transplantation, especially in control group. Knockdown of CNTF expression in rMSCs failed to block muscle preservation, although administration of CNTF protein alone inhibited muscle atrophy, which indicating that delivery of rMSCs could preserve gastrocnemius muscle function following denervation and post-junctional mechanisms involved in the repairing capability of rMSCs.

Increased expression of IL-6 family members in tendon pathology.

OBJECTIVES: Histological examination of pathological tendon generally does not reveal signs of inflammation. However, the inflammatory cytokine IL-6 has been shown to be expressed in ruptured rotator cuff tendon. The aim of this study was to investigate the expression of IL-6 family members in painful posterior tibialis tendon (PTT) and in painful and ruptured Achilles tendon (AT) compared with normal tendon. METHODS: AT samples were obtained from cadavers (normal) or from patients undergoing surgical procedures to treat chronic painful tendinopathy or ruptured tendon. PTT samples were obtained from patients undergoing surgery for other reasons (normal) and from patients with PTT dysfunction (painful). Total RNA was extracted and mRNA expression was analysed by quantitative real-time PCR. RESULTS: Collagen type I α-chain I (COL1A1) expression was increased in both painful PTT and AT compared with normal. Ciliary neurotrophic factor levels were increased in painful PTT only. In the painful AT, cyclooxygenase-2 (COX2) and IL-6 expression increased compared with normal. In the ruptured AT, levels of VEGF A, COX2, oncostatin-M, leukaemia inhibitory factor and IL-6 expression were higher compared with both normal and painful AT. IL-6R expression decreased in both painful and ruptured AT compared with normal. CONCLUSION: Painful AT and PTT show different expression patterns, indicating a substantial difference between those two tendinopathies. Inflammatory markers are up-regulated in painful and particularly in ruptured AT, pointing towards a role of inflammation not only in rupture healing, but also in Achilles tendinopathy.

Non-virally modified human mesenchymal stem cells produce ciliary neurotrophic factor in biodegradable fibrin-based 3D scaffolds.

We report the adaptation of dendrimer-based nonviral expression system for ciliary neurotrophic factor (CNTF) overproduction in human mesenchymal stem cells (hMSCs) embedded into fibrin-based three-dimensional (3D) matrix. Time-restricted neurotrophin expression enables autologous adult stem cells for additional trophic support and increases their therapeutic potential in neuroregeneration applications. Polyamidoamine (PAMAM)-NH(2) dendrimers of fourth generation effectively provided virus-free delivery and expression of CNTF-internal ribosome entry site-green fluorescent protein cassette with a transfection efficiency in hMSCs over 11%. CNTF levels in transfected cultures were 10-fold higher as compared with the control cells. Dendrimer-driven CNTF expression also persisted in hMSCs embedded into fibrin-based 3D matrix, an emerging vehicle for cell delivery or bioartificial organ formation. Nonviral modification of autologous adult stem cells with use of dendrimers is a novel tool perspective in terms of biosafety and technological availability.