Altered Brain Expression of DNA Methylation and Hydroxymethylation Epigenetic Enzymes in a Rat Model of Neuropathic Pain.

The role of epigenetics in chronic pain at the supraspinal level is yet to be fully characterized. DNA histone methylation is crucially regulated by de novo methyltransferases (DNMT1-3) and ten-eleven translocation dioxygenases (TET1-3). Evidence has shown that methylation markers are altered in different CNS regions related to nociception, namely the dorsal root ganglia, the spinal cord, and different brain areas. Decreased global methylation was found in the DRG, the prefrontal cortex, and the amygdala, which was associated with decreased DNMT1/3a expression. In contrast, increased methylation levels and mRNA levels of TET1 and TET3 were linked to augmented pain hypersensitivity and allodynia in inflammatory and neuropathic pain models. Since epigenetic mechanisms may be responsible for the regulation and coordination of various transcriptional modifications described in chronic pain states, with this study, we aimed to evaluate the functional role of TET1-3 and DNMT1/3a genes in neuropathic pain in several brain areas. In a spared nerve injury rat model of neuropathic pain, 21 days after surgery, we found increased TET1 expression in the medial prefrontal cortex and decreased expression in the caudate-putamen and the amygdala; TET2 was upregulated in the medial thalamus; TET3 mRNA levels were reduced in the medial prefrontal cortex and the caudate-putamen; and DNMT1 was downregulated in the caudate-putamen and the medial thalamus. No statistically significant changes in expression were observed with DNMT3a. Our results suggest a complex functional role for these genes in different brain areas in the context of neuropathic pain. The notion of DNA methylation and hydroxymethylation being cell-type specific and not tissue specific, as well as the possibility of chronologically differential gene expression after the establishment of neuropathic or inflammatory pain models, ought to be addressed in future studies.

Umbilical cord tissue-derived mesenchymal stromal cells maintain immunomodulatory and angiogenic potencies after cryopreservation and subsequent thawing.

BACKGROUND AIMS: The effect of cryopreservation on mesenchymal stromal cell (MSC) therapeutic properties has become highly controversial. However, data thus far have indiscriminately involved the assessment of different types of MSCs with distinct production processes. This study assumed that MSC-based products are affected differently depending on the tissue source and manufacturing process and analyzed the effect of cryopreservation on a specific population of umbilical cord tissue-derived MSCs (UC-MSCs), UCX®. METHODS: Cell phenotype was assessed by flow cytometry through the evaluation of the expression of relevant surface markers such as CD14, CD19, CD31, CD34, CD44, CD45, CD90, CD105, CD146, CD200, CD273, CD274 and HLA-DR. Immunomodulatory activity was analyzed in vitro through the ability to inhibit activated T cells and in vivo by the ability to reverse the signs of inflammation in an adjuvant-induced arthritis (AIA) model. Angiogenic potential was evaluated in vitro using a human umbilical vein endothelial cell-based angiogenesis assay, and in vivo using a mouse model for hindlimb ischemia. RESULTS: Phenotype and immunomodulatory and angiogenic potencies of this specific UC-MSC population were not impaired by cryopreservation and subsequent thawing, both in vitro and in vivo. DISCUSSION: This study suggests that potency impairment related to cryopreservation in a given tissue source can be avoided by the production process. The results have positive implications for the development of advanced-therapy medicinal products.

Self-assembled 3D spheroids and hollow-fibre bioreactors improve MSC-derived hepatocyte-like cell maturation in vitro.

3D cultures of human stem cell-derived hepatocyte-like cells (HLCs) have emerged as promising models for short- and long-term maintenance of hepatocyte phenotype in vitro cultures by better resembling the in vivo environment of the liver and consequently increase the translational value of the resulting data. In this study, the first stage of hepatic differentiation of human neonatal mesenchymal stem cells (hnMSCs) was performed in 2D monolayer cultures for 17 days. The second stage was performed by either maintaining cells in 2D cultures for an extra 10 days, as control, or alternatively cultured in 3D as self-assembled spheroids or in multicompartment membrane bioreactor system. All systems enabled hnMSC differentiation into HLCs as shown by positive immune staining of hepatic markers CK-18, HNF-4α, albumin, the hepatic transporters OATP-C and MRP-2 as well as drug-metabolizing enzymes like CYP1A2 and CYP3A4. Similarly, all models also displayed relevant glucose, phase I and phase II metabolism, the ability to produce albumin and to convert ammonia into urea. However, EROD activity and urea production were increased in both 3D systems. Moreover, the spheroids revealed higher bupropion conversion, whereas bioreactor showed increased albumin production and capacity to biotransform diclofenac. Additionally, diclofenac resulted in an IC50 value of 1.51 ± 0.05 and 0.98 ± 0.03 in 2D and spheroid cultures, respectively. These data suggest that the 3D models tested improved HLC maturation showing a relevant biotransformation capacity and thus provide more appropriate reliable models for mechanistic studies and more predictive systems for in vitro toxicology applications.

Activation of dopaminergic D2/D3 receptors modulates dorsoventral connectivity in the hippocampus and reverses the impairment of working memory after nerve injury.

Dopamine plays an important role in several forms of synaptic plasticity in the hippocampus, a crucial brain structure for working memory (WM) functioning. In this study, we evaluated whether the working-memory impairment characteristic of animal models of chronic pain is dependent on hippocampal dopaminergic signaling. To address this issue, we implanted multichannel arrays of electrodes in the dorsal and ventral hippocampal CA1 region of rats and recorded the neuronal activity during a food-reinforced spatial WM task of trajectory alternation. Within-subject behavioral performance and patterns of dorsoventral neuronal activity were assessed before and after the onset of persistent neuropathic pain using the Spared Nerve Injury (SNI) model of neuropathic pain. Our results show that the peripheral nerve lesion caused a disruption in WM and in hippocampus spike activity and that this disruption was reversed by the systemic administration of the dopamine D2/D3 receptor agonist quinpirole (0.05 mg/kg). In SNI animals, the administration of quinpirole restored both the performance-related and the task-related spike activity to the normal range characteristic of naive animals, whereas quinpirole in sham animals caused the opposite effect. Quinpirole also reversed the abnormally low levels of hippocampus dorsoventral connectivity and phase coherence. Together with our finding of changes in gene expression of dopamine receptors and modulators after the onset of the nerve injury model, these results suggest that disruption of the dopaminergic balance in the hippocampus may be crucial for the clinical neurological and cognitive deficits observed in patients with painful syndromes.

Impaired spatial memory performance in a rat model of neuropathic pain is associated with reduced hippocampus-prefrontal cortex connectivity.

Chronic pain patients commonly complain of working memory deficits, but the mechanisms and brain areas underlying this cognitive impairment remain elusive. The neuronal populations of the mPFC and dorsal CA1 (dCA1) are well known to form an interconnected neural circuit that is crucial for correct performance in spatial memory-dependent tasks. In this study, we investigated whether the functional connectivity between these two areas is affected by the onset of an animal model of peripheral neuropathic pain. To address this issue, we implanted two multichannel arrays of electrodes in the mPFC and dCA1 of rats and recorded the neuronal activity during a food-reinforced spatial working memory task in a reward-based alternate trajectory maze. Recordings were performed for 3 weeks, before and after the establishment of the spared nerve injury model of neuropathy. Our results show that the nerve lesion caused an impairment of working memory performance that is temporally associated with changes in the mPFC populational firing activity patterns when the animals navigated between decision points-when memory retention was most needed. Moreover, the activity of both recorded neuronal populations after the nerve injury increased their phase locking with respect to hippocampal theta rhythm. Finally, our data revealed that chronic pain reduces the overall amount of information flowing in the fronto-hippocampal circuit and induces the emergence of different oscillation patterns that are well correlated with the correct/incorrect performance of the animal on a trial-by-trial basis. The present results demonstrate that functional disturbances in the fronto-hippocampal connectivity are a relevant cause for pain-related working memory deficits.

Orbitostriatal encoding of reward delayed gratification and impulsivity in chronic pain.

Central robust network functional rearrangement is a characteristic of several neurological conditions, including chronic pain. Preclinical and clinical studies have shown the importance of pain-induced dysfunction in both orbitofrontal cortex (OFC) and nucleus accumbens (NAc) brain regions for the emergence of cognitive deficits. Outcome information processing recruits the orbitostriatal circuitry, a pivotal pathway regarding context-dependent reward value encoding. The current literature reveals the existence of structural and functional changes in the orbitostriatal crosstalk in chronic pain conditions, which have emerged as a possible underlying cause for reward and time discrimination impairments observed in individuals affected by such disturbances. However, more comprehensive investigations are needed to elucidate the underlying disturbances that underpin disease development. In this review article, we aim to provide a comprehensive view of the orbitostriatal mechanisms underlying time-reward dependent behaviors, and integrate previous findings on local and network malplasticity under the framework of the chronic pain sphere.

The role of human umbilical cord tissue-derived mesenchymal stromal cells (UCX®) in the treatment of inflammatory arthritis.

BACKGROUND: ECBio has developed proprietary technology to consistently isolate, expand and cryopreserve a well-characterized population of stromal cells from human umbilical cord tissue (UCX® cells). The technology has recently been optimized in order to become compliant with Advanced Medicine Therapeutic Products. In this work we report the immunosuppressive capacity of UCX® cells for treating induced autoimmune inflammatory arthritis. METHODS: UCX® cells were isolated using a proprietary method (PCT/IB2008/054067) that yields a well-defined number of cells using a precise proportion between tissue digestion enzyme activity units, tissue mass, digestion solution volume and void volume. The procedure includes three recovery steps to avoid non-conformities related to cell recovery. UCX® surface markers were characterized by flow cytometry and UCX® capacity to expand in vitro and to differentiate into adipocyte, chondrocyte and osteoblast-like cells was evaluated. Mixed Lymphocyte Reaction (MLR) assays were performed to evaluate the effect of UCX® cells on T-cell activation and Treg conversion assays were also performed in vitro. Furthermore, UCX® cells were administered in vivo in both a rat acute carrageenan-induced arthritis model and rat chronic adjuvant induced arthritis model for arthritic inflammation. UCX® anti-inflammatory activity was then monitored over time. RESULTS: UCX® cells stained positive for CD44, CD73, CD90 and CD105; and negative for CD14, CD19 CD31, CD34, CD45 and HLA-DR; and were capable to differentiate into adipocyte, chondrocyte and osteoblast-like cells. UCX® cells were shown to repress T-cell activation and promote the expansion of Tregs better than bone marrow mesenchymal stem cells (BM-MSCs). Accordingly, xenogeneic UCX® administration in an acute carrageenan-induced arthritis model showed that human UCX® cells can reduce paw edema in vivo more efficiently than BM-MSCs. Finally, in a chronic adjuvant induced arthritis model, animals treated with intra-articular (i.a.) and intra-peritoneal (i.p.) infusions of UCX® cells showed faster remission of local and systemic arthritic manifestations. CONCLUSION: The results suggest that UCX® cells may be an effective and promising new approach for treating both local and systemic manifestations of inflammatory arthritis.

Role of Glutamatergic Projections from Lateral Habenula to Ventral Tegmental Area in Inflammatory Pain-Related Spatial Working Memory Deficits.

The lateral habenula (LHb) and the ventral tegmental area (VTA), which form interconnected circuits, have important roles in the crucial control of sensory and cognitive motifs. Signaling in the LHb-VTA pathway can be exacerbated during pain conditions by a hyperactivity of LHb glutamatergic neurons to inhibit local VTA DAergic cells. However, it is still unclear whether and how this circuit is endogenously engaged in pain-related cognitive dysfunctions. To answer this question, we modulated this pathway by expressing halorhodopsin in LHb neurons of adult male rats, and then selectively inhibited the axonal projections from these neurons to the VTA during a working memory (WM) task. Behavioral performance was assessed after the onset of an inflammatory pain model. We evaluated the impact of the inflammatory pain in the VTA synapses by performing immunohistochemical characterization of specific markers for GABAergic (GAD65/67) and dopaminergic neurons (dopamine transporter (DAT), dopamine D2 receptor (D2r) and tyrosine hydroxylase (TH)). Our results revealed that inhibition of LHb terminals in the VTA during the WM delay-period elicits a partial recovery of the performance of pain animals (in higher complexity challenges); this performance was not accompanied by a reduction of nociceptive responses. Finally, we found evidence that the pain-affected animals exhibit VTA structural changes, namely with an upregulation of GAD65/67, and a downregulation of DAT and D2r. These results demonstrate a role of LHb neurons and highlight their responsibility in the stability of the local VTA network, which regulates signaling in frontal areas necessary to support WM processes.

Dynamics of Lateral Habenula-Ventral Tegmental Area Microcircuit on Pain-Related Cognitive Dysfunctions.

Chronic pain is a health problem that affects the ability to work and perform other activities, and it generally worsens over time. Understanding the complex pain interaction with brain circuits could help predict which patients are at risk of developing central dysfunctions. Increasing evidence from preclinical and clinical studies suggests that aberrant activity of the lateral habenula (LHb) is associated with depressive symptoms characterized by excessive negative focus, leading to high-level cognitive dysfunctions. The primary output region of the LHb is the ventral tegmental area (VTA), through a bidirectional connection. Recently, there has been growing interest in the complex interactions between the LHb and VTA, particularly regarding their crucial roles in behavior regulation and their potential involvement in the pathological impact of chronic pain on cognitive functions. In this review, we briefly discuss the structural and functional roles of the LHb-VTA microcircuit and their impact on cognition and mood disorders in order to support future studies addressing brain plasticity during chronic pain conditions.

Corrigendum: Consistent long-term therapeutic efficacy of human umbilical cord matrix-derived mesenchymal stromal cells after myocardial infarction despite individual differences and transient engraftment.

[This corrects the article DOI: 10.3389/fcell.2021.624601.].

Altered prefrontal-striatal theta-band oscillatory dynamics underlie working memory deficits in neuropathic pain rats.

BACKGROUND: Prelimbic medial prefrontal cortex (PL-mPFC) and nucleus accumbens core region (NAcc) play an important role in supporting several executive cognitive mechanisms, such as spatial working memory (WM). Recently, this circuit has been also associated with both sensory and affective components of pain. However, it is still unclear whether this circuit is endogenously engaged in neuropathic pain-related cognitive dysfunctions. METHODS: To answer this question, we induced the expression of halorhodopsin in local PL-mPFC neurons projecting to NAcc, and then selectively inhibited the terminals of these neurons in the NAcc while recording neural activity during the performance of a delayed non-match to sample (DNMS) spatial WM task. Within-subject behavioural performance and PL-mPFC to NAcc circuit neural activity was assessed after the onset of a persistent rodent neuropathic pain model-spared nerve injury (SNI). RESULTS: Our results revealed that the induction of the neuropathy reduced WM performance, and altered the interplay between PL-mPFC and NAcc neurons namely in increasing the functional connectivity from NAcc to PL-mPFC, particularly in the theta-band spontaneous oscillations; in addition, these behavioural and functional perturbations were partially reversed by selective optogenetic inhibition of PL-mPFC neuron terminals into the NAcc during the DNMS task delay-period, without significant antinociceptive effects. CONCLUSIONS: Altogether, these results strongly suggest that the PL-mPFC excitatory output into the NAcc plays an important role in the deregulation of WM under pain conditions. SIGNIFICANCE: Selective optogenetic inhibition of prefrontal-striatal microcircuit reverses pain-related working memory deficits but has no significant impact on pain responses. Neuropathic pain underlies an increase of functional connectivity between the nucleus accumbens core area and the prelimbic medial prefrontal cortex mediated by theta-band activity.

A serum-free media formulation for cultured meat production supports bovine satellite cell differentiation in the absence of serum starvation.

Cultured meat production requires the robust differentiation of satellite cells into mature muscle fibres without the use of animal-derived components. Current protocols induce myogenic differentiation in vitro through serum starvation, that is, an abrupt reduction in serum concentration. Here we used RNA sequencing to investigate the transcriptomic remodelling of bovine satellite cells during myogenic differentiation induced by serum starvation. We characterized canonical myogenic gene expression, and identified surface receptors upregulated during the early phase of differentiation, including IGF1R, TFRC and LPAR1. Supplementation of ligands to these receptors enabled the formulation of a chemically defined media that induced differentiation in the absence of serum starvation and/or transgene expression. Serum-free myogenic differentiation was of similar extent to that induced by serum starvation, as evaluated by transcriptome analysis, protein expression and the presence of a functional contractile apparatus. Moreover, the serum-free differentiation media supported the fabrication of three-dimensional bioartificial muscle constructs, demonstrating its suitability for cultured beef production.

Instability of spatial encoding by CA1 hippocampal place cells after peripheral nerve injury.

Several authors have shown that the hippocampus responds to painful stimulation and suggested that prolonged painful conditions could lead to abnormal hippocampal functioning. The aim of the present study was to evaluate whether the induction of persistent peripheral neuropathic pain would affect basic hippocampal processing such as the spatial encoding performed by CA1 place cells. These place cells fire preferentially in a certain spatial position in the environment, and this spatial mapping remains stable across multiple experimental sessions even when the animal is removed from the testing environment. To address the effect of prolonged pain on the stability of place cell encoding, we chronically implanted arrays of electrodes in the CA1 hippocampal region of adult rats and recorded the multichannel neuronal activity during a simple food-reinforced alternation task in a U-shaped runway. The activity of place cells was followed over a 3-week period before and after the establishment of an animal model of neuropathy, spared nerve injury. Our results show that the nerve injury increased the number of place fields encoded per cell and the mapping size of the place fields. In addition, there was an increase in in-field coherence while the amount of spatial information content that a single spike conveyed about the animal location decreased over time. Other measures of spatial tuning (in-field firing rate, firing peak and number of spikes) were unchanged between the experimental groups. These results demonstrate that the functioning of spatial place cells is altered during neuropathic pain conditions.

Consistent Long-Term Therapeutic Efficacy of Human Umbilical Cord Matrix-Derived Mesenchymal Stromal Cells After Myocardial Infarction Despite Individual Differences and Transient Engraftment.

Human mesenchymal stem cells gather special interest as a universal and feasible add-on therapy for myocardial infarction (MI). In particular, human umbilical cord matrix-derived mesenchymal stromal cells (UCM-MSC) are advantageous since can be easily obtained and display high expansion potential. Using isolation protocols compliant with cell therapy, we previously showed UCM-MSC preserved cardiac function and attenuated remodeling 2 weeks after MI. In this study, UCM-MSC from two umbilical cords, UC-A and UC-B, were transplanted in a murine MI model to investigate consistency and durability of the therapeutic benefits. Both cellular products improved cardiac function and limited adverse cardiac remodeling 12 weeks post-ischemic injury, supporting sustained and long-term beneficial therapeutic effect. Donor associated variability was found in the modulation of cardiac remodeling and activation of the Akt-mTOR-GSK3ß survival pathway. In vitro, the two cell products displayed similar ability to induce the formation of vessel-like structures and comparable transcriptome in normoxia and hypoxia, apart from UCM-MSCs proliferation and expression differences in a small subset of genes associated with MHC Class I. These findings support that UCM-MSC are strong candidates to assist the treatment of MI whilst calling for the discussion on methodologies to characterize and select best performing UCM-MSC before clinical application.

Comparison of anesthetic depth indexes based on thalamocortical local field potentials in rats.

BACKGROUND: Local field potentials may allow a more precise analysis of the brain electrical activity than the electroencephalogram. In this study, local field potentials were recorded in the thalamocortical axis of rats to (i) compare the performance of several indexes of anesthetic depth and (ii) investigate the existence of thalamocortical correlated or disrupted activity during isoflurane steady-state anesthesia. METHODS: Five rats chronically implanted with microelectrodes were used to record local field potentials in the primary somatosensory cortex and ventroposterolateral thalamic nuclei at six periods: before induction of anesthesia; in the last 5 min of randomized 20-min steady-state end-tidal 0.8, 1.1, 1.4, and 1.7% isoflurane concentrations; and after recovery. The approximate entropy, the index of consciousness, the spectral edge frequency, and the permutation entropy were estimated using epochs of 8 s. A correction factor for burst suppression was applied to the spectral edge frequency and to the permutation entropy. The correlation between the derived indexes and the end-tidal isoflurane was calculated and compared for the two studied brain regions indexes. Coherence analysis was also performed. RESULTS: The burst suppression-corrected permutation entropy showed the highest correlation with the end-tidal isoflurane concentration, and a high coherence was obtained between the two studied areas. CONCLUSIONS: The permutation entropy corrected with the classic burst suppression ratio is a promising alternative to other indexes of anesthetic depth. Furthermore, high coherence level of activity exists between the somatosensory cortical and thalamic regions, even at deep isoflurane stages.

Animal models of congenital hypoalgesia: Untapped potential for assessing pain-related plasticity.

Over the last 20 years a large number of transgenic mouse models have been produced showing different degrees of congenital hypoalgesia; some of these models mimic known human conditions while others seemingly have no human counterpart. However, very little significant contributions to our understanding of pain neurobiology were obtained from this multitude of animal models; in most cases the study of these animals was limited to the characterization of its pain perception without addressing the long term consequences of their hypoalgesic condition. In this review we discuss the untapped potential that these animal models of congenital hypoalgesia hold for future studies addressing brain plasticity during permanent conditions of reduced pain perception, and that may result in important insights on the interplay between pain, emotion, and cognition. Revisiting hypoalgesia using modern techniques of functional neurophysiology in awake animals may complement the recent literature of functional clinical and preclinical studies that improve our understanding of the central malplasticity caused by pain.

Bidirectional optogenetic modulation of prefrontal-hippocampal connectivity in pain-related working memory deficits.

Dysfunction of the prefrontal-hippocampal circuit has been identified as a leading cause to pain-related working-memory (WM) deficits. However, the underlying mechanisms remain poorly determined. To address this issue, we implanted multichannel arrays of electrodes in the prelimbic cortex (PL-mPFC), and in the dorsal hippocampal CA1 field (dCA1) to record the neural activity during the performance of a delayed non-match to sample (DNMS) task. The prefrontal-hippocampal connectivity was selectively modulated by bidirectional optogenetic inhibition or stimulation of local PL-mPFC glutamatergic calcium/calmodulin-dependent protein kinase-II alpha (CaMKIIα) expressing neurons during the DNMS task delay-period. The within-subject behavioral performance was assessed using a persistent neuropathic pain model - spared nerve injury (SNI). Our results showed that the induction of the neuropathic pain condition affects the interplay between PL-mPFC and dCA1 regions in a frequency-dependent manner, and that occurs particularly across theta oscillations while rats performed the task. In SNI-treated rats, this disruption was reversed by the selective optogenetic inhibition of PL-mPFC CaMKIIα-expressing neurons during the last portion of the delay-period, but without any significant effect on pain responses. Finally, we found that prefrontal-hippocampal theta connectivity is strictly associated with higher performance levels. Together, our findings suggest that PL-mPFC CaMKIIα-expressing neurons could be modulated by painful conditions and their activity may be critical for prefrontal-hippocampal connectivity during WM processing.

Selective optogenetic inhibition of medial prefrontal glutamatergic neurons reverses working memory deficits induced by neuropathic pain.

Stability of local medial prefrontal cortex (mPFC) network activity is believed to be critical for sustaining cognitive processes such as working memory (WM) and decision making. Dysfunction of the mPFC has been identified as a leading cause to WM deficits in several chronic pain conditions; however, the underlying mechanisms remain largely undetermined. Here, to address this issue, we implanted multichannel arrays of electrodes in the prelimbic region of the mPFC and recorded the neuronal activity during a food-reinforced delayed nonmatch to sample (DNMS) task of spatial WM. In addition, we used an optogenetic technique to selectively suppress the activity of excitatory pyramidal neurons that are considered the neuronal substrate for memory retention during the delay period of the behavioral task. Within-subject behavioral performance and pattern of neuronal activity were assessed after the onset of persistent pain using the spared nerve injury model of peripheral neuropathy. Our results show that the nerve lesion caused a disruption in WM and prelimbic spike activity and that this disruption was reversed by the selective inhibition of prelimbic glutamatergic pyramidal neurons during the delay period of the WM task. In spared nerve injury animals, photoinhibition of excitatory neurons improved the performance level and restored neural activity to a similar profile observed in the control animals. In addition, we found that selective inhibition of excitatory neurons does not produce antinociceptive effects. Together, our findings suggest that disruption of balance in local prelimbic networks may be crucial for the neurological and cognitive deficits observed during painful syndromes.

The Secretome Derived From 3D-Cultured Umbilical Cord Tissue MSCs Counteracts Manifestations Typifying Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disorder whose treatment is mostly restricted to pain and symptom management and to the delay of joint destruction. Mesenchymal stem/stromal cells from the umbilical cord tissue (UC-MSCs) have previously been proven to be immunomodulatory and more efficient than bone marrow-derived MSCs in causing remission of local and systemic arthritic manifestations in vivo. Given the paracrine nature of UC-MSC activity, their application as active substances can be replaced by their secretome, thus avoiding allogeneic rejection and safety issues related to unwanted grafting. In this work, we aimed at demonstrating the viability of applying the 3D-primed UC-MSC secretome for the amelioration of arthritic signs. A proteomic analysis was performed to both, media conditioned by UC-MSC monolayer (CM2D) and 3D cultures (CM3D). The analysis of relevant trophic factors confirmed secretome profiles with very significant differences in terms of therapeutic potential. Whereas, CM3D was characterised by a prevailing expression of anti-inflammatory cytokines such as IL-10 and LIF, along with trophic factors involved in different mechanisms leading to tissue regeneration, such as PDGF-BB, FGF-2, I-309, SCF, and GM-CSF; CM2D presented relatively higher levels of IL-6, MCP-1, and IL-21, with recognised pro-inflammatory roles in joint disease and pleiotropic effects in the progression of rheumatoid arthritis (RA). Accordingly, different motogenic effects over mouse chondrocytes and distinct capacities of inducing glycosaminoglycan synthesis in vitro were observed between CM3D and CM2D. Finally, the evaluation of arthritic manifestations in vivo, using an adjuvant-induced model for arthritis (AIA), suggested a significantly higher therapeutic potential of CM3D over CM2D and even UC-MSCs. Histological analysis confirmed a faster remission of local and systemic arthritic manifestations of CM3D-treated animals. Overall, the results show that the use of UC-MSC CM3D is a viable and better strategy than direct UC-MSC administration for counteracting AIA-related signs. This strategy represents a novel MSC-based but nonetheless cell-free treatment for arthritic conditions such as those characterising RA.

Identification and characterization of merozoite antigens of a Theileria species highly pathogenic for small ruminants in China.

A new pathogenic Theileria species transmitted by Haemaphysalis qinghaiensis was identified in the Northwestern part of China and was shown to be highly pathogenic for small ruminants. The present article aimed at identifying merozoite antigens that might be suitable for developing diagnostic methods and designing a potential vaccine. Absence of other theilerial or babesial infections was confirmed by reverse line blot in all antigen samples used. Extensive Western blot analyses using serum from infected and noninfected animals led to the identification of four potential merozoite immunoreactive proteins at different molecular weights. Further protein characterization using peptide mass mapping by matrix-assisted laser desorption/ionization (MALDI) followed by database searching resulted in two significant hits that identified two proteins of parasite origin, one homologous to a possible MO25-family protein from Cryptosporidium parvum and the other with an HSP70 from Theileria annulata. Another protein was also identified as a parasite protein but without significant homology. Immunization of rabbits with selected proteins produced antisera that reacted specifically on Western blots with merozoite antigens of the corresponding sizes. This article represents the first identification and characterization of potential antigenic proteins of Theileria sp. (China) for veterinary purposes.