Development of Good Manufacturing Practice-Compatible Isolation and Culture Methods for Human Olfactory Mucosa-Derived Mesenchymal Stromal Cells.

Demyelination in the central nervous system (CNS) resulting from injury or disease can cause loss of nerve function and paralysis. Cell therapies intended to promote remyelination of axons are a promising avenue of treatment, with mesenchymal stromal cells (MSCs) a prominent candidate. We have previously demonstrated that MSCs derived from human olfactory mucosa (hOM-MSCs) promote myelination to a greater extent than bone marrow-derived MSCs (hBM-MSCs). However, hOM-MSCs were developed using methods and materials that were not good manufacturing practice (GMP)-compliant. Before considering these cells for clinical use, it is necessary to develop a method for their isolation and expansion that is readily adaptable to a GMP-compliant environment. We demonstrate here that hOM-MSCs can be derived without enzymatic tissue digestion or cell sorting and without culture antibiotics. They grow readily in GMP-compliant media and express typical MSC surface markers. They robustly produce CXCL12 (a key secretory factor in promoting myelination) and are pro-myelinating in in vitro rodent CNS cultures. GMP-compliant hOM-MSCs are comparable in this respect to those grown in non-GMP conditions. However, when assessed in an in vivo model of demyelinating disease (experimental autoimmune encephalitis, EAE), they do not significantly improve disease scores compared with controls, indicating further pre-clinical evaluation is necessary before their advancement to clinical trials.

Fibroblast growth factor 9 (FGF9)-mediated neurodegeneration: Implications for progressive multiple sclerosis?

AIMS: Fibroblast growth factor (FGF) signalling is dysregulated in multiple sclerosis (MS) and other neurological and psychiatric conditions, but there is little or no consensus as to how individual FGF family members contribute to disease pathogenesis. Lesion development in MS is associated with increased expression of FGF1, FGF2 and FGF9, all of which modulate remyelination in a variety of experimental settings. However, FGF9 is also selectively upregulated in major depressive disorder (MDD), prompting us to speculate it may also have a direct effect on neuronal function and survival. METHODS: Transcriptional profiling of myelinating cultures treated with FGF1, FGF2 or FGF9 was performed, and the effects of FGF9 on cortical neurons investigated using a combination of transcriptional, electrophysiological and immunofluorescence microscopic techniques. The in vivo effects of FGF9 were explored by stereotactic injection of adeno-associated viral (AAV) vectors encoding either FGF9 or EGFP into the rat motor cortex. RESULTS: Transcriptional profiling of myelinating cultures after FGF9 treatment revealed a distinct neuronal response with a pronounced downregulation of gene networks associated with axonal transport and synaptic function. In cortical neuronal cultures, FGF9 also rapidly downregulated expression of genes associated with synaptic function. This was associated with a complete block in the development of photo-inducible spiking activity, as demonstrated using multi-electrode recordings of channel rhodopsin-transfected rat cortical neurons in vitro and, ultimately, neuronal cell death. Overexpression of FGF9 in vivo resulted in rapid loss of neurons and subsequent development of chronic grey matter lesions with neuroaxonal reduction and ensuing myelin loss. CONCLUSIONS: These observations identify overexpression of FGF9 as a mechanism by which neuroaxonal pathology could develop independently of immune-mediated demyelination in MS. We suggest targeting neuronal FGF9-dependent pathways may provide a novel strategy to slow if not halt neuroaxonal atrophy and loss in MS, MDD and potentially other neurodegenerative diseases.

Targeting 3D chromosomal architecture at the RANK loci to suppress myeloma-driven osteoclastogenesis.

Bone disease represents a major cause of morbidity and mortality in Multiple Myeloma (MM); primarily driven by osteoclasts whose differentiation is dependent on expression of RANKL by MM cells. Notably, costimulation by ITAM containing receptors (i.e., FcγR) can also play a crucial role in osteoclast differentiation. Modeling the pathology of the bone marrow microenvironment with an ex vivo culture system of primary human multiple myeloma cells, we herein demonstrate that FcγR-mediated signaling, via staphylococcal protein A (SpA) IgG immune-complexes, can act as a critical negative regulator of MM-driven osteoclast differentiation. Interrogation of the mode-of-action revealed that FcγR-mediated signaling causes epigenetic modulation of chromosomal 3D architecture at the RANK promoter; with altered spatial orientation of a proximal super enhancer. Combined this leads to substantial down-regulation of RANK at a transcript, protein, and functional level. These observations shed light on a novel mechanism regulating RANK expression and provide a rationale for targeting FcγR-signaling for the amelioration of osteolytic bone pathology in disease.

Myelinated axons are the primary target of hemin-mediated oxidative damage in a model of the central nervous system.

Iron released from oligodendrocytes during demyelination or derived from haemoglobin breakdown products is believed to amplify oxidative tissue injury in multiple sclerosis (MS). However, the pathophysiological significance of iron-containing haemoglobin breakdown products themselves is rarely considered in the context of MS and their cellular specificity and mode of action remain unclear. Using myelinating cell cultures, we now report the cytotoxic potential of hemin (ferriprotoporphyrin IX chloride), a major degradation product of haemoglobin, is 25-fold greater than equimolar concentrations of free iron in myelinating cultures; a model that reproduces the complex multicellular environment of the CNS. At low micro molar concentrations (3.3 - 10 µM) we observed hemin preferentially binds to myelin and axons to initiate a complex detrimental response that results in targeted demyelination and axonal loss but spares neuronal cell bodies, astrocytes and the majority of oligodendroglia. Demyelination and axonal loss in this context are executed by a combination of mechanisms that include iron-dependent peroxidation by reactive oxygen species (ROS) and ferroptosis. These effects are microglial-independent, do not require any initiating inflammatory insult and represent a direct effect that compromises the structural integrity of myelinated axons in the CNS. Our data identify hemin-mediated demyelination and axonal loss as a novel mechanism by which intracerebral degradation of haemoglobin may contribute to lesion development in MS.

Lipid-specific IgMs induce antiviral responses in the CNS: implications for progressive multifocal leukoencephalopathy in multiple sclerosis.

Progressive multi-focal leukoencephalopathy (PML) is a potentially fatal encephalitis caused by JC polyomavirus (JCV). PML principally affects people with a compromised immune system, such as patients with multiple sclerosis (MS) receiving treatment with natalizumab. However, intrathecal synthesis of lipid-reactive IgM in MS patients is associated with a markedly lower incidence of natalizumab-associated PML compared to those without this antibody repertoire. Here we demonstrate that a subset of lipid-reactive human and murine IgMs induce a functional anti-viral response that inhibits replication of encephalitic Alpha and Orthobunyaviruses in multi-cellular central nervous system cultures. These lipid-specific IgMs trigger microglia to produce IFN-ß in a cGAS-STING-dependent manner, which induces an IFN-α/ß-receptor 1-dependent antiviral response in glia and neurons. These data identify lipid-reactive IgM as a mediator of anti-viral activity in the nervous system and provide a rational explanation why intrathecal synthesis of lipid-reactive IgM correlates with a reduced incidence of iatrogenic PML in MS.

Polarizing receptor activation dissociates fibroblast growth factor 2 mediated inhibition of myelination from its neuroprotective potential.

Fibroblast growth factor (FGF) signaling contributes to failure of remyelination in multiple sclerosis, but targeting this therapeutically is complicated by its functional pleiotropy. We now identify FGF2 as a factor up-regulated by astrocytes in active inflammatory lesions that disrupts myelination via FGF receptor 2 (FGFR2) mediated activation of Wingless (Wnt) signaling; pharmacological inhibition of Wnt being sufficient to abrogate inhibition of myelination by FGF2 in tissue culture. Using a novel FGFR1-selective agonist (F2 V2) generated by deleting the N-terminal 26 amino acids of FGF2 we demonstrate polarizing signal transduction to favor FGFR1 abrogates FGF mediated inhibition of myelination but retains its ability to induce expression of pro-myelinating and immunomodulatory factors that include Cd93, Lif, Il11, Hbegf, Cxcl1 and Timp1. Our data provide new insights into the mechanistic basis of remyelination failure in MS and identify selective activation of FGFR1 as a novel strategy to induce a neuroprotective signaling environment in multiple sclerosis and other neurological diseases.

An in vitro model for studying CNS white matter: functional properties and experimental approaches.

The normal development and maintenance of CNS white matter, and its responses to disease and injury, are defined by synergies between axons, oligodendrocytes, astrocytes and microglia, and further influenced by peripheral components such as the gut microbiome and the endocrine and immune systems. Consequently, mechanistic insights, therapeutic approaches and safety tests rely ultimately on in vivo models and clinical trials. However, in vitro models that replicate the cellular complexity of the CNS can inform these approaches, reducing costs and minimising the use of human material or experimental animals; in line with the principles of the 3Rs. Using electrophysiology, pharmacology, time-lapse imaging, and immunological assays, we demonstrate that murine spinal cord-derived myelinating cell cultures recapitulate spinal-like electrical activity and innate CNS immune functions, including responses to disease-relevant myelin debris and pathogen associated molecular patterns (PAMPs).  Further, we show they are (i) amenable to siRNA making them suitable for testing gene-silencing strategies; (ii) can be established on microelectrode arrays (MEAs) for electrophysiological studies; and (iii) are compatible with multi-well microplate formats for semi-high throughput screens, maximising information output whilst further reducing animal use. We provide protocols for each of these. Together, these advances increase the utility of this in vitro tool for studying normal and pathological development and function of white matter, and for screening therapeutic molecules or gene targets for diseases such as multiple sclerosis, motor neuron disease or spinal cord injury, whilst avoiding in vivo approaches on experimental animals.

Fibroblast growth factor signalling in multiple sclerosis: inhibition of myelination and induction of pro-inflammatory environment by FGF9.

Remyelination failure plays an important role in the pathophysiology of multiple sclerosis, but the underlying cellular and molecular mechanisms remain poorly understood. We now report actively demyelinating lesions in patients with multiple sclerosis are associated with increased glial expression of fibroblast growth factor 9 (FGF9), which we demonstrate inhibits myelination and remyelination in vitro. This inhibitory activity is associated with the appearance of multi-branched 'pre-myelinating' MBP+ / PLP+ oligodendrocytes that interact with axons but fail to assemble myelin sheaths; an oligodendrocyte phenotype described previously in chronically demyelinated multiple sclerosis lesions. This inhibitory activity is not due to a direct effect of FGF9 on cells of the oligodendrocyte lineage but is mediated by factors secreted by astrocytes. Transcriptional profiling and functional validation studies demonstrate that these include effects dependent on increased expression of tissue inhibitor of metalloproteinase-sensitive proteases, enzymes more commonly associated with extracellular matrix remodelling. Further, we found that FGF9 induces expression of Ccl2 and Ccl7, two pro-inflammatory chemokines that contribute to recruitment of microglia and macrophages into multiple sclerosis lesions. These data indicate glial expression of FGF9 can initiate a complex astrocyte-dependent response that contributes to two distinct pathogenic pathways involved in the development of multiple sclerosis lesions. Namely, induction of a pro-inflammatory environment and failure of remyelination; a combination of effects predicted to exacerbate axonal injury and loss in patients.

Notch signaling drives multiple myeloma induced osteoclastogenesis.

Multiple myeloma (MM) is closely associated with bone destruction. Once migrated to the bone marrow, MM cells unbalance bone formation and resorption via the recruitment and maturation of osteoclast precursors. The Notch pathway plays a key role in different types of cancer and drives several biological processes relevant in MM, including cell localization within the bone marrow, proliferation, survival and pharmacological resistance. Here we present evidences that MM can efficiently drive osteoclastogenesis by contemporaneously activating Notch signaling on tumor cells and osteoclasts through the aberrant expression of Notch ligands belonging to the Jagged family. Active Notch signaling in MM cells induces the secretion of the key osteoclastogenic factor, RANKL, which can be boosted in the presence of stromal cells. In turn, MM cells-derived RANKL causes the upregulation of its receptor, RANK, and Notch2 in pre-osteoclasts. Notch2 stimulates osteoclast differentiation by promoting autocrine RANKL signaling. Finally, MM cells through Jagged ligands expression can also activate Notch signaling in pre-osteoclast by direct contact. Such synergism between tumor cells and pre-osteoclasts in MM-induced osteoclastogenesis can be disrupted by silencing tumor-derived Jagged1 and 2. These results make the Jagged ligands new promising therapeutic targets in MM to contrast bone disease and the associated co-morbidities.

miR-142-3p is involved in CD25+ CD4 T cell proliferation by targeting the expression of glycoprotein A repetitions predominant.

Because of the numerous targets of microRNAs (miRNAs), functional dissection of specific miRNA/mRNA interactions is important to understand the complex miRNA regulatory mechanisms. Glycoprotein A repetitions predominant (GARP) is specifically expressed on regulatory CD25(+) CD4 T cells upon their activation. GARP has a long 3' untranslated region containing five highly conserved regions suggesting miRNA regulation of its expression. Although GARP is physiologically expressed on a cell subset characterized by stringent control of proliferation, amplification of the GARP gene has been found in many tumors characterized by uncontrolled proliferation. In this study, we investigated in detail miRNA regulation of GARP expression, in particular by miR-142-3p, and dissected the functional outcome of miR-142-3p/GARP mRNA interaction. We demonstrate that miR-142-3p binds directly to the 3' untranslated region of GARP and represses GARP protein expression by Argonaute 2-associated degradation of GARP mRNA. Functionally, miR-142-3p-mediated regulation of GARP is involved in the expansion of CD25(+) CD4 T cells in response to stimulation. The data indicate that miR-142-3p regulates GARP expression on CD25(+) CD4 T cells and, as a result, their expansion in response to activation. Our data provide novel insight into the molecular mechanisms controlling regulatory T cell expansion. They may also have implications for understanding tumor cell biology.

Co-opting endogenous immunoglobulin for the regulation of inflammation and osteoclastogenesis in humans and mice.

OBJECTIVE: Cells of the monocytic lineage play fundamental roles in the regulation of health, ranging from the initiation and resolution of inflammation to bone homeostasis. In rheumatoid arthritis (RA), the inflamed synovium exhibits characteristic infiltration of macrophages along with local osteoclast maturation, which, together, drive chronic inflammation and downstream articular destruction. The aim of this study was to explore an entirely novel route of immunoglobulin-mediated regulation, involving simultaneous suppression of the inflammatory and erosive processes in the synovium. METHODS: Using in vivo and in vitro studies of human cells and a murine model of RA, the ability of staphylococcal protein A (SPA) to interact with and modulate cells of the monocytic lineage was tested. In addition, the efficacy of SPA as a therapeutic agent was evaluated in murine collagen-induced arthritis (CIA). RESULTS: SPA showed a capacity to appropriate circulating IgG, by generating small immunoglobulin complexes that interacted with monocytes, macrophages, and preosteoclasts. Formation of these complexes resulted in Fcγ receptor type I-dependent polarization of macrophages to a regulatory phenotype, rendering them unresponsive to activators such as interferon-γ. The antiinflammatory complexes also had the capacity to directly inhibit differentiation of preosteoclasts into osteoclasts in humans. Moreover, administration of SPA in the early stages of disease substantially alleviated the clinical and histologic erosive features of CIA in mice. CONCLUSION: These findings demonstrate the overarching utility of immunoglobulin complexes for the prevention and treatment of inflammatory diseases. The results shed light on the interface between immunoglobulin complex-mediated pathways, osteoclastogenesis, and associated pathologic processes. Thus, therapeutic agents designed to harness all of these properties may be an effective treatment for arthritis, by targeting both the innate inflammatory response and prodestructive pathways.

Immune regulation by peripheral suppressor T cells induced upon homotypic T cell/T cell interactions.

We have shown previously that homotypic interaction of resting memory CD4 T cells with activated T cells induces the production of cytokines with immunoregulatory potential (IL-10, IL-4) from the former. Here, we analyzed the effector functions of these T cells stimulated by homotypic T cell interaction. T cells induced upon homotypic T cell interaction expressed CD25 and reduced levels of CD127 and produced TGF-ß. Functionally, homotypic T cell interaction-induced T cells were anergic and inhibited the proliferation of CD25-negative T cells as potently as naturally occurring CD25-positive Tregs in vitro. They also prevented clonotypic expansion of OVA TCR tg T cells in BALB/c mice upon antigenic challenge in vivo. The generation of suppressor T cells by homotypic T cell contact is anchored and tuned through interactions of LFA-1 and its ligands ICAM-1, ICAM-2, and ICAM-3. Together, the data suggest a negative-feedback mechanism of specific immunity involving bystander-activated memory T cells.

Surfactant-free poly(styrene-co-glycidyl methacrylate) particles with surface-bound antibodies for activation and proliferation of human T cells.

In this article, we present our results on the design of new polymeric carriers for antibodies. Polymer colloids based on poly(styrene-co-glycidyl methacrylate) were synthesized by surfactant-free emulsion polymerization. Obtained polymer particles stabilized by grafted poly(ethylene glycol) (PEG) chains and carrying active epoxy groups were used for the covalent immobilization of activating antibodies against the human surface proteins CD (cluster of differentiation) 3 and CD28. The particle-antibody conjugates were employed for the stimulation of human CD4 memory T cells. This was analyzed by the up-regulation of the activation markers CD69 and CD25 on T cells and T cell proliferation as assessed by the dilution of a fluorescent dye on dividing daughter T cells. The particle-antibody conjugates were able to stimulate T cells at least as efficiently as conventional methods, e.g., surface-immobilized antibodies. Furthermore, an increase of the PEG chain length of the particles decreased the efficiency of the particle-antibody conjugates to activate T cells.

Homotypic T-cell/T-cell interaction induces T-cell activation, proliferation, and differentiation.

Activated CD4 T cells might induce T-cell activation from CD4 resting T cells in the absence of antigen presenting cells through interaction of activation-induced surface molecules (e.g., CD80, CD86, CD70, major histocompatibility complex class II) and their ligands constitutively expressed on resting T cells. Supporting this hypothesis, CD4 memory T cells proliferated in response to contact with activated T cells and expressed activation markers, such as CD25, CD30, and CD69. Analysis of their cytokine profile revealed differentiation of interleukin (IL)-10 and interferon-gamma double-producing cells in response to contact with activated T helper (Th) 1 effector cells, and interleukin (IL)-4-producing cells in response to contact with activated Th2 effector cells. Whereas neutralization of interferon-gamma or IL-4 during co-culture did not diminish the frequency of the arising cytokine-producing cells, separation of the responder cells from effector cells significantly decreased cytokine secretion. Specific blocking of particular receptor/ligand interactions denoted above could not prevent cytokine production induced by T-cell/T-cell interaction. However, blockade of all of the receptor/counterreceptor pairs significantly inhibited cytokine production, although not completely. Given the immunomodulatory capacity of IL-4 and IL-10, these findings might indicate a novel contact dependent negative feedback mechanism to control T-cell-driven immunity.

Classification of malignant gliomas by infrared spectroscopy and linear discriminant analysis.

Gliomas are the most frequent primary brain tumors. Their malignancies are graded from 1 to 4. Malignant gliomas are astrocytoma grade 3 and glioblastoma grade 4. An IR spectroscopic approach is presented to diagnose brain tissue at the molecular level probing chemical and structural properties without external markers. IR spectroscopic maps were recorded in transmission mode by sequential acquisition of IR spectra. Training spectra of various tissue types are selected from IR spectroscopic maps in accordance with histological assessment of hematoxylin and eosin stained parallel tissue sections. A decrease of the lipid-to-protein ratio in IR spectra is correlated with the malignancy of gliomas. This chemical property is described by the band intensity ratio 2850 to 1655 cm(-1). Two additional molecular descriptors are identified at 1545 cm(-1)/1655 cm(-1) and (1231 + 1450) cm(-1)/1655 cm(-1), which are associated with hemoglobin and collagen, respectively. This metric is used to train a classification model based on linear discriminant analysis. The model is applied to classify normal brain tissue, astrocytoma grade 2, astrocytoma grade 3, glioblastoma, hemorrhage, and leptomeninges in IR spectroscopic maps of cryosections from two glioma patients. As independent test samples, single IR spectra from cryosections of 51 patients are subjected to the classification model. Normal brain tissue is assigned with 100% accuracy; malignant gliomas are assigned with 93% accuracy. The high success rate demonstrates that IR spectroscopy can complement established methods such as histopathology or immunohistochemistry to characterize dried cryosections.