Search

We report the analysis of 1 year of data from the first cohort of 15 patients enrolled in an open-label, first-in-human, dose-escalation phase I study (ClinicalTrials.gov: NCT03282760, EudraCT2015-004855-37) to determine the feasibility, safety, and tolerability of the transplantation of allogeneic human neural stem/progenitor cells (hNSCs) for the treatment of secondary progressive multiple sclerosis. Participants were treated with hNSCs delivered via intracerebroventricular injection in combination with an immunosuppressive regimen. No treatment-related deaths nor serious adverse events (AEs) were observed. All participants displayed stability of clinical and laboratory outcomes, as well as lesion load and brain activity (MRI), compared with the study entry. Longitudinal metabolomics and lipidomics of biological fluids identified time- and dose-dependent responses with increased levels of acyl-carnitines and fatty acids in the cerebrospinal fluid (CSF). The absence of AEs and the stability of functional and structural outcomes are reassuring and represent a milestone for the safe translation of stem cells into regenerative medicines., Competing Interests: Declaration of interests S.P. is founder, CSO, and shareholder (>5%) of CITC Ltd. and Chair of the Scientific Advisory Board at ReNeuron plc., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) mediated by blood-derived immune cells invading the CNS. This invasion could be determined by chemokines, and their role within the MS-affected brain is still poorly defined. We investigated the expression by RT-PCR and protein release by ELISA of the interferon-gamma (IFN-gamma)-inducible chemokines in human brain microvascular endothelial cells (HBMECs) and astrocytes. The monokine induced by IFN-gamma (Mig) behaves as a homing chemokine constitutively expressed in HBMECs and astrocytes, whereas the IFN-gamma-inducible 10-kDa protein (IP-10) and IFN-inducible T cell alpha-chemoattractant (I-TAC) are induced only after inflammatory stimuli. The biologic activity of IFN-gamma-inducible chemokines from an endothelial source was analyzed, and the transendothelial migration of activated lymphocytes was partly antagonized by specific antibodies, especially anti-Mig antibody. Our data highlight the capability of cells of the CNS to activate the chemoattractant machinery in a proinflammatory environment and in MS.

Background: Intravenous methylprednisolone hemisuccinate is administered to patients with multiple sclerosis (MS) during exacerbations to improve the rate of recovery. Corticosteroids could be beneficial in MS exacerbations also by decreasing transmigration of peripheral blood mononuclear cells (PBMNCs) through the blood-brain barrier., Objectives: To evaluate how in vivo intravenous methylprednisolone treatment in patients with MS could influence transmigration of PBMNCs in an in vitro model; to perform transmigration experiments through a methylprednisolone-treated endothelium with PBMNCs from untreated healthy control subjects to evaluate putative selective effects of corticosteroids on endothelium; concomitantly, to quantify the concentration of matrix metalloproteinases 2 and 9 in supernatants of PBMNCs and in serum samples from methylprednisolone-treated patients with MS; to evaluate monokine induced by interferon-gamma release in the supernatants of human umbilical vein endothelial cells treated with interferon-gamma alone or interferon-gamma and methylprednisolone; and to perform gene expression studies of matrix metalloproteinases 2 and 9 in human umbilical vein endothelial cells and PBMNCs from methylprednisolone-treated patients with MS., Patients: Eight patients with MS in exacerbation were studied before and 3 and 24 hours after intravenous methylprednisolone treatment, 1 g., Results: The absolute number of transmigrated PBMNCs from methylprednisolone-treated patients with MS significantly (P<.01) decreased at 3 hours and increased again at 24 hours, reaching values higher than those before treatment onset. Methylprednisolone was also able to significantly (P<.03) reduce the number of PBMNCs from healthy controls migrating through interferon-gamma-stimulated or unstimulated endothelium. In vitro methylprednisolone treatment decreased monokine induced by interferon-gamma production in human umbilical vein endothelial cells., Conclusions: Methylprednisolone may be able to decrease transmigration of PBMNCs through the blood-brain barrier, exerting its inhibitory effects on PBMNCs and endothelium. A "rebound" of transmigration at 24 hours suggests that a single infusion is not optimal for achieving a persistent reduction in transmigration.

To evaluate the effects of in vivo beta-IFN-1b treatment on transmigration of mononuclear cells, we monitored for one year in vitro mononuclear cells trafficking through HUVECs monolayers stratified over a collagen gel during beta-IFN-1b treatment of 7 RR MS patients. The number of transmigrated cells was analysed before treatment (T0) and after 3 (T3), 6 (T6) and 12 months (T12); at the same time, levels of serum MMP-9 were quantified. The number of transmigrated cells decreased during treatment compared to pre-treatment values: the lowest number of transmigrated cells was detected at T3, and, although transmigration was still lower at T12, there was a trend to a return to pre-treatment levels over time. The amount of MMP-9 also decreased during therapy, although we could not find an absolute correlation between transmigration and levels of MMP-9, nor between either parameter and the clinical course of patients.

The long-term immunological effects of azathioprine treatment have been investigated in 8 multiple sclerosis patients with different course of disease, chronic progressive (CP) or relapsing progressive (RP). We studied fluctuations in peripheral blood mononuclear cell subsets, IgG, IgM and soluble vascular cell adhesion molecule-1 (sVCAM-1), before and after 2 (T24) and 3 (T36) years of therapy. We observed a significant decrease in CD8+ cells over time and a trend to lower percentage of CD3-CD56+ cells at T24 and T36. CD4+CD45RA+ cells in MS patients were lower than in healthy controls before therapy and reached values similar to those of healthy controls at T24 and T36. The remaining immunological parameters did not show any significant fluctuations.

Ten patients with multiple sclerosis and treated with interferon-beta1b (IFN-beta1b) were followed-up for 1 year with quantitation of serum VCAM-1 and ICAM-1 levels, mean fluorescence intensity of HLA-DR, VLA-4, CD11a, and CD18 on peripheral blood monocytes and lymphocytes, and adhesion of peripheral blood monocytes and CD45+ cells on endothelial cell monolayers. Adhesion molecule expression and adhesion of peripheral blood monocytes to endothelium were also monitored in healthy controls. No differences in adhesion were detected between MS patients before treatment and healthy controls, while after 1 year a marked decrease in the number of monocytes and mononuclear cells adhering to human umbilical vein endothelial cell monolayers was observed in patients treated with IFN-beta1b. After 1 year of treatment a significant increase in HLA-DR on peripheral blood monocytes was also detected. Our findings regarding lowered adhesion add information to available evidence of the mechanisms of action of IFN-beta1b in MS.

Endothelia from the brains of four patients undergoing neurosurgery, including one multiple sclerosis (MS) patient, were studied in vitro to determine cytokine and chemokine production; the release of soluble adhesion molecules was also investigated. The same procedure was repeated on human umbilical vein endothelial cells (HUVECs) in order to detect possible district-specific differences. After isolation, the endothelium was cultured and stimulated with gamma-interferon (IFN), tumour necrosis factor alpha (TNF-alpha) and LPS. The results showed that brain endothelium, in our experimental conditions, does not produce interleukin (IL)-10 and produces lower amounts of IL-1beta and soluble intercellular adhesion molecule-(sICAM-1) than HUVECs do; no differences were detected in soluble vascular cell adhesion molecule-(sVCAM-1) production. MCP-1 mRNA was detected both without and after stimulation with TNF-alpha and gamma-IFN in HUVECs and MS human brain endothelial cells (HBECs), while in non-MS-HBECs it was found only after gamma-IFN stimulation.

CSF and serum levels of soluble Fas were studied in MS patients, in patients with various neurological diseases and in healthy controls. We did not detect differences in serum sFas levels between MS patients and controls. In CSF, despite sFas levels being similar in all patients studied, a statistically significant correlation between sFas CSF/sFas serum ratio and BBB damage (expressed as albumin CSF/albumin serum ratio) was detected in non-MS neurological disease, but not in MS patients. The normalized ratio (sFas CSF/sFas serum)/(Alb CSF/Alb serum) was significantly increased in MS patients compared to patients with non-inflammatory neurological disease suggesting an intrathecal synthesis of soluble Fas in MS. The percentage of apoptotic mononuclear cells was higher in CSF as compared to peripheral blood; moreover a lower proportion of apoptotic cells was found in CSF of MS patients. The findings lend support to the involvement of sFas in MS pathogenesis and suggest that a lower apoptosis in CSF may be a feature of the disease.

Methylprednisolone (MP) is a synthetic steroid commonly used in the treatment of multiple sclerosis (MS) relapses. It has a wide spectrum of activities on immune cells: it might also act by preventing mononuclear cell/endothelium adhesion. We studied adhesion phenomena between cultured human umbilical vein endothelial cells (HUVECs) and PBMNCs (CD45+, CD14+) from 6 MS patients treated in vivo with MP. We also studied fluctuations in CD11a and CD18 levels on lymphocytes and monocytes, as well as changes in serum sICAM-1 and sVCAM-1 concentrations. After MP treatment, PBMNCs adhesion to endothelium decreased at 3 h, while it went back to baseline levels at 24 h. A tendency to increase in both CD11a and CD18 on the surface of lymphocytes was detected, while an increase in serum sVCAM-1 was seen at 3 h.

The in vivo effects on the expression of adhesion molecules and on the adhesion between mononuclear cells and multiple sclerosis human brain endothelial cells (MS-HBECs) were investigated at the beginning of beta-IFN-1b treatment of MS patients. MS-HBECs were isolated from a surgical specimen obtained from an MS patient undergoing brain surgery for vascular aneurysm. 48 h after the first single administration of beta-IFN-1b, PBMNCs of 10 MS patients were analyzed for HLA-DR, CD11a, CD18 and VLA-4 expression and the adhesion between PBMNCs and both stimulated and unstimulated MS-HBECs evaluated. sICAM-1 and sVCAM-1 dosage in the serum of the patients was checked as well. The experiments were repeated using HUVECs in order to detect possible endothelial organ-specific differences. The experiments were also performed after six months of beta-INF-1b treatment on HUVECs. No significant effects on mononuclear cells/endothelium adhesion were detected at 48 h, but adhesion of PBMNCs to HUVECs decreased at six months. An increase in HLA-DR and VLA-4 and a decrease of CD18 was detected in monocytes. The serum level of sVCAM-1 increased at T2 and was still higher than at T0 at six months. The effect of the beta-IFN-1b treatment on both MS-HBECs and HUVECs, was selectively studied in vitro by testing the expression of cytokine-induced adhesion molecules HLA-DR, ICAM-1 and VCAM-1. The in vitro experiments confirmed that beta-IFN-1b is able to antagonize gamma-IFN-induced HLA-DR expression on MS human brain endothelial cells without relevant effects on VCAM-1 and ICAM-1.

IL-10 is a cytokine with suppressive effects on (auto) antigen presentation and T-cell-mediated immune reactions, but is also capable of stimulating polyclonal IgG synthesis. Recent evidence suggests its involvement in multiple sclerosis (MS) and systemic lupus erythematosus (SLE). We assessed PBMNC IL-10 release in MS and SLE patients before and after in vivo prednisone or methylprednisolone treatment. The SLE patients showed a high level of IL-10 release by unstimulated PBMNCs, whereas the PHA-stimulated PBMNCs from MS patients produced large amounts of the cytokine.

Ten patients with multiple sclerosis and treated with interferon-β1b (IFN-β1b) were followed-up for 1 year with quantitation of serum VCAM-1 and ICAM-1 levels, mean fluorescence intensity of HLA-DR, VLA-4, CD11a, and CD18 on peripheral blood monocytes and lymphocytes, and adhesion of peripheral blood monocytes and CD45+ cells on endothelial cell monolayers. Adhesion molecule expression and adhesion of peripheral blood monocytes to endothelium were also monitored in healthy controls. No differences in adhesion were detected between MS patients before treatment and healthy controls, while after 1 year a marked decrease in the number of monocytes and mononuclear cells adhering to human umbilical vein endothelial cell monolayers was observed in patients treated with IFN-β1b. After 1 year of treatment a significant increase in HLA-DR on peripheral blood monocytes was also detected. Our findings regarding lowered adhesion add information to available evidence of the mechanisms of action of IFN-β1b in MS. [ABSTRACT FROM AUTHOR]