Effects of Mesenchymal Stem Cell Transplantation on Cerebrospinal Fluid Biomarkers in Progressive Multiple Sclerosis.

BACKGROUND: Neurofilament light chains (NF-L) were shown to serve as a reliable biomarker of neurodegeneration in multiple sclerosis (MS). The chemokine receptor CXCL13 was shown to correlate with CNS inflammatory activity and to predict the future progression of MS. OBJECTIVE: To evaluate the levels of NF-L and CXCL13 in the cerebrospinal fluid (CSF) following treatment with mesenchymal stem cells (MSC) in patients with progressive MS. METHODS: The CSF samples were obtained from 48 patients with progressive MS who participated in a double-blind randomized phase II clinical trial that tested the effects of intrathecal (IT) or intravenous (IV) transplantation of mesenchymal stem cells (MSC), at baseline (before the first injection of the MSC) and at 6 months following treatment with MSC, or sham treatment. The CSF specimens were tested in a blinded way, using a single-molecule array (SIMOA) technique. FINDINGS: The CSF levels of NF-L were significantly lower at 6 months following treatment with MSC-IT when compared with the baseline, pre-treatment measurements (P = .026, Wilcoxon paired test). Nine out of 15 tested patients in the MSC-IT group had a reduction in NF-L levels of more than 50% (median decrease: -4449 pg/mL) when compared with 5/15 in the MSC-IV group (median decrease: -151 pg/mL) and 1/15 in the placebo group (median increase: +2450 pg/mL) (P = .001 for MSC-IT vs. placebo, chi-square test). CXCL13 levels were also reduced at 6 months following MSC-IT treatment but not to a statistically significant level. CONCLUSIONS: Our findings indicate possible neuroprotective effects of MSC transplantation in patients with MS. CLINICAL TRIAL REGISTRATION: NCT02166021.

A phase II clinical trial with repeated intrathecal injections of autologous mesenchymal stem cells in patients with amyotrophic lateral sclerosis.

Background: Mesenchymal stem cells (MSC) were shown to induce beneficial effects in animal models of neurodegeneration and in pilot human trials in multiple sclerosis and amyotrophic lateral sclerosis (ALS). Aim: An open-label, clinical trial to evaluate the safety and efficacy of repeated intrathecal administrations of autologous-MSC in ALS-patients. Methods: The study included 20 subjects (age: 20-70) with definite diagnosis of ALS and Amyotrophic Lateral Sclerosis Functional Rating Scale Revised (ALSFRS-R) score of >20. The patients were treated with 1-4 intrathecal injections of MSC, at intervals of 3-6 months. The primary endpoints were safety and tolerability. Efficacy measurements including ALSFRS-R score and forced vital capacity (FVC), were assessed as secondary endpoints. Results: No serious adverse events were observed during the whole period of the trial. One patient withdrew from the study before the first injection. The monthly rate of progression in ALSFRS-R was ameliorated by more than 25% in 15/19 patients between the 1st and 2nd injection (mean improvement of 107.1%); in 11/12 between the 2nd and 3rd injection and in 8/10 between the 3rd and 4th injection. Overall, during the whole period till the last transplantation 13 patients had a >25% improvement in the slope of progression of ALSFRS-R (mean improvement of 47.4%, p < 0.0038, Wilcoxon rank signed test). 7 out of 19 patients actually improved clinically (range of increase in ALSFRS-R: +1 to +4 degrees) after the first transplantation and 5 remained improved after the second cycle. The response rate correlated with the time-intervals between the injections. Conclusion: The results of our study show that repeated intrathecal injections of autologous MSC was safe in patients with ALS and provide indications of medium-term clinical benefits that were related to the intervals between the administrations of the cells. Larger studies are needed to confirm these observations.

Beneficial effects of a nano formulation of pomegranate seed oil, GranaGard, on the cognitive function of multiple sclerosis patients.

BACKGROUND: Though often neglected, cognitive impairment is a common feature of multiple sclerosis in 43-70% of patients. None of the novel MS treatment seems to substantially affect or restore cognitive disability in MS. GranaGard (Granalix Bio Technologies LTD) is a food supplement shown to prevent neuronal death in several animal models of neurological diseases. Capsules of GranaGard comprise a self-emulsion nano formulation of pomegranate seed oil (PSO). This oil contains 80-90% of Punicic Acid (PA), one of the strongest natural antioxidants. In animal experiments, administration of GranaGard results in conjugation with linoleic acid (CLA), the main metabolite of PA, which is a well-known neuroprotective agent. AIMS: To investigate whether GranaGard administration has an effect on the cognitive state of MS patients. METHODS: This is a single center, randomized double blind clinical trial that started in May 2018. The study included 30 MS patients; half of them (Group-A) were given GranaGard for the first three months and then placebo pills containing soybean oil for additional three months. Patients in Group-B received placebo for the first three months, and GranaGard for the following three months. GranaGard was administrated in addition to their immunomodulatory MS-treatments. Subsequently, all patients received GranaGard for additional six months. Patients were required to visit the neurologist at baseline (inclusion, visit 1) and at 3 months after treatment-initiation at each cycle of the trial (visits 2 and 3). During the follow up visits, clinical and cognitive examinations were performed, including Expanded Disability Status Scale (EDSS), Multiple Sclerosis Functional Composite (MSFC: 25 ft walking test, 9 PEG hole test & PASAT). Cognitive tests included The Brief International Cognitive Assessment for Multiple Sclerosis (BICAMS) battery: 1) Symbol Digit Modalities Test (SDMT); 2) California Verbal Learning Test - Second Edition (CVLT-II); 3) and Brief Visuospatial Memory Test - Revised (BVMT-R). Cognitive outcomes were normalized to the healthy population and expressed as z-scores, depended on age, gender and education. Short quality of life and fatigue questionnaires (SF-12, MFIS-5) were also provided by the participants. RESULTS: No serious adverse effects, related to the product, were observed during the study period. All patients receiving GranaGard reported a ''positive'' effect in their ADL while using the product. While there were no significant differences in the clinical parameters of disability (EDSS scores) between the treatment groups, there was a trend of beneficial effect of GranaGard, on the verbal testing during the first 3-month period of treatment. The z score for CVLT-II, significantly increased (from 0.891 to 1.415, p = 0.012, Wilcoxon rank test) at 3-months in the group of patients who were treated with GranaGard, as compared to baseline. A similar (but not statistically significant) trend was seen also in the BVMTr testing during the same 3 months-period, whereas there was no change in the SDMT. The overall average z-score of all three cognitive functions was significantly improved in the three months of Granagard treatment (-0.0077 at 3 months vs 0.462 at baseline, p = 0.034, Wilcoxon rank test). During the same 3-months period there were no significant changes in the placebo-treated group. For the patients receiving GranaGard in the initial 3 months, the value of z score of CVLT-II remained high (z = 1.415) also at the following three months (while they received placebo), suggesting a longer lasting effect for at least 3 months after discontinuation of the drug. CONCLUSION: This is the first study in which GranaGard, a brain targeted nano-formulation of PSO, was tested in humans. Our results in this small pilot, controlled trial provide indications that GranaGard administration to MS patients might improve/stabilize cognitive disability. Larger studies with longer duration, are needed to confirm these initial observations.

Long-Term Clinical and Immunological Effects of Repeated Mesenchymal Stem Cell Injections in Patients With Progressive Forms of Multiple Sclerosis.

Background: Mesenchymal stem cells (MSC) were shown to possess immunomodulatory and neurotrophic effects. Our previous trials, have shown that intrathecal (IT) and intravenous (IV) administration of MSCs were safe and provided indications of beneficial clinical effects. Methods: This is an open prospective study to evaluate the safety and the long-term clinical and immunological effects of multiple injections of autologous MSCs in 24 patients with active-progressive MS. At inclusion, the mean age of the patients was 47.0 ± 9.22, and the mean EDSS score was 6.75 ± 0.68 (range: 5.5-7.5). Patients were initially treated with 1 ×106 MSCS/kg of body weight (IT + IV) and subsequently with up to additional eight courses of MSCs, at intervals of 6-12 months. The duration of the trial was 4 years. Results: No serious, treatment-related adverse events were observed during the follow-up period. Twenty-two of the 24 patients were either stable or improved at the last follow-up visit. Ten patients had a lower than baseline EDSS at the last follow-up (nine were among those who received >2 treatments and one in the subgroup of ≤ 2 treatments, p = 0.04). The mean EDSS score reduced from 6.75 ± 0.68 at baseline to 6.42 ± 0.84 at the last visit, during a median follow-up period of 27.8 months (p = 0.028). Immunological follow-up showed a transient upregulation of CD4+CD25+FoxP3+ cells and downregulation of the proliferative ability of lymphocytes. Conclusions: Repeated MSC treatments in patients with progressive MS were shown safe at the short/intermediate term and induced clinical benefits (especially in patients treated with >2 injections) that lasted for up to 4 years, paralleled by short-term immunomodulatory effects. Clinical Trial Registration: www.ClinicalTrials.gov, identifier: NCT04823000.

Beneficial effects of autologous mesenchymal stem cell transplantation in active progressive multiple sclerosis.

In this study (trial registration: NCT02166021), we aimed to evaluate the optimal way of administration, the safety and the clinical efficacy of mesenchymal stem cell (MSC) transplantation in patients with active and progressive multiple sclerosis. Forty-eight patients (28 males and 20 females) with progressive multiple sclerosis (Expanded Disability Status Scale: 3.0-6.5, mean : 5.6 ± 0.8, mean age: 47.5 ± 12.3) and evidence of either clinical worsening or activity during the previous year, were enrolled (between 2015 and 2018). Patients were randomized into three groups and treated intrathecally (IT) or intravenously (IV) with autologous MSCs (1 × 106/kg) or sham injections. After 6 months, half of the patients from the MSC-IT and MSC-IV groups were retreated with MSCs, and the other half with sham injections. Patients initially assigned to sham treatment were divided into two subgroups and treated with either MSC-IT or MSC-IV. The study duration was 14 months. No serious treatment-related safety issues were detected. Significantly fewer patients experienced treatment failure in the MSC-IT and MSC-IV groups compared with those in the sham-treated group (6.7%, 9.7%, and 41.9%, respectively, P = 0.0003 and P = 0.0008). During the 1-year follow-up, 58.6% and 40.6% of patients treated with MSC-IT and MSC-IV, respectively, exhibited no evidence of disease activity compared with 9.7% in the sham-treated group (P < 0.0001 and P < 0.0048, respectively). MSC-IT transplantation induced additional benefits on the relapse rate, on the monthly changes of the T2 lesion load on MRI, and on the timed 25-foot walking test, 9-hole peg test, optical coherence tomography, functional MRI and cognitive tests. Treatment with MSCs was well-tolerated in progressive multiple sclerosis and induced short-term beneficial effects regarding the primary end points, especially in the patients with active disease. The intrathecal administration was more efficacious than the intravenous in several parameters of the disease. A phase III trial is warranted to confirm these findings.

Can CD44 Be a Mediator of Cell Destruction? The Challenge of Type 1 Diabetes.

CD44 is a multi-functional receptor with multiple of isoforms engaged in modulation of cell trafficking and transmission of apoptotic signals. We have previously shown that injection of anti-CD44 antibody into NOD mice induced resistance to type 1 diabetes (T1D). In this communication we describe our efforts to understand the mechanism underlying this effect. We found that CD44-deficient NOD mice develop stronger resistance to T1D than wild-type littermates. This effect is not explained by the involvement of CD44 in cell migration, because CD44-deficient inflammatory cells surprisingly had greater invasive potential than the corresponding wild type cells, probably owing to molecular redundancy. We have previously reported and we show here again that CD44 expression and hyaluronic acid (HA, the principal ligand for CD44) accumulation are detected in pancreatic islets of diabetic NOD mice, but not of non-diabetic DBA/1 mice. Expression of CD44 on insulin-secreting ß cells renders them susceptible to the autoimmune attack, and is associated with a diminution in ß-cells function (e.g., less insulin production and/or insulin secretion) and possibly also with an enhanced apoptosis rate. The diabetes-supportive effect of CD44 expression on ß cells was assessed by the TUNEL assay and further strengthened by functional assays exhibiting increased nitric oxide release, reduced insulin secretion after glucose stimulation and decreased insulin content in ß cells. All these parameters could not be detected in CD44-deficient islets. We further suggest that HA-binding to CD44-expressing ß cells is implicated in ß-cell demise. Altogether, these data agree with the concept that CD44 is a receptor capable of modulating cell fate. This finding is important for other pathologies (e.g., cancer, neurodegenerative diseases) in which CD44 and HA appear to be implicated.

Oligosaccharide conjugates of Bordetella pertussis and bronchiseptica induce bactericidal antibodies, an addition to pertussis vaccine.

Pertussis is a highly contagious respiratory disease that is especially dangerous for infants and children. Despite mass vaccination, reported pertussis cases have increased in the United States and other parts of the world, probably because of increased awareness, improved diagnostic means, and waning vaccine-induced immunity among adolescents and adults. Licensed vaccines do not kill the organism directly; the addition of a component inducing bactericidal antibodies would improve vaccine efficacy. We investigated Bordetella pertussis and Bordetella bronchiseptica LPS-derived core oligosaccharide (OS) protein conjugates for their immunogenicity in mice. B. pertussis and B. bronchiseptica core OS were bound to aminooxylated BSA via their terminal Kdo residues. The two conjugates induced similar anti-B. pertussis LPS IgG levels in mice. B. bronchiseptica was investigated because it is easier to grow than B. pertussis. Using B. bronchiseptica genetically modified strains deficient in the O-specific polysaccharide, we isolated fractions of core OS with one to five repeats of the terminal trisaccharide, having at the nonreducing end a GlcNAc or GalNAc, and bound them to BSA at different densities. The highest antibody levels in mice were elicited by conjugates containing an average of 8-17 OS chains per protein and with one repeat of the terminal trisaccharide. Conjugate-induced antisera were bactericidal against B. pertussis, and the titers correlated with ELISA-measured antibody levels (r = 0.74). Such conjugates are easy to prepare and standardize; added to a recombinant pertussis toxoid, they may induce antibacterial and antitoxin immunity.

Effect of drying on the biological activities of a red microalgal polysaccharide.

The red microalga Porphyridium sp. produces a polysaccharide exhibiting a variety of biological activities with potential for medical and cosmetic uses. For this reason, it is important that the drying process, which is the end point of production, should not destroy the natural characteristics of the material. The objective of this study was to evaluate the effect of drying at temperatures ranging from 40 to 140 degrees C on the bioactivities of the polysaccharide. Drying the polysaccharide at temperatures above 90 degrees C caused a significant decline in its biological activities (antiviral and anti-cell proliferation) and reduced elasticity, viscosity, and intrinsic viscosity relative to lyophilized polysaccharide and to the starting product. The relationship between molecular weight and intrinsic viscosity indicated that the polysaccharide takes a rigid coil conformation, which stiffens as a result of drying. FTIR analysis revealed that drying caused both significant conformational alterations in the polymer chains and changes in the interaction between the polysaccharide and the glycoprotein to which it is noncovalently associated. Differential scanning calorimetry analysis of the water adsorbed on the charged groups of the polysaccharide showed that drying at higher temperatures increased the bound water content due to dissociation of the polymer chains. Thus, it is recommended that the polysaccharide be dried in a two-step process in which free water is removed by convection and bound freezing water is removed by lyphophilization.