Maria Antonietta Avanzini

Human Bone Marrow–Derived Mesenchymal Stem Cells Do Not Undergo Transformation after Long-term In vitro Culture and Do Not Exhibit Telomere Maintenance Mechanisms

Significant improvement in the understanding of mesenchymal stem cell (MSC) biology has opened the way to their clinical use. However, concerns regarding the possibility that MSCs undergo malignant transformation have been raised. We investigated the susceptibility to transformation of human bone marrow (BM)-derived MSCs at different in vitro culture time points. MSCs were isolated from BM of 10 healthy donors and propagated in vitro until reaching either senescence or passage (P) 25. MSCs in the senescence phase were closely monitored for 8 to 12 weeks before interrupting the cultures. The genetic characterization of MSCs was investigated through array-comparative genomic hybridization (array-CGH), conventional karyotyping, and subtelomeric fluorescent in situ hybridization analysis both before and after prolonged culture. MSCs were tested for the expression of telomerase activity, human telomerase reverse transcriptase (hTERT) transcripts, and alternative lengthening of telomere (ALT) mechanism at different passages. A huge variability in terms of proliferative capacity and MSCs life span was noted between donors. In eight of 10 donors, MSCs displayed a progressive decrease in proliferative capacity until reaching senescence. In the remaining two MSC samples, the cultures were interrupted at P25 to pursue data analysis. Array-CGH and cytogenetic analyses showed that MSCs expanded in vitro did not show chromosomal abnormalities. Telomerase activity and hTERT transcripts were not expressed in any of the examined cultures and telomeres shortened during the culture period. ALT was not evidenced in the MSCs tested. BM-derived MSCs can be safely expanded in vitro and are not susceptible to malignant transformation, thus rendering these cells suitable for cell therapy approaches.

Autologous bone marrow-derived mesenchymal stromal cells in the treatment of fistulising Crohn's disease

Objective External fistulas represent a disabling manifestation of Crohns disease with a difficult curability and a high relapse rate despite a large therapeutic armamentarium. Stem cell therapy is a novel and promising approach for treatment of chronic inflammatory conditions. We therefore investigated the feasibility, safety and efficacy of serial intrafistular injections of autologous bone marrow-derived mesenchymal stromal cells (MSCs) in the treatment of fistulising Crohns disease. Patients and methods We enrolled 12 consecutive outpatients (eight males, median age 32 years) refractory to or unsuitable for current available therapies. MSCs were isolated from bone marrow and expanded ex vivo to be used for both therapeutic and experimental purposes. Ten patients (two refused) received intrafistular MSC injections (median 4) scheduled every 4 weeks, and were monitored by surgical, MRI and endoscopic evaluation for 12 months afterwards. The feasibility of obtaining at least 50×106 MSCs from each patient, the appearance of adverse events, and the efficacy in terms of fistula healing and reduction of both Crohns disease and perianal disease activity indexes were evaluated. In addition, the percentage of both mucosal and circulating regulatory T cells expressing FoxP3, and the ability of MSCs to influence mucosal T cell apoptosis were investigated. Results MSC expansion was successful in all cases; sustained complete closure (seven cases) or incomplete closure (three cases) of fistula tracks with a parallel reduction of Crohns disease and perianal disease activity indexes (p<0.01 for both), and rectal mucosal healing were induced by treatment without any adverse effects. The percentage of mucosal and circulating regulatory T cells significantly increased during the treatment and remained stable until the end of follow up (p<0.0001 and p<0.01, respectively). Furthermore, MSCs have been proven to affect mucosal T cell apoptotic rate. Conclusions Locally injected MSCs represent a feasible, safe and beneficial therapy in refractory fistulising Crohns disease.

Mutations of CD40 gene cause an autosomal recessive form of immunodeficiency with hyper IgM

CD40 is a member of the tumor necrosis factor receptor superfamily, expressed on a wide range of cell types including B cells, macrophages, and dendritic cells. CD40 is the receptor for CD40 ligand (CD40L), a molecule predominantly expressed by activated CD4+ T cells. CD40/CD40L interaction induces the formation of memory B lymphocytes and promotes Ig isotype switching, as demonstrated in mice knocked-out for either CD40L or CD40 gene, and in patients with X-linked hyper IgM syndrome, a disease caused by CD40L/TNFSF5 gene mutations. In the present study, we have identified three patients with an autosomal recessive form of hyper IgM who fail to express CD40 on the cell surface. Sequence analysis of CD40 genomic DNA showed that one patient carried a homozygous silent mutation at the fifth base pair position of exon 5, involving an exonic splicing enhancer and leading to exon skipping and premature termination; the other two patients showed a homozygous point mutation in exon 3, resulting in a cysteine to arginine substitution. These findings show that mutations of the CD40 gene cause an autosomal recessive form of hyper IgM, which is immunologically and clinically undistinguishable from the X-linked form.

Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors

Umbilical cord blood is an attractive source of stem cells for several cell-based therapies. In this paper, it is shown that umbilical cord blood-derived mesenchymal stroma cells, cultured in the presence of platelet lysate, have an increased proliferative potential but comparable immunomodulatory functions relative to their bone marrow-derived counterparts. Background Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However, relatively little is known about the mechanisms responsible for their immunomodulatory effects, which could be influenced by both the cell source and culture conditions. Design and Methods We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells, in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells. Results The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology, immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency, umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition, these cells do not express hTERT and telomerase activity, do express p16ink4a protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses, umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation, (ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10, while decreasing interferon-γ secretion, in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects, a prostaglandin E2-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA-G. Conclusions Umbilical cord blood- and bone marrow-mesenchymal stromal cells may differ in terms of clonogenic efficiency, proliferative capacity and immunomodulatory properties; these differences may be relevant for clinical applications.

Serum IgG subclass concentrations in healthy subjects at different age: Age normal percentile charts

IgG subclass levels were determined in 448 normal children from 6 months to 18 years of age and in 141 healthy adults by radial immunodiffusion using monoclonal antibodies. Age-normal percentile values were calculated for each year of age up to 18 years for IgG1, IgG2, IgG3 and in adults for all four subclasses. The broad spread of IgG4 values in children did not permit calculation of reference values.

Antibody response to pneumococcal vaccine in children receiving bone marrow transplantation

Fifty-three pediatric patients given an allogeneic or an autologous bone marrow transplantation (BMT) were immunized with a polyvalent pneumococcal capsular polysaccharide vaccine (Pneumovax II). Vaccine was administered six months or more after BMT and the pneumococcal IgM, total IgG, and IgG subclasses levels were evaluated before and three weeks after immunization. Immunization promoted a significant rise in antibody serum levels (P<0.000001), and all children vaccinated more than two years after transplantation responded to pneumococcal polysaccharides, whereas only 20–30% and 50% of patients given BMT between six months and one year and one and two years, respectively, mounted an eifective antibody production (P<0.0001). In univariate analysis, lapse of time from BMT to vaccination, chronic graft-versus-host disease occurrence, and female sex influenced the response rate. However, in multivariate analysis, only time between marrow transplant and immunization was a powerful predictor of response. Interestingly, four of 11 patients with IgG2 deficiency before immunization normalized serum levels of this IgG subclass after the pneumococcal antigenic challenge. Our study suggests that time after transplant is the major factor influencing the recovery of immune reactivity to polysaccharide antigens. This seems to confirm the hypothesis that ontogeny of the B-cell repertoire follows a predetermined sequential program in which polysaccharide antigens are some of the last to evoke an antibody response.

Mesenchymal Stem/Stromal Cells: A New "Cells as Drugs" Paradigm. Efficacy and Critical Aspects in Cell Therapy

Mesenchymal stem cells (MSCs) were first isolated more than 50 years ago from the bone marrow. Currently MSCs may also be isolated from several alternative sources and they have been used in more than a hundred clinical trials worldwide to treat a wide variety of diseases. The MSCs mechanism of action is undefined and currently under investigation. For in vivo purposes MSCs must be produced in compliance with good manufacturing practices and this has stimulated research on MSCs characterization and safety. The objective of this review is to describe recent developments regarding MSCs properties, physiological effects, delivery, clinical applications and possible side effects.

Comparison between metalloproteinases-2 and -9 in healthy subjects, diabetics, and subjects with acute coronary syndrome

We hypothesized that matrix metalloproteinase (MMP)-2, -9, and tissue inhibitor metalloproteinase-1, -2 (TIMP-1, -2) would be abnormal in diabetes and in acute coronary syndromes (ACS). We measured MMP-2, -9, and TIMP-1, -2 plasma levels in healthy subjects (controls), in type 2 diabetic patients, in nondiabetic patients with ACS (ACS) and in diabetic patients with ACS (DACS). We enrolled 165 controls, 181 diabetic patients, 78 ACS, and 46 DACS. We measured also BMI (body mass index), HbA1c (glycated hemoglobin) FPG (fasting plasma glucosa), FPI (fasting plasma insulin), HOMA index (homeostasis model assessment index), SBP (systolic blood pressure), DBP (diastolic blood pressure), TC (total cholesterol), LDL-C (low density lipoprotein cholesterol), HDL-C (high-density lipoprotein cholesterol), Tg (triglycerides), Lp(a) (lipoprotein(a)) PAI-1 (plasminogen activator inhibitor-1), Hct (homocysteine), Fg (fibrinogen), and hs-CRP (high-sensitivity C-reactive protein). A significant increase of BMI was observed in the diabetic group, in ACS and DACS patients compared to controls. A significant increase of SBP and DBP resulted in the diabetic and DACS groups, while only SBP improvement was present in ACS patients with respect to controls. A decrease in SBP and DBP was observed in the ACS group, while SBP variation was present in DACS patients compared to diabetics, and DBP increase was obtained in the DACS group with respect to ACS patients. TC, LDL-C, Tg, and Lp(a) increase was present in diabetics, while TC, Tg, and Lp(a) improvement was present in ACS and DACS patients with a significant decrease of HDL-C levels in diabetic, ACS, and DACS groups compared to controls. A decrease in LDL-C was obtained in ACS and DACS groups, while HDL-C increase was observed in these patients with respect to diabetics. Tg levels were higher in the DACS group compared to diabetics and ACS patients, respectively. Increases in PAI-1, Hct, Fg, and hs-CRP were present in diabetic and DACS groups, while PAI-1, Hct, and hs-CRP improvement was obtained in ACS patients with respect to controls. Higher PAI-1 levels came about in ACS and DACS groups, while HCT and Fg levels were lower in ACS patients compared to diabetics. An increase in Fg was present in the DACS group with respect to ACS patients. A decrease in Hs-CRP was observed in DACS patients compared to diabetics and the ACS group, respectively. Higher MMP-2, MMP-9, TIMP-1, and TIMP-2 levels were present in diabetic, ACS, and DACS patients compared to controls. Significant MMP-2, TIMP-1, and TIMP-2 increases were observed in ACS and DACS groups, while MMP-9 decreased in these patients compared to diabetics. In conclusion, MMP-2, MMP-9, TIMP-1, and TIMP-2 plasma levels were higher in diabetic, ACS, and DACS patients, which may reflect abnormal extracellular matrix metabolism in diabetes and in acute coronary syndrome.

A comparison of secretory antibodies in breast-fed and formula-fed infants over the first six months of life

In the present study salivary IgA, anti‐Escherichia coli, anti‐β‐lactoglobulin and anti‐poliovirus type 1 IgA and IgM in serum and saliva were evaluated longitudinally in 13 breast‐fed and 14 formula‐fed infants over the first six months of life. Salivary IgA was quantified by electroimmunodiffusion; specific IgA and IgM antibodies were determined in serum and saliva by ELISA. Salivary IgA was significantly lower at age one month in breast‐fed compared with formula‐fed infants but in breast‐fed infants salivary IgA increased with age and was significantly higher at six months than at one month. In both groups of infants, at the age of six months, salivary IgA levels were significantly lower than in adult controls. No significant differences in secretory anti‐E. coli were observed between the two groups of infants. Salivary anti‐poliovirus IgA and IgM antibodies increased transiently only to disappear in most babies at age six months, while anti‐β lactoglobulin IgA and IgM, present in saliva at all ages, showed a wide scatter. No important differences in specific serum IgA or IgM antibodies were observed either between the groups or at different times within the groups.

IgG subclass deficiency in patients with Down's syndrome and aberrant hepatitis B vaccine response.

Seventeen adult patients with Downs syndrome (DS) and 19 adult healthy references were vaccinated with a hepatitis B vaccine in order to study the IgG subclass response. An enzyme‐linked immunosorbent assay (ELISA) using monoclonal antibodies specific for IgG subclasses was employed. In spite of normal levels of total IgG1 and normal or even high levels of IgG3 in the DS patients, a significantly lower IgG1 response to the vaccine was observed in trisomic patients than in the references.