In vivo mononuclear cell tracking using superparamagnetic particles of iron oxide: feasibility and safety in humans.

BACKGROUND: Cell therapy is an emerging and exciting novel treatment option for cardiovascular disease that relies on the delivery of functional cells to their target site. Monitoring and tracking cells to ensure tissue delivery and engraftment is a critical step in establishing clinical and therapeutic efficacy. The study aims were (1) to develop a Good Manufacturing Practice-compliant method of labeling competent peripheral blood mononuclear cells with superparamagnetic particles of iron oxide (SPIO), and (2) to evaluate its potential for magnetic resonance cell tracking in humans. METHODS AND RESULTS: Peripheral blood mononuclear cells 1-5 × 10(9) were labeled with SPIO. SPIO-labeled cells had similar in vitro viability, migratory capacity, and pattern of cytokine release to unlabeled cells. After intramuscular administration, up to 10(8) SPIO-labeled cells were readily identifiable in vivo for at least 7 days using magnetic resonance imaging scanning. Using a phased-dosing study, we demonstrated that systemic delivery of up to 10(9) SPIO-labeled cells in humans is safe, and cells accumulating in the reticuloendothelial system were detectable on clinical magnetic resonance imaging. In a healthy volunteer model, a focus of cutaneous inflammation was induced in the thigh by intradermal injection of tuberculin. Intravenously delivered SPIO-labeled cells tracked to the inflamed skin and were detectable on magnetic resonance imaging. Prussian blue staining of skin biopsies confirmed iron-laden cells in the inflamed skin. CONCLUSIONS: Human peripheral blood mononuclear cells can be labeled with SPIO without affecting their viability or function. SPIO labeling for magnetic resonance cell tracking is a safe and feasible technique that has major potential for a range of cardiovascular applications including monitoring of cell therapies and tracking of inflammatory cells. Clinical Trial Registration- URL: http://www.clinicaltrials.gov; Unique identifier: NCT00972946, NCT01169935.

Dendritic cells previously exposed to mannan-binding lectin enhance cytokine production in allogeneic mononuclear cell cultures.

Mannan (or mannose)-binding lectin (MBL) can bind to monocytes and dendritic cells, but the significance of such interactions is unknown. We hypothesized that the presence of MBL might prevent the differentiation of monocytes into monocyte-derived dendritic cells or interfere with the development of dendritic cells in some way. We therefore investigated the influence of recombinant human MBL on surface antigen expression and on secretion of selected cytokines. By these means, no direct influence of rhMBL on dendritic cell differentiation or maturation was detected. However, mature dendritic cells prepared in the presence of rhMBL and subsequently co-cultured with allogeneic mononuclear cells, markedly promoted production of interleukin-1ß, interleukin-6, and tumor necrosis factor-α in vitro. In most dendritic cell-mononuclear cell combinations, IFN-γ production was also enhanced. This influence required the presence of rhMBL during dendritic cell maturation and was critically dependent on the presence of monocytes. This observation provides evidence that MBL can influence cellular immunity in addition to its established role as an opsonin.

L-ficolin (ficolin-2) insufficiency is associated with combined allergic and infectious respiratory disease in children.

We previously reported an association between relative L-ficolin deficiency and recurrent respiratory infections co-existing with allergic disorders in children. To confirm and extend this preliminary finding, we performed a prospective study on children of a similar age (mean 8.9 years) designed to establish whether the principal relationship was with infection or allergy. Serum L-ficolin values in healthy children were normally distributed with a mean value of 3838 ng/ml. L-ficolin concentrations were generally lower in patients with asthma and/or allergic rhinitis with (mean 3413 ng/ml; p=0.02) or without (3512 ng/ml; p<0.07) respiratory infections, but not in patients with respiratory infections without allergic disease (3623 ng/ml; p=0.2). The lower average values in the group comprised of children with respiratory allergy and infections were largely due to a high proportion of very low values: 18.3% had values below 2150 ng/ml compared to only 5.5% of healthy controls (OR=3.9; p=0.01). This relationship was not apparent in the groups characterized by allergy without infection or infections without allergy. An association between mannan-binding lectin (MBL) insufficiency and recurrent respiratory infections was also confirmed. One of the patients was MASP-2 deficient, evidenced both by MASP2 genotyping and by lectin pathway activity measurement. In conclusion, L-ficolin may confer some protection from microorganisms that exacerbate allergic inflammation in the lung and its relative deficiency may contribute to enhanced susceptibility to respiratory infections. MBL insufficiency and MASP-2 deficiency are risk factors for recurrence of infections independently of allergic disease.

Mannan-binding lectin-associated serine protease-2 (MASP-2) in a large cohort of neonates and its clinical associations.

One collectin (mannan-binding lectin, MBL) and three ficolins (M-ficolin/ficolin-1, L-ficolin/ficolin-2 and H-ficolin/ficolin-3) share the capability to activate complement via the lectin pathway. This property depends on the ability of these lectins to form complexes with MBL-associated serine proteases (MASPs), particularly MASP-2. We report the results of an investigation of cord blood MASP-2 concentrations in a large, ethnically homogeneous cohort (n=1788) of neonates. The median value of MASP-2 in cord sera was determined to be 93 ng/ml (range <25-812). Serum MASP-2 concentrations correlated with gestational age and birthweight and were significantly lower in premature babies and other pre-term babies compared with term babies. Neonates with MASP-2 concentrations below 42 ng/ml were deemed to be MASP-2 deficient. That group had a shorter mean gestational age and a higher incidence of premature and low birthweight babies, but not of perinatal infections when compared with the others. Indeed, there was a trend towards higher MASP-2 concentrations amongst babies with infections. Among 362 samples tested for the D120G single nucleotide polymorphism (SNP) of the MASP2 gene, no homozygote for that mutation was found. Heterozygosity for this allele significantly influenced the protein concentration, but not the lectin pathway of complement activity (MBL-MASP-2 complex activity). Moreover, no association of this SNP was apparent with prematurity, low birthweight or perinatal infections.

Mannan-binding lectin genotypes and genotype-phenotype relationships in a large cohort of Polish neonates.

Circulating mannan (or mannose)-binding lectin (MBL) is genetically determined. Low MBL concentrations are associated with certain point mutations in the human MBL2 gene. Here we report the full MBL2 genotypes of 1800 Polish neonates and relate individual genotypes to serum MBL and MBL-dependent activity of the lectin pathway of complement activation. The seven acknowledged common haplotypes were found, plus the uncommon LYPD haplotype, combining to form 33 genotypes in this population. As expected, a strong correlation existed between genotypes and serum MBL or lectin pathway activity, and the latter two entities correlated strongly with each other. However, serum MBL values varied up to greater than 90-fold within genotypes. Unexpectedly, higher lectin pathway activity was found in association with the P allele relative to the Q allele. These data from a large cohort of neonates, representing an ethnically homogenous population, suggest that the current knowledge of the genetics of MBL2 is inadequate to predict serum MBL concentration and MBL-dependent lectin pathway activity in individual subjects.

Two factors of the lectin pathway of complement, l-ficolin and mannan-binding lectin, and their associations with prematurity, low birthweight and infections in a large cohort of Polish neonates.

Ficolins and one collectin, mannan-binding lectin (MBL), are the only factors known to activate the lectin pathway (LP) of complement. There is considerable circumstantial evidence that MBL insufficiency can increase susceptibility to various infections and influence the course of several non-infectious diseases complicated by infections. Much less information is available concerning l-ficolin. We report the results of a prospective study to investigate any association between either MBL deficiency or l-ficolin deficiency with prematurity, low birthweight or perinatal infections in a large cohort of Polish neonates, representing an ethnically homogenous population (n=1832). Cord blood samples were analysed to determine mbl-2 gene variants, MBL concentrations and MBL-MASP-2 complex activities (MBL-dependent lectin pathway activity) as well as l-ficolin levels. Median concentrations of l-ficolin and MBL were 2500 and 1124 ng/ml, respectively, while median LP activity was 272 mU/ml. After genotyping, 60.6% of babies were mbl-2 A/A, 35.4% were A/O and 4% were O/O genotypes. We found relative l-ficolin deficiency to be associated with prematurity, low birthweight and infections. l-Ficolin concentration correlated with gestational age and with birthweight, independently of gestational age. Preterm deliveries (<38 weeks) occurred more frequently among neonates with low LP activity but not with those having low serum MBL levels. Similarly, no association of serum MBL deficiency with low birthweight was found, but there was a correlation between LP activity and birthweight. Genotypes conferring very low serum MBL concentrations were associated with perinatal infections, and high-MBL-conferring genotypes were associated with prematurity. Our findings suggest that l-ficolin participates in host defence during the perinatal period and constitute the first evidence that relative l-ficolin deficiency may contribute to the adverse consequences of prematurity. Some similar trends were found with facets of MBL deficiency, but the observed relationships were weaker and less consistent.