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BACKGROUND AIMS: Autologous macrophage therapy represents a potentially significant therapeutic advance for the treatment of severe progressive liver cirrhosis. Administration of macrophages has been shown to reduce inflammation and drive fibrotic scar breakdown and tissue repair in relevant models. This therapeutic approach is being assessed for safety and feasibility in a first-in-human trial (MAcrophages Therapy for liver CirrHosis [MATCH] trial).METHODS: We outline the development and validation phases of GMP production. This includes use of the CliniMACS Prodigy cell sorting system to isolate CD14(+) cells; optimizing macrophage culture conditions, assessing cellular identity, product purity, functional capability and determining the stability of the final cell product.RESULTS: The GMP-compliant macrophage products have a high level of purity and viability, and have a consistent phenotypic profile, expressing high levels of mature macrophage markers 25F9 and CD206 and low levels of CCR2. The macrophages demonstrate effective phagocytic capacity, are constitutively oriented to an anti-inflammatory profile and remain responsive to cytokine and TLR stimulation. The process validation shows that the cell product in excipient is remarkably robust, consistently passing the viability and phenotypic release criteria up to 48 hours after harvest.CONCLUSIONS: This is the first report of validation of a large-scale, fully Good Manufacturing Practice-compliant, autologous macrophage cell therapy product for the potential treatment of cirrhosis. Phenotypic and functional assays confirm that these cells remain functionally viable for up to 48 h, allowing significant flexibility in administration to patients.

Many drugs have been reported to convert dendritic cells (DCs) into a tolerogenic phenotype in vitro. However, there is evidence that an additional stimulus, such as lipopolysaccharide (LPS), may also be necessary for tolerogenic function in vivo. Little is known concerning the effects of drug modification on LPS-prestimulated DCs. In this study, monocyte-derived immature DCs were stimulated with LPS first and the influence investigated of six different agents on surface antigen expression, cytokine production and lymphocyte proliferation and cytotoxicity. Mycophenolic acid- and rapamycin-exposed DCs had little effect on surface antigen expression or functional activity towards lymphocytes. In contrast, treatment of immature dendritic cells with aspirin, dexamethasone, 1alpha,25-dihydroxyvitamin D3 (VD3) or butyric acid was associated with diminished expression of CD1a, CD1c, CD40, CD80 and CD83. Dendritic cell modification by aspirin, dexamethasone and VD3 were all associated with decreased production of tumour necrosis factor-alpha (TNFalpha). Furthermore, VD3 treatment was associated with a consistent and significant elevation of IL-6 production. Aspirin-, dexamethasone- VD3- and butyric acid-modified DCs suppressed interferon-gamma production, proliferation and cytotoxicity in co-culture with allogeneic mononuclear cells, but inconsistent results were obtained with different allogeneic combinations. Different drugs show varying effects on DC phenotype. No single agent was consistently effective in suppressing the stimulation of allogeneic mononuclear cells and future work is needed to explore drug combinations.

Udgivelsesdato: 2009-May 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

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 (

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.

Background aimsMacrophages have complex roles in the liver. The aim of this study was to compare profiles of human monocyte-derived macrophages between controls and cirrhotic patients, to determine whether chronic inflammation affects precursor number or the phenotype, with the eventual aim to develop a cell therapy for cirrhosis.MethodsInfusion of human macrophages in a murine liver fibrosis model demonstrated a decrease in markers of liver injury (alanine transaminase, bilirubin, aspartate transaminase) and fibrosis (transforming growth factor-β, α-smooth muscle actin, phosphatidylserine receptor) and an increase in markers of liver regeneration (matrix metalloproteinases [MMP]-9, MMP-12 and TNF-related weak inducer of apoptosis). CD14+ monocytes were then isolated from controls. Monocytes were matured into macrophages for 7 days using a Good Manufacturing Practice–compatible technique.ResultsThere was no significant difference between the mean number of CD14+ monocytes isolated from cirrhotic patients (n = 9) and controls (n = 10); 2.8 ± SEM 0.54 × 108 and 2.5 ± 0.56 × 108, respectively. The mean yield of mature macrophages cultured was also not significantly different between cirrhotic patients and controls (0.9 × 108 ± 0.38 × 108, with more than 90% viability and 0.65 × 108 ± 0.16 × 108, respectively. Maturation to macrophages resulted in up-regulation of a number of genes (MMP-9, CCL2, interleukin [IL]-10 and TNF-related weak inducer of apoptosis). A cytokine and chemokine polymerase chain reaction array, comparing the control and cirrhotic macrophages, revealed no statistically significant differences.ConclusionsMacrophages can be differentiated from cirrhotic patients' apheresis-derived CD14 monocytes and develop the same pro-resolution phenotype as control macrophages, indicating their suitability for clinical therapy.

SUMMARY The lectin pathway of complement activation is used by a collectin, mannan-binding lectin (MBL), and two ficolins, L-ficolin and H-ficolin, to opsonize microorganisms for phagocytosis. We published evidence recently that MBL insufficiency is associated with recurrent respiratory infections in childhood. We have now measured serum L-ficolin in 313 respiratory infection patients and 74 healthy control children. L-ficolin concentrations below the lower limit of the control group were found in 6% of the patients (P

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-1β, 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.

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