Lost therapeutic potential of monocyte-derived dendritic cells through lost tissue homing: stable restoration of gut specificity with retinoic acid.

Human monocyte-derived dendritic cells (DC) (MoDC) are utilized for immunotherapy. However, in-vitro immunological effects are often not mirrored in vivo. We studied the tissue-homing potential of MoDC. Circulating monocytes and DC expressed different tissue-homing markers and, during in-vitro development of MoDC, homing marker expression was lost resulting in a 'homeless' phenotype. Retinoic acid (RA) induced gut-homing markers (ß7 and CCR9) and a regulatory phenotype and function [decreased human leucocyte antigen D-related (HLA-DR) and increased ILT3 and fluorescein isothiocyanate (FITC-dextran uptake) in MoDC]. RA-MoDC were less stimulatory and primed conditioned T cells with a gut-homing profile (ß7(+)CLA(-)). Unlike the normal intestinal microenvironment, that from inflamed colon of ulcerative colitis (UC) patients did not induce regulatory properties in MoDC. However, RA-MoDC maintained their regulatory gut-specific properties even in the presence of UC microenvironment. Therefore, MoDC may be ineffectual for immunotherapy because they lack tissue-homing and tissue-imprinting specificity. However, MoDC rehabilitation with gut-homing potential by RA could be useful in promoting immunotherapy in pathologies such as UC.

Variable alterations of the microbiota, without metabolic or immunological change, following faecal microbiota transplantation in patients with chronic pouchitis.

Faecal microbiota transplantation (FMT) is effective in the treatment of Clostridium difficile infection, where efficacy correlates with changes in microbiota diversity and composition. The effects of FMT on recipient microbiota in inflammatory bowel diseases (IBD) remain unclear. We assessed the effects of FMT on microbiota composition and function, mucosal immune response, and clinical outcome in patients with chronic pouchitis. Eight patients with chronic pouchitis (current PDAI ≥7) were treated with FMT via nasogastric administration. Clinical activity was assessed before and four weeks following FMT. Faecal coliform antibiotic sensitivities were analysed, and changes in pouch faecal and mucosal microbiota assessed by 16S rRNA gene pyrosequencing and (1)H NMR spectroscopy. Lamina propria dendritic cell phenotype and cytokine profiles were assessed by flow cytometric analysis and multiplex assay. Following FMT, there were variable shifts in faecal and mucosal microbiota composition and, in some patients, changes in proportional abundance of species suggestive of a "healthier" pouch microbiota. However, there were no significant FMT-induced metabolic or immunological changes, or beneficial clinical response. Given the lack of clinical response following FMT via a single nasogastric administration our results suggest that FMT/bacteriotherapy for pouchitis patients requires further optimisation.

Subpopulations of peripheral blood dendritic cells show differential susceptibility to infection with a lymphotropic strain of HIV-1.

Blood dendritic cells (DC) express CD4 and are susceptible to HIV infection. By electron microscopy two morphologically distinct types of DC were identified in peripheral blood. Only one of these two types was susceptible to infection with a lymphotropic strain of HIV-1. By FACS two populations could be defined based on the expression of CD11c. The morphology of cultured FACS-purified CD11c negative DC was similar to that DC population shown to be susceptible to infection with the lymphotropic strain of HIV. Furthermore after several hours in culture CXCR4, the co-receptor for lymphotropic strains of HIV-1, was expressed at a significantly higher level on the CD11c negative DC than on the CD11c positive cells. This study suggests that there are subpopulations of DC that show differences in susceptibility to infection with some strains of HIV-1.

Relationship between V3 sequences of HIV type 1 from plasma, T cells, and dendritic cells suggests that plasma virus may arise from different cell sources.

Nucleotide sequences of HIV-1 from plasma virus RNA and proviral DNA extracted from blood dendritic cells (DCs) and from T cells were analyzed to determine whether blood DCs may harbor a restricted population of virus variants. The sequence of the V3 loop and 51 bases from the 3' flanking region were determined in four patients not receiving antiviral therapy. There was no evidence of a unique or more restricted population of variants in DCs for any of the four patients studied. However, for one patient there was evidence of differences between plasma virus and virus in the T cell population, with virus in the plasma showing a closer relationship to DC-derived sequences.

Measurement of L-carnitine and acylcarnitines in body fluids and tissues in children and in adults.

We have developed a reliable and validated radio-enzymatic method for the assay of L-carnitine and acylcarnitines, using a modification of existing methods. The sensitivity of the assay is 10 mumol/l using 10 microliters of plasma or urine. It is also suitable for measurements of carnitine in a 10 mg sample of liver or muscle obtained by percutaneous biopsy. The use of N-ethylmaleimide in the reaction mixture together with an excess of [1-14C]acetyl CoA ensures that the reaction proceeds to completion and a linear response is obtained. Using this method control ranges have been established for plasma and urine carnitine concentrations in healthy children and adults, and for the carnitine content of liver and muscle in adults. No significant difference was found between fasting and post-prandial plasma carnitine levels. An age-related increase was found in urinary total carnitine and acylcarnitine concentration throughout childhood. These data provide a reliable basis for studies of patients with abnormal carnitine and acylcarnitine metabolism, distribution and excretion.

A mechanistic role for leptin in human dendritic cell migration: differences between ileum and colon in health and Crohn's disease.

Dendritic cells (DC) migrate to lymph nodes on expression of C-C motif chemokine receptor 7 (CCR7) and control immune activity. Leptin, an immunomodulatory adipokine, functions via leptin receptors, signaling via the long isoform of receptor, LepRb. Leptin promotes DC maturation and increases CCR7 expression on blood DC. Increased mesenteric fat and leptin occur early in Crohn's disease (CD), suggesting leptin-mediated change in intestinal CCR7 expression on DC as a pro-inflammatory mechanism. We have demonstrated CCR7 expression and capacity to migrate to its ligand macrophage inflammatory protein 3ß in normal human ileal DC but not colonic or blood DC. In CD, functional CCR7 was expressed on DC from all sites. Only DC populations containing CCR7-expressing cells produced LepRb; in vitro exposure to leptin also increased expression of functional CCR7 in intestinal DC in a dose-dependent manner. In conclusion, leptin may regulate DC migration from gut, in homeostatic and inflammatory conditions, providing a link between mesenteric obesity and inflammation.

Inhaled allergen-driven CD1c up-regulation and enhanced antigen uptake by activated human respiratory-tract dendritic cells in atopic asthma.

BACKGROUND: Dendritic cells (DC) mediate inflammation in rodent models of allergic airway disease, but the role played by human respiratory-tract DC (hRTDC) in atopic asthma remains poorly defined. Recent data suggest that CD1 antigen presentation by hRTDC may contribute to asthma pathogenesis. OBJECTIVE: To investigate the influence of hRTDC on the balance between atopy and allergic asthma in human subjects and to determine whether CD1 expression by hRTDC is modulated during asthmatic inflammation. METHODS: Sputum cells were induced from steroid-naïve, allergen-challenged and allergen-naïve subjects (atopic asthmatics, atopic non-asthmatics and non-atopic controls). hRTDC were identified using monoclonal antibody labelling and analysis by flow cytometry. RESULTS: hRTDC stained HLA-DR(+) (negative for markers of other cell lineages) were predominantly myeloid and comprised approximately 0.5% of viable sputum cells. Sputum cells were potent stimulators of allogeneic CD4(+) naïve T cells and enrichment/depletion experiments correlated stimulatory potency with DC numbers. Sputum contained cells that exhibited typical dendritic morphology when analysed by electron microscopy. Myeloid hRTDC were endocytically active, but uptake of FITC-dextran was enhanced in cells from asthmatics (P<0.001). Despite their increased endocytic capacity, asthmatic myeloid hRTDC appeared mature and expressed increased levels of maturation markers (P<0.05-P<0.001), CD1c, CD1d and langerin (P<0.05). CD1c expression by asthmatic myeloid hRTDC was enhanced upon in vivo allergen challenge (three to ninefold within 24 h; P<0.05). CD11c(-)CD123(high) hRTDC were only detected in asthmatic sputum and were increased in number following allergen challenge. CONCLUSION: Despite limited cell numbers, it proved possible to analyse human RTDC in induced sputum, providing evidence that increased antigen uptake and enhanced CD1 presentation by activated hRTDC may contribute to allergic airway disease. CD1 presentation by hRTDC in atopic asthma may therefore constitute a novel target for future intervention strategies.

The effect of AZT on dendritic cell number and provirus load in the peripheral blood of AIDS patients: a preliminary study.

In this pilot study, the numbers of dendritic cells (DC) in peripheral blood of AIDS patients and the level of infection with HIV1 were determined before and after AZT treatment. Mononuclear cells were cultured overnight and DC were identified by their lack of labelling with antibodies specific for T, B and natural killer (NK) cells and monocytes and by their high level of staining with antibodies for MHC class II molecules. Although the numbers of DC identified by this method were lower than those identified morphologically in earlier studies (Macatonia et al., 1990), the numbers in three untreated AIDS patients were below the range seen in normals. There was also a marked rise in DC number in patients given AZT therapy. In two patients, there was a significant provirus load in the DCs which was decreased two to three weeks after the commencement of AZT therapy. The studies suggest that DC numbers and their infection levels may be markers of disease in HIV infection.

Analysis of human immunodeficiency virus type 1 (HIV-1) variants and levels of infection in dendritic and T cells from symptomatic HIV-1-infected patients.

Dendritic cells (DC) are required to initiate primary cellular immune responses. Human immunodeficiency virus type 1 (HIV-1) infection of DC may be central to transmission and persistence of virus and in the pathogenesis of AIDS. In symptomatic HIV-1-infected patients the proportion of DC in the mononuclear cell population was reduced. Provirus load in the T cells was 3-100 times higher than in DC and there was no correlation between the levels of infection in the two cell types. Phylogenetic analysis of amino acids in the V3 loop and flanking regions indicated intermingling of sequences and thus provides the first evidence for transfer of virus between DC and T cells in vivo. In one of three patients analysed there were significant differences in amino acid residues in the V3 region. This may reflect reduced interactions between DC and T cells in infected individuals and for the existence of variants with a stronger tropism for DC, which could play a role in transmission by initiating infection in mucosal DC.

Detection of HIV DNA in peripheral blood dendritic cells of HIV-infected individuals.

Previous studies from this laboratory indicated infection of dendritic cells (DC) from peripheral blood of individuals infected with HIV1. Here, further evidence for the infection of peripheral blood DC with HIV1 is presented. Low-density cells (LDC) were prepared from blood mononuclear cells of HIV-infected individuals at different clinical stages of disease. These cells are enriched (10-40%) for MHC class-II-bearing DC, while most of the remaining cells are monocytes, and 2-10% are lymphocytes. A quantitative polymerase chain technique (PCR) was used to estimate the HIV provirus load in LDC and lymphocytes of patients in different disease categories. HIV provirus was detected in every LDC preparation, and for many individuals, particularly CDC stage IV patients, the load was higher in the LDC than in the lymphocyte fraction. These findings suggested that patient DC are infected with HIV. In order to provide confirmatory evidence for this conclusion, PCR was performed on DC that were highly purified from LDC by panning to remove contaminating T, B, natural killer and monocytic cells. High levels of HIV provirus were found in these purified DC. These findings suggest that DC provide a reservoir of HIV and that the consequences of such infection may be relevant to the development of disease.

Increased production of immature myeloid cells in cancer patients: a mechanism of immunosuppression in cancer.

Defective dendritic cell (DC) function caused by abnormal differentiation of these cells is an important mechanism of tumor escape from immune system control. Previously, we have demonstrated that the number and function of DC were dramatically reduced in cancer patients. This effect was closely associated with accumulation of immature cells (ImC) in peripheral blood. In this study, we investigated the nature and functional role of those ImC. Using flow cytometry, electron microscopy, colony formation assays, and cell differentiation in the presence of different cell growth factors, we have determined that the population of ImC is composed of a small percentage (<2%) of hemopoietic progenitor cells, with all other cells being represented by MHC class I-positive myeloid cells. About one-third of ImC were immature macrophages and DC, and the remaining cells were immature myeloid cells at earlier stages of differentiation. These cells were differentiated into mature DC in the presence of 1 microM all-trans-retinoic acid. Removal of ImC from DC fractions completely restored the ability of the DC to stimulate allogeneic T cells. In two different experimental systems ImC inhibited Ag-specific T cell responses. Thus, immature myeloid cells generated in large numbers in cancer patients are able to directly inhibit Ag-specific T cell responses. This may represent a new mechanism of immune suppression in cancer and may suggest a new approach to cancer treatment.