Targeting gene-modified hematopoietic cells to the central nervous system: use of green fluorescent protein uncovers microglial engraftment.

Gene therapy in the central nervous system (CNS) is hindered by the presence of the blood-brain barrier, which restricts access of serum constituents and peripheral cells to the brain parenchyma. Expression of exogenously administered genes in the CNS has been achieved in vivo using highly invasive routes, or ex vivo relying on the direct implantation of genetically modified cells into the brain. Here we provide evidence for a novel, noninvasive approach for targeting potential therapeutic factors to the CNS. Genetically-modified hematopoietic cells enter the CNS and differentiate into microglia after bone-marrow transplantation. Up to a quarter of the regional microglial population is donor-derived by four months after transplantation. Microglial engraftment is enhanced by neuropathology, and gene-modified myeloid cells are specifically attracted to the sites of neuronal damage. Thus, microglia may serve as vehicles for gene delivery to the nervous system.

Left Shift 1+ flag for the detection of band neutrophils: interlaboratory variations and recommendations for the routine laboratory.

INTRODUCTION: Microscopic differential analysis of leukocytes is a time-consuming activity for routine diagnostic laboratories. The criteria used to decide whether a manual differential should be performed should therefore be as strict as possible. The goal of this investigation was to give recommendations for the use of the left shift (LS) 1+ flag, which signals the presence of band neutrophils. METHODS: The LS1+ flag of the ADVIA 120 and 2120 hematology analyzers was evaluated in 6 peripheral hospital laboratories in the Netherlands. In 2683 samples with exclusively a LS1+ flag, the percentage of band neutrophils were determined microscopically. A set of photographs of neutrophils were used to examine the differences between laboratories in the assessment of band cells. RESULTS: In 18% of all samples with only a LS1+ flag, 5% or more band neutrophils were found. However, this percentage differed greatly between laboratories, as did the proportion of samples that received a LS1+ flag. Several factors were found to influence the amount and accuracy of the LS1+ alarm, i.e. band neutrophil counting by microscopists, specificity of request for leukocyte differentials, percentage of general practitioners requesting a leukocyte differential, and sample storage. Based on these findings, a number of recommendations were formulated. CONCLUSION: Critical control of the factors influencing the LS1+ flag can significantly decrease the number of microscopic samples to be reviewed and may be valuable for every laboratory performing routine differentials, using any type of hematology analyzer.

In vitro production of IgG4 by peripheral blood mononuclear cells (PBMC): the contribution of committed B cells.

IgG4 and IgE isotypes contribute marginally to the pool of circulating antibodies in healthy individuals, but are elevated during atopic diseases and particularly upon helminth infections. To examine whether the high levels of these isotypes in circulation are reflected in a higher capacity of PBMC to produce IgG4 and IgE, we examined cells from patients infected with filarial nematodes that exhibit high levels of IgG4 and IgE. Indeed, IgG4 production by PBMC correlated strongly with plasma levels of IgG4 (r=0.534, P=0.002), but such correlation was not found for IgE. The replacement of CD19+ cells from PBMC by IgD+ cells abrogated the high capacity of PBMC to make IgG4. This indicates that an altered B cell compartment accounts for the high IgG4-producing capacity of the PBMC. The high production of IgG4 in vitro was not dependent on IL-4 and IL-13, as neutralizing antibodies to these cytokines did not inhibit IgG4. However, IgE release by PBMC was dependent on IL-4 and IL-13. Antifilarial IgG4 was detected in culture supernatants from filarial patients and its production was independent of IL-4 and IL-13. These results demonstrate that in individuals with elevated IgG4. the B cell compartment in PBMC carries cells that are already committed to IgG4 production and are independent of IL-4 and IL-13.

Interleukin-12 suppresses immunoglobulin E production but enhances immunoglobulin G4 production by human peripheral blood mononuclear cells.

The effect of interleukin-12 (IL-12) on human immunoglobulin G4 (IgG4) and IgE production was examined with cells derived from filarial patients and European controls. IL-12 inhibited IgE release but enhanced IgG4 production in cultures of peripheral blood mononuclear cells stimulated with anti-CD2 plus IL-2. When purified T- and B-cell cocultures were examined, IL-12 again markedly enhanced IgG4, whereas IgE production was no longer inhibited.

Antigen-stimulated IL-4, IL-13 and IFN-gamma production by human T cells at a single-cell level.

To understand the intricate balance and the coordinate expression of the Th1 and Th2 cytokines following a natural mode of T cell triggering, antigen-stimulated IL-4, IL-13 and IFN-gamma production was studied in primary peripheral blood mononuclear cell cultures at a single-cell level. Cells from filariasis patients who respond to parasite antigen by producing not only IFN-gamma but also IL-4 and IL-13 were stimulated with Brugia malayi adult worm antigen and analyzed for co-expression of cytokines by intracellular staining. IL-4 and IL-13 were frequently co-expressed (54% of IL-4+ cells stained for IL-13 and 29% of IL-13+ cells expressed IL-4 at all time points), whereas IFN-gamma expression was totally segregated from both IL-4 and IL-13. These data indicate that in human peripheral T cells the co-expression of the dominant Th1 and Th2 cytokines within a single cell is a rare event and that IL-13 is clearly more frequently associated with a Th2 than a Th1 type response in primary T cell cultures.

CXCR5-deficient mice develop functional germinal centers in the splenic T cell zone.

The chemokine receptor CXCR5 is thought to be essential for the migration of B cells into the network of follicular dendritic cells in the spleen. However, as shown here, B cells and follicular dendritic cells do co-localize, albeit aberrantly, even in the absence of CXCR5. In mice lacking CXCR5 both cell types are found in a broad ring around the sinuses of the marginal zones. Upon immunization with the T cell-dependent antigen 2-phenyl-oxazolone, ectopic germinal centers develop in the periarteriolar lymphocyte sheath. A network of follicular dendritic cells forms in the vicinity of the central arteriole within which the antigen-activated B cells proliferate. The analysis of the expressed V gene repertoire revealed that during B cell proliferation, hypermutation is activated and V region genes accumulate somatic mutations. The pattern of somatic mutations suggests that affinity selection may occur. This analysis confirms that in CXCR5-deficient mice, the organization of splenic primary follicles is severely impaired. However, within the T cell zone a micro-environment is built up, which provides all requirements needed for the affinity maturation to take place.