Antigen-specific T cells demonstrate several potent effector functions during immune responses. Direct killing of infected cells is crucial for clearing viruses and other intracellular pathogens, but it has been difficult to measure the frequency of cytolytic cells. We have now developed a single-cell assay to measure the number of cytotoxic cells in a population, using a herpes simplex virus amplicon vector to express Escherichia coli beta-galactosidase in mouse or human target cells, and an Elispot to detect release of beta-galactosidase from killed target cells. This antigen-specific, perforin-dependent Lysispot assay has been combined with a cytokine Elispot in a two-color assay to confirm that cytotoxicity and interferon-gamma secretion are regulated independently. The simultaneous enumeration of cytokine-secreting and cytotoxic cells should be invaluable for ex vivo analysis of immune responses during infection and autoimmunity.

Tetracyclines have recently been shown to exert a number of pleiotropic anti-inflammatory and immunomodulatory activities, independent of their antibiotic properties. These include the ability to inhibit metalloproteinases (MP), a class of enzymes involved in crucial cellular functions such as the shedding of soluble mediators and their receptors from the cell surface, as well as interaction with, and remodeling of, the extracellular matrix. Here we report that doxycycline at therapeutic concentrations (1--5 microg/ml) significantly suppresses Ig secretion and class switching by in vitro activated murine B cells. Suppression of Ig secretion correlates with a decrease in levels of mRNA for the terminal B cell differentiation-associated genes Blimp-1 and mad-4, as well as to a reduction in expression of the plasma cell markers Syndecan-1 and J chain. Inhibition of class switching occurs at the recombination stage and is also induced by other MP inhibitors, including tetracycline analogs lacking antibiotic activity and the chemically unrelated hydroxamate KB8301. These novel, direct effects of MP inhibitors on B lymphocytes suggest an intrinsic role for MP in B cell activation and likely explain some of the observed in vivo immunomodulatory properties of tetracyclines. Moreover, these findings have significant implications for tetracycline therapy in Ig-mediated autoimmune or allergic diseases and raise questions about the use of doxycycline-inducible transgenic systems for the study of B cell function.

Exposure to the well known environmental toxicant lead is typically assessed by blood and/or bone levels and has been implicated in the onset of a variety of diseases affecting multiple human systems. However, there are conflicting data regarding the efficiency of in utero versus lactational transfer of lead to offspring, and the immunomodulatory effects of lead in early life have not been well defined. Pregnant BALB/c mice were exposed to lead acetate in their drinking water beginning at approximately day 15 of gestation, and cross-fostering of exposed/nonexposed litters was performed at parturition. Significant increases of blood lead levels of all exposed offspring were found at 1 week of age with evidence for both transplacental and lactational transfer. Additionally, mice exposed to lead continuously beginning at approximately 6 days prior to birth showed significant decreases in their blood lead levels 2 weeks after weaning, despite continued exposure as adults. This result suggests maternal transfer of lead is more efficient than oral adult exposure and that substantial lead transfer occurs both transplacentally and lactationally. The incidence of childhood atopic responses including asthma has risen considerably in recent years, particularly within areas containing higher levels of environmental pollutants. Plasma IgE levels of 2-week-old neonates exposed to lead before and/or after birth were measured as an index of atopy. Neonates exposed to lead transplacentally and/or lactationally had significantly higher plasma IgE levels, a biomarker of atopy, and lower splenic white blood cell numbers than age-matched controls. These results resemble the lag in immunocompetency and increase in serum IgE noted in atopic children and suggest a role for environmental toxicants and non-allergen-specific immunology in the prevalence of atopy and asthma in children.

Despite the importance of dendritic targeting in neural circuit assembly, the mechanisms by which it is controlled still remain incompletely understood. We previously showed that in the developing Drosophila antennal lobe, the Wnt5 protein forms a gradient that directs the~45r otation of a cluster of projection neuron (PN) dendrites, including the adjacent DA1 and VA1d dendrites. We report here that the Van Gogh (Vang) transmembrane planar cell polarity (PCP) protein is required for the rotation of the DA1/VA1d dendritic pair. Cell type-specific rescue and mosaic analyses showed that Vang functions in the olfactory receptor neurons (ORNs), suggesting a codependence of ORN axonal and PN dendritic targeting. Loss of Vang suppressed the repulsion of the VA1d dendrites by Wnt5, indicating that Wnt5 signals through Vang to direct the rotation of the DA1 and VA1d glomeruli. We observed that the Derailed (Drl)/Ryk atypical receptor tyrosine kinase is also required for the rotation of the DA1/VA1d dendritic pair. Antibody staining showed that Drl/Ryk is much more highly expressed by the DA1 dendrites than the adjacent VA1d dendrites. Mosaic and epistatic analyses showed that Drl/Ryk specifically functions in the DA1 dendrites in which it antagonizes the Wnt5-Vang repulsion and mediates the migration of the DA1 glomerulus towards Wnt5. Thus, the nascent DA1 and VA1d glomeruli appear to exhibit Drl/Ryk-dependent biphasic responses to Wnt5. Our work shows that the final patterning of the fly olfactory map is the result of an interplay between ORN axons and PN dendrites, wherein converging preand postsynaptic processes contribute key Wnt5 signaling components, allowing Wnt5 to orient the rotation of nascent synapses through a PCP mechanism.