Neutrophil chemotaxis is a critical component of the innate immune response. Neutrophils can sense an extremely shallow gradient of chemoattractants and produce relatively robust chemotactic behavior. This directional migration requires cell polarization with actin polymerization occurring predominantly in the leading edge. Synthesis of phosphatidylinositol (3,4,5) trisphosphate (PIP3) by phosphoinositide 3-kinase (PI3K) contributes to asymmetric F-actin synthesis and cell polarization during neutrophil chemotaxis. To determine the contribution of the hemopoietic cell-restricted PI3Kδ in neutrophil chemotaxis, we have developed a potent and selective PI3Kδ inhibitor, IC87114. IC87114 inhibited polarized morphology of neutrophils, fMLP-stimulated PIP3 production and chemotaxis. Tracking analysis of IC87114-treated neutrophils indicated that PI3Kδ activity was required for the directional component of chemotaxis, but not for random movement. Inhibition of PI3Kδ, however, did not block F-actin synthesis or neutrophil adhesion. These results demonstrate that PI3Kδ can play a selective role in the amplification of PIP3 levels that lead to neutrophil polarization and directional migration.

Several different repetitive DNA sequences have been isolated from the pathogenic yeast Candida albicans. These include two families of large dispersed repeat sequences (Ca3, Ca24) and a short (23-bp) tandemly repeated element (Ca7) associated with C. albicans telomeres. In addition, a large subtelomeric repeat (WOL17) has been cloned. DNA fragments containing the telomeric repeats are highly variable among different C. albicans strains. We have shown that the Ca3 repeat is relatively more stable and is suitable for use as a species-specific and strain-specffic probe for C. albicans.Candida albicans and related species are responsible for the majority of oral and vaginal yeast infections (la, 26) and continue to increase in frequency as a major cause of systemic infections in immunocompromised hosts (1,4,5,25,30). Because C. albicans and related species lack sexual cycles (46), genetic studies have been difficult. Analyses of mitotic recombination of auxotrophic markers (47, 48), DNA content (32, 42), and complexity (32) and chromosome organization by orthogonal field alternating gel electrophoresis (21) indicate that C. albicans possesses a diploid genome organized into about eight chromosome pairs (16,20), with a DNA content and complexity similar to those of bakers' yeast, Saccharomyces cerevisiae (17).The lack of a sexual phase in C. albicans and related species presumably prevents opportunities for recombination between strains, leading to a direct clonal relationship in the descendants. This characteristic may be useful in clinical analyses of colonization and epidemiological studies, since it allows direct determination of strain relatedness by employing genomic markers. Distinguishing between species and strains is of paramount importance in such studies, since it has been demonstrated that more than one Candida species and more than one strain of a single species can colonize or infect the same individual (9,23,27,28,44). In addition, multiple "switch phenotypes" (growth forms with distinctive cell types and colony morphologies [40,41]) of the same strain can be found at the same site of infection (43). Sugar assimilation and biotyping methods are normally accurate in distinguishing species and, in many cases, strains (15, 29, 31) but can provide false distinctions between switch phenotypes of the same strain (1). Serological typing methods can discriminate between only two C. albicans subtypes (8). Even electrophoretic karyotyping can be misleading, since subclones of single strains can exhibit significant changes in chromosome size or structure over relatively short periods of culture (36,45 Repetitive DNA sequences provide useful markers for strain identification and also provide keys to understanding chromosome structure and organization. Scherer and Stevens (38) reported the isolation of a dispersed middle repetitive DNA sequence from C. albicans and showed that it could be used for fingerprinting strains of that species. We have also generated species-specific repetitive DNA probes that have been ...

No abstract

CD11c, a member of the leukointegrin family, is expressed prominently on tissue macrophages and dendritic cells and binds to complement fragment (iC3b), provisional matrix molecules (fibrinogen), and the Ig superfamily cell adhesion molecule, ICAM-1. CD11c has been proposed to function in phagocytosis, cell migration, and cytokine production by monocytes/macrophages as well as induction of T cell proliferation by Langerhans cells. Using assays to quantify CD11c-mediated cell adhesion, we demonstrate that CD11c recognizes ICAM-2 and VCAM-1. The CD11c-binding site on VCAM-1 appears to be different from that used by the integrin alpha4. CD11c and alpha4beta1 contributed to monocyte capture and transmigration on inflamed human aortic endothelial cells. We discovered that the anti-mouse CD11c mAb N418 blocks CD11c binding to iC3b, ICAM-1, and VCAM-1. Treatment of mice with N418 reduced SRBC-induced delayed-type hypersensitivity significantly. CD11c appeared to contribute predominantly to the sensitization phase and somewhat less to the response to SRBC challenge. This suggests a novel role for CD11c during leukocyte recruitment, antigen uptake, and the survival of APC.

We have isolated a gene, designated CAG1, from Candida albicans by using the G-protein alpha-subunit clone SCG1 of Saccharomyces cerevisiae as a probe. Amino acid sequence comparison revealed that CAG1 is more homologous to SCG1 than to any other G protein reported so far. Homology between CAG1 and SCG1 not only includes the conserved guanine nucleotide binding domains but also spans the normally variable regions which are thought to be involved in interaction with the components of the specific signal transduction pathway. Furthermore, CAG1 contains a central domain, previously found only in SCG1. cag1 null mutants of C. albicans created by gene disruption produced no readily detectable phenotype. The C. albicans CAG1 gene complemented both the growth and mating defects of S. cerevisiae scg1 null mutants when carried on either a low- or high-copy-number plasmid. In diploid C. albicans, the CAG1 transcript was readily detectable in mycelial and yeast cells of both the white and opaque forms. However, the CAG1-specific transcript in S. cerevisiae transformants containing the C. albicans CAG1 gene was observed only in haploid cells. This transcription pattern matches that of SCG1 in S. cerevisiae and is caused by a1-alpha 2 mediated repression in diploid cells. That is, CAG1 behaves as a haploid-specific gene in S. cerevisiae, subject to control by the a1-alpha 2 mating-type regulation pathway. We infer from these results that C. albicans may have a signal transduction system analogous to that controlling mating type in S. cerevisiae or possibly even a sexual pathway that has so far remained undetected.