Pathology

Circulating lymphocytes home to the mucosal lymphoid organs, Peyer's patches (PP), through high endothelial venules (HEV). In situ analyses revealed that transfused lymph node cells (LNCs) interact with PP-HEV In a series of overlapping adhesion events: L-selectin (CD62L) >a467 Initiates interaction, L-selectin and a467 both participate in rolling, and Gai-linked activation triggers arrest that requires both a467 and LFA-1. a467 dramatically reduces rolling velocity, and appears to be required for engagement of LFA-1. In contrast with resting LNC, preactlvated LNC or ~467~ lymphoma cells require only a467for arrest in PP-HEV. The predominant PP-HEV ligand for a467 but also apparently for L-se&tin is the mucosal addressin MAd-CAM-l. These results validate the concept of multlmolecular adhesion/decision cascades in physiologic lymphocyte-endothelial recognition, define a novel role for a4 in&grins as a 'bridge" between selectln and 62 integrin-dependent events, and reemphasize the potential for direct adhesion through preactlvated a4 integrins alone.

The malaria parasite Plasmodium falciparum replicates within an intraerythrocytic parasitophorous vacuole (PV). Rupture of the host cell allows release (egress) of daughter merozoites, which invade fresh erythrocytes. We previously showed that a subtilisin-like protease called PfSUB1 regulates egress by being discharged into the PV in the final stages of merozoite development to proteolytically modify the SERA family of papain-like proteins. Here, we report that PfSUB1 has a further role in ‘priming' the merozoite prior to invasion. The major protein complex on the merozoite surface comprises three proteins called merozoite surface protein 1 (MSP1), MSP6 and MSP7. We show that just before egress, all undergo proteolytic maturation by PfSUB1. Inhibition of PfSUB1 activity results in the accumulation of unprocessed MSPs on the merozoite surface, and erythrocyte invasion is significantly reduced. We propose that PfSUB1 is a multifunctional processing protease with an essential role in both egress of the malaria merozoite and remodelling of its surface in preparation for erythrocyte invasion.

Toxoplasma gondii is a member of the phylum Apicomplexa that includes several important human pathogens, such as Cryptosporidium and Plasmodium falciparum, the causative agent of human malaria. It is an obligate intracellular parasite that can cause severe disease in congenitally infected neonates and immunocompromised individuals. Despite the importance of attachment and invasion to the success of the parasite, little is known about the underlying mechanisms that drive these processes. Here we describe a screen to identify small molecules that block the process of host cell invasion by the T. gondii parasite. We identified a small molecule that specifically and irreversibly blocks parasite attachment and subsequent invasion of host cells. Using tandem orthogonal proteolysis-activity-based protein profiling, we determined that this compound covalently modifies a single cysteine residue in a poorly characterized protein homologous to the human protein DJ-1. Mutation of this key cysteine residue in the native gene sequence resulted in parasites that were resistant to inhibition of host cell attachment and invasion by the compound. Further analysis of the invasion phenotype confirmed that modification of Cys127 on TgDJ-1 resulted in a block of microneme secretion and motility, even in the presence of direct stimulators of calcium release. Together, our results suggest that TgDJ-1 plays an important role that is likely downstream of the calcium flux required for microneme secretion, parasite motility, and subsequent invasion of host cells.

The malaria merozoite invades erythrocytes in the vertebrate host. Iterative rounds of asexual intraerythrocytic replication result in disease. Proteases play pivotal roles in erythrocyte invasion, but little is understood about their mode of action. The Plasmodium falciparum malaria merozoite surface sheddase, PfSUB2, is one such poorly characterized example. We have examined the molecular determinants that underlie the mechanisms by which PfSUB2 is trafficked initially to invasion-associated apical organelles (micronemes) and then across the surface of the free merozoite. We show that authentic promoter activity is important for correct localization of PfSUB2, likely requiring canonical features within the intergenic region 5 of the pfsub2 locus. We further demonstrate that trafficking of PfSUB2 beyond an early compartment in the secretory pathway requires autocatalytic protease activity. Finally, we show that the PfSUB2 transmembrane domain is required for microneme targeting, while the cytoplasmic domain is essential for surface translocation of the protease to the parasite posterior following discharge from micronemes. The interplay of pre-and post-translational regulatory elements that coordinate subcellular trafficking of PfSUB2 provides the parasite with exquisite control over enzyme-substrate interactions.

Although there have been numerous advances in our understanding of how apicomplexan parasites such as Toxoplasma gondii enter host cells, many of the signaling pathways and enzymes involved in the organization of invasion mediators remain poorly defined. We recently performed a forward chemical-genetic screen in T. gondii and identified compounds that markedly enhanced infectivity. Although molecular dissection of invasion has benefited from the use of small-molecule inhibitors, the mechanisms underlying induction of invasion by small-molecule enhancers have never been described. Here we identify the Toxoplasma ortholog of human APT1, palmitoyl protein thioesterase-1 (TgPPT1), as the target of one class of small-molecule enhancers. Inhibition of this uncharacterized thioesterase triggered secretion of invasion-associated organelles, increased motility and enhanced the invasive capacity of tachyzoites. We demonstrate that TgPPT1 is a bona fide depalmitoylase, thereby establishing an important role for dynamic and reversible palmitoylation in host-cell invasion by T. gondii.

Chemical biology and the techniques the field encompasses provide scientists with the means to address biological questions in ever-evolving and technically sophisticated ways. They facilitate the dissection of molecular mechanisms of cell phenomena on timescales not achievable by other means. Libraries of small molecules, bioorthogonal chemistries and technical advances in mass-spectrometry techniques enable the modern chemical biologist to tackle even the most difficult of biological questions. It is because of their broad applicability that these approaches are well suited to systems less tractable to more classical genetic methods. As such, the parasite community has embraced them with great success. Some of these successes and the continuing evolution of chemical biology applied to apicomplexans will be discussed.

Recent studies have implicated reduced thiols (cysteine ؊SH) in the function of individual cell surface proteins. Studies presented here demonstrate that the overall level of reduced thiols on cell surface molecules differs on individual subsets of peripheral blood mononuclear cells and that these levels can be manipulated in vitro by altering the level of intracellular glutathione (iGSH). To quantitate cell surface thiols, we have developed a Hi-D (11-color) fluorescence-activated cell sorter method in which we covalently couple a fluorescent molecule, Alexa-maleimide, to free (reduced) -SH groups on proteins or other molecules exposed on the cell surface (exofacial membrane). In addition, to reveal changes in cell surface thiol levels in response to various in vitro treatments, we used a pair of fluorescent Alexa dyes with distinct excitation and emission spectra to stain the cells before and after treatments. These in vitro studies demonstrate that decreasing iGSH, by specifically inhibiting its synthesis, decreases cell surface molecule thiols (csm؊SH) and that preventing loss of iGSH also prevents loss of csm؊SH. However, examination of peripheral blood mononuclear cell subsets tested immediately after isolation from healthy or HIVinfected subjects failed to reveal a similar relationship between internal iGSH and csm؊SH. Although there is a relatively wide variation between individuals in both csm؊SH and iGSH, there is no correlation between median iGSH and csm؊SH compared for 22 healthy and 36 HIV-infected subjects. Collectively, our findings indicate that local environment plays a greater role in determining the redox status of cell surface molecules than the internal redox status of the cells.

There is an overwhelming interest in the study of the redox status of the cell surface aVecting redox signaling in the cells and also predicting the total redox status of the cells. Measuring the total surface thiols (cell surface molecule thiols, csm-SH) we have shown that the overall level of surface thiols is tightly controlled. In vitro, the total concentration of intracellular glutathione (iGSH) seems to play a regulatory role in determination of the amounts of reduced proteins on cells. In addition, short term exposure of the cell surface to glutathione disulWde (GSSG, oxidized GSH) seems to reduce the overall levels of csm-SH suggesting that the function of some cysteine containing proteins on the cell surface may be regulated by the amount of GSSG secreted from the cells or the GSSG available in the extracellular environment. Examination of peripheral blood mononuclear cells (PBMCs) from healthy or HIVinfected subjects failed to reveal a similar correlation between the intra-and extracellular thiol status of cells. Although there is a relatively wide variation between individuals in both csm-SH and iGSH there is no correlation between the iGSH and csm-SH levels measured for healthy and HIV-infected individuals. There are many reports suggesting diVerent redox active proteins on the cell surface to be the key players in the total cell surface redox regulation. However, we suggest that the redox status of the cells is regulated through a complex and tightly regulated mechanism that needs further investigation. In the mean time, overall surface thiol measurements together with case speciWc protein determinations may oVer the most informative approach. In this review, we discuss our own results as well as results from other laboratories to argue that the overall levels of surface thiols on the exofacial membrane are regulated primarily by redox status of the cell surface microenvironment.  2004 Elsevier Inc. All rights reserved.

In this presentation, we outline ways in which current users of Fluorescence Activated Cell Sorting (FACS) can get more from their FACS work without undue effort. FACS technology development, and the emergence of new software support for various aspects of this technology are now cooperating in this effort.

Allergen-specific IgE production is a hallmark of allergic diseases like asthma, rhinitis or eczema. This could be due to a high number of allergen-specific B cells, Tcells (helping B cells differentiate into allergen-specific IgE-producing plasma cells) or other mechanisms. Here we compared the number of allergen-specific B cells and CD4 T (Th) cells between 48 patients with allergic asthma/rhinitis/eczema (allergic individuals) and 34 nonallergic individuals (asymptomatic and with negative skin prick test (SPT)). Allergen-specific B and Th cells were enumerated by culturing CFSE-loaded PBMNCs for 7 days with allergen (cat, dog, D.pteronyssimus, timothy or birch), and determining by flow cytometry the number of B or Th cells that had proliferated (diluted CFSE). The quantities of B cells specific for each of the 5 allergens were similar in individuals allergic to the allergen (per SPT) compared to nonallergic individuals. The quantity of allergen-specific Th cells for timothy (p = 0.003), cat (p = 0.05) and birch (p = 0.04) was significantly higher in timothy-, cat-and birch-allergic individuals compared to nonallergic individuals. In contrast, the quantity of allergen-specific Th cells for dog and DP was similar in allergic and nonallergic individuals. We conclude that for some allergens (eg, timothy, cat and birch), a high number of allergen-specific Th cells, but not B cells, may play a role in the pathogenesis of allergic asthma/rhinitis/eczema. For other allergens (eg, dog and DP), the pathogenesis is likely different.