Toxoplasma gondii sequesters lysosomes from mammalian hosts in the vacuolar space

The intracellular compartment harboring Toxoplasma gondii satisfies the parasite's nutritional needs for rapid growth in mammalian cells. We demonstrate that the parasitophorous vacuole (PV) of T. gondii accumulates material coming from the host mammalian cell via the exploitation of the host endo-lysosomal system. The parasite actively recruits host microtubules, resulting in selective attraction of endo-lysosomes to the PV. Microtubule-based invaginations of the PV membrane serve as conduits for the delivery of host endo-lysosomes within the PV. These tubular conduits are decorated by a parasite coat, including the tubulogenic protein GRA7, which acts like a garrote that sequesters host endocytic organelles in the vacuolar space. These data define an unanticipated process allowing the parasite intimate and concentrated access to a diverse range of low molecular weight components produced by the endo-lysosomal system. More generally, they identify a unique mechanism for unidirectional transport and sequestration of host organelles.     

The phylogenetic position of Rhopalura ophiocomae (Orthonectida) based on 18S ribosomal DNA sequence analysis

The Orthonectida is a small, poorly known phylum of parasites of marine invertebrates. Their phylogenetic placement is obscure; they have been considered to be multicellular protozoans, primitive animals at a "mesozoan" grade of organization, or secondarily simplified flatworm- like organisms. The best known species in the phylum, Rhopalura ophiocomae, was collected on San Juan Island, Wash. and a complete 18S rDNA sequence was obtained. Using the models of minimum evolution and parsimony, phylogenetic analyses were undertaken and the results lend support to the following hypotheses about orthonectids: (1) orthonectids are more closely aligned with triploblastic metazoan taxa than with the protist or diploblastic metazoan taxa considered in this analysis; (2) orthonectids are not derived members of the phylum Platyhelminthes; and (3) orthonectids and rhombozoans are not each other's closest relatives, thus casting further doubt on the validity of the phylum Mesozoa previously used to encompass both groups.      

Distribution of Clara cell secretory protein expression in the tracheobronchial airways of rhesus monkeys

Clara cell secretory protein (CCSP) is a protective lung protein that is believed to have antioxidant, immunomodulatory, and anticarcinogenic properties; to be present in all adult mammals; and to be well conserved in rodents, humans, and nonhuman primates. The rationale for this study is to define the distribution and abundance of CCSP in the airway epithelium and lavage fluid of the adult rhesus monkey and to provide information for evaluating CCSP as a marker of Clara cells and as a biomarker of lung health. Lung tissue and lavage fluid from 3-yr-old rhesus monkeys were examined using histopathology and immunohistochemistry. Proximal bronchi, midlevel bronchi, and terminal/respiratory bronchioles were compared for immunohistochemical localization of CCSP in three-dimensional whole mounts as well as in paraffin and Araldite sections. Immunoreactive CCSP was found in nonciliated cells throughout the airway epithelium. Proximal and midlevel airways had the highest labeling. CCSP decreased in distal airways, and respiratory bronchioles had little to no CCSP. CCSP in the most distal airways was in tall cuboidal cells adjacent to the pulmonary artery. Although a large number of cells were present in the terminal bronchioles that would be classified as Clara cells based on morphology (nonciliated cells with apical protrusions), only a small number stained positively for immunoreactive CCSP. Semiquantitative analysis of Western blots indicated that changes in lavage CCSP are consistent with, and may be predictive of, overall CCSP levels in the airway epithelium in this primate species that is phylogenetically similar to humans.     

A cleavable propeptide influences Toxoplasma infection by facilitating the trafficking and secretion of the TgMIC2-M2AP invasion complex

Propeptides regulate protein function and trafficking in many eukaryotic systems and have emerged as important features of regulated secretory proteins in parasites of the phylum Apicomplexa. Regulated protein secretion from micronemes and host cell invasion are inextricably linked and essential processes for the apicomplexan parasite Toxoplasma gondii. TgM2AP is a propeptide-containing microneme protein found in a heterohexameric complex with the microneme protein TgMIC2, a protein that has a demonstrated fundamental role in gliding motility and invasion. TgM2AP function is also central to these processes, because disruption of TgM2AP (m2apKO) results in secretory retention of TgMIC2, leading to reduced TgMIC2 secretion from the micronemes and impaired invasion. Because the TgM2AP propeptide is predicted to be processed in an intracellular site near where TgMIC2 is retained in m2apKO parasites, we hypothesized that the propeptide and its proteolytic removal influence trafficking and secretion of the complex. We found that proTgM2AP traffics through endosomal compartments and that deletion of the propeptide leads to defective trafficking of the complex within or near this site, resulting in aberrant processing and decreased secretion of TgMIC2, impaired invasion, and reduced virulence in vivo, mirroring the phenotypes observed in m2apKO parasites. In contrast, mutation of several cleavage site residues resulted in normal localization, but it affected the stability and secretion of the complex from the micronemes. Therefore, the propeptide and its cleavage site influence distinct aspects of TgMIC2-M2AP function, with both impacting the outcome of infection.     

New clinically relevant,orthotopic mouse models of human chondrosarcoma with spontaneous metastasis

Background  

Chondrosarcoma responds poorly to adjuvant therapy and new, clinically relevant animal models are required to test targeted therapy.     

Low production of reactive oxygen species in granulocytes is associated with organ damage in systemic lupus erythematosus

Introduction

Polymorphonuclear leukocytes (PMN) are main effector cells in the acute immune response. While the specific role of PMN in systemic lupus erythematosus (SLE) and autoimmunity is still unclear, their importance in chronic inflammation is gaining more attention. Here we investigate aspects of function, bone marrow release and activation of PMN in patients with SLE.

Methods

The following PMN functions and subsets were evaluated using flow cytometry; (a) production of reactive oxygen species (ROS) after ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA) or Escherichia coli (E. coli); (b) capacity to phagocytose antibody-coated necrotic cell material; (c) PMN recently released from bone marrow, defined as percentage of CD10⁻D16^low in peripheral blood, and (d) PMN activation markers; CD11b, CD62L and C5aR.

Results

SLE patients (n = 92) showed lower ROS production compared with healthy controls (n = 38) after activation ex vivo. The ROS production was not associated with corticosteroid dose or other immunotherapies. PMA induced ROS production was significantly reduced in patients with severe disease. In contrast, neither ROS levels after E. coli activation, nor the capacity to phagocytose were associated with disease severity. This suggests that decreased ROS production after PMA activation is a sign of changed PMN behaviour rather than generally impaired functions. The CD10⁻CD16^low phenotype constitute 2% of PMN in peripheral blood of SLE patients compared with 6.4% in controls, indicating a decreased release of PMN from the bone marrow in SLE. A decreased expression of C5aR on PMN was observed in SLE patients, pointing towards in vivo activation.

Conclusions

Our results indicate that PMN from SLE patients have altered function, are partly activated and are released abnormally from bone marrow. The association between low ROS formation in PMN and disease severity is consistent with findings in other autoimmune diseases and might be considered as a risk factor.     

Molecular evolution of the ZFY and ZNF6 gene families

Members of the ZFY and ZNF6 gene families have been cloned from species representing different taxa and different modes of sex determination. Comparisons of these genes show the ZFY-like and ZNF6 sequences to be strongly conserved across marsupials, birds, and lepidosaurians. Sequence analyzed by neighbor-joining indicated that both gene families are monophyletic with a high bootstrap value. Pairing of sequences from males and females of nonmammalian species showed there to be no significant difference between male and female sequences from a single species, consistent with autosomal locations. The molecular distances between murine Zfy-1, Zfy-2, and other ZFY-like sequences suggested that Zfy genes have undergone a period of rapid evolutionary change not seen in human ZFY.