Visualization of tumors and metastases in live animals with bacteria and vaccinia virus encoding light-emitting proteins

We have shown that bacteria injected intravenously into live animals entered and replicated in solid tumors and metastases. The tumor-specific amplification process was visualized in real time using luciferase-catalyzed luminescence and green fluorescent protein fluorescence, which revealed the locations of the tumors and metastases. Escherichia coli and three attenuated pathogens (Vibrio cholerae, Salmonella typhimurium, and Listeria monocytogenes) all entered tumors and replicated. Similarly, the cytosolic vaccinia virus also showed tumor-specific replication, as visualized by real-time imaging. These findings indicate that neither auxotrophic mutations, nor vaccinia virus deficient for the thymidine kinase gene, nor anaerobic growth conditions were required for tumor specificity and intratumoral replication. We observed localization of tumors by light-emitting microorganisms in immunocompetent and in immunocompromised rodents with syngeneic and allogeneic tumors. Based on their 'tumor-finding' nature, bacteria and viruses may be designed to carry multiple genes for detection and treatment of cancer.     

Lipid rafts in cytokine signaling

Lipid rafts are established as critical structures for a variety of cellular processes, including immune cell activation. Beyond their importance for initial immune cell activation at the immunological synapse, lipid rafts are now also being recognized as important sites for cytokine and growth factor signal transduction, both in immune cells as part of secondary regulatory processes, and in non-immune cells. This review summarizes current knowledge regarding the roles of rafts in cytokine signaling and emphasizes the need for measures to better standardize the study of rafts.     

T-cell senescence: a culprit of immune abnormalities in chronic inflammation and persistent infection

Long-lived clonal T cells deficient in CD28 expression are commonly found in patients with inflammatory syndromes and persistent infections. Considering that CD28 loss is the most consistent immunological marker of aging, we propose that, in pathological states, CD28(null) T cells represent prematurely senescent cells resulting from persistent immune activation. These unusual lymphocytes have aberrant functions that contribute to disease-related immune abnormalities, and the degree of accumulation of CD28(null) T cells predicts the severity of clinical manifestations. We suggest that understanding of the biological properties of T cells that have reached replicative senescence will influence the future management of certain diseases. Indeed, studies on the molecular basis for the loss of CD28 are already providing information on methods to functionally rescue senescent T cells.     

Identification of a novel proliferation-inducing determinant using lentiviral expression cloning

One of the major challenges in the post-genome era is the correlation between genes and function or phenotype. We have pioneered a strategy for screening of cDNA libraries, which is based on sequential combination of lentiviral and oncoretroviral expression systems and can be used to identify proliferation-modulating genes. Screening of a lentiviral expression library derived from adult human brain cDNA resulted in cloning of the potent proliferation-inducing determinant termed pi1 (proliferation inducer 1). Transduction experiments using GFP-expressing oncoretroviruses to target proliferation-competent cells suggested that overexpression of pi1 initiates proliferation of human umbilical vein endothelial cells (HUVECs). Growth induction of HUVECs as well as Swiss3T3 fibroblasts was confirmed by Brd-uridine incorporation assays, which correlated increased DNA synthesis with expression of pi1. The identified pi1 cDNA is 297 bp long and encodes a 10 kDa polypeptide. Since deregulation of proliferation control accounts for a number of today’s untreatable human diseases such as neurodegenerative disorders and cancer, discovery of novel proliferation-modulating genes is essential for developing new strategies for gene therapy and tissue engineering.     

Synoviocyte-mediated expansion of inflammatory T cells in rheumatoid synovitis is dependent on CD47-thrombospondin 1 interaction

Fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis elicit spontaneous proliferation of autologous T cells in an HLA-DR and CD47 costimulation-dependent manner. T cell costimulation through CD47 is attributed to specific interaction with thrombospondin-1 (TSP1), a CD47 ligand displayed on FLS. CD47 binding by FLS has broad biological impact that includes adhesion and the triggering of specific costimulatory signals. TSP1(+) FLS are highly adhesive to T cells and support their aggregation and growth in situ. Long-term cultures of T cells and FLS form heterotypic foci that are amenable to propagation without exogenous growth factors. T cell adhesion and aggregate formation on TSP1(+) FLS substrates are inhibited by CD47-binding peptides. In contrast, FLS from arthroscopy controls lack adhesive or T cell growth-promoting activities. CD47 stimulation transduces a costimulatory signal different from that of CD28, producing a gene expression profile that included induction of ferritin L chain, a component of the inflammatory response. Ferritin L chain augments CD3-induced proliferation of T cells. Collectively, these results demonstrate the active role of FLS in the recruitment, activation, and expansion of T cells in a CD47-dependent manner. Because TSP1 is abundantly expressed in the rheumatoid synovium, CD47-TSP1 interaction is proposed to be a key component of an FLS/T cell regulatory circuit that perpetuates the inflammatory process in the rheumatoid joint.     

MAHRP-1, a novel Plasmodium falciparum histidine-rich protein,binds ferriprotoporphyrin IX and localizes to the Maurer's clefts

Using a stage-specific cDNA library from Plasmodium falciparum we have identified a gene coding for a novel histidine-rich protein (MAHRP-1). The gene is exclusively transcribed during early erythrocyte stages and codes for a small transmembrane protein. The C-terminal region contains a polymorphic stretch of histidine-rich repeats. Fluorescence microscopy studies using polyclonal mouse sera revealed that MAHRP-1 is located at the Maurer's clefts, which represent parasite-induced structures within the cytosol of infected erythrocytes. Biochemical studies showed that recombinant MAHRP-1 binds the toxic hemoglobin degradation product, ferriprotoporphyrin (FP) with a submicromolar dissociation constant and a stoichiometry determined by the number of DHGH motifs. The bound FP has increased peroxidase-like activity and is 10-fold more susceptible to H2O2-induced degradation compared with unbound FP. These properties of MAHRP-1 suggest it may play a protective role against oxidative stress, and its location at the Maurer's clefts suggests a function in promoting the correct trafficking of exported proteins, such as P. falciparum erythrocyte membrane protein-1.     

Angiopoietin-1 and angiopoietin-2 share the same binding domains in the Tie-2 receptor involving the first Ig-like loop and the epidermal growth factor-like repeats

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) have been identified as ligands with different effector functions of the vascular assembly and maturation-mediating receptor tyrosine kinase Tie-2. To understand the molecular interactions of the angiopoietins with their receptor, we have studied the binding of Ang-1 and Ang-2 to the Tie-2 receptor. Enzyme-linked immunosorbent assay-based competition assays and co-immunoprecipitation experiments analyzing the binding of Ang-1 and Ang-2 to truncation mutants of the extracellular domain of Tie-2 showed that the first Ig-like loop of Tie-2 in combination with the epidermal growth factor (EGF)-like repeats (amino acids 1-360) is required for angiopoietin binding. The first Ig-like domain or the EGF-like repeats alone are not capable of binding Ang-1 and Ang-2. Concomitantly, we made the surprising finding that Tie-2 exon-2 knockout mice do express a mutated Tie-2 protein that lacks 104 amino acids of the first Ig-like domain. This mutant Tie-2 receptor is functionally inactive as shown by the lack of ligand binding and receptor phosphorylation. Collectively, the data show that the first 104 amino acids of the Tie-2 receptor are essential but not sufficient for angiopoietin binding. Conversely, the first 360 amino acids (Ig-like domain plus EGF-like repeats) of the Tie-2 receptor are necessary and sufficient to bind both Ang-1 and Ang-2, which suggests that differential receptor binding is not likely to be responsible for the different functions of Ang-1 and Ang-2.     

Functional aspects of strepsirrhine lumbar vertebral bodies and spinous processes

The relationship between form and function in the lumbar vertebral column has been well documented among platyrrhines and especially catarrhines, while functional studies of postcranial morphology among strepsirrhines have concentrated predominantly on the limbs. This morphometric study investigates biomechanically relevant attributes of the lumbar vertebral morphology of 20 species of extant strepsirrhines. With this extensive sample, our goal is to address the influence of positional behavior on lumbar vertebral form while also assessing the effects of body size and phylogenetic history. The results reveal distinctions in lumbar vertebral morphology among strepsirrhines in functional association with their habitual postures and primary locomotor behaviors. In general, strepsirrhines that emphasize pronograde posture and quadrupedal locomotion combined with leaping (from a pronograde position) have the relatively longest lumbar regions and lumbar vertebral bodies, features promoting sagittal spinal flexibility. Indrids and galagonids that rely primarily on vertical clinging and leaping with orthograde posture share a relatively short (i.e., stable and resistant to bending) lumbar region, although the length of individual lumbar vertebral bodies varies phylogenetically and possibly allometrically. The other two vertical clingers and leapers, Hapalemur and Lepilemur, more closely resemble the pronograde, quadrupedal taxa. The specialized, suspensory lorids have relatively short lumbar regions as well, but the lengths of their lumbar regions are influenced by body size, and Arctocebus has dramatically longer vertebral bodies than do the other lorids. Lumbar morphology among galagonids appears to reflect a strong phylogenetic signal superimposed on a functional one. In general, relative length of the spinous processes follows a positively allometric trend, although lorids (especially the larger-bodied forms) have relatively short spinous processes for their body size, in accordance with their positional repertoire. The results of the study broaden our understanding of postcranial adaptation in primates, while providing an extensive comparative database for interpreting vertebral morphology in fossil primates.