Experimental Salmonellosis VIII. Postinfective Immunity and Its Significance for Conferring Cellular Immunity

In the process of live-vaccine immunization of Salmonella enteritidis infection in mice, the relation between the number of bacteria in the organs of mice and their protecting effect was studied. Treatment with antibiotics was used to control the number of immunizing bacteria in the tissues. Mice, which were infected with 10(-5) mg (1,000 mouse MLD) of virulent S. enteritidis and treated with kanamycin simultaneously, acquired high antilethal resistance against infection with the same organisms. However, the administration of large amounts of kanamycin, which caused a rapid decrease in bacterial numbers in the organs of infected mice, was incapable of conferring immunity. This indicated the necessity of persistence of live bacteria in the host for the production of immunity. A large number of microorganisms were maintained for 53 weeks in a diffusion chamber inserted into the mouse abdominal cavity. The mice implanted with diffusion chambers containing large numbers of virulent S. enteritidis did not acquire antilethal resistance against infection with the same organisms, although agglutinins against S. enteritidis were observed in these mice. Agglutinin was also found in the fluid contained in diffusion chambers inserted into mice immunized with a killed vaccine of S. enteritidis. This indicated that antibody penetrated the membrane filter of diffusion chambers from outside to inside and vice versa. From these results, it is suggested that contact of live microorganisms with the host cell is necessary for conferring postinfective immunity in salmonellosis.     

Novel migrating mouse neural crest cell assay system utilizing P0-Cre/EGFP fluorescent time-lapse imaging

Background

Heparan sulfate (HS) is present on the surface of virtually all mammalian cells and is a major component of the extracellular matrix (ECM), where it plays a pivotal role in cell-cell and cell-matrix cross-talk through its large interactome. Disruption of HS biosynthesis in mice results in neonatal death as a consequence of malformed lungs, indicating that HS is crucial for airway morphogenesis. Neonatal mortality (~50%) in newborns with congenital diaphragmatic hernia (CDH) is principally associated with lung hypoplasia and pulmonary hypertension. Given the importance of HS for lung morphogenesis, we investigated developmental changes in HS structure in normal and hypoplastic lungs using the nitrofen rat model of CDH and semi-synthetic bacteriophage ('phage) display antibodies, which identify distinct HS structures.

Results

The pulmonary pattern of elaborated HS structures is developmentally regulated. For example, the HS4E4V epitope is highly expressed in sub-epithelial mesenchyme of E15.5 - E17.5 lungs and at a lower level in more distal mesenchyme. However, by E19.5, this epitope is expressed similarly throughout the lung mesenchyme. We also reveal abnormalities in HS fine structure and spatiotemporal distribution of HS epitopes in hypoplastic CDH lungs. These changes involve structures recognised by key growth factors, FGF2 and FGF9. For example, the EV3C3V epitope, which was abnormally distributed in the mesenchyme of hypoplastic lungs, is recognised by FGF2.

Conclusions

The observed spatiotemporal changes in HS structure during normal lung development will likely reflect altered activities of many HS-binding proteins regulating lung morphogenesis. Abnormalities in HS structure and distribution in hypoplastic lungs can be expected to perturb HS:protein interactions, ECM microenvironments and crucial epithelial-mesenchyme communication, which may contribute to lung dysmorphogenesis. Indeed, a number of epitopes correlate with structures recognised by FGFs, suggesting a functional consequence of the observed changes in HS in these lungs. These results identify a novel, significant molecular defect in hypoplastic lungs and reveals HS as a potential contributor to hypoplastic lung development in CDH. Finally, these results afford the prospect that HS-mimetic therapeutics could repair defective signalling in hypoplastic lungs, improve lung growth, and reduce CDH mortality.     

Crystal structure of Ufc1, the Ufm1-conjugating enzyme

Ubiquitin and ubiquitin-like protein-conjugating enzymes play central roles in posttranslational modification processes. The ubiquitin-fold modifier 1 (Ufm1), one of a variety of ubiquitin-like modifiers, is covalently attached to target proteins via Uba5 and Ufm1-conjugating enzyme 1 (Ufc1), which are analogous to the E1 and E2 ubiquitylation enzymes. As Ufm1-related proteins are conserved in metazoa and plants, the Ufm1 system likely plays important roles in various multicellular organisms. Herein, we report the X-ray structure of human Ufc1 determined at 1.6 A resolution. The Ufc1 structure comprises a canonical E2 domain and an additional N-terminal domain. The Uba5 binding site on Ufc1 was assigned by structural comparison of Ufc1 and Ubc12 and related mutational analyses. In addition, we show that the N-terminal unique domain of Ufc1 contributes to thermal stability.     

Stable integration and conditional expression of electroporated transgenes in chicken embryos

The in ovo electroporation in chicken embryos has widely been used as a powerful tool to study roles of genes during embryogenesis. However, the conventional electroporation technique fails to retain the expression of transgenes for more than several days because transgenes are not integrated into the genome. To overcome this shortcoming, we have developed a transposon-mediated gene transfer, a novel technique in chicken manipulations. It was previously reported that the transposon Tol2, originally found in medaka fish, facilitates an integration of a transgene into the genome when co-acting with Tol2 transposase. In this study, we co-electroporated a plasmid containing a CAGGS-EGFP cassette cloned in the Tol2 construct along with a transposase-encoding plasmid into early presomitic mesoderm or optic vesicles of chicken embryos. This resulted in persistent expression of EGFP at least until embryonic day 8 (E8) and E12 in somite-derived tissues and developing retina, respectively. The integration of the transgene was confirmed by genomic Southern blotting using chicken cultured cells. We further combined this transposon-mediated gene transfer with the tetracycline-dependent conditional expression system that we also developed recently. With this combined method, expression of a stably integrated transgene could be experimentally induced upon tetracycline administration at relatively late stages such as E6, where a variety of organogenesis are underway. Thus, the techniques proposed in this study provide a novel approach to study the mechanisms of late organogenesis, for which chickens are most suitable model animals.     

Mature and juvenile tissue models of regeneration in small fish species

The multitude of cells constituting organisms are fragile and easily damaged day by day. Therefore, maintenance of tissue morphology and function is fundamental for multicellular organisms to attain long life. For proper maintenance of tissue integrity, organisms must have mechanisms that detect the loss of tissue mass, activate the de novo production of cells, and organize those cells into functional tissues. However, these processes are only poorly understood. Here we give an overview of adult and juvenile tissue regeneration models in small fish species, such as zebrafish and medaka, and highlight recent advances at the molecular level. From these advances, we have come to realize that the epidermal and mesenchymal parts of the regenerating fish fin-that is, the wound epidermis and blastema, respectively-comprise heterogeneous populations of cells with different molecular identities that can be termed "compartments." These compartments and their mutual interactions are thought to play important roles in promoting the proper progression of tissue regeneration. We further describe the current understanding of these compartments and discuss the possible approaches to affording a better understanding of their roles and interactions during regeneration.     

Fabrication of endothelialized tube in collagen gel as starting point for self-developing capillary-like network to construct three-dimensional organs in vitro

A possible strategy for creating three-dimensional (3D) tissue-engineered organs in vitro with similar volumes to the primary organs is to develop a capillary network throughout the constructs to provide sufficient oxygenation and nutrition to the cells composing them. Here, we propose a novel approach for the creation of a capillary-like network in vitro, based on the spontaneous tube-forming activity of vascular endothelial cells (ECs) in collagen gel. We fabricated a linear tube of 500 microm in diameter, the inner surface of which was filled with bovine carotid artery vascular endothelial cells (BECs), in type I collagen gel as a starting point for the formation of a capillary-like network. The BECs exposed to a medium containing vascular endothelial growth factor (VEGF) migrated into the ambient gel around the tube. After 2 weeks of VEGF exposure, the distance of the migration into the ambient gel in the radial direction of the tube reached approximately 800 microm. Cross-sections of capillary-like structures composed of the migrating BECs, with a lumen-like interior space, were observed in slices of the gel around the tube stained with hematoxylin-eosin (H&E). These results demonstrate that this approach using a pre-established tube, which is composed of ECs, as a starting point for a self-developing capillary-like network is potentially useful for constructing 3D organs in vitro.     

Differences in lectin binding patterns of normal human endometrium between proliferative and secretory phases

Lectin binding patterns in normal human endometrium were examined by light and electron microscopy using seven different lectins (ConA, WGA, RCA, PNA, UEA-1, DBA, and SBA). For light microscopic observations, criteria based on the incidence and intensity of cells positive for the lectin staining were adopted to evaluate the different staining patterns of the proliferative and secretory endometria obtained by the avidin-biotin-peroxidase complex (ABC) technique. At the light microscopic level, ConA, WGA, and RCA stained endometrial glandular cells in both phases. The number of PNA-positive cells with the binding sites entirely limited to the apical surface tended to be reduced slightly in the secretory phase. UEA-1 weakly stained the apical surface of glandular cells in the proliferative phase but not in the secretory phase. Among the lectins used in this study, DBA and SBA displayed remarkable changes between the phases. That is, in the proliferative phase they produced only a faint or slight positive stain at the apical surface, but the incidence and intensity of DBA- and the SBA-positive glandular cells increased in the secretory phase. By electron microscopy, the reaction product of ConA was observed in the plasma membrane, endoplasmic reticulum, nuclear envelope, and the Golgi apparatus, and the binding sites of RCA and DBA were observed in the plasma and Golgi membranes. Between both phases, the reactivity of ConA and RCA showed almost no change. However, the secretory endometrial cells containing the DBA-positive Golgi apparatus were markedly increased in number compared with the proliferative ones bearing the lectin-positive organelles.(ABSTRACT TRUNCATED AT 250 WORDS)