A phage display system for detection of T cell receptor-antigen interactions

The process of T cell recognition involves a complex set of interactions between the various components of the TCR/MHC/peptide trimolecular complex. We have developed a system for exploring the specific binding interactions contributed by the constituent subunits of TCR complexes for components of their ligands. We utilized an M13 phage display system, designed for multivalent receptor display, to explore specific binding interactions between various TCRα chains and specific antigen in the absence of MHC. The multivalent TCR-phage display system was sensitive enough to reveal some TCRα chains capable of binding directly to antigen with the same fine specificity shown by the MHC-restricted T cells from which the α chains were derived. Cross-specificity analysis using two antigen-binding TCRα chains derived from T cells with different polypeptide antigen specificities confirmed the fidelity of this binding. In mixtures of antigen-binding and non-binding TCRα-displaying phage, specific selection was achieved at a starting frequency of 1/1000, suggesting that this system can be employed for selection and analysis of TCR-displaying phage libraries. While the binding specificities exhibited by these TCRs are unusual, they provide a novel perspective from which to study the specific binding interactions that constitute TCR antigen binding.     

Lon,a stress-induced ATP-dependent protease,is critically important for systemic Salmonella enterica serovar typhimurium infection of mice

Studies on the pathogenesis of Salmonella enterica serovar Typhimurium infections in mice have revealed the presence of two prominent virulence characteristics-the invasion of the nonphagocytic cells to penetrate the intestinal epithelium and the proliferation within host phagocytic cells to cause a systemic spread and the colonization of host organs. We have recently demonstrated that the ATP-dependent Lon protease of S. enterica serovar Typhimurium negatively regulates the efficiency of invasion of epithelial cells and the expression of invasion genes (A. Takaya et al., J. Bacteriol. 184:224-232, 2002). This study was performed to reveal the contribution of the Lon protease to the virulence of S. enterica serovar Typhimurium in mice. Determination of 50% lethal doses for the lon disruption mutant and wild-type strain revealed that the mutant was highly attenuated when administered either orally or intraperitoneally to BALB/c mice. The mutant was also found to be able to reach extraintestinal sites but unable to proliferate efficiently within the spleen and cause lethal systemic disease of mice. Macrophage survival assays revealed that the lon disruption mutant could not survive or proliferate within murine macrophages. In addition, the mutant showed extremely increased susceptibility to hydrogen peroxide, which contributes to the bactericidal capacity of phagocytes. The mutant also showed increased sensitivity to acidic conditions. Taken together, the impaired ability of the lon disruption mutant to survive and grow in macrophages could be due to the enhanced susceptibility to the oxygen-dependent killing mechanism associated with respiratory burst and the low phagosomal pH. These results suggest that the Lon protease is essentially involved in the systemic infection of mice with S. enterica serovar Typhimurium, which can be fatal. Of further interest is the finding that the lon disruption mutant persists in the BALB/c mice for long periods without causing an overwhelming systemic infection.     

Neurotoxicity of Clostridium perfringens Epsilon-Toxin for the Rat Hippocampus via the Glutamatergic System

The neurotoxicity of epsilon-toxin, one of the major lethal toxins produced by Clostridium perfringens type B, was studied by histological examination of the rat brain. When the toxin was injected intravenously at a lethal dose (100 ng/kg), neuronal damage was observed in many areas of the brain. Injection of the toxin at a sublethal dose (50 ng/kg) caused neuronal damage predominantly in the hippocampus: pyramidal cells in the hippocampus showed marked shrinkage and karyopyknosis, or so-called dark cells. The dark cells lost the immunoreactivity to microtubule-associated protein-2, a postsynaptic somal and dendric marker, while acetylcholinesterase-positive fibers were not affected. Timm’s zinc staining revealed that zinc ions were depleted in the mossy layers of the CA3 subfield containing glutamate as a synaptic transmitter. The cerebral blood flow in the hippocampus was not altered significantly before or after administration of the toxin, as measured by laser-Doppler flowmetry, excluding the possibility that the observed histological change was due to a secondary effect of ischemia in the hippocampus. Prior injection of either a glutamate release inhibitor or a glutamate receptor antagonist protected the hippocampus from the neuronal damage caused by epsilon-toxin. These results suggest that epsilon-toxin acts on the glutamatergic system and evokes excessive release of glutamate, leading to neuronal damage.     

LIGHT AND ELECTRON MICROSCOPIC STUDY OF NONFUNCTIONING PARATHYROID CARCINOMA

A case of nonfunctioning parathyroid carcinoma in a 69-year-old female has been studied by light and electron microscopy. The tumor, located on the left side of the anterior neck, was well encapsulated by connective tissue but showed invasion to the capsule and to the thyroid. The tumor cells exhibited a trabecular arrangement surrounded by capillary networks but focally showed several ductal structures. They were polygonal in shape, had a large nucleus showing frequent mitosis and poor cytoplasm containing glycogen. Some tumor cells had clear and abundant cytoplasm, and resembled water-clear cells of the parathyroid. Immunohistochemically, no thyroglobulin was demonstrated in the tumor tissue. Electronmicroscopically, the tumor cells with high N/C ratio contained poorly developed cell organelles and abundant glycogen particles. They were poor in secretory granules and had no conglomeration of lipid. Desmosomes and tonoflbrils were observed. The ratio of the reported number of nonfunctioning parathyroid carcinoma to that of functioning one in Japan was compared with that in western countries. No difference of the ratio was found between these two, when identical criteria were employed.     

Biomimetic porous scaffolds with high elasticity made from mineralized collagen--an animal study

Histological investigations of a new hydroxyapatite-collagen composite material were carried out to evaluate its possible suitability as a bone substitute. The three-dimensional scaffolds made from biomimetically mineralized collagen exhibit an interconnecting pore structure and elastic mechanical properties. They were implanted into the subcutaneous tissue and bone defects made in the femur of rats and harvested with the surrounding tissue at 1, 2, 4, 8, and 12 weeks after surgery. The materials implanted in the subcutaneous tissue were covered by fibrous connective tissue with a slight inflammatory response, and many foreign-body giant cells were observed on the surface of the scaffolds. Most of the material implanted in the subcutaneous tissue was resorbed at 8 weeks by phagocytosis. In the bone defects, new bone formation was observed on the surface of the material at 1 week. New bone increased with time, and osteoclasts were seen on the surface of the scaffolds at 2 weeks. Resorption and replacement by new bone of many parts of the materials implanted in the femur were observed by 12 weeks. These responses occurred faster than those of other hydroxyapatite-collagen composites. The results suggested that the new biomimetically mineralized collagen scaffolds were suitable as an implant material for bone-tissue reconstruction.     

Massive apoptosis in infantile myofibromatosis. A putative mechanism of tumor regression.

Two cases of solitary infantile myofibromatosis (IM) are presented. Solitary IM are tumors prone to spontaneous regression. Histopathologically, several tumor lobules in our IM cases had central areas of massive cell death, with nuclear pyknosis, cytoplasmic hyalinization and nuclear fragmentation but without lymphoid or neutrophilic cell infiltration. These central cell death areas consisted of about 40% in case 2 and 50% in case 1 of the entire tumor tissues, respectively. Electron microscopy revealed that the condensed nuclei and cytoplasm were fragmented into "apoptotic bodies", with or without phagocytosis by histiocytes. DNA fragmentation, as evidenced by the terminal deoxy transferase-mediated uptake of biotinylated dUTP, was identified at massive cell death areas on paraffin sections from both cases. A characteristic 180- to 190-bp nucleosomal ladder was detected in DNA obtained from the tumor cells in case 1. The collective evidence suggested that these tumors underwent a central, massive apoptosis. As massive cell death similar to that seen in the present cases has been described in other documented cases of IM, we propose that the spontaneous regression that frequently occurs with this type of tumor may be mediated by massive apoptotic cell death.     

Cloning of a Novel Babesia equi Gene Encoding a 158-Kilodalton Protein Useful for Serological Diagnosis

In this study, we characterized a Babesia equi Be158 gene obtained by immunoscreening a B. equi cDNA expression phage library with B. equi-infected horse serum. The Be158 gene consists of an open reading frame of 3,510 nucleotides. The recombinant Be158 gene product was produced in Escherichia coli and used for the immunization of mice. In Western blot analysis, mouse immune serum against the Be158 gene product recognized 75- and 158-kDa proteins from the lysate of B. equi-infected erythrocytes. In an indirect fluorescent-antibody test with the mouse immune serum, the Be158 antigen appeared in the cytoplasm of Maltese cross-forming parasites (which consist of four merozoites) and was located mainly in the extraerythrocytic merozoite body. When the recombinant Be158 gene product was used in an enzyme-linked immunosorbent assay as a serological antigen, it was found to react to B. equi-infected horse sera, indicating that the Be158 gene product is useful as a serologically diagnostic antigen for B. equi infection.     

Epidemiology of Campylobacter jejuni isolated from patients with Guillain-Barré and Fisher syndromes in Japan

Campylobacter jejuni isolation is the standard for the diagnosis of this type of bacterial infection, but there have been no epidemiological studies of a large number of C. jejuni isolates from patients with Guillain-Barre syndrome (GBS) and Fisher syndrome (FS). For 13 years, stool specimens from GBS/FS patients have been sent from 378 hospitals throughout Japan to the Tokyo Metropolitan Institute of Public Health. A total of 113 strains (11%) were isolated from the stool specimens from 1,049 patients. The isolation rate did not differ by region. The rates were 22% for 449 patients with a history of diarrhea and 2% for the others. An additional 18 isolates were provided by various hospitals. There was no noticeable seasonal distribution in the onset of C. jejuni isolated from patients with GBS/FS. The male/female ratios were 1.7:1 for GBS and 2.2:1 for FS. The patient age range showed a peak in 10- to 30-year-old subjects who had GBS and in 10- to 20-year-old subjects who had FS. The predominance of young adults and male patients who had C. jejuni-associated GBS/FS may be related to the preponderance of young adults and male patients who had C. jejuni enteritis. The median interval from diarrhea onset to neurologic symptom onset was 10 days for GBS/FS. Penner's C. jejuni serotype HS:19 was more frequently present in GBS (67%) than in enteritis (6%) patients. HS:2 was more frequent in FS (41%) than in enteritis (14%) patients. These findings suggest that certain C. jejuni strains specifically trigger GBS and that others specifically trigger FS.