Split tolerance between spleen and lymph node cells in severe combined immunodeficiency mice grafted with AKR fetal liver cells

Severe combined Immunodeficient (SCID) mice defective in stemcells for T and B cells appear to be an ideal host for constructionof chimeric mice. When bone marrow cells are used as a sourceof stem cells, however, host SCID mice do not always show sufficientreconstitutlon. In this study, fetal liver cells from AKR embryoswere transplanted into SCID mice without prior irradiation.This treatment induced full reconstltution of lymphopoiesisas evaluated by flow cytometry analysis and serum Ig production2 months after transplantation. Thus, fetal liver cells seemto be a better source for reconstitutlon of SCID mice than bonemarrow cells. Lymph node (LN) cells of these mice (FLT mice)had no proliferatlve or cytotoxlc activities against eitherhost-type (C.B-17) or donor-type (AKR) spleen cells. However,spleen cells from FLT mice exhibited marked proliferatlve andcytotoxlc activities against C.B-17 cells, with no activitiesagainst AKR cells. Spirt tolerance against C.B-17 cells In spleenand LN cells was not a transient phenomenon, since similar resultswere obtained from a cytotoxic T lymphocyte assay 4 months later.In spite of the strong host reactivity in vitro, aberrationof clonal deletion or development of a graft-versus-host diseasewas not seen in FLT mice. As IL-2 induced the host reactivityof LN cells in a mixed lymphocyte reaction, potentially host-reactiveT cells were present in LN but were rendered anerglc. Tolerancein FLT mice seems to be regulated by a peripheral mechanism.We supposed that the split tolerance in FLT mice was inducedby the different antigenicity between the spleen and LN.     

Rapid diagnosis of human herpesvirus 6 infection by a novel DNA amplification method, loop-mediated isothermal amplification

A novel nucleic acid amplification method, termed loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions, may be a valuable tool for the rapid detection of infectious agents. LAMP was developed for human herpesvirus 6 (HHV-6), and its reliability was evaluated in this study. Although LAMP products were detected in HHV-6 B and HHV-6 A DNA, they were not detected in HHV-7 and human cytomegalovirus DNA. The sensitivity of the original HHV-6 LAMP protocol was 50 copies/tube. In order to increase the method's sensitivity, HHV-6 LAMP was modified by increasing the primer concentration. As a result of the modification, sensitivity increased to 25 copies/tube. After these initial validation studies, 13 patients with fever were tested for HHV-6 by viral isolation, serological analysis, and HHV-6 LAMP. In three of the eight patients with primary HHV-6 infection, HHV-6 DNA was detected in whole blood by the original HHV-6 LAMP protocol in not only the acute phase but also the convalescent phase. HHV-6 DNA was detected by modified HHV-6 LAMP in all eight plasma samples collected in the acute phase; however, no HHV-6 DNA was detected in plasma samples collected in the convalescent phase. Although HHV-6 DNA was detected in both the acute and convalescent phases of whole-blood samples in patients with past HHV-6 infection, it was not detected in plasma samples that did not contain latent viral DNA. Thus, detection of HHV-6 DNA in plasma by using this modified HHV-6 LAMP protocol is appropriate for diagnosis of active HHV-6 infection.     

Saliva-binding region of Streptococcus mutans surface protein antigen.

A 190-kDa surface protein antigen (PAc) of Streptococcus mutans binds to human salivary components. For detection of specific binding of the PAc protein to human salivary components, a simple sandwich assay was used. Microtiter plates precoated with recombinant PAc (rPAc), PAc fragments, or S. mutans whole cells were allowed to react with human whole saliva and then were incubated with biotinylated rPAc. The biotinylated rPAc bound to salivary components was detected by use of alkaline phosphatase-conjugated streptavidin and p-nitrophenylphosphate. In this assay, the binding of whole cells of S. mutans and purified rPAc to salivary components was confirmed. For determination of a saliva-binding region of the PAc molecule, 14 truncated PAc fragments were constructed by use of the polymerase chain reaction and an expression vector, pAX4a+. The binding of these truncated PAc fragments to human salivary components was determined by the sandwich assay. Among the truncated PAc fragments, fragments corresponding to residues 39 to 864 and residues 39 to 1000 of PAc showed a high ability to bind to salivary components. Shorter recombinant fragments corresponding to residues 39 to 217, residues 200 to 481, residues 470 to 749, and residues 688 to 864 did not exhibit any binding ability. The fragment that corresponds to a proline-rich repeating region (residues 828 to 1000) bound directly to the PAc protein. These results suggest that residues 39 864 of the PAc molecule are important in the binding of the surface protein to human salivary components, and the proline-rich repeating region of the PAc protein may contribute to spontaneous self-aggregation of the PAc protein.     

Alpha-fetoprotein-producing immature mediastinal teratoma showing rapid and massive recurrent growth in an adult.

A case of immature mediastinal teratoma in a 43-year-old Japanese man is reported. The tumor, which was multicystic with solid zones and measured 12 x 6 x 8 cm, arose in the anterior mediastinum. The serum alpha-fetoprotein (AFP) level was elevated to 5,114 ng/ml before surgery. Histologically, the solid zones showed an admixture of irregular glands lined by columnar or cuboidal epithelium set in a spindle cell stroma, some foci of primitive neural tissue, and scattered small nests of hepatoid cells. Immunohistochemically, the hepatoid cells and epithelia lining some of the cysts showed a strongly positive reaction for AFP. Eight months after surgery, the patient died of respiratory failure caused by a rapidly growing massive recurrent tumor, which measured 40 x 24 x 13 cm, in the left thoracic cavity. However, the elevated serum AFP level had been decreasing during the course of the recurrence in response to chemotherapy. The recurrent tumor showed remarkable proliferation of loose mesenchymal tissue without primitive neural tissue. These findings suggest that immature mediastinal teratoma in adults is highly malignant, and that non-AFP-producing mesenchymal tissue played a critical role in forming the rapidly growing massive recurrent tumor in the present case.     

Method for In Situ Evaluation of Superoxide Production by Pulmonary Macrophages in the Rat

In order to investigate superoxide production by pulmonary macrophages in the rat, a route was created by ligating both the inferior and superior venae cavae and resecting the aorta after cannulation through the inferior vena cava into the right atrium of the heart. Lung perfusion was performed via this route with nitro blue tetrazolium. Although there was no formazan deposition throughout the lung, it became detectable in both alveolar and interstitial macrophages when phorbol myristate acetate was added to the perfusate. This deposition was markedly enhanced by previous injection of Corynebacterium parvum. The deposition disappeared after further addition of Cu(Lys)₂, a scavenger of superoxide anions. This procedure may be useful for estimating in situ the ability of pulmonary macrophages to produce superoxide in the rat.     

Association of Actinobacillus actinomycetemcomitans leukotoxin with nucleic acids on the bacterial cell surface.

Actinobacillus actinomycetemcomitans, a periodontopathic gram-negative bacterium, produces a leukotoxin that is a member of the RTX cytotoxin family. Although genes may function in toxin secretion, the leukotoxin is not secreted extracellularly but remains associated with the bacterial cell surface. We report here that this toxin-cell surface association is mediated by nucleic acids and directly demonstrate that the extracellular secretion of toxin occurs in growing cultures with increased ionic strength of medium. All examinations were performed with freshly harvested A. actinomycetemcomitans 301-b from anaerobic fructose-limited chemostat cultures. The occurrence of cell surface-localized DNA was shown by directly digesting whole cells with the restriction endonuclease EcoRI or HindIII, which yielded many DNA fragments. The cell surface DNA constituted about 20% of the total cellular DNA. The leukotoxin was released from the whole cells by digestion with DNase I as well as restriction endonucleases. Because the leukotoxin binds ionically to DNA, it is dependent on the ionic strength of buffers or media. Accordingly, the toxin was released from cells suspended in saline at pH 7.5 in the presence of increasing amounts of MgCl2 (0 to 10 mM) or NaCl (0 to 50 mM). Moreover, a considerable quantity of leukotoxin was detected in the culture supernatant of fructose-limited chemostat cultures when sodium succinate solution was pumped into the steady state as an additional salt (30 and then 50 mM). This toxin-DNA association was also found in well-characterized strains including not only the leukotoxin-producing ATCC 29522 but also the toxin production-variable ATCC 29523 and the non-leukotoxin-producing ATCC 33384 when these strains were grown in the chemostat culture.     

Inherited partial duplication of chromosome No. 15

A boy with unusual facial appearance and mental retardation was found to have duplication for the distal half of the long arm of chromosome No. 15 and possibly deficiency for the distal end of the long arm of No. 21. The chromosome abnormality was inherited from his mother, who had a translocation involving chromosomes Nos. 15 and 21. Giemsa-banding localized the break point in chromosome No. 15 just distal to the intense band at the midportion of the long arm. The break point in chromosome No. 21 appeared to be at the distal end of the long arm. The difficulty encountered in cytogenetic analysis of the propositus with conventional staining, the importance of chromosome analysis of the parents, and the application of differential staining techniques are also presented.     

Functional expression of the ascofuranone-sensitive Trypanosoma brucei brucei alternative oxidase in the cytoplasmic membrane of Escherichia coli

Trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms (LS forms) of African trypanosoma, which causes sleeping sickness in human and nagana in cattle. TAO is a cytochrome-independent, cyanide-insensitive quinol oxidase and these properties are quite different from those of the bacterial quinol oxidase which belongs to the heme-copper terminal oxidase superfamily. Only little information concerning the molecular structure and enzymatic features of TAO have been available, whereas the bacterial enzyme has been well characterized. In this study, a cDNA encoding TAO from Trypanosoma brucei brucei was cloned into the expression vector pET15b (pTAO) and recombinant TAO was expressed in Escherichia coli. The growth of the transformant carrying pTAO was cyanide-resistant. A peptide with a molecular mass of 37 kDa was found in the cytoplasmic membrane of E. coli, and was recognized by antibodies against plant-type alternative oxidases from Sauromatum guttatum and Hansenula anomala. Both the ubiquinol oxidase and succinate oxidase activities found in the membrane of the transformant were insensitive to cyanide, while those of the control strain, which contained vector alone, were inhibited. This cyanide-insensitive growth of the E. coli carrying pTAO was inhibited by the addition of ascofuranone, a potent and specific inhibitor of TAO ubiquinol oxidase. The ubiquinol oxidase activity of the membrane from the transformant was sensitive to ascofuranone. These results clearly show the functional expression of TAO in E. coli and indicate that ubiquinol-8 in the E. coli membrane is able to serve as an electron donor to the recombinant enzyme and confer cyanide-resistant and ascofuranone-sensitive growth to E. coli. This system will facilitate the biochemical characterization of the novel terminal oxidase, TAO, and the understanding on the mechanism of the trypanocidal effect of ascofuranone.