Natural killer cells in the thymus. Studies in mice with severe combined immune deficiency

The relationship between NK cell and T cell progenitors was investigated by using mice with severe combined immune deficiency (scid). Scid mice are devoid of mature T and B cells because they cannot rearrange their Ig and TCR genes. However, they have normal splenic NK cells. Thymus of scid mice, although markedly hypocellular, contains cells that lyse YAC-1, an NK-sensitive tumor cell. By flow cytometry, two populations of cells were identified in the scid thymus. Eighty percent of the cells were Thy-1+, IL-2R(7D4)+, J11d+, CD3-, CD4-, CD8- whereas the remaining were IL-2R-, J11d-, CD3-, CD4-, and CD8-. By cell sorting, all NK activity was found in the latter population, which is phenotypically similar to splenic NK cells. To determine if the thymus contains a bipotential NK/T progenitor cell, J11d+, IL-2R+ cells were cultured and analyzed for the generation of NK cells in vitro. These cells were used because they resemble 15-day fetal and adult CD4- CD8- thymocytes that are capable of giving rise to mature T cells. Cultured J11d+ thymocytes acquired non-MHC-restricted cytotoxicity, but in contrast to mature NK cells, the resulting cells contained mRNA for the gamma, delta, and epsilon-chains of CD3. This suggests that J11d+ cells are early T cells that can acquire the ability to kill in a non-MHC-restricted manner, but which do not give rise to NK cells in vitro. The differentiative potential of scid thymocytes was also tested in vivo. Unlike bone marrow cells, scid thymocytes containing 80% J11d+ cells failed to give rise to NK cells when transferred into irradiated recipients. Together these results suggest that mature NK cells reside in the thymus of scid mice but are not derived from a common NK/T progenitor.     

Adoptive transfer of CD8+ T cells from immune animals does not transfer immunity to blood stage Plasmodium yoelii malaria

The malaria parasite, Plasmodium yoelii 17X, causes a self-limited, nonlethal infection characterized, in the blood stage, by preferential invasion of reticulocytes. Previous studies have suggested that immunity to the blood stage infection may be related to enhanced levels of class I MHC Ag on the parasitized reticulocyte surface and can be adoptively transferred to immunodeficient mice by immune CD8+ T cells in the absence of CD4+ T cells. To further examine the mechanisms of CD8+ T cell involvement in immunity to blood stage P. yoelii infection, we performed in vivo CD8 depletion and adoptive transfer experiments. Depletion of CD8+ T cells during primary blood stage infection in BALB/c mice did not diminish the ability of the mice to resolve their infections. Spleen cells from immune BALB/c and C57BL/10 mice were transferred to BALB/c-nu/nu and C57BL/10-nu/nu mice, respectively. The recipient mice were CD4 depleted in vivo to kill any transferred CD4+ T cells. The mice failed to control the infection. Populations of CD4-, CD8+ T cells were transferred from immune CBA/CaJ donors to in vivo CD4-depleted CBA/CaJ recipients. The mice were unable to control the infection. Although immune unfractionated spleen cells transferred rapid protection in all three mouse strains and immune CD4+ T cells transferred immunity in the two mouse strains studied, CD8+ T cells by themselves were neither protective nor did they enhance immunity.     

Structure of testicular angiotensin-converting enzyme. A segmental mosaic isozyme

The complete amino acid sequence of rabbit testicular angiotensin-converting enzyme has been deduced from the sequence of the corresponding cDNA clone. A protein of the expected molecular weight of 84,000 was translated in vitro from the mRNA encoded by this cDNA. All of the previously determined sequences of seven tryptic peptides from the enzyme are present in the deduced sequence, thus confirming the identity of the protein. From the deduced sequence it appears that the protein contains a signal peptide at the amino terminus and a hydrophobic anchoring domain near the carboxyl terminus. Northern analysis with oligonucleotide probes, whose sequences represented different regions of the cDNA, revealed not only the regions of extensive homology between the mRNAs encoding the testicular and the pulmonary isozymes but also a stretch of sequence near the 5' end unique to the testicular mRNA.     

Spectral studies of bovine dopamine beta-hydroxylase. Absence of covalently bound pyrroloquinoline quinone

Bovine dopamine beta-hydroxylase was examined spectroscopically for the presence of covalently bound pyrroloquinoline quinone (PQQ). Pure dopamine beta-hydroxylase had a featureless UV-visible spectrum above 300 nm. An equimolar solution of dopamine beta-hydroxylase and exogenously added PQQ (1 PQQ/active site) had a strong absorption maximum at 333 nm. Dialysis removed the added PQQ, indicating that dopamine beta-hydroxylase does not bind PQQ irreversibly. Reaction of dopamine beta-hydroxylase with 6 mM phenylhydrazine in the presence of 15 mM ascorbate caused 96% inactivation within 20 min and did not produce any spectrally detectable amounts of the phenylhydrazone adduct of PQQ, as reported by van der Meer et al. (van der Meer, R.A., Jongejan, J.A., and Duine, J.A. (1988) FEBS Lett. 231, 303-307). The peptide profile of phenylhydrazine inactivated dopamine beta-hydroxylase was monitored at 316 nm and did not reveal any peptides that might contain a PQQ-phenylhydrazone adduct. Thus, the absence of any spectrally detectable PQQ-phenylhydrazone adducts under these conditions demonstrates that the mechanism of phenylhydrazine inactivation does not involve covalent modification of PQQ at the active site of dopamine beta-hydroxylase and provides strong evidence that the native enzyme does not contain PQQ.     

Effector cell expression of NK1.1, a murine natural killer cell-specific molecule, and ability of mice to reject bone marrow allografts

The rejection of Hh-1 incompatible bone marrow cells in irradiated mice is mediated by NK cells and is genetically regulated. We tested the role of the NK-specific gene, NK1.1, in regulating the rejection of allogeneic bone marrow cell grafts. NK1.1+ mice, that are known to display strong resistance against Hh-1 incompatible grafts, were crossed to H-2/Hh-1 identical NK1.1-, poor responder mice, and the progeny were backcrossed to the poor responder parent. The segregating mice were individually typed for their expression of NK1.1 and the ability to resist Hh-1 incompatible bone marrow cells (BMC). A strong correlation was noted between expression of NK1.1 and rejection of H-2d/Hh-1d BMC. Our results support the idea that NK1.1 is one of the genes responsible for strong resistance to Hh-1d (determinant 2) but not for Hh-1j (determinant 3) BMC grafts. We suggest that the NK1.1 molecule functions as an accessory molecule in the cellular interactions involving the recognition of Hh-1 determinants.     

Nucleotide sequence and distinctive characteristics of the env gene of endogenous feline leukemia provirus.

Nucleotide sequence analysis of the env gene of two different endogenous feline leukemia virus (FeLV) loci, CFE-6 and CFE-16, of domestic cats revealed the following characteristics. (i) Both proviruses contain an open reading frame in the env region; (ii) whereas the full complement of the exogenous FeLV env is generally present in CFE-6 DNA, it is truncated in CFE-16 DNA such that the 5' half of the gp70 domain and the untranslated region 3' to the p15E domain have been fused by an internal deletion, resulting in loss of the C-terminal half of the gp70- and all of the p15E-coding sequences; (iii) endogenous env is highly homologous to large sequence domains conserved in all three exogenous FeLV subgroups (A, B, and C) but is similar to FeLV-B sequence domains in the variable regions detected in these viruses; and (iv) there are four other sequence domains, one residing at the C terminus of gp70 and three scattered in p15E, which are unique for the endogenous env, thereby distinguishing it from the FeLV-B gene.     

Colloid carcinoma of the breast with concomitant metastasis and a tuberculous lesion in the axillary lymph nodes. A case report.

A 30-year-old woman presented with a lump in the left breast and left axillary lymphadenopathy that, on fine needle aspiration cytology (FNAC), proved to be duct cell carcinoma with metastasis. Histology of the radical mastectomy specimen showed a mixed colloid carcinoma. Axillary lymph nodes revealed a variety of pathologic changes consisting of reactive hyperplasia, tuberculosis and metastasis. A combination of a tuberculous lesion and metastasis in the same lymph nodes was also found. During follow-up, after radiotherapy, the patient developed left supraclavicular and right cervical lymphadenopathy that, on FNAC, revealed a tuberculous lesion and metastasis, respectively. The rarity of this condition with double pathology is highlighted, and the reason behind the limitations of FNA in subtyping the primary malignancy and its failure to detect the tuberculous lesion in the axillary lymph node are discussed.     

Extracellular protease from the antarctic yeast Candida humicola.

The psychrotrophic, dimorphic yeast Candida humicola, isolated from Antarctic soil, secretes an acidic protease into the medium. The secretion of this protease by C. humicola was found to be dependent on the composition of the medium. In YPD or yeast nitrogen base medium containing either amino acids or ammonium sulfate as the nitrogen source, the activity of the protease in the medium was low (basal level). However, when yeast nitrogen base medium was depleted of amino acids or ammonium sulfate and supplemented with proteins, the activity of the enzyme increased. The secretion of the enzyme was greater during exponential growth at low temperatures than during growth at higher temperatures. The purified protease had a molecular mass of 36,000 Da and was inhibited by pepstatin, iodoacetamide, and sodium dodecyl sulfate. Despite the prevalent cold temperatures in Antarctica, this extracellular protease of the psychrotrophic yeast C. humicola was active at temperatures ranging from 0 to 45 degrees C, with an optimum activity at 37 degrees C.     

Cross-linking activity of the 14-kilodalton beta-galactoside-specific vertebrate lectin with asialofetuin: comparison with several galactose-specific plant lectins.

We have previously shown that plant lectins with a wide range of carbohydrate binding specificities can bind and cross-link (precipitate) specific multiantennary oligosaccharides and glycopeptides [cf. Bhattacharyya, L., Fant, J., Lonn, H., & Brewer, C. F. (1990) Biochemistry 29, 7523-7530]. This leads to a new source of binding specificity: namely, the formation of homogeneous cross-linked lattices between lectins and carbohydrates. Recently, we have demonstrated the existence of highly ordered cross-linked lattices that form between the D-Man/D-Glc-specific plant lectin concanavalin A and the soybean agglutinin which is a tetrameric glycoprotein possessing a single Man9 oligomannose chain per monomer [Khan, M. I., Mandal, D. K., & Brewer, C. F. (1991) Carbohydr. Res. 213, 69-77]. In the present study, we have compared the ability of the 14-kDa beta-galactoside-specific lectin from calf spleen, a dimeric S-type animal lectin, and several galactose-specific plant lectins from Erythrina indica, Erythrina cristagalli, and Glycine max (soybean agglutinin) to form specific cross-linked complexes with asialofetuin (ASF), a 48-kDa monomeric glycoprotein, using quantitative precipitation analyses. The results show the formation of 1:9 and 1:3 stoichiometric cross-linked complexes (per monomer) of ASF to the 14-kDa lectin, depending on their relative ratio in solution. Evidence indicates that the three triantennary N-linked complex-type oligosaccharide chains of ASF mediate the cross-linking interactions and that each chain expresses either trivalency in the 1:9 cross-linked complex or univalency in the 1:3 complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretion of a platelet-derived growth factor homologue by rat alveolar macrophages exposed to particulates in vitro

Lung macrophages secrete a homologue of platelet-derived growth factor (PDGF) which induces the proliferation of fibroblasts in vitro. In previous studies, we showed that such a PDGF homologue is produced by rat alveolar macrophages and that rat lung fibroblasts have specific receptors for the macrophage-derived PDGF. In this study, we demonstrate the biological and physicochemical properties of the growth factor, as well as the time-related production of this factor following macrophage activation in vitro by organic and inorganic particles. Alveolar macrophages (AMs) collected by saline lavage from the lungs of rats were cultured in serum-free Dulbecco's modified Eagle's medium (SF-DMEM) for varying periods of time up to 72 h. The SF-DMEM "conditioned" by the AMs was used to treat early passage rat lung fibroblasts (RLFs), which were rendered quiescent by culturing in 2% platelet-poor plasma (PPP). Alveolar macrophage conditioned media (AMCM) in the presence of PPP caused increases in the number of fibroblasts, the percent of labeled fibroblast nuclei and tritiated [3H]thymidine incorporation. AMCM alone caused no detectable changes in fibroblast growth rate. These results indicate that AMs release a "competence-like" growth factor. The AMs were left untreated or were exposed to opsonized zymosan, carbonyl iron spheres or chrysotile asbestos fibers. Macrophages attached to a plastic substrate spontaneously produced the factor, and subsequent addition of the organic and inorganic particles to the macrophage cultures significantly increased the fibroblast-stimulating activity of the AMCM. The growth factor was stable after concentration (100-fold), lyophilization and reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)