L3T4 effector cells in multiple organ-localized autoimmune disease in nude mice grafted with embryonic rat thymus

Rat thymic grafts reconstituted T cell functions of BALB/c nude (nu/nu) mice to a considerable degree, but multiple organ-localized autoimmune diseases such as oophoritis and thyroiditis generally developed. The effector cell population in this autoimmune model was studied by adoptive transfer of the lesions into syngeneic nude mice. The transfer activity was not diminished when spleen cells were incubated with antiserum against rat cell antigen and C, but the activity was completely vanished by incubation with anti-Thy-1.2 plus C, indicating that the effector cells are T cells of mouse origin. Elimination of the L3T4+ subset virtually abolished the transfer activity, whereas that of the Lyt-2+ subset did not, indicating that the effector cells are L3T4+. Positive selection experiments by FACS also demonstrated that L3T4+ cells, but not Lyt-2+ cells, were capable of inducing the lesion, confirming the results with depletion experiments described above.     

Clinical-scale expansion of human cytomegalovirus-specific cytotoxic T lymphocytes from peripheral blood mononuclear cells requiring single-peptide stimulation and feeder cells but not additional antigen-presenting cells

BACKGROUND: The aim of this study was to find a simple and feasible method for ex vivo expansion of human cytomegalovirus (HCMV)-specific cytotoxic T cells from peripheral blood mononuclear cells (PBMNCs) without the aid of exogenous antigen-presenting cells (APCs) such as cultured dendritic cells. STUDY DESIGN AND METHODS: PBMNCs from three HLA-A*2402-seropositive donors were stimulated with HCMV pp65(341-350) peptide on Day 1 and then cultured with interleukin-2 and allogeneic feeder cells for 3 to 4 weeks. HCMV peptide-specific T cells were purified with HLA-A*2402/pp65(341-350) tetramer on Days 12 to 13 and harvested on Days 23 to 27. RESULTS: The initial numbers of PBMNCs were 2 x 10(7), 1.5 x 10(7), and 2.5 x 10(7) and the increases in HCMV peptide-specific T cells were 3.5 x 10(4)-, 2.0 x 10(3)-, and 1.1 x 10(3)-fold, respectively. The estimated final numbers of tetramer-positive cells were 9.1 x 10(7), 9.0 x 10(6), and 5.3 x 10(6), respectively. The purities of the tetramer-positive cell population in culture were 72.6, 75.0, and 80.9 percent, respectively. The cells killed peptide-pulsed B-lympoblastoid cell lines and secreted interferon-gamma in a HLA-restricted manner. They did not have natural killer cell activity or lymphokine activated killer cell activity. Most of them had an effector-memory phenotype. They did not express killer inhibitory receptors. CONCLUSION: This method makes it possible to obtain more than 1 x 10(7) HCMV-specific T cells from approximately 2 x 10(7) to 5 x 10(7) PBMNCs without exogenous APCs such as cultured dendritic cells.     

Lobar dysmorphism of the kidney: reevaluation of junctional parenchyma using helical CT

BACKGROUND: The term "junctional parenchyma" (JP) has been used to represent many renal anomalies including lobar dysmorphism; however, it has not been evaluated with modalities other than ultrasound (US). METHODS: Twenty-two kidneys with lobar dysmorphism incidentally found on helical computed tomography (CT) were studied. In all cases, axial, multiplanar reformation, and three-dimensional images on corticomedullary phase scans were analyzed. Fifteen additional kidneys were prospectively examined with US, and we compared those sonograms with helical CT findings. RESULTS: Comparison of the US and helical CT findings showed that the JP defect and the JP line corresponded anatomically to the upper aspect of the renal sinus and to the thick mural cortex originating from that point, extending inferiorly, respectively. The lesions of lobar dysmorphism were situated deep in the medulla, adjacent to the cortex; however, findings on helical CT did not indicate JP. CONCLUSIONS: Although JP may have been seen on US in this study, it did not show fusion remnants of subkidneys but a combination of the upper aspect of the renal sinus, the mural cortex, and the lesion of lobar dysmorphism.     

Effects of N(G)-monomethyl-L-arginine on Ca(2+) current and nitric-oxide synthase in rat ventricular myocytes

The effects of N(G)-monomethyl-L-arginine (L-NMMA), a nitric-oxide synthase (NOS) inhibitor, on the L-type Ca(2+) current (ICa) and NO effects on NOS were determined in rat ventricular myocytes. L-NMMA (10 and 100 microM) had no significant effect on basal ICa, but in a cAMP-stimulated condition due to forskolin (1 microM) or milrinone (10 microM), a cGMP-inhibited cAMP-phosphodiesterase (PDE), L-NMMA (10 and 100 microM) concentration dependently augmented ICa. The enhancing effects of L-NMMA (10 and 100 microM) on ICa were not seen in the presence of either a nonselective inhibitor of PDE, 3-isobutyl-1-methylxanthine (20 microM), resulting in a stimulated ICa condition or a cGMP-dependent protein kinase activator, 8-bromo-cGMP (200 microM). 8-Bromo-cGMP (200 microM) inhibited 100 microM L-NMMA-induced ICa increase in the simultaneous application of forskolin (1 microM). Acetylcholine (ACh; 1 and 3 microM) inhibited 1 microM forskolin-stimulated ICa in a concentration-dependent manner, but this inhibitory action of ACh was significantly attenuated by the additional application of L-NMMA (100 microM). In the continuing presence of both L-NMMA (100 microM) and forskolin (1 microM), ACh (6 microM) had no inhibitory effect on ICa. In another series of experiments with isolated ventricular myocytes, we obtained both the positive staining of NADPH-diaphorase activity and the expression of the endothelial isoform of NOS. These data suggest that the effect of L-NMMA on ICa in a cAMP-stimulated condition with or without cholinergic inhibition is due to inhibition (acute effects) of a cGMP-stimulated cAMP-PDE via inhibition of the endothelial isoform of NOS.     

Molecular cloning and nucleotide sequence of complementary DNAs encoding human short chain acyl-coenzyme A dehydrogenase and the study of the molecular basis of human short chain acyl-coenzyme A dehydrogenase deficiency.

Complementary DNAs encoding the precursor of human placental short chain acyl-coenzyme A (CoA) dehydrogenase (SCAD) (EC 1.3.99.2) were cloned and sequenced. The cDNA inserts in these clones were 1,852 bases in length combined, and encoded the entire 412-amino acid precursor SCAD (mol wt 44,303). This sequence included the 24-amino acid leader peptide moiety (mol wt 2,576) and 388 amino acids corresponding to the mature protein (mol wt 41,727). The comparison of SCAD and medium chain acyl-CoA dehydrogenase sequences revealed a high degree of homology, suggesting that these enzymes evolved from a common ancestral gene and belong to a gene family. We also studied mutant human SCAD in cultured skin fibroblasts from three patients with hereditary SCAD deficiency. Labeling fibroblast cultures with [35S]-methionine followed by immunoprecipitation with anti-SCAD antibody revealed that a normal size variant SCAD protein was synthesized. In all of the three SCAD-deficient cell lines, the size of variant SCAD mRNA as determined by Northern blotting using one of the normal SCAD cDNA as a probe was also normal, and no difference was observed on Southern blots in the restriction patterns of mutant genomic DNA using EcoRI, TaqI, HincII, and BamHI. These results suggest that the defects in SCAD in these cell lines are caused by a point mutation.