Role of cytotoxic T lymphocytes in the prevention of lupus-like disease occurring in a murine model of graft-vs-host disease

The inoculation of B6D2F1 mice with T lymphocytes from the C57BL/6 parental strain induces an "immunosuppressive" graft-vs-host reaction (B6 GVH), whereas inoculation of T cells from the other, DBA/2 parental strain induces an "immunostimulatory" GVH reaction and a lupus-like disease (DBA GVH). The present study compares cytotoxic T lymphocyte (CTL) function in the spleens of these GVH mice as well as differences in the donor inoculum that could account for these different types of GVH. We observed that the B6 GVH induces an immunodeficiency that encompasses CTL precursors (and possibly T helper cells) and results in suppressor cells that abrogate responses to both trinitrophenyl (TNP)-modified self and third party alloantigens. In contrast, the DBA GVH induces only a T helper cell immunodeficiency and results in suppressor cells selective for class II restricted L3T4+ T helper cells. Chimeric T cells were detected in both types of GVH. In the B6 GVH both L3T4+ and Lyt-2+ donor cells were observed, although Lyt-2+ cells predominated. In the DBA GVH, donor T cells were almost exclusively of the L3T4+ phenotype. The lack of appreciable donor Lyt-2+ cells in the DBA GVH can be explained by a defect in the DBA donor inoculum manifested by a naturally occurring two-fold reduction in Lyt-2+ cell numbers as well as a nine-fold reduction in CTL precursors with anti-F1 specificity. T cells in the DBA inoculum, therefore, are predominantly L3T4+. A similar defect induced in B6 donor cells by anti-Lyt2 antibody and complement not only converted the suppressive GVH to a stimulatory GVH, as measured by anti-DNA antibodies, but also resulted in a T cell immune deficiency characteristic of the DBA GVH, i.e., a selective loss of the TNP-self CTL response. Thus the presence or absence of adequate numbers of functioning Lyt-2+ cells in the donor inoculum is correlated with the development of either a suppressive or stimulatory GVH, respectively. That donor Lyt-2+ cells mediate a suppressive GVH through cytolytic mechanisms is evidenced by greater than 70% reduction in B6 GVH spleen cell numbers and readily demonstrable anti-F1 CTL activity by these spleen cells despite an inability to generate anti-allogeneic or anti-TNP self CTL activity even in the presence of added T helper factors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isotype concentrations of human antibodies to Haemophilus influenzae type b polysaccharide (Hib) in young adults immunized with the polysaccharide as such or conjugated to a protein (diphtheria toxoid)

Antibody responses of young adults to Haemophilus influenzae type b polysaccharide (Hib) or its protein conjugate were studied with special attention to the isotype composition of the antibodies. Three conclusions of interest can be made: 1) Immunoglobulin G (IgG) antibodies in polysaccharide-immunized volunteers displayed the subclass pattern previously found in antibodies to meningococcal type A polysaccharide. IgG1 was the predominant subclass in IgG antibodies of some individuals, IgG2 in others. Still others had the two subclasses in varying but more even proportions. 2) The conjugate vaccine induced a geometric mean response 2 to 3 times higher and an IgG response 4 times higher to Hib than the polysaccharide vaccine. 3) Anti-Hib antibodies induced by the conjugate vaccine still had essentially the same IgG subclass composition as anti-Hib antibodies induced by the polysaccharide. This composition was strikingly different from the composition of the anti-diphtheria toxoid response induced by the same conjugate vaccine.     

Proton magnetic resonance study of lysine-binding to the kringle 4 domain of human plasminogen. The structure of the binding site

The binding of L-Lys, D-Lys and epsilon-aminocaproic acid (epsilon ACA) to the kringle 4 domain of human plasminogen has been investigated via one and two-dimensional 1H-nuclear magnetic resonance spectroscopy at 300 and 600 MHz. Ligand-kringle association constants (Ka) were determined assuming single site binding. At 295 K, pH 7.2, D-Lys binds to kringle 4 much more weakly (Ka = 1.2 mM-1) than does L-Lys (Ka = 24.4 mM-1). L-Lys binding to kringle 4 causes the appearance of ring current-shifted high-field resonances within the -1 approximately less than delta approximately less than 0 parts per million range. The ligand origin of these signals has been confirmed by examining the spectra of kringle 4 titrated with deuterated L-Lys. A systematic analysis of ligand-induced shifts on the aromatic resonances of kringle 4 has been carried out on the basis of 300 MHz two-dimensional chemical shift correlated (COSY) and double quantum correlated spectroscopies. Significant differences in the effect of L-Lys and D-Lys binding to kringle 4 have been observed in the aromatic COSY spectrum. In particular, the His31 H4 and Trp72 H2 singlets and the Phe64 multiplets appear to be the most sensitive to the particular enantiomers, indicating that these residues are in proximity to the ligand C alpha center. In contrast, the rest of the indole spectrum of Trp72 and the aromatic resonances of Trp62 and Tyr74, which are affected by ligand presence, are insensitive to the optical nature of the ligand isomer. These results, together with two-dimensional proton Overhauser studies and ligand-kringle saturation transfer experiments reported previously, enabled us to generate a model of the kringle 4 ligand-binding site from the crystallographic co-ordinates of the prothrombin kringle 1. The latter, although lacking recognizable lysine-binding capability, is otherwise structurally homologous to the plasminogen kringles.     

Selection of DNA binding sites by regulatory proteins. Statistical-mechanical theory and application to operators and promoters

We present a statistical-mechanical selection theory for the sequence analysis of a set of specific DNA regulatory sites that makes it possible to predict the relationship between individual base-pair choices in the site and specific activity (affinity). The theory is based on the assumption that specific DNA sequences have been selected to conform to some requirement for protein binding (or activity), and that all sequences that can fulfil this requirement are equally likely to occur. In most cases, the number of specific DNA sequences that are known for a certain DNA-binding protein is very small, and we discuss in detail the small-sample uncertainties that this leads to. When applied to the binding sites for cro repressor in phage lambda, the theory can predict, from the sequence statistics alone, their rank order binding affinities in reasonable agreement with measured values. However, the statistical uncertainty generated by such a small sample (only 6 sites known) limits the result to order-of-magnitude comparisons. When applied to the much larger sample of Escherichia coli promoter sequences, the theory predicts the correlation between in vitro activity (k2KB values) and homology score (closeness to the consensus sequence) observed by Mulligan et al. (1984). The analysis of base-pair frequencies in the promoter sample is consistent with the assumption that base-pairs at different positions in the sites contribute independently to the specific activity, except in a few marginal cases that are discussed. When the promoter sites are ordered according to predicted activities, they seem to conform to the Gaussian distribution that results from a requirement for maximal sequence variability within the constraint of providing a certain average activity. The theory allows us to compare the number of specific sites with a certain activity to the number that would be expected from random occurrence in the genome. While strong promoters are "overspecified", in the sense that their probability of random occurrence is very low, random sequences with weak promoter-like properties are expected to occur in very large numbers. This leads to the conclusion that functional specificity is based on other properties in addition to primary sequence recognition; some possibilities are discussed. Finally, we show that the sequence information, as defined by Schneider et al. (1986), can be used directly (at least in the case of equilibrium binding sites) to estimate the number of protein molecules that are specifically bound at random "pseudosites" in the genome.(ABSTRACT TRUNCATED AT 400 WORDS)     

Polypeptides of the Golgi Apparatus of Neurons from Rat Brain

An antiserum was raised against fractions of the Golgi apparatus of neurons from rat brain. Immunoblots of these fractions with the antiserum showed two principal bands of 185 and 150 kilodaltons (kd) in apparent molecular mass. The antiserum reacted with five or six bands of 200, 150, 130, 100-110, 64, and 40 kd in apparent molecular mass in immunoblots of several crude brain membrane fractions. Affinity-purified antibodies from the different gel bands transferred to nitrocellulose paper were used in immunoblot and immunocytochemical studies. Antibodies eluted from the 200-, 150-, 100-110-, and 64-kd bands reacted not only with the corresponding band but also with the other three bands. Antibodies eluted from the 40-kd band stained only the corresponding band. On light and/or electron microscopic immunocytochemistry, the antiserum stained the Golgi apparatus of rat neurons, glia, liver, and kidney tubule cells. Weaker, segmented, and less consistent staining was observed in nuclear envelopes, rough endoplasmic reticulum, and plasma membranes of neurons. Antibodies eluted from the bands at 200, 150, 100-110, and 64 kd stained intermediate cisterns of the Golgi apparatus of neurons. These findings suggest that a group of related polypeptides of brain membranes is preferentially expressed or enriched in the Golgi apparatus of neurons. Polypeptides with apparent molecular masses of 185 and 150 kd probably represent moieties endogenous to membranes of the neuronal Golgi apparatus.     

γ-Hydroxybutyric Acid in Subcellular Fractions of Rat Brain

The presence of gamma-hydroxybutyric acid (GHB) in synaptosome-enriched fractions of rat brain was ascertained using a GLC technique. The stability of GHB in synaptosomes was evaluated by addition of various gamma-aminobutyric acid (GABA) transaminase (GABA-T) inhibitors, GHB, or ethosuximide to the homogenizing medium. Furthermore, changes in whole brain GHB levels were compared with those in the synaptosomal fraction in animals treated with GABA-T inhibitors, GABA, or ethosuximide. GHB was present in synaptosome-enriched fractions in concentrations ranging from 40 to 70 pmol/mg of protein. There was no evidence for redistribution, leakage, or metabolism of GHB during the preparation of synaptosomes. The elevations of whole brain GHB level associated with GABA-T or ethosuximide treatment were reflected by a parallel increase in synaptosomal GHB content. These data add to the growing evidence that GHB may have neurotransmitter or neuromodulator function.     

Immunoperoxidase confirmation of parathyroid origin of ultrasound-guided fine needle aspirates of the parathyroid glands

It may be difficult to differentiate between cells of parathyroid and thyroid origin in ultrasound-guided fine needle aspirations of the neck region, even in patients with a clinical history of hyperparathyroidism. A parathyroid hormone antibody was used in an immunohistochemical system to confirm a parathyroid origin in fine needle aspirate smears from nine patients with hyperparathyroidism. Immunoperoxidase positivity for parathyroid hormone confirmed a parathyroid origin in six of nine cases and was strongly suggestive, although equivocal, in the remaining three cases. Technical problems included nonuniform staining and background staining.     

Thermochemistry of aqueous solutions of alkylated nucleic acid bases. VI. Enthalpies of hydration of 2-alkyl-9-methyladenines

Enthalpies of solution in water, delta H0sol, and vant'Hoff enthalpies of sublimation, delta H0subl, were determined experimentally for a number of crystalline 2-alkyl derivatives of 9-methyladenine: m2(2,9)Ade, e2m9Ade, pr2m9Ade and but2m9Ade. Standard enthalpies of hydration, delta H0hydr derived from these data were corrected for the calculated cavity terms, delta H0cav, to yield enthalpies of interaction, delta H0int, of the solutes with their hydration shells. The apparent residual contribution of alkyl groups, R, to the enthalpy of interaction delta delta H0int (R) was found to increase linearly with the number of CH2 groups added upon alkyl substitution, whereas this contribution calculated per unit area of the water-accessible molecular surface, SB, of alkyl residues delta delta H0int (R): delta SB(R) appeared constant over the whole series of the compounds investigated. This indicates that alkyl groups substituted at the C(2) carbon atom of the adenine contribute additively to the van der Waals' part of the enthalpy of interaction and do not affect the electrostatic part of the energy of interaction of the solutes with their hydration shells.     

Histochemical and ultrastructural data on the adrenergic and cholinergic innervation of the epididymis in the mouse

The adrenergic and cholinergic innervation of the epididymal duct in the mouse has been investigated using histochemical methods and electron microscopy. This study demonstrates that the epididymal innervation of the mouse does not really differ from that of other mammals. Cholinergic nerves are mainly vascular, even in the cauda where cholinesterase activity is increased within the tubular musculature. Catecholamine fibres ensure the motricity of the wall of the canalicular system, particularly the terminal segment where the smooth muscle fibres are specifically differentiated.