Kinetics of gene expression in murine cutaneous graft-versus-host disease

The kinetics of gene expression associated with the development of cutaneous graft-versus-host disease (GVHD) were examined in a mouse model of MHC-matched allogeneic hematopoietic stem cell transplantation. Ear skin was obtained from recipient mice with or without GVHD between 7 and 40 days after transplantation for histopathological analysis and gene expression profiling. Gene expression patterns were consistent with early infiltration and activation of CD8(+) T and mast cells, followed by CD4(+) T, natural killer, and myeloid cells. The sequential infiltration and activation of effector cells correlated with the histopathological development of cutaneous GVHD and was accompanied by up-regulated expression of many chemokines and their receptors (CXCL-1, -2, -9, and -10; CCL-2, -5, -6, -7, -8, -9, -11, and -19; CCR-1 and CCR-5), adhesion molecules (ICAM-1, CD18, Ly69, PSGL-1, VCAM-1), molecules involved in antigen processing and presentation (TAP1 and TAP2, MHC class I and II, CD80), regulators of apoptosis (granzyme B, caspase 7, Bak1, Bax, and BclII), interferon-inducible genes (STAT1, IRF-1, IIGP, GTPI, IGTP, Ifi202A), stimulators of fibroblast proliferation and matrix synthesis (interleukin-1beta, transforming growth factor-beta1), and markers of keratinocyte proliferation (keratins 5 and 6), and differentiation (small proline-rich proteins 2E and 1B). Many acute-phase proteins were up-regulated early in murine cutaneous GVHD including serum amyloid A2 (SAA2), SAA3, serpins a3g and a3n, secretory leukocyte protease inhibitor, and metallothioneins 1 and 2. The kinetics of gene expression were consistent with the evolution of cutaneous pathology as well as with current models of disease progression during cutaneous GVHD.     

Afferent volleys in limb nerves influencing impulse discharges in cerebellar cortex I. In mossy fibers and granule cells

Summary This paper is the first of a series in which the processing of information in the cerebellum has been studied by investigating the effects that known inputs from limb nerves produce on the unitary spike potentials in the cerebellar cortex. These spikes have been recorded extracellularly at all depths along microelectrode tracks in the 5th, 4th and 3rd lobules of the anterior lobe in the lateral vermis or in the pars intermedia. These units have a background frequency of discharge, often very irregular, and computer averaging techniques have been employed in order to derive reliable information on the time course and intensity of the excitatory and/or inhibitory actions produced by the input against this background.Most of the spike responses recorded from the granular layer fall into two classes, one characteristic of impulses in mossy fibers, and the other of impulse discharges from granule cells. Both in the spontaneous background and in the response to afferent volleys in limb nerves the mossy fibers exhibit a performance in close accord with that described for the discharges up the spino-cerebellar tracts. The short latency of 6–9 msec for hindlimb stimuli and the high frequency burst response of 2–4 impulses are characteristic. The mossy fibers displayed a wide variety of responses to the wide range of testing inputs, there being various combinations of excitatory and inhibitory responses and also delayed excitatory actions, all of which must be assumed to be reflections of synaptic influences on the cells of origin of the mossy fibers in the spinal cord.Granule cells have a longer latency by several milliseconds, 9–20 msec for the hindlimb, and a slower frequency in their burst response which tended to be longer and more irregular. The small unitary spike potentials are more difficult to isolate. Also with repetitive stimulation granule cells are more readily depressed than are mossy fibers.Usually a granule cell exhibits a wider range of response to the various cutaneous and muscular afferents of a limb. Both mossy fibers and granule cells may display reciprocal responses to volleys from muscle nerves to antagonistic muscles. This attempt to define properties of the mossy fiber and granule cell spike potentials should help in their identification in future investigations.Post-Doctoral Fellow NINDS (1F2NB40,544101 NSRB).Post-Doctoral Fellow UHF Grant No. FTF-3-UB-70.     

The effects of reverse monocular deprivation in monkeys II. Electrophysiological and anatomical studies

Summary Monkeys had one eye closed at about 30 days of age for 14, 30, 60, or 90 days, then opened, and the fellow eye closed for another 120 days. The animals then had at least 10 months of binocular visual experience before extensive behavioral training and testing were carried out. In terminal experiments concluded more than 18 months later, microelectrode investigations of the striate cortex demonstrated that there was almost a complete absence of binocular neurons in all animals. The initially deprived eyes (IDEs) dominated the majority of cortical neurons, even when soma size measurements of lateral geniculate neurons indicated that the LGN cells driven by the IDE had not regained their normal size. The monkeys which had significant interocular differences in spatial vision also exhibited abnormalities in the distribution of the metabolic enzyme, cytochrome oxidase (CO), within the striate cortex. These results demonstrate that many of the severe alterations in cortical physiology and eye dominance produced by early monocular form deprivation can be reversed, with recovery of normal cortical function, via the reverse-deprivation procedure.Supported by National Eye Institute grants R01 EY01120, R01 EY03611, R01 EY01139, and EY02520     

Heterogeneity of monoclonal antibody-reactive epitopes on mycobacterial 30-kilodalton-region proteins and the secreted antigen 85 complex and demonstration of antigen 85B on the Mycobacterium leprae cell wall surface.

Proteins of the antigen 85 complex in the 30-kDa region secreted by live mycobacteria are important in the immune response against mycobacterial infections and may play an important biological role in the host-parasite interaction. In the present study, we have characterized epitopes of the 30-kDa-region proteins and the antigen 85 complex by using a panel of 13 monoclonal antibodies (MAbs) reacting with these antigens, 6 of which have not been described before. By using five previously characterized related secreted proteins of Mycobacterium tuberculosis, MPT44 (85A), MPT59 (85B), MPT45 (85C), MPT51 (27 kDa), and MPT64 (26 kDa), we have identified at least 10 different MAb-reactive epitopes on the proteins of the antigen 85 complex. A heterogeneous distribution of epitopes was observed within the components of the antigen 85 complex. Two distinct epitopes specific for antigen 85B and two other epitopes restricted to the 85A and 85B components were recognized. Two of them were shared with a previously unidentified 27-kDa protein present in M. tuberculosis culture fluid from which all MPT proteins were derived. The rest of the MAb-reactive epitopes were found to be present mostly in antigens 85A and 85B and to a lesser extent in antigen 85C. None of these MAbs recognized component 85C alone nor did they bind to the related MPT51 and MPT64 proteins. Interestingly, most of the MAbs reacted with purified native proteins of the antigen 85 complex but not to them in their denatured forms. In contrast, reactivity of the MAbs with the cytosol fraction of M. tuberculosis in immunoblotting revealed that they bound to a closely related cytosolic 30-kDa protein(s) even when they were denatured. Heterogeneity of these MAb-reactive epitopes of the antigen 85 complex was further evident as they were found to be distributed in various patterns among 19 different mycobacterial species. By using fusion proteins of the Mycobacterium leprae 30/31-kDa antigen 85 complex, we have localized at least six different epitopes within amino acid residues 55 to 266 of the M. leprae antigen 85 complex. Finally, by immunohistochemical analysis, we have demonstrated the in situ expression of one of the novel MAb-reactive epitopes specific for antigen 85B on the cell wall surface of M. leprae within macrophages in lepromatous leprosy lesions and thus provide direct evidence for the presence of the B component of the antigen 85 complex on the surface of intact M. leprae.     

Quantitative nucleic acid sequence-based assay as a new molecular tool for detection and quantification of Leishmania parasites in skin biopsy samples

Currently available methods for the diagnosis of cutaneous leishmaniasis (CL) have low sensitivities or are unable to quantify the number of viable parasites. This constitutes a major obstacle for the diagnosis of the disease and for the study of the effectiveness of treatment schedules and urges the development of improved detection methods. In this study, quantitative nucleic acid sequence-based amplification (QT-NASBA) technology was used to detect and quantify Leishmania parasites in skin biopsy samples from CL patients. The assay is based on the detection of a small subunit rRNA (18S rRNA), which may allow for the detection of viable parasites. The QT-NASBA assay was evaluated using in vitro-cultured promastigotes and amastigotes and 2-mm skin biopsy samples from Old and New World CL patients. The study demonstrated that the lower detection limit of the QT-NASBA was two parasites per biopsy sample. Parasites could be quantified in a range of 2 to 11,300,000 parasites per biopsy sample. The QT-NASBA could detect levels of parasites 100-fold lower than those detected by conventional PCR. Test evaluation revealed that the QT-NASBA had a sensitivity of 97.5% and a specificity of 100% in the present study. The QT-NASBA is a highly sensitive and specific method that allows quantification of both Old and New World Leishmania parasites in skin biopsy samples and may provide an important tool for diagnosis as well as for monitoring the therapy of CL patients.     

Transfer of hydrophilic molecules by placenta and yolk sac of the guinea pig.

Prenatal transfer of hydrophilic materials was studied in chronic guinea pig preparations of ca. 30-65 days gestation. In most preparations the vitelline (yolk sac placenta) circulation of one of the fetuses was ligated between 1 and 20 days before the experiment. Fetal and maternal serum protein concentrations were recorded as a function of the number of days after ligation. Prenatal transfer of hydrophilic materials of 60-5,500 daltons molecular wt was consistent with diffusion, with some evidence of steric restriction of the larger molecules. There was no difference between operated and sham-operated or control fetuses. However, the transfer of radiolabeled homologous gamma globulin ceased after ligation of the yolk sac vessels. The transfer in control fetuses accounted for most of the prenatal requirements of this protein, as calculated from the increases in fetal weight and serum gamma globulin concentrations as a function of gestational age. There was evidence of a nonspecific suppression of yolk sac function by surgery but no evidence of fetal catabolism of gamma globulin.     

New approaches to the prevention and eradication of rabies

Despite significant progress in improving the pre- and postexposure prophylaxis of human rabies, the development of better and more cost-effective vaccines and antiviral therapeutics remains a major goal for the treatment of human rabies, the control of animal rabies and particularly for the eradication of rabies virus reservoirs in terrestrial wildlife. In this review, we discuss the structural requirements for an effective rabies vaccine, as well as new strategies currently in use for the development of safer and more potent rabies vaccines for rabies prophylaxis and eradication. Finally, we discuss new immune therapeutics aimed at replacing the conventional administration of antirabies immunoglobulin used in rabies post-exposure prophylaxis in humans.     

Differences in host susceptibility to disease progression in the human challenge model of Haemophilus ducreyi infection

With human volunteers inoculated at two sites with Haemophilus ducreyi, outcomes for a subject were not independent. In a reinfection trial, 2 of 11 previous pustule formers and 6 of 10 previous resolvers resolved all sites of infection. There was no correlation between serum bactericidal or phagocytic activity and outcome in the trial. These data indicate that different hosts are differentially susceptible to disease progression versus resolution in the model.     

Foetal placental blood flow in the lamb

1. Fifteen sheep foetuses of 1.5-5.2 kg body weight were prepared with indwelling arterial and venous catheters for experimentation one to six days later.2. Unanaesthetized foetuses were found to have mean arterial and central venous blood pressures of 40 +/- 1.5 (S.E. of mean) and 2.0 +/- 0.3 (S.E. of mean) mm Hg respectively, compared to intra-uterine pressure. Intra-uterine pressure was 16 +/- 0.8 (S.E. of mean) mm Hg with respect to atmospheric pressure at mid-uterine level.3. Mean placental blood flow of the foetuses was 199 +/- 20 (S.E. of mean) ml./(min.kg body wt.). Mean cardiac output in eleven of the foetuses was 658 +/- 102 (S.E. of mean) ml./(min.kg).4. Mean foetal and maternal colloid osmotic pressures were 17.5 +/- 0.7 (S.E. of mean) and 20.5 +/- 0.6 (S.E. of mean) mm Hg respectively at 38 degrees C.5. Intravenous infusions into six ewes of 1.8 mole of mannitol and 0.4 mole of NaCl resulted in significant increases in foetal plasma osmolarity, sodium, potassium, and haemoglobin concentrations, without detectable transfer of mannitol to the foetal circulation.6. In the sheep placenta there is osmotic and hydrostatic equilibration of water. As a consequence, there should be an interaction between foetal placental blood flow and foetal water exchange with the maternal circulation. It was concluded that this interaction tends to stabilize foetal placental blood flow.     

A characterization of glycinergic receptors present in cultured rat medullary neurons.

1. Whole-cell current responses to bath application of glycine, beta-alanine, and taurine were studied in medullary neurons cultured from embryonic rats. 2. Two current components were seen in the responses to bath application of agonist, one component that desensitized and another that did not. 3. The two current components have different dose-response characteristics, with the nondesensitizing component being activated more effectively at lower concentrations than the desensitizing component and also reaching its peak at lower concentrations. The agonist concentrations producing half-maximal responses are 26 +/- 4 (SE, n = 6) and 69 +/- 17 (n = 7) microM for the nondesensitizing and desensitizing components, respectively, for glycine; 54 +/- 7 (n = 9) and 127 +/- 37 (n = 7) microM for beta-alanine; and 153 +/- 24 (n = 9) 443 +/- 99 (n = 3) microM for taurine. Thus, for each component, the order of potency is glycine greater than beta-alanine greater than taurine. 4. When total responses to glycine, beta-alanine, and taurine are compared in the same cells, taurine and beta-alanine are less potent agonists than glycine, with relative potencies of 1:0.4:0.1 for glycine-beta-alanine-taurine. 5. The desensitizing component is more sensitive to strychnine than the nondesensitizing one. The strychnine concentrations that block 50% of the response to a control dose of agonist are 15 and 500 nM for the desensitizing and nondesensitizing components, respectively, for glycine; 60 nM and 1 microM for beta-alanine; and 18 and 500 nM for taurine. 6. The complete occlusion between the responses to glycine and beta-alanine or glycine and taurine suggests that these agonists activate the same receptors. 7. The two current components may be manifestations of one receptor population with complicated kinetics or two independent receptor populations.