Inhibition of experimental autoimmune encephalomyelitis in Lewis rats by nasal administration of encephalitogenic MBP peptides: synergistic effects of MBP 68-86 and 87-99

Induction of mucosal tolerance by inhalation of soluble peptides with defined T cell epitopes is receiving much attention as a means of specifically down-regulating pathogenic T cell reactivities in autoimmune and allergic disorders. Experimental autoimmune encephalomyelitis (EAE) induced in the Lewis rat by immunization with myelin basic protein (MBP) and Freund's adjuvant (CFA) is mediated by CD4+ T cells specific for the MBP amino acid sequences 68-86 and 87-99. To further define the principles of nasal tolerance induction, we generated three different MBP peptides (MBP 68-86, 87-99 and the non-encephalitogenic peptide 110-128), and evaluated whether their nasal administration on day -11, -10, -9, -8 and -7 prior to immunization with guinea pig MBP (gp-MBP) + CFA confers protection to Lewis rat EAE. Protection was achieved with the encephalitogenic peptides MBP 68-86 and 87-99, MBP 68-86 being more potent, but not with MBP 110-128. Neither MBP 68-86 nor 87-99 at doses used conferred complete protection to gp-MBP-induced EAE. In contrast, nasal administration of a mixture of MBP 68-86 and 87-99 completely blocked gp-MBP-induced EAE even at lower dosage compared to that being used for individual peptides. Rats tolerized with MBP 68-86 + 87-99 nasally showed decreased T cell responses to MBP reflected by lymphocyte proliferation and IFN-gamma ELISPOT assays. Rats tolerized with MBP 68-86 + 87-99 also had abrogated MBP-reactive IFN-gamma and tumor necrosis factor-alpha mRNA expression in lymph node cells compared to rats receiving MBP 110-128 nasally, while similar low levels of MBP-reactive transforming growth factor-beta and IL-4 mRNA expressing cells were observed in the two groups. Nasal administration of MBP 68-86 + 87-99 only slightly inhibited guinea pig spinal cord homogenate-induced EAE, and passive transfer of spleen mononuclear cells from MBP 68-86 + 87-99-tolerized rats did not protect na?ve rats from EAE. Finally, we show that nasal administration of MBP 68-86 + 87-99 can reverse ongoing EAE induced with gp-MBP, although higher doses are required compared to the dosage needed for prevention. In conclusion, nasal administration of encephalitogenic MBP peptides can induce antigen-specific T cell tolerance and confer incomplete protection to gp-MBP-induced EAE, and MBP 68-86 and 87-99 have synergistic effects. Non-regulatory mechanisms are proposed to be responsible for tolerance development after nasal peptide administration.     

Evaluation of titanium brackets for orthodontic treatment: part I. The passive configuration

The static and kinetic frictional coefficients of commercially pure titanium brackets were evaluated in the passive configuration in the dry and wet states against stainless steel, nickel-titanium, and beta-titanium arch wires. For comparison, stainless steel brackets were evaluated under identical conditions. Titanium brackets were grayer in color and rougher in texture than the stainless steel brackets. Bracket slots were up to 0.002 inch greater than the nominally stated values. Remarkably, the static and kinetic frictional coefficients of the couples formed by titanium and stainless steel brackets were comparable. When evaluated against stainless steel and nickel-titanium arch wires in the dry state at 34 degrees C, the static coefficient averaged.12 and.20, respectively, independent of bracket alloy. When evaluated against stainless steel and nickel-titanium wires in the wet state at 34 degrees C using human saliva, the static coefficient averaged.15 and.20, respectively, independent of bracket alloy. Only the beta-titanium arch wires increased by about 15%, when tested in either the dry or the wet state against titanium versus stainless steel brackets. Noteworthy, too, was the decrease of both coefficients in the beta-titanium wire couples from their previously reported values. Analyses of electron spectroscopy for chemical analysis spectra and depth profiles show that these new brackets are titanium only in the bulk. Indeed the immediate surfaces are composed of, at least, 80 atomic percent (at.%) carbon and oxygen; whereas, the titanium that is present (>11 at.%) is mostly in the form of titanium dioxide. The presence of this quite thin passivating layer, which resides on top of an oxygen-hardened titanium substrate, reduces the galling and fretting that would normally be expected in such materials. Pending the outcome of future angulation tests, these frictional measurements show that titanium brackets are not only comparable to stainless steel brackets but also are more biocompatible with nickel having been eliminated from their constitution.     

Oxidative stress, mutant SOD1, and neurofilament pathology in transgenic mouse models of human motor neuron disease

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that primarily affects motor neurons in the spinal cord and brain stem. About 10% of all ALS cases are familial (FALS), inherited in an autosomal dominant manner. One fifth of FALS patients carry mutations in the Cu/Zn superoxide dismutase (SOD1) gene, and several lines of transgenic mice have been engineered to express mutant forms of the SOD1 gene that are linked to FALS. Significantly, many of these transgenic lines of mice develop a motor neuron disease (MND) that resembles human FALS. Oxidative stress induced by human SOD1 mutations is believed to play an important role in the pathogenesis of FALS and the FALS-like MND seen in the mutant SOD1 transgenic mice. For example, two lines of these mice showed prominent degeneration of mitochondria and endoplasmic reticulum in spinal cord neurons. Furthermore, recent studies have shown that neurofilament (NF)-rich spheroids. Lewy body-like NF inclusions, altered ubiquitin immunoreactivity, and Golgi fragmentation occur in the spinal cord motoneurons of these mutant SOD1 transgenic mice. Because these lesions recapitulate hallmark abnormalities of human ALS, mutant SOD1 transgenic mice provide a useful model for studies designed to elucidate the pathogenesis of ALS. Furthermore, transgenic mice that overexpress NF proteins also develop a clinical and pathologic phenotype similar to human MND, and polymorphisms in an NF gene have been linked to patients with ALS. Collectively, these observations implicate NF protein abnormalities in the pathogenesis of this disorder. Accordingly, this review summarizes recent insights into mechanisms of motor neuron degeneration in ALS that have emerged from studies of these new animal models of this neurodegenerative disease.     

Circadian rhythm in the hypothermic response to serotonin1A receptor agonist 8-OH-DPAT in rats

The gene encoding the CD2 mouse cell surface antigen was retrovirally transduced into cord blood CD34+ cells. On infection by culture at the contact of retrovirus-packaging cells, the mCD2 marker was expressed by 30-40% CD34+ cells, that included the most primitive stem cell-enriched Thy-1+ and CD38- subsets. Accordingly, sorted cord blood CD34+Thy-1+ cells could be directly infected in the same conditions. mCD2- transgenic cord blood CD34+ cells were then used to reconstitute human fetal thymus implanted in SCID mice. Five to 8 weeks later, the mCD2 antigen was detected on approximately 10% of the human thymocytes repopulating the thymus grafts and the transgene genome was detected in graft cell DNA by Southern blot. These results demonstrate efficient gene transfer into primitive cord blood hematopoietic cells endowed with lymphoid potential and suggest gene therapy schemes in neonates suffering inherited or acquired-such as HIV infection-disorders of the T-cell lineage.     

Functional association between Arf and RalA in active phospholipase D complex

Activation of phospholipase D1 (PLD1) by Arf has been implicated in vesicle transport and membrane trafficking. PLD1 has also been shown to be associated with the small GTPase RalA, which functions downstream from Ras in a Ras-RalA GTPase cascade that facilitates intracellular signal transduction. Although PLD1 associates directly with RalA, RalA has no effect upon the activity of PLD1. However, PLD1 precipitated from cell lysates with immobilized glutathione S-transferase-RalA fusion protein is active. This suggests the presence of an additional activating factor in the active RalA-PLD1 complexes. Because Arf stimulates PLD1, we looked for the presence of Arf in the active RalA-PLD1 complexes isolated from v-Src- and v-Ras-transformed cell lysates. Low levels of Arf protein were detected in RalA-PLD1 complexes; however, if guanosine 5'-[gamma-thio]triphosphate was added to activate Arf and stimulate translocation to the membrane, high levels of Arf were precipitated by RalA from cell lysates. Interestingly, deletion of 11 amino-terminal amino acids unique to Ral GTPases, which abolished the ability of RalA to precipitate PLD activity, prevented the association between RalA and Arf. Brefeldin A, which inhibits Arf GDP-GTP exchange, inhibited PLD activity in v-Src- and v-Ras-transformed cells but not in the nontransformed cells, suggesting that the association of Arf with RalA is required for the increased PLD activity induced by v-Src and v-Ras. These data implicate Arf in the transduction of intracellular signals activated by v-Src and mediated by the Ras-RalA GTPase cascade. Because both Arf and PLD1 stimulate vesicle formation in the Golgi, these data raise the possibility that vesicle formation and trafficking may play a role in the transduction of intracellular signals.     

Two-year follow-up of directly-observed intermittent regimens for smear-positive pulmonary tuberculosis in China

SETTING: The tuberculosis component of the Infectious and Endemic Disease Control Project in the People's Republic of China is the largest single tuberculosis control project in the world using directly-observed therapy and standardized intermittent regimens. OBJECTIVE: To determine the two-year relapse and mortality rates following completion of treatment. DESIGN: A prospective cohort study of 649 cases cured in this project. The 306 new and 343 retreatment cases were treated under field conditions with 2H3R3Z3S3/4H3R3 and 2H3R3Z3E3S3/6H3R3E3, respectively. Following treatment completion, two sputum samples were collected every six months for two years and examined for acid-fast bacilli. Causes of death were identified. RESULTS: The two-year relapse rates for new and retreatment cases were 3.3% and 5.6%, respectively. Retreatment cases with delayed sputum conversion had a greater risk for subsequent relapse. The two-year mortality rate for new and retreatment cases was 3.3% and 8.5%, respectively. The higher mortality rate in retreatment cases was not attributable to relapse of disease, but rather to non-infectious sequelae of tuberculosis. CONCLUSION: The use of directly-observed intermittent regimens is effective in permanently removing infectious tuberculosis cases from the community.     

Purification and characterization of the catalytic subunit of human DNA polymerase delta expressed in baculovirus-infected insect cells

The catalytic subunit of human DNA polymerase delta has been overexpressed in insect cells by a recombinant baculovirus. The recombinant protein has a Mr = approximately 125,000 and is recognized by polyclonal antisera against N-terminal and C-terminal peptides of the catalytic subunit of human DNA polymerase delta. The recombinant protein was purified to near homogeneity (approximately 1200-fold) from insect cells by chromatography on DEAE-cellulose, phosphocellulose, heparin-agarose, and single-stranded DNA-cellulose. The purified protein had both DNA polymerase and 3'-5' exonuclease activities. The properties of the recombinant catalytic subunit were compared with those of the native heterodimeric DNA polymerase delta isolated from fetal calf thymus, and the enzymes were found to differ in several respects. Although the native heterodimer is equally active with either Mn2+ or Mg2+ as divalent cation activator, the recombinant catalytic subunit is approximately 5-fold more active in Mn2+ than in Mg2+. The most striking difference between the two proteins is the response to the proliferating cell nuclear antigen (PCNA). The activity and processivity of native DNA polymerase delta are markedly stimulated by PCNA whereas it has no effect on the recombinant catalytic subunit. These results suggest that the small subunit of DNA polymerase delta is essential for functional interaction with PCNA.