Analysis of the tyrosine phosphorylation and calcium fluxing of human CD6 isoforms with different cytoplasmatic domains

CD6 is a cell surface glycoprotein that functions both as a co-stimulatory and adhesion receptor on T cells. Recently we have described CD6 isoforms (CD6a, b, c, d, e) that arise via alternative splicing of exons encoding the cytoplasmic region of the molecule. CD6 becomes phosphorylated on tyrosine (Tyr) residues following stimulation through the T cell receptor (TCR) complex. Since the phosphorylation of Tyr residues renders some cell surface receptors competent for interactions with proteins of intracellular signaling pathways, we wanted to determine which region(s) and residues in the cytoplasmic domain of CD6 were important for phosphorylation on Tyr residues. We engineered and stably expressed chimeric receptors that consisted of the extracellular region of mouse CD6 and the cytoplasmic regions of either naturally occurring isoforms of human CD6, truncated proteins, or point mutants. We were able to demonstrate that of the nine Tyr residues in the cytoplasmic domain of the largest isoform CD6a, the two C-terminal Tyr residues (Tyr 629/662) are critical for the phosphorylation of CD6 following TCR cross-linking. Isoform CD6e, which is missing a region that contains two proline-rich motifs, is not phosphorylated. We further analyzed the ability of the different CD6 isoforms and truncated receptors to mobilize intracellular calcium after CD6/TCR co-ligation. All CD6 isoforms, including CD6e, as well as the truncation mutant Δ 555, which is missing approximately the C-terminal half of the cytoplasmic domain, are able to increase Ca²⁺ influx. Taken together, these results suggest that the region of CD6 which is critical for Ca²⁺ mobilization is located N-terminal from amino acid 555 and is therefore different from the region located at the C terminus of CD6, which is necessary for tyrosine phosphorylation.     

Intervertebral Disc Mechanics Are Restored Following Cyclic Loading and Unloaded Recovery

The objectives of this study were (1) to quantify changes in the mechanical behavior of the intervertebral disc in response to cyclic compressive loading and (2) to determine whether mechanical behavior would be restored following a period of unloading. The elastic and viscoelastic compressive mechanical behaviors of adult sheep motion segments were assessed. Ten thousand cycles of compressive loading resulted in increased elastic stiffness and decreased stress-relaxation. After 18 h of unloading in a PBS bath stiffness and relaxation were fully restored. Cyclic loading did not cause structural damage as determined by radiographs and magnetic resonance images. After cyclic loading, average stiffness increased from 603 to 800 N/mm (p = 0.015) and returned to initial levels after the recovery period. Cyclic loading caused a decrease in total relaxation (from 92 to 38 N, p < 0.001) that also returned to initial levels after recovery. The reversible, repeatable effects of cyclic loading and recovery demonstrated in this in vitro study may be attributed to fluid flow. Intervertebral disc fluid transport during the diurnal recovery cycle may be key to understanding intervertebral disc degeneration, as fluid exudation and recovery may be integral to maintaining adequate disc nutrition.     

Macromolecular synthesis in cells infected with frog virus 3: V. The absence of polyadenylic acid in the majority of frog virus 3-specific mRNA species

Most of the virus-specific RNA molecules isolated from the cytoplasm of BHK-21 cells infected with frog virus 3 (FV 3) did not contain poly(A) tracts of sufficient length to permit the RNA to bind to oligo(dT) cellulose. The 14% of [³H]adenosine-labeled RNA molecules that did bind were 40–55% RNAse-resistant and sedimented at about 6 S. In contrast, 40–70% of the pulse-labeled RNA isolated from vaccinia virus-infected BHK cells was bound to oligo(dT) cellulose, and the bound RNA was representative of the whole spectrum of vaccinia virus RNA size classes.     

Modulation of T-cell function by (R)- and (S)-isomers of albuterol: anti-inflammatory influences of (R)-isomers are negated in the presence of the (S)-isomer

BACKGROUND: beta(2)-Adrenergic agonists interact with specific receptors on T lymphocytes to mediate anti-inflammatory activities. However, anti-inflammatory effects are not observed when beta(2)-adrenergic agonists are administered in vivo as racemates. OBJECTIVE: We hypothesized that anti-inflammatory influences are mediated by the (R)-isomer and are masked in the additional presence of the (S)-isomer. METHODS: Antigen-specific T-cell lines were generated in the presence of recombinant human IL-2 and tetanus with or without varying concentrations of (R)- and (S)-isomers of albuterol alone or in combination. Parallel lines were generated in the presence of propranolol. Cells were briefly pulsed with PHA and evaluated for proliferation, apoptosis, and cytokine secretion. RESULTS: (R)-Albuterol significantly inhibited T-cell proliferation (77.0% +/- 9.7% of control at 10(-8) mol/L and 61.1% +/- 9.0% at 10(-7) mol/L). No influence was observed with (S)-albuterol alone. However, the addition of (S)-albuterol to (R)-albuterol mediated a dose-dependent increase in proliferation. At equivalent concentrations of the 2 isomers, proliferation was unchanged from the control, whereas at 10(-6) mol/L (S)-albuterol, proliferation was enhanced. Both the inhibitory effects of (R)-albuterol alone and the stimulating influence of (R)- plus (S)-albuterol were blocked in the additional presence of propranolol. (R)-Albuterol at 10(-8) mol/L inhibited IL-2 and IFN-gamma production. Racemic albuterol (10(-8) mol/L each) had no influence on cytokine production; however, the combination of 10(-8) mol/L (R)-albuterol with 10(-6) (S)-albuterol stimulated production of IL-2 and IL-13. No effects were observed on apoptosis or cell viability. CONCLUSION: These studies confirm the beta-adrenergic receptor-specific anti-inflammatory effects of (R)-albuterol. The racemate had minimal influences on proliferation or cytokine production. The presence of excess (S)-albuterol resulted in proinflammatory influences. We hypothesize that the (S)-isomer functions as an inverse agonist to switch the function of the beta(2)-adrenergic receptor.     

Plasmacytoid dendritic cells prime IFN-gamma-secreting melanoma-specific CD8 lymphocytes and are found in primary melanoma lesions

Plasmacytoid dendritic cells (PDC) are a small population of leukocytes specialized in the production of type I IFN. It has been shown that PDC have a potent T cell stimulatory capacity in allogeneic mixed lymphocyte reaction, However, their role in initiating primary immune responses remains elusive. We report that blood PDC efficiently prime naive CD8(+) lymphocytes specific for the melan-A(26-35) epitope to become IFN-gamma producing cells in vitro. In addition, we found that CD40L-stimulated PDC induce expression on primed melan-A-specific T cells of cutaneous lymphocyte antigen and L-selectin (CD62L), homing receptors that allow the migration of effector cells to the inflamed skin. Finally, we show that PDC can be found in the peri-tumoral area of most primary cutaneous melanomas in vivo and that type I IFN-containing supernatants derived from PDC increase melanoma cell surface expression of CD95 and MHC class I and class II molecules in vitro. Our results suggest a new immunomodulatory role for tissue infiltrating PDC, which may prime tumor-specific T cell responses and affect tumor growth via soluble factors.     

Clinical and immunologic progression in HIV-infected US women before and after the introduction of highly active antiretroviral therapy

OBJECTIVE: To examine factors associated with clinical and immunologic HIV disease progression in a cohort of US women. DESIGN: Analysis of data from a prospective, longitudinal, case-control study of HIV-infected women followed every 6 months for 7 years. SETTING: Four urban clinical centers in the United States. PARTICIPANTS: 648 HIV-infected women who did not have AIDS at time of entry into the study. MEASUREMENTS: Structured clinical and behavioral interviews; protocol-directed physical examinations; CD4 lymphocyte counts; plasma HIV RNA; infectious pathogen serologies.RESULTS With 2304 women-years of follow-up, 46.1% of the women developed AIDS; however, 93.3% of the diagnoses were based on CD4 counts dropping to <200 cells/mm(3). Only 10.6% of the women with CD4 counts <200 cells/mm(3) developed an opportunistic infection. Baseline CD4 count was the strongest predictor of subsequent clinical progression. Illicit substance use, multiple pregnancies, demographic variables, and other infections were not associated with progression. Among women with CD4 counts >500 cells/mm(3) at baseline, those who were anemic or had hepatitis C were more likely to progress to AIDS. By the end of the study, only 52% of the participants were on highly active antiretroviral therapy (HAART). CONCLUSIONS: Despite underutilization of HAART in this multicenter cohort of urban women, opportunistic infections were uncommon, despite CD4 declines.     

Impairment of synaptic vesicle exocytosis and recycling during neuromuscular weakness produced in mice by 2,4-dithiobiuret

Chronic treatment of rodents with 2,4-dithiobiuret (DTB) induces a neuromuscular syndrome of flaccid muscle weakness that mimics signs seen in several human neuromuscular disorders such as congenital myasthenic syndromes, botulism, and neuroaxonal dystrophy. DTB-induced muscle weakness results from a reduction of acetylcholine (ACh) release by mechanisms that are not yet clear. The objective of this study was to determine if altered release of ACh during DTB-induced muscle weakness was due to impairments of synaptic vesicle exocytosis, endocytosis, or internal vesicular processing. We examined motor nerve terminals in the triangularis sterni muscles of DTB-treated mice at the onset of muscle weakness. Uptake of FM1-43, a fluorescent marker for endocytosis, was reduced to approximately 60% of normal after either high-frequency nerve stimulation or K(+) depolarization. Terminals ranged from those with nearly normal fluorescence ("bright terminals") to terminals that were poorly labeled ("dim terminals"). Ultrastructurally, the number of synaptic vesicles that were labeled with horseradish peroxidase (HRP) was also reduced by DTB to approximately 60%; labeling among terminals was similarly variable. A subset of DTB-treated terminals having abnormal tubulovesicular profiles in their centers did not respond to stimulation with increased uptake of HRP and may correspond to dim terminals. Two findings suggest that posttetanic "slow endocytosis" remained qualitatively normal: the rate of this type of endocytosis as measured with FM1-43 did not differ from normal, and HRP was observed in organelles associated with this pathway- coated vesicles, cisternae, as well as synaptic vesicles but not in the tubulovesicular profiles. In DTB-treated bright terminals, end-plate potential (EPP) amplitudes were decreased, and synaptic depression in response to 15-Hz stimulation was increased compared with those of untreated mice; in dim terminals, EPPs were not observed during block with D-tubocurarine. Nerve-stimulation-induced unloading of FM1-43 was slower and less complete than normal in bright terminals, did not occur in dim terminals, and was not enhanced by alpha-latrotoxin. Collectively, these results indicate that the size of the recycling vesicle pool is reduced in nerve terminals during DTB-induced muscle weakness. The mechanisms by which this reduction occurs are not certain, but accumulated evidence suggests that they may include defects in either or both exocytosis and internal vesicular processing.     

Effects of Modified Instructions on Peabody Developmental Motor Scales,Second Edition,Gross Motor Scores in Children with Typical Development

Aims: The current study assessed whether modifying instructions on the Peabody Developmental Motor Scales, Second Edition (PDMS-2) affected scores in children with typical development. Methods: The gross motor portion of the PDMS-2 was administered twice, 2–10?days apart, to 38 children. Age- and gender-matched groups received instructions in both standard and modified formats, with order depending on group assignment. Results: Gross Motor Quotient results showed an effect for instruction type (p?=?.03) and an interaction between instruction type and order (p?=?.02). Improved scores for those given modified instructions during the second session indicated the interaction favored modifications. Stationary scores showed an effect for instruction type (p?=?.01) and an interaction between instruction type and age (p?=?.02). Object Manipulation scores showed an interaction between instruction type and order only (p =.002); Locomotion scores showed no significant changes (p?=?.25). Percentile rank changes ranged from 9% to 22% across subtests. Conclusions: Findings suggested instruction modifications may change PDMS-2 gross motor scores, even in children with typical development. Findings also suggested normative scores should not be reported if modifications were used during testing. Research is needed to determine optimal cues for the best representation of true motor ability during standardized assessment.