Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance

Introduction  

Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology.     

Selective inhibition of inducible NO synthase activity in vivo reverses inflammatory abnormalities in surfactant protein D-deficient mice

Surfactant protein D (SP-D)-deficient (SP-D-/-) mice exhibit early development of emphysema. Previously we have shown that SP-D deficiency results in increased production and activity of inducible NO synthase (iNOS). In this study, we examined whether treatment with the iNOS inhibitor 1400W could inhibit the inflammatory phenotype. Mice were treated with 1400W systemically for 7 wk from 3 wk of age. Treatment reduced total lung NO synthase activity to 14.7+/-6.1% of saline-treated 10-wk-old SP-D-/- littermates. Long-term administration of 1400W reduced lung inflammation and cellular infiltration; and significantly attenuated the increased levels of matrix metalloproteinases 2 and 9, chemokines (KC, TARC), and cytokines (IFN-gamma) seen in bronchoalveolar lavage (BAL) of SP-D-/- mice. Abrogation of these levels was associated with decreasing BAL chemotactic activity for RAW cells. Two weeks of treatment with 1400W reduced total lung NO synthase (NOS) activity to 12.7+/-6.3% of saline-treated SP-D-/- mice. Short-term iNOS inhibition resulted in attenuation of pulmonary inflammation within SP-D-/- mice as shown by decreases in total BAL cell count (63+/-6% of SP-D-/- control), macrophage size (>25 microm) within the BAL (62+/-10% of SP-D-/- control), and a percentage of BAL macrophages producing oxidants (76+/-9% of SP-D-/- control). These studies showed that s.c. delivery of 1400W can be achieved in vivo and can attenuate the inflammatory processes within SP-D deficiency. Our results represent the first report linking defects in the innate immune system in the lung with alterations in NO homeostasis.     

Activatory and Inhibitory Fcγ Receptors Augment Rituximab-mediated Internalization of CD20 Independent of Signaling via the Cytoplasmic Domain

Type I anti-CD20 mAb such as rituximab and ofatumumab engage with the inhibitory FcγR, FcγRIIb on the surface of B cells, resulting in immunoreceptor tyrosine-based inhibitory motif (ITIM) phosphorylation. Internalization of the CD20·mAb·FcγRIIb complex follows, the rate of which correlates with FcγRIIb expression. In contrast, although type II anti-CD20 mAb such as tositumomab and obinutuzumab also interact with and activate FcγRIIb, this interaction fails to augment the rate of CD20·mAb internalization, raising the question of whether ITIM phosphorylation plays any role in this process. We have assessed the molecular requirements for the internalization process and demonstrate that in contrast to internalization of IgG immune complexes, FcγRIIb-augmented internalization of rituximab-ligated CD20 occurs independently of the FcγRIIb ITIM, indicating that signaling downstream of FcγRIIb is not required. In transfected cells, activatory FcγRI, FcγRIIa, and FcγRIIIa augmented internalization of rituximab-ligated CD20 in a similar manner. However, FcγRIIa mediated a slower rate of internalization than cells expressing equivalent levels of the highly homologous FcγRIIb. The difference was maintained in cells expressing FcγRIIa and FcγRIIb lacking cytoplasmic domains and in which the transmembrane domains had been exchanged. This difference may be due to increased degradation of FcγRIIa, which traffics to lysosomes independently of rituximab. We conclude that the cytoplasmic domain of FcγR is not required for promoting internalization of rituximab-ligated CD20. Instead, we propose that FcγR provides a structural role in augmenting endocytosis that differs from that employed during the endocytosis of immune complexes.     

Hypobaric impact on clinical tolerance and 24-h patterns in iron metabolism markers and plasma proteins in men

Long-distance flights can cause a number of clinical problems due to mild hypoxia resulting from cabin pressurization. Using a chronobiological approach, the aim of this work was to assess the clinical tolerance and biological impact of daytime exposure to hypobaric hypoxia on markers of iron metabolism and plasma proteins. Fourteen healthy, male volunteers, ages 23 to 39 yrs, spent 8.5 h in a hypobaric chamber (from 07:45 to 16:15 h) simulating an altitude of 8000 ft. This was followed by another 8.5-h session 4 wks later simulating conditions at an altitude of 12,000 ft. Biological variables were assayed every 2 h over two 24-h spans (control and hypoxia spans, respectively) per simulated altitude. Whereas most of the subjects tolerated the 8000 ft exposure well, eight subjects (57%) presented clear clinical signs of hypoxic intolerance at 12,000 ft. The 24-h blood iron profile showed a biphasic pattern at both altitude simulations, with a significant (～40%) increase during hypoxia, followed by a (～25%) decrease during the first hours of recovery. The iron circadian rhythm showed a significant phase delay during the hypoxic exposure at 8000 ft vs. reference. Mean 24-h ferritin levels decreased at both altitudes, but mainly during the nighttime after the 12,000 ft exposure in accordance with Cosinor analysis. The transferrin and total plasma proteins 24-h profiles did not show significant change. Moreover, significant differences, mainly in iron, ferritin, and transferrin, were found at 12,000 ft according to the clinical tolerance to hypoxia, and significant correlations were found between the mid-range crossing times, i.e., here half-descent times (d-T(50)), for ferritin and total plasma proteins and the reported level of clinical discomfort under hypoxia. This study shows that an 8.5-h exposure to mild hypoxia is able to alter very quickly the 24-h pattern of iron and ferritin. These alterations seem to depend, at least in part, on the clinical tolerance to hypoxia and may help explain the interindividual differences observed in the tolerance to hypoxia.     

Effect of one night of sleep loss on changes in tumor necrosis factor alpha (TNF-α) levels in healthy men

Total sleep deprivation in humans is associated with increased daytime sleepiness, decreased performance, elevations in inflammatory cytokines, and hormonal/metabolic disturbances.To assess the effects of 40 h of total sleep deprivation (TSD) under constant and well controlled conditions, on plasma levels of TNF-α and its receptor (TNFR1), interleukin-6 (IL-6), cortisol and C-reactive protein (CRP), sleepiness and performance, 12 healthy men (29 ± 3 years) participated in a 5-days sleep deprivation experiment (two control nights followed by a night of sleep loss and one recovery night). Between 0800 and 2300 (i.e. between 25 and 40 h of sleep deprivation), a serial of blood sampling, multiple sleep latency, subjective levels of sleepiness and reaction time tests were completed before (day 2: D2) and after (day 4: D4) one night of sleep loss. We showed that an acute sleep deprivation (i.e. after 34 and 37 h of sleep deprivation) induced a significant increase in TNF-α (P < 0.01), but there were no significant changes in TNFR1, IL-6, cortisol and CRP. In conclusion, our study in which constant and controlled experimental conditions were realized with healthy subjects and in absence of psychological or physical stressors, an acute total sleep deprivation (from 34 h) was sufficient to induce secretion of pro-inflammatory cytokine such as TNF-α, a marker more described in chronic sleep restriction or deprivation and as mediators of excessive sleepiness in humans in pathological conditions.     

Hypoxic alterations of cortisol circadian rhythm in man after simulation of a long duration flight

Fatigue is often reported after long duration flights. Mild hypobaric hypoxia caused by pressurisation may be involved in this effect through disruption of circadian rhythms, independently of the number of time zones crossed. In this controlled crossover study, we assessed the effects of two levels of hypoxia equivalent to 8000 and 12,000 ft on the circadian rhythm of plasma cortisol, a marker of the circadian time structure. Sixteen healthy young male volunteers (23-39 years) were exposed in a hypobaric chamber for 8 h (08:00-16:00 h) to 8000 ft, followed 4 weeks later to 12,000 ft. Plasma cortisol was assayed during two 24-h cycles (control and hypoxic exposure) every 2h in all subjects. We found a significant change in the pattern of cortisol secretion during both hypoxic exposures, with an initial fall in cortisol followed by a transient rebound, whereas the phase and the 24-h mean level remained unchanged. The change in cortisol pattern followed the alterations in autonomic balance assessed by heart rate variability (HRV) spectral analysis. The normalised high frequencies and the low-to-high frequencies ratio showed a significant shift toward sympathetic dominance with some differences in time course for both altitudes studied. HRV analysis improved the interpretation of cortisol 24-h profiles. Our data, which strongly suggest that prolonged mild hypoxia alters the expression of cortisol circadian rhythm, should be taken into account to interpret secretory rhythm changes after transmeridian flights.     

Coexpression of RTI40 with alveolar epithelial type II cell proteins in lungs following injury: identification of alveolar intermediate cell types

Injured alveolar epithelial type (AT) I cells are replaced following the proliferation and transformation of ATII cells to new ATI cells. RTI(40) is an ATI cell-specific protein required for normal lung development. We hypothesized that intermediate cell types in the ATII-to-ATI cell transformation would coexpress RTI(40) and ATII cell-selective proteins. To test this hypothesis, we used a rat model of Staphylococcus aureus-induced acute lung injury and a panel of ATI and ATII cell-specific and -selective antibodies. S. aureus induced an acute inflammatory reaction that was resolving by day 3 postinoculation. At day 3 postinoculation, the alveolar wall was thickened secondary to ATII cell hyperplasia. With the use of confocal microscopy, there was a fivefold increase in the fractional surface area of alveolar walls stained with ATII cell membrane proteins (RTII(70) and MMC4) and a decrease in the fractional surface area associated with RTI(40)-expressing cells. S. aureus-treated lungs also contained unique cell types that coexpressed the RTI(40) and ATII markers RTI(40)/MMC4/RTII(70)- and RTI(40)/MMC4-positive cells. These cells were not observed in control lungs. RTI(40)/MMC4-positive cells were also found in cultured ATII cells before they transformed to an ATI-like phenotype. Our data suggest that RTI(40)/MMC4/RTII(70)- and RTI(40)/MMC4-positive cells are intermediates in the ATII-to-ATI cell transformation. These data also suggest that the coexpression of RTI(40) with ATII cell proteins may be used to identify and investigate ATII cell transdifferentiation to ATI cells following injury.     

Expression profiles of hydrophobic surfactant proteins in children with diffuse chronic lung disease

Background

Abnormalities of the intracellular metabolism of the hydrophobic surfactant proteins SP-B and SP-C and their precursors may be causally linked to chronic childhood diffuse lung diseases. The profile of these proteins in the alveolar space is unknown in such subjects.

Methods

We analyzed bronchoalveolar lavage fluid by Western blotting for SP-B, SP-C and their proforms in children with pulmonary alveolar proteinosis (PAP, n = 15), children with no SP-B (n = 6), children with chronic respiratory distress of unknown cause (cRD, n = 7), in comparison to children without lung disease (n = 15) or chronic obstructive bronchitis (n = 19).

Results

Pro-SP-B of 25–26 kD was commonly abundant in all groups of subjects, suggesting that their presence is not of diagnostic value for processing defects. In contrast, pro-SP-B peptides cleaved off during intracellular processing of SP-B and smaller than 19–21 kD, were exclusively found in PAP and cRD. In 4 of 6 children with no SP-B, mutations of SFTPB or SPTPC genes were found. Pro-SP-C forms were identified at very low frequency. Their presence was clearly, but not exclusively associated with mutations of the SFTPB and SPTPC genes, impeding their usage as candidates for diagnostic screening.

Conclusion

Immuno-analysis of the hydrophobic surfactant proteins and their precursor forms in bronchoalveolar lavage is minimally invasive and can give valuable clues for the involvement of processing abnormalities in pediatric pulmonary disorders.     

In vivo and in vitro characterization of novel neuronal plasticity factors identified following spinal cord injury

Following spinal cord injury, there are numerous changes in gene expression that appear to contribute to either neurodegeneration or reparative processes. We utilized high density oligonucleotide microarrays to examine temporal gene profile changes after spinal cord injury in rats with the goal of identifying novel factors involved in neural plasticity. By comparing mRNA changes that were coordinately regulated over time with genes previously implicated in nerve regeneration or plasticity, we found a gene cluster whose members are involved in cell adhesion processes, synaptic plasticity, and/or cytoskeleton remodeling. This group, which included the small GTPase Rab13 and actin-binding protein Coronin 1b, showed significantly increased mRNA expression from 7-28 days after trauma. Overexpression in vitro using PC-12, neuroblastoma, and DRG neurons demonstrated that these genes enhance neurite outgrowth. Moreover, RNAi gene silencing for Coronin 1b or Rab13 in NGF-treated PC-12 cells markedly reduced neurite outgrowth. Coronin 1b and Rab13 proteins were expressed in cultured DRG neurons at the cortical cytoskeleton, and at growth cones along with the pro-plasticity/regeneration protein GAP-43. Finally, Coronin 1b and Rab13 were induced in the injured spinal cord, where they were also co-expressed with GAP-43 in neurons and axons. Modulation of these proteins may provide novel targets for facilitating restorative processes after spinal cord injury.     

Depletion of the co-chaperone CDC-37 reveals two modes of PAR-6 cortical association in C. elegans embryos

PAR proteins play roles in the establishment and maintenance of polarity in many different cell types in metazoans. In C. elegans, polarity established in the one-cell embryo determines the anteroposterior axis of the developing animal and is essential to set the identities of the early blastomeres. PAR-1 and PAR-2 colocalize at the posterior cortex of the embryo. PAR-3, PAR-6 and PKC-3 (aPKC) colocalize at the anterior cortex of the embryo. A process of mutual exclusion maintains the anterior and posterior protein domains. We present results indicating that a homolog of the Hsp90 co-chaperone Cdc37 plays a role in dynamic interactions among the PAR proteins. We show that CDC-37 is required for the establishment phase of embryonic polarity; that CDC-37 reduction allows PAR-3-independent cortical accumulation of PAR-6 and PKC-3; and that CDC-37 is required for the mutual exclusion of the anterior and posterior group PAR proteins. Our results indicate that CDC-37 acts in part by maintaining PKC-3 levels and in part by influencing the activity or levels of other client proteins. Loss of the activities of these client proteins reveals that there are two sites for PAR-6 cortical association, one dependent on CDC-42 and not associated with PAR-3, and the other independent of CDC-42 and co-localizing with PAR-3. We propose that, in wild-type embryos, CDC-37-mediated inhibition of the CDC-42-dependent binding site and PAR-3-mediated release of this inhibition provide a key mechanism for the anterior accumulation of PAR-6.