Roles of TNF-related apoptosis-inducing ligand in experimental autoimmune encephalomyelitis

TRAIL, the TNF-related apoptosis-inducing ligand, induces apoptosis of tumor cells, but not normal cells; the roles of TRAIL in nontransformed tissues are unknown. Using a soluble TRAIL receptor, we examined the consequences of TRAIL blockade in an animal model of multiple sclerosis. We found that chronic TRAIL blockade in mice exacerbated experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein. The exacerbation was evidenced primarily by increases in disease score and degree of inflammation in the CNS. Interestingly, the degree of apoptosis of inflammatory cells in the CNS was not affected by TRAIL blockade, suggesting that TRAIL may not regulate apoptosis of inflammatory cells in experimental autoimmune encephalomyelitis. By contrast, myelin oligodendrocyte glycoprotein-specific Th1 and Th2 cell responses were significantly enhanced in animals treated with the soluble TRAIL receptor. Based on these observations, we conclude that unlike TNF, which promotes autoimmune inflammation, TRAIL inhibits autoimmune encephalomyelitis and prevents activation of autoreactive T cells.     

Blunted Sodium Excretion in Response to a Saline Load in 5 Year Old Female Sheep Following Fetal Uninephrectomy

Previously, we have shown that fetal uninephrectomy (uni-x) causes hypertension in female sheep by 2 years of age. Whilst the hypertension was not exacerbated by 5 years of age, these uni-x sheep had greater reductions in renal blood flow (RBF). To further explore these early indications of a decline in renal function, we investigated the renal response to a saline load (25 ml/kg/40 min) in 5-year old female uni-x and sham sheep. Basal mean arterial pressure was ∼15 mmHg greater (P_Group<0.001), and sodium excretion (∼50%), glomerular filtration rate (∼30%, GFR) and RBF (∼40%) were all significantly lower (P_Group<0.01) in uni-x compared to sham animals. In response to saline loading, sodium excretion increased significantly in both groups (P_Time<0.001), however this response was blunted in uni-x sheep (P_GroupxTime<0.01). This was accompanied with an attenuated increase in GFR and fractional sodium excretion (both P_GroupxTime<0.05), and reduced activation of the renin-angiotensin system (both P<0.05), as compared to the sham group. The reduction in sodium excretion was associated with up-regulations in the renal gene expression of NHE3 and Na⁺/K⁺ ATPase α and β subunits in the kidney cortex of the uni-x compared to the sham animals (P<0.05). Notably, neither group completely excreted the saline load within the recovery period, but the uni-x retained a higher percentage of the total volume (uni-x: 48±7%; sham: 22±9%, P<0.05). In conclusion, a reduced ability to efficiently regulate extracellular fluid homeostasis is evident in female sheep at 5 years of age, which was exacerbated in animals born with a congenital nephron deficit. Whilst there was no overt exacerbation of hypertension and renal insufficiency with age in the uni-x sheep, these animals may be more vulnerable to secondary renal insults.     

Implications in the difference of anti-Mi-2 and -p155/140 autoantibody prevalence in two dermatomyositis cohorts from Mexico City and Guadalajara

Introduction

Autoantibodies and clinical manifestations in polymyositis/dermatomyositis (PM/DM) are affected by both genetic and environmental factors. The high prevalence of DM and anti-Mi-2 in Central America is thought to be associated with the high UV index of the area. The prevalences of autoantibodies and the clinical manifestations of PM/DM were evaluated comparing two cohorts in Mexico.

Methods

Ninety-five Mexican patients with PM/DM (66 DM, 29 PM; 67 Mexico City, 28 Guadalajara) were studied. Autoantibodies were characterized by immunoprecipitation using ³⁵S-methionine labeled K562 cell extract. Clinical information was obtained from medical records.

Results

DM represented 69% of PM/DM and anti-Mi-2 was the most common autoantibody (35%), followed by anti-p155/140 (11%); however, anti-Jo-1 was only 4%. The autoantibody profile in adult-onset DM in Mexico City versus Guadalajara showed striking differences: anti-Mi-2 was 59% versus 12% (P = 0.0012) whereas anti-p155/140 was 9% versus 35% (P = 0.02), respectively. A strong association of anti-Mi-2 with DM was confirmed and when clinical features of anti-Mi-2 (+) DM (n = 30) versus anti-Mi-2 (-) DM (n = 36) were compared, the shawl sign (86% versus 64%, P < 0.05) was more common in the anti-Mi-2 (+) group (P = 0.0001). Levels of creatine phosphokinase (CPK) were higher in those who were anti-Mi-2 (+) but they responded well to therapy.

Conclusions

Anti-Mi-2 has a high prevalence in Mexican DM and is associated with the shawl sign and high CPK. The prevalence of anti-Mi-2 and anti-p155/140 was significantly different in Mexico City versus Guadalajara, which have a similar UV index. This suggests roles of factors other than UV in anti-Mi-2 antibody production.     

Rpp25 is a major target of autoantibodies to the Th/To complex as measured by a novel chemiluminescent assay

Introduction

Autoantibodies to the Th/To antigen have been described in systemic sclerosis (SSc) and several proteins of the macromolecular Th/To complex have been reported to react with anti-Th/To antibodies. However, anti-Th/To has not been clinically utilized due to unavailability of commercial tests. The objective of the present study is to evaluate the newly developed ELISA and chemiluminescent immunoassay (CLIA) to measure autoantibodies to Rpp25 (a component of the Th/To complex) using immunoprecipitation (IP) as the reference method.

Methods

The first cohort consisted of 123 SSc patients including 7 anti-Th/To positive samples confirmed by IP. Additional seven anti-Th/To positive samples from non-SSc patients were also tested. For evaluation of the QUANTA Flash Rpp25 CLIA (research use only), 8 anti-Th/To IP positives, a cohort of 70 unselected SSc patients and sera from various disease controls (n = 357) and random healthy individuals (n = 10) were studied.

Results

Anti-Rpp25 antibodies determined by ELISA were found in 11/14 anti-Th/To IP positive but only in 1/156 (0.6%) negative samples resulting in a positive percent agreement of 78.6% (95% confidence interval [CI] 49.2, 95.3%) and a negative percent agreement of 99.4% (95% CI 96.4, 100.0%). To verify the results using a second method, 53 samples were tested by ELISA and CLIA for anti-Rpp25 reactivity and the results were highly correlated (rho = 0.71, 95% CI 0.56, 0.81; P < 0.0001). To define the cutoff of the CLIA, anti-Th/To IP positive and negative sera were tested using the anti-Rpp25 CLIA. At the cutoff selected by receiver operating characteristic (ROC) analysis 8/8 (100.0%) of the anti-Th/To positive sera but only 2/367 (0.5%) of the controls were positive for anti-Rpp25 antibodies. The positive and negative percent agreements were 100.0% (95% CI 63.1, 100.0%) and 99.5% (95% CI 98.0, 99.9%), respectively. In the disease cohorts 2/70 (2.9%) of the SSc patients were positive for anti-Rpp25 antibodies compared to 2/367 (0.5%) of the controls (P = 0.032). ROC analysis showed discrimination between SSc patients and controls with an area under the curve value of 0.732 (95% CI 0.655, 0.809).

Conclusion

Rpp25 is a major target of autoantibodies to the Th/To autoantigen complex. Further studies are needed to evaluate the clinical utility of the new assays.     

Herpes simplex virus 1 ICP0 phosphorylation site mutants are attenuated for viral replication and impaired for explant-induced reactivation

In cell culture experiments, phosphorylation appears to be a critical regulator of the herpes simplex virus 1 (HSV-1) immediate-early (IE) protein, ICP0, which is an E3 ubiquitin ligase that transactivates viral gene expression. Three major regions of phosphorylation in ICP0 (amino acids 224 to 232, 365 to 371, and 508 to 518) have been identified, and mutant viruses that block phosphorylation sites within each region (termed Phos 1, 2, and 3, respectively) have been constructed. Previous studies indicated that replication of Phos 1 is significantly reduced compared to that of wild-type virus in cell culture (C. Boutell, et al., J. Virol. 82:10647-10656, 2008). To determine the effects these phosphorylation site mutations have on the viral life cycle in vivo, mice were ocularly infected with wild-type HSV-1, the Phos mutants, or their marker rescue counterparts. Subsequently, viral replication, establishment of latency, and viral explant-induced reactivation of these viruses were examined. Relative to wild-type virus, Phos 1 eye titers were reduced as much as 7- and 18-fold on days 1 and 5 postinfection, respectively. Phos 2 eye titers showed a decrease of 6-fold on day 1 postinfection. Titers of Phos 1 and 2 trigeminal ganglia were reduced as much as 16- and 20-fold, respectively, on day 5 postinfection. Additionally, the reactivation efficiencies of Phos 1 and 2 were impaired relative to wild-type HSV-1, although both viruses established wild-type levels of latency in vivo. The acute replication, latency, and reactivation phenotypes of Phos 3 were similar to those of wild-type HSV-1. We conclude from these studies that phosphorylation is likely a key modulator of ICP0's biological activities in a mouse ocular model of HSV-1 infection.