Modeling the heterogeneity of multiple sclerosis in animals.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS) manifested with varying clinical course, pathology, and inflammatory patterns. There are multiple animal models that reflect different aspects of this heterogeneity. Collectively, these models reveal a balance between pathogenic and regulatory CD4(+) T cells, CD8(+) T cells, and B cells that influences the incidence, timing, and severity of CNS autoimmunity. In this review we discuss experimental autoimmune encephalomyelitis (EAE) models that have been used to study the pathogenic and regulatory roles of these immune cells; models that recapitulate different aspects of the disease seen in patients with MS, and questions remaining for future studies.

Prospective evaluation of a protocol for using transabdominal ultrasound to screen for short cervix.

OBJECTIVE: We sought to evaluate a recently proposed protocol whereby transabdominal ultrasound of the cervix might be used as a prescreen to select women to undergo or to forgo measurement of cervical length via transvaginal ultrasound (CLvag). STUDY DESIGN: This was a prospective cohort study. Measurements of cervical length via transabdominal ultrasound (CLabd) and CLvag were made in women with singleton pregnancy during routine obstetrical ultrasound examination at 18(0/7) to 23(6/7) weeks of gestation. The transabdominal screen was considered positive if CLabd was ≤36 mm with the maternal bladder full or ≤35 mm with the bladder empty, or adequate imaging of the cervix could not be obtained. Sensitivity, specificity, predictive values, and likelihood ratios of a positive screen to detect a short cervix (CLvag ≤25 mm) were calculated. RESULTS: An interim analysis identified several technical problems with CLabd measurements, so the protocol was extensively revised. Under the revised protocol, 1580 women were included. Adequate views of the cervix were obtained via transabdominal imaging in 46% of subjects with the bladder empty and 56% with the bladder full. The correlation between CLabd and CLvag was poor (r = 0.38). Of the 17 patients with a short cervix, 15 had suboptimal transabdominal exams (screen positive) and 2 had CLabd ≤35 mm with bladder empty (screen positive). Sensitivity of the screen was 100% (95% confidence interval, 80.5-100%) but specificity was only 32.2% (95% confidence interval, 29.9-34.6%) and screen positive rate was 66.3%. Several technical problems and limitations of transabdominal imaging of the cervix are shown. CONCLUSION: Using modern, high-resolution ultrasound equipment, we were unable to adequately image the cervix via transabdominal ultrasound in half the cases. Although we confirmed that a CLabd cutoff value of 35-36 mm is appropriate for detection of short cervix, the technique for measuring CLabd is fraught with technical problems. Practitioners must validate the technique in their own practice before adopting this or similar prescreening protocols. We decided not to adopt this protocol.

The influence of T cell Ig mucin-3 signaling on central nervous system autoimmune disease is determined by the effector function of the pathogenic T cells.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the CNS mediated by self-reactive, myelin-specific T cells. Both CD4(+) and CD8(+) T cells play important roles in the pathogenesis of MS. MS is studied using experimental autoimmune encephalomyelitis (EAE), an animal model mediated by myelin-specific T cells. T cell Ig mucin-3 (Tim-3) is a cell surface receptor expressed on CD4(+) IFN-γ-secreting Th1 cells, and triggering Tim-3 signaling ameliorated EAE by inducing death in pathogenic Th1 cells in vivo. This suggested that enhancing Tim-3 signaling might be beneficial in patients with MS. However, Tim-3 is also expressed on activated CD8(+) T cells, microglia, and dendritic cells, and the combined effect of manipulating Tim-3 signaling on these cell types during CNS autoimmunity is unknown. Furthermore, CD4(+) IL-17-secreting Th17 cells also play a role in MS, but do not express high levels of Tim-3. We investigated Tim-3 signaling in EAE models that include myelin-specific Th17, Th1, and CD8(+) T cells. We found that preventing Tim-3 signaling in CD4(+) T cells altered the inflammatory pattern in the CNS due to differential effects on Th1 versus Th17 cells. In contrast, preventing Tim-3 signaling during CD8(+) T cell-mediated EAE exacerbated disease. We also analyzed the importance of Tim-3 signaling in EAE in innate immune cells. Tim-3 signaling in dendritic cells and microglia did not affect the manifestation of EAE in these models. These results indicate that the therapeutic efficacy of targeting Tim-3 in EAE is dependent on the nature of the effector T cells contributing to the disease.

Complete but curtailed T-cell response to very low-affinity antigen.

After an infection, T cells that carry the CD8 marker are activated and undergo a characteristic kinetic sequence of rapid expansion, subsequent contraction and formation of memory cells. The pool of naive T-cell clones is diverse and contains cells bearing T-cell antigen receptors (TCRs) that differ in their affinity for the same antigen. How these differences in affinity affect the function and the response kinetics of individual T-cell clones was previously unknown. Here we show that during the in vivo response to microbial infection, even very weak TCR-ligand interactions are sufficient to activate naive T cells, induce rapid initial proliferation and generate effector and memory cells. The strength of the TCR-ligand interaction critically affects when expansion stops, when the cells exit lymphoid organs and when contraction begins; that is, strongly stimulated T cells contract and exit lymphoid organs later than weakly stimulated cells. Our data challenge the prevailing view that strong TCR ligation is a prerequisite for CD8(+) T-cell activation. Instead, very weak interactions are sufficient for activation, but strong TCR ligation is required to sustain T-cell expansion. We propose that in response to microbial challenge, T-cell clones with a broad range of avidities for foreign ligands are initially recruited, and that the pool of T cells subsequently matures in affinity owing to the more prolonged expansion of high-affinity T-cell clones.

Blood-brain barrier permeability is increased after acute adult stroke but not neonatal stroke in the rat.

The immaturity of the CNS at birth greatly affects injury after stroke but the contribution of the blood-brain barrier (BBB) to the differential response to stroke in adults and neonates is poorly understood. We asked whether the structure and function of the BBB is disrupted differently in neonatal and adult rats by transient middle cerebral artery occlusion. In adult rats, albumin leakage into injured regions was markedly increased during 2-24 h reperfusion but leakage remained low in the neonates. Functional assays employing intravascular tracers in the neonates showed that BBB permeability to both large (70 kDa dextran) and small (3 kDa dextran), gadolinium (III)-diethyltriaminepentaacetic acid tracers remained largely undisturbed 24 h after reperfusion. The profoundly different functional integrity of the BBB was associated with the largely nonoverlapping patterns of regulated genes in endothelial cells purified from injured and uninjured adult and neonatal brain at 24 h (endothelial transcriptome, 31,042 total probe sets). Within significantly regulated 1266 probe sets in injured adults and 361 probe sets in neonates, changes in the gene expression of the basal lamina components, adhesion molecules, the tight junction protein occludin, and matrix metalloproteinase-9 were among the key differences. The protein expression of collagen-IV, laminin, claudin-5, occludin, and zonula occludens protein 1 was also better preserved in neonatal rats. Neutrophil infiltration remained low in acutely injured neonates but neutralization of cytokine-induced neutrophil chemoattractant-1 in the systemic circulation enhanced neutrophil infiltration, BBB permeability, and injury. The markedly more integrant BBB in neonatal brain than in adult brain after acute stroke may have major implications for the treatment of neonatal stroke.

IL-6 trans-signaling system in intra-amniotic inflammation, preterm birth, and preterm premature rupture of the membranes.

Classic IL-6 signaling is conditioned by the transmembrane receptor (IL-6R) and homodimerization of gp130. During trans-signaling, IL-6 binds to soluble IL-6R (sIL-6R), enabling activation of cells expressing solely gp130. Soluble gp130 (sgp130) selectively inhibits IL-6 trans-signaling. To characterize amniotic fluid (AF) IL-6 trans-signaling molecules (IL-6, sIL-6R, sgp130) in normal gestations and pregnancies complicated by intra-amniotic inflammation (IAI), we studied 301 women during second trimester (n = 39), third trimester (n = 40), and preterm labor with intact (n = 131, 85 negative IAI and 46 positive IAI) or preterm premature rupture of membranes (PPROM; n = 91, 61 negative IAI and 30 positive IAI). ELISA, Western blotting, and real-time RT-PCR were used to investigate AF, placenta, and amniochorion for protein and mRNA expression of sIL-6R, sgp130, IL-6R, and gp130. Tissues were immunostained for IL-6R, gp130, CD15(+) (polymorphonuclear), and CD3(+) (T cell) inflammatory cells. The ability of sIL-6R and sgp130 to modulate basal and LPS-stimulated release of amniochorion matrix metalloprotease-9 was tested ex vivo. We showed that in physiologic gestations, AF sgp130 decreases toward term. AF IL-6 and sIL-6R were increased in IAI, whereas sgp130 was decreased in PPROM. Our results suggested that fetal membranes are the probable source of AF sIL-6R and sgp130. Immunohistochemistry and RT-PCR revealed increased IL-6R and decreased gp130 expression in amniochorion of women with IAI. Ex vivo, sIL-6R and LPS augmented amniochorion matrix metalloprotease-9 release, whereas sgp130 opposed this effect. We conclude that IL-6 trans-signaling molecules are physiologic constituents of the AF regulated by gestational age and inflammation. PPROM likely involves functional loss of sgp130.

A short educational intervention measurably benefits keloid-prone individuals' knowledge of prevention and treatment.

BACKGROUND: The Internet is a commonly utilized health information resource that provides access to information of varying quality. OBJECTIVE: We sought to evaluate the use of the Internet as a health information resource within a keloid patient population and the effects of an educational intervention on patient knowledge about keloids.
METHODS: A consecutive convenience sample of subjects completed a questionnaire on keloid-related Internet use and on personal and family history of keloids. Participants listened to a short educational intervention on keloid-related topics followed by assessment of relevant knowledge at baseline, immediately postintervention, and 3 months after the intervention. RESULTS: Among 40 participants, 55% reported having used the Internet to obtain keloid-related information. Subjects who had used the Internet to obtain keloid-related information had baseline knowledge similar to those who had not. When subjects were assessed immediately and 3 months postintervention, the intervention improved knowledge that not all raised scars are keloids, that keloids are not cancerous, and that certain areas of the body are more prone to keloid formation. The proportion of subjects who reported being less likely to obtain piercings or tattoos because of the intervention was 80% and 75%, respectively. LIMITATIONS: This study was performed at a single academic center. CONCLUSION: The Internet is a commonly used information resource for keloid-prone individuals, but keloid-related knowledge was not greater among Internet keloid-related information seekers. A very short educational intervention benefits keloid-prone individuals by improving knowledge about keloid prevention and treatment and by discouraging them from obtaining piercings and tattoos.

Cord blood erythropoietin and interleukin-6 for prediction of intraventricular hemorrhage in the preterm neonate.

OBJECTIVE: To evaluate cord blood erythropoietin (EPO) and interleukin-6 (IL-6) levels to predict preterm infants at risk of developing intraventricular hemorrhage (IVH). METHODS: Levels of umbilical cord EPO, acid-base status and IL-6 were analyzed in 116 consecutive, preterm newborns (GA at delivery: 29 [23-34 ] weeks) born to mothers who had a clinically indicated amniocentesis to rule out infection. Early-onset neonatal sepsis (EONS) was diagnosed using symptoms, hematological criteria and blood cultures. RESULTS: IVH was diagnosed by cranial ultrasounds. The prevalence of IVH in our population was 25% (29/116). There was a direct relationship between cord blood EPO and cord blood IL-6 concentration (r = 0.225, p = 0.014), independent of GA at birth. Elevated cord blood EPO levels (r = 0.182, p = 0.016) and GA at birth (r =  -0.236, p = 0.004) remained significant independent factors associated with the risk of IVH, when evaluated with stepwise logistic regression analyses. Cord blood IL-6, pH, and EONS were not associated with IVH. These relationships remained following correction for GA at birth (p = 0.027). CONCLUSIONS: Our results suggest that elevation in cord blood EPO may predict newborns at risk for IVH, independent of fetal inflammatory status. Further studies are warranted to confirm this association.

Activation of the receptor for advanced glycation end products system in women with severe preeclampsia.

CONTEXT: Activation of the receptor for advanced glycation end products (RAGE) mediates cellular injury. Soluble forms of RAGE [soluble RAGE (sRAGE), endogenous secretory (esRAGE)] bind RAGE ligands, thereby preventing downstream signaling and damage. OBJECTIVES: The objective of the study was to characterize the changes in maternal serum, amniotic fluid, and cord blood soluble receptor for advanced glycation end products (sRAGE) during physiological gestation and to provide insight into mechanisms responsible for RAGE activation in preeclampsia. DESIGN AND SETTINGS: This was a cross-sectional study at a tertiary university hospital. PATIENTS: We studied 135 women in the following groups: nonpregnant controls (n = 16), healthy pregnant controls (n = 68), pregnant women with chronic hypertension (n = 13), or pregnant women with severe preeclampsia (sPE; n = 38). INTERVENTIONS AND MAIN OUTCOME MEASURES: sRAGE and esRAGE levels were evaluated in vivo by ELISA in maternal serum, amniotic fluid, and cord blood and in vitro after stimulation of the amniochorion and placental explants with lipopolysaccharide or xanthine/xanthine oxidase. Placenta and amniochorion were immunostained for RAGE. Real-time quantitative PCR measured RAGE mRNA. RESULTS: Pregnant women had significantly decreased serum sRAGE compared with nonpregnant subjects (P < 0.001). sPE women had higher serum and amniotic fluid sRAGE and esRAGE relative to those expected for gestational age (P < 0.001). Cord blood sRAGE remained unaffected by sPE. RAGE immunoreactivity and mRNA expression appeared elevated in the amniochorion of sPE women. Xanthine/xanthine oxidase (but not lipopolysaccharide) significantly up-regulated the release of sRAGE (P < 0.001) in the amniochorion explant system. CONCLUSIONS: Fetal membranes are a rich source of sRAGE. Elevated maternal serum and amniotic fluid sRAGE and esRAGE, paralleled by increased RAGE expression in the amniochorion, suggest activation of this system in sPE.

Macrophages are comprised of resident brain microglia not infiltrating peripheral monocytes acutely after neonatal stroke.

Macrophages can be both beneficial and detrimental after CNS injury. We previously showed rapid accumulation of macrophages in injured immature brain acutely after ischemia-reperfusion. To determine whether these macrophages are microglia or invading monocytes, we subjected post-natal day 7 (P7) rats to transient 3 h middle cerebral artery (MCA) occlusion and used flow cytometry at 24 and 48 h post-reperfusion to distinguish invading monocytes (CD45high/CD11b+) from microglia (CD45low/medium/CD11b+). Inflammatory cytokines and chemokines were determined in plasma, injured and contralateral tissue 1-24 h post-reperfusion using ELISA-based cytokine multiplex assays. At 24 h, the number of CD45+/CD11b+ cells increased 3-fold in injured compared to uninjured brain tissue and CD45 expression shifted from low to medium with less than 10% of the population expressing CD45high. MCA occlusion induced rapid and transient asynchronous increases in the pro-inflammatory cytokine IL-beta and chemokines cytokine-induced neutrophil chemoattractant protein 1 (CINC-1) and monocyte-chemoattractant protein 1 (MCP-1), first in systemic circulation and then in injured brain. Double immunofluorescence with cell-type specific markers showed that multiple cell types in the injured brain produce MCP-1. Our findings show that despite profound increases in MCP-1 in injured regions, monocyte infiltration is low and the majority of macrophages in acutely injured regions are microglia.

Aminoguanidine inhibits caspase-3 and calpain activation without affecting microglial activation following neonatal transient cerebral ischemia.

Microglial cells, the resident macrophages of the CNS, can be both beneficial and detrimental to the brain. These cells play a central role as mediators of neuroinflammation associated with many neurodegenerative states, including cerebral ischemia. Because microglial cells are both a major source of inducible nitric oxide synthase (iNOS)/nitric oxide (NO) production locally in the injured brain and are activated by NO-mediated injury, we tested whether iNOS inhibition reduces microglial activation and ischemic injury in a neonatal focal ischemia-reperfusion model. Post-natal day 7 rats were subjected to a 2 h transient middle cerebral artery (MCA) occlusion. Pups with confirmed injury on diffusion-weighted magnetic resonance imaging (MRI) during occlusion were administered 300 mg/kg/dose aminoguanidine (AG) or vehicle at 0, 4 and 18 h after reperfusion, and animals were killed at 24 or 72 h post-reperfusion. The effect of AG on microglial activation as judged by the acquisition of ED1 immunoreactivity and proliferation of ED1-positive cells, on activation of cell death pathways and on injury volume, was determined. The study shows that while AG attenuates caspase 3 and calpain activation in the injured tissue, treatment does not affect the rapidly occurring activation and proliferation of microglia following transient MCA occlusion in the immature rat, or reduce injury size.