Diagnosis of hepatic fibrosis and cirrhosis by transient elastography in HIV/hepatitis C virus-coinfected patients

BACKGROUND: Chronic hepatitis C in HIV-infected patients is an increasing cause of death dependent on the development of liver fibrosis, which is currently assessed by liver biopsy despite its limitations. Liver stiffness measurement, a new noninvasive method, allows the evaluation of liver fibrosis. The aim of this prospective study was to assess the accuracy of liver stiffness measurement for the detection of fibrosis and cirrhosis in HIV/hepatitis C virus (HCV)-coinfected patients and to compare its accuracy with other noninvasive methods. METHODS: We studied 72 consecutive HIV patients with chronic hepatitis C who had a simultaneous liver biopsy and liver stiffness measurement by transient elastography (FibroScan; Echosens, Paris, France) for the assessment of liver fibrosis. RESULTS: Liver stiffness values ranged from 3.0 to 46.4 kilopascal. Liver stiffness was significantly correlated to fibrosis stage (Kendall tau-b = 0.48; P < 0.0001). The area under the receiver operating characteristic (AUROC) curve of liver stiffness measurement was 0.72 for F > or = 2 and 0.97 for F = 4. For the diagnosis of cirrhosis, AUROC curves of liver stiffness measurement were significantly higher than those for platelet count (P = 0.02), aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio (P = 0.0001), Aspartate aminotransferase-to-Platelet Ratio Index (APRI) (P = 0.01), and FIB-4 (P = 0.004). CONCLUSION: Liver stiffness measurement is a promising noninvasive method for the assessment of fibrosis in HIV-infected patients with chronic HCV infection. Its use for the follow-up of these patients should be further evaluated.     

Robust coupling of body movement and gaze in young infants

In the first few months after birth, rapid bursts of body movement precede and possibly facilitate shifts of gaze during free looking, with potential consequences for perception and cognition. Here we report that the characteristic features of movement-gaze coupling found during free looking are preserved when attention is perturbed by a salient change in the visual environment. Twenty-four 3-month-olds looked at two attractive 3-dimensional objects while body movement and corneal reflections of the objects were recorded. Lateral head movement was measured offline. After approximately 2 s of looking at one stimulus, the nonfixated stimulus either began to rotate back and forth (distracter events) or remained motionless (control events). In distracter events, the motion of the nonfixated stimulus triggered substantial motor quieting, shortened the duration of the look, and shortened the time to reorient gaze compared to control events. Abbreviated motor quieting and small increases in lateral head movement occurred during control events at the same time in the look as the protracted motor quieting and increased head movement in distracter events. Despite these perturbations, the characteristic bursts of body movement that precede shifts of gaze during free looking occurred in both distracter and control events. The results demonstrate the robust nature of early movement-gaze coupling, raise questions about the specific role of attention in the dynamic links between body movement and gaze, and highlight the potential short and long term functional significance of movement-gaze-attention coupling.     

Combining EL4-B5-based B-cell stimulation and phage display technology for the successful isolation of human anti-Scl-70 autoantibody fragments

Scl-70 is the major antigen recognised by autoantibodies in the sera of patients with systemic sclerosis (SSc). The autoantibodies that specifically react with Scl-70 are highly characteristic of the disease and represent valuable markers for the diagnosis of SSc. We describe a novel strategy for cloning autoantibody fragments starting with a small blood sample from an SSc patient. B cells isolated from the collected peripheral blood mononuclear cells (PBMCs) were cultured in vitro using the EL4-B5 system. Anti-Scl-70 IgG-producing cells were pooled for RNA preparation followed by the generation of phagemid libraries of approximately 10(7) independent single-chain Fvs (scFvs). The screening of these libraries by phage display allowed us to isolate four anti-Scl-70 scFvs following three rounds of biopanning. About 10 times more starting blood material was needed to generate scFv libraries of similar size from PBMCs of an SSc patient and only two anti-Scl-70 scFvs were isolated after three rounds of phage selection. Together, this work shows that functional autoantibody fragments can be advantageously cloned after in vitro expansion of B cells. The isolated anti-Scl-70 autoantibody fragments represent useful tools for calibrating SSc diagnostic assays.     

Environmental allergens and asthma in urban elementary schools.

BACKGROUND: Asthma in school children is rising, and indoor allergens are very common triggers of asthma attacks; however, the risk of the school environment on asthma has not been well studied. OBJECTIVE: To determine the presence and the levels of common aeroallergens in schools, where asthma prevalence rates are high. METHODS: Settled dust samples were collected from 12 Baltimore City public elementary schools, and they were analyzed for the following allergens: cockroaches (Bla g 1/2), dust mites (Der f 1/p 1), dog (Can f 1), cat (Fel d 1), and mouse (Mus m 1). School asthma prevalence rates were correlated with allergen levels, and association between allergen levels and other risk factors present in the schools' environment was examined. RESULTS: The mean and range levels were 1.49 U/g (0 to 8) for Bla g 1/2; 0.38 microg/g (0 to 11.9) for the Der f 1/p 1; 1.44 microg/g (0.1 to 9.6) for Can f 1; 1.66 microg/g (0.2 to 12) for Fel d 1; and 6.24 microg/g (0.3 to 118.3) for Mus m 1. Dust mite, cat and dog allergens were significantly in rooms with carpet and/or area rugs, compared to rooms with bare floors (P < 0.05). Asthma prevalence rates varied from 11.8 to 20.8% between schools and positively correlation with the mean levels of Bla g 1/2 in the schools (P = 0.001). CONCLUSIONS: Common allergens that are known to trigger asthma were detected in all school environments, where asthma prevalence rates were high. However, the overall allergen levels were low, indicating that other factors, including exposures in the homes of asthmatic patients, may have more relevance to sensitization and symptoms than school exposures.     

IL1 receptor accessory protein like,a protein involved in X-linked mental retardation,interacts with Neuronal Calcium Sensor-1 and regulates exocytosis

Previously, human genetics-based approaches allowed us to show that mutations in the IL-1 receptor accessory protein-like gene (IL1RAPL) are responsible for a non-specific form of X-linked mental retardation. This gene encodes a predicted protein of 696 amino acids that belongs to a novel class of the IL-1/Toll receptor family. In addition to the extracellular portion consisting of three Ig-like domains and the intracellular TIR domain characteristic of the IL-1/Toll receptor family, IL1RAPL contains a specific 150 amino acid carboxy terminus that has no significant homology with any protein of known function. In order to begin to elucidate the function of this IL-1/Toll receptor-like protein, we have assessed the effect of recombinant IL1RAPL on the binding affinity of type I IL-1R for its ligands IL-1alpha and beta and searched for proteins interacting with the specific carboxy terminus domain of IL1RAPL. Our results show that IL1RAPL is not a protein receptor for IL-1. In addition we present here the identification of Neuronal Calcium Sensor-1 (NCS-1) as an IL1RAPL interactor. Remarkably, although NCS-1 and its non-mammalian homologue, frequenin, are members of a highly conserved EF-hand Ca(2+) binding protein family, our data show that IL1RAPL interacts only with NCS-1 through its specific C-terminal domain. The functional relevance of IL1RAPL activity was further supported by the inhibitory effect on exocytosis in PC12 cells overexpressing IL1RAPL. Taken together, our data suggest that IL1RAPL may regulate calcium-dependent exocytosis and provide insight into the understanding of physiopathological mechanisms underlying cognitive impairment resulting from IL1RAPL dysfunction.     

Cigarette Smoke-Related Hydroquinone Induces Filamentous Actin Reorganization and Heat Shock Protein 27 Phosphorylation through p38 and Extracellular Signal-Regulated Kinase 1/2 in Retinal Pigment Epithelium : Implications for Age-Related Macular Degeneration

Retinal pigment epithelium (RPE)-derived membranous debris named blebs, may accumulate and contribute to sub-RPE deposit formation, which is the earliest sign of age-related macular degeneration (AMD). Oxidative injury to the RPE might play a significant role in AMD. However, the underlying mechanisms are unknown. We previously reported that hydroquinone (HQ), a major pro-oxidant in cigarette smoke, foodstuff, and atmospheric pollutants, induces actin rearrangement and membrane blebbing in RPE cells as well as sub-RPE deposits in mice. Here, we show for the first time that phosphorylated Heat shock protein 27 (Hsp27), a key regulator of actin filaments dynamics, is up-regulated in RPE from patients with AMD. Also, HQ-induced nonlethal oxidative injury led to Hsp27mRNA up-regulation, dimer formation, and Hsp27 phosphorylation in ARPE-19 cells. Furthermore, we found that a cross talk between p38 and extracellular signal-regulated kinase (ERK) mediates HQ-induced Hsp27 phosphorylation and actin aggregate formation, revealing ERK as a novel upstream mediator of Hsp27 phosphorylation. Finally, we demonstrated that Hsp25, p38, and ERK phosphorylation are increased in aging C57BL/6 mice chronically exposed to HQ, whereas Hsp25 expression is decreased. Our data suggest that phosphorylated Hsp27 might be a key mediator in AMD and HQ-induced oxidative injury to the RPE, which may provide helpful insights into the early cellular events associated with actin reorganization and bleb formation involved in sub-RPE deposits formation relevant to the pathogenesis of AMD.Age-related macular degeneration (AMD) is the main cause of blindness in the elderly population in the Western societies and has a devastating impact on people’s quality of life.^1,2,3,4 An estimated 13 million Americans have some degree of AMD and unless better preventive treatments emerge, this number is expected to climb and even reach epidemic proportions with the overall aging demographics. Although the cause of AMD has not yet been fully determined, this complex multifactorial disease clearly results from the interplay of multiple genetic and environmental risk factors^2,5 that lead to progressive destruction of retinal pigment epithelial (RPE) cells and photoreceptors and ultimately to vision loss.Accumulation of deposits between the RPE and its basement membrane (called basal laminar deposits) is part of the numerous biochemical and anatomical changes associated with aging retina. However, the presence of large numbers and extensive areas of additional deposits (named basal linear deposits and drusen) beneath the RPE and within the inner collagenous layer of Bruch’s membrane (BrM) is a defining histopathological landmark associated with dry AMD,⁶ the most common type of the disease that affects the greatest number of people. Until now, the underlying pathogenic mechanisms of sub-RPE deposits formation and accumulation as well as their relative contribution to the pathogenesis of AMD are still obscure. However, a growing body of evidence suggests that cumulative oxidative injury is implicated in the pathogenesis of the disease^7,8,9 and plays a critical role in deposits formation.¹⁰ Cigarette smoking is a major source of oxidative stress and has unequivocally been established as the single greatest environmental risk factor in the onset and development of AMD.^11,12,13,14 It has been suggested that cigarette smoking might contribute to the pathogenesis of AMD by causing oxidative damage to the RPE.¹⁵ Tar within cigarette smoke contains a large number of pro-oxidant compounds among which hydroquinone (HQ) is the most abundant. HQ is an oxidant of special relevance due to its presence not only in cigarette smoke but also in processed foods, plastic containers, and atmospheric pollutants as well as its widespread occurrence in nature.^16,17 Detectable levels of HQ have been measured in the plasma of many nonsmoker individuals living a Western life style.¹⁶We embrace the pathogenic paradigm based on the response-to-injury hypothesis, which proposes that sub-RPE deposits originate from RPE-derived cell membrane blebs^18,19 induced by chronic nonlethal injury to the RPE in response to oxidative damage. Clinical studies performed on eyes from patients with AMD have revealed that membranous debris from the basolateral RPE surface traversed the RPE membrane and deposited in the inner and outer collagenous layers of BrM.²⁰ We previously found that exposure to cigarette smoke and HQ resulted in RPE membrane blebbing and sub-RPE deposits in mice.^21,22 We also demonstrated that nonlethal HQ-induced oxidative injury in cultured RPE cells results in reorganization of actin cytoskeleton and blebs formation^22,23,24,25 relevant to the accumulation of deposits. Blebbing of the plasma membrane is an early morphological sign of cell injury, which occurs immediately after exposure to a wide variety of toxic agents. Oxidative stress induces profound rearrangements of the actin cytoskeleton^26,27,28 leading to membrane blebbing through the activation of p38 mitogen activated protein kinase (MAPK)/Heat shock protein 27 (Hsp27) pathway.^26,29Hsp27 (Hsp25 in mice) is a phosphorylation-regulated filamentous actin (F-actin) cap binding protein that plays a key role in modulating actin microfilaments dynamics.³⁰ We previously reported that ARPE-19 cells constitutively express high levels of Hsp27, which are regulated by oxidant-mediated injury.³¹ We also found that Hsp27 is expressed in extruded blebs confirming that Hsp27 plays a role in actin filaments dynamics. In addition, we demonstrated that prolonged exposure of RPE cells to HQ results in actin rearrangements into aggregates and membrane blebbing, with a concomitant activation of the p38 MAPK pathway showed by immunohistochemistry.²⁵ However, whether HQ-induced oxidative injury can activate other MAPK signaling pathways, including extracellular signal-regulated kinase 1/2 (ERK1/2) cascade, has not been investigated. In addition, this study did not address the question of any posttranslational modifications such as dimerization and phosphorylation of Hsp27 known to regulate F-actin polymerization. Taken together, these data suggest that Hsp27 may be an important mediator of RPE response to HQ-induced oxidative damage and may contribute to injury-induced actin rearrangement and blebbing, which may be crucial in the formation of sub-RPE deposits. The aim of this study was to evaluate the expression of phosphorylated Hsp27 in RPE from patients with AMD and further characterize the contribution of phosphorylated Hsp27 in mediating F-actin cytoskeleton reorganization during nonlethal HQ-induced oxidative stress as well as the involvement of p38 and ERK MAPK pathways. A greater understanding of the sequence of early cellular events associated with HQ-induced F-actin dysregulation in RPE cells is of critical importance to further identify its role in the regulation of RPE blebbing and define its contribution to the accumulation of sub-RPE deposits relevant to the pathophysiology of AMD.     

T- and B-Cell Immune Responses of Patients Who Had Undergone Colectomies to Oral Administration of Salmonella enterica Serovar Typhi Ty21a Vaccine

The capacity of an oral live attenuated Salmonella enterica serovar Typhi Ty21a vaccine to induce immune responses in patients who had undergone colectomies because of ulcerative colitis was evaluated, and these responses were compared with those of healthy volunteers. Purified CD4⁺ and CD8⁺ T cells from peripheral blood were stimulated in vitro by using the heat-killed Ty21a vaccine strain, and the proliferation and gamma interferon (IFN-γ) production were measured before and 7 or 8 days after vaccination. Salmonella-specific immunoglobulin A (IgA) and IgG antibody responses in serum along with IgA antibody responses in ileostomy fluids from the patients who had undergone colectomies were also evaluated. Three doses of vaccine given 2 days apart failed to induce proliferative T-cell responses in all the six patients who had undergone colectomies, and increases in IFN-γ production were found only among the CD8⁺ cells from three of the patients. In contrast, both proliferative responses and increased IFN-γ production were observed among CD4⁺ and CD8⁺ T cells from 3 and 6 of 10 healthy volunteers, respectively. Salmonella-specific IgA and/or IgG antibody responses in serum were observed for five (56%) of nine patients who had undergone colectomies and in 15 (88%) of 17 healthy volunteers. In ileostomy fluids, significant anti-Salmonella IgA antibody titer increases were detected in six (67%) of nine patients who had undergone colectomies. The impaired T- and B-cell immune responses found after vaccination in the circulation of patients who have undergone colectomies may be explained by a diminished colonization of the Ty21a vaccine strain due to the lack of a terminal ileum and colon.     

PRQFVamide,a novel pentapeptide identified from the CNS and gut of Aplysia

We have purified a novel pentapeptide from the Aplysia nervous system using bioassay on gut contractions. The structure of the peptide is Pro-Arg-Gln-Phe-Val-amide (PRQFVa). The precursor for PRQFVa was found to code for 33 copies of PRQFVamide and four related pentapeptides. Peaks corresponding to the predicted masses of all five pentapeptides were detected in Aplysia neurons by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Northern analysis revealed that expression of the precursor is abundant in the abdominal ganglion, much less in the pedal and cerebral ganglia, and rarely seen in the buccal and pleural ganglia. PRQFVa-positive neurons, mapped by immunohistochemistry and in situ hybridization, were present in all the central ganglia. PRQFVa immunopositive processes were observed in the gut, particularly in association with the vasculature. Some arteries and other highly vascularized tissues, such as the gill and the kidney, also contain numerous PRQFVa immunopositive processes. Application of synthetic PRQFVa suppresses not only contractions of the gut but also contractions of vasculature. PRQFVa is expressed in some of the neurons within the feeding circuitry and application of synthetic PRQFVa was found to decrease the excitability of some (B4/5 and B31/32) but not all (B8) neurons of the buccal feeding circuit. Our findings suggest that PRQFVa may act as a modulator within the feeding system as well as in other systems of Aplysia.     

The gamma interferon (IFN-gamma)-inducible GTP-binding protein IGTP is necessary for toxoplasma vacuolar disruption and induces parasite egression in IFN-gamma-stimulated astrocytes

Toxoplasma gondii is a common central nervous system infection in individuals with immunocompromised immune systems, such as AIDS patients. Gamma interferon (IFN-γ) is the main cytokine mediating protection against T. gondii. Our previous studies found that IFN-γ significantly inhibits T. gondii in astrocytes via an IFN-γ-inducible GTP-binding protein (IGTP)-dependent mechanism. The IGTP-dependent-, IFN-γ-stimulated inhibition is not understood, but recent studies found that IGTP induces disruption of the parasitophorous vacuole (PV) in macrophages. In the current study, we have further investigated the mechanism of IFN-γ inhibition and the role of IGTP in the vacuolar disruption in murine astrocytes. Vacuolar disruption was found to be dependent upon IGTP, as PV disruption was not observed in IGTP-deficient (IGTP^−/−) astrocytes and PV disruption could be induced in IGTP^−/− astrocytes transfected with IGTP. Live-cell imaging studies using green fluorescent protein-IGTP found that IGTP is delivered to the PV via the host cell endoplasmic reticulum (ER) early after invasion and that IGTP condenses into vesicle-like structures on the vacuole just prior to PV disruption, suggesting that IGTP is involved in PV disruption. Intravacuolar movement of the parasite occurred just prior to PV disruption. In some instances, IFN-γ induced parasite egression. Electron microscopy and immunofluorescence studies indicate that the host cell ER fuses with the PV prior to vacuolar disruption. On the basis of these results, we postulate a mechanism by which ER/PV fusion is a crucial event in PV disruption. Fusion of the ER with the PV, releasing calcium into the vacuole, may also be the mechanism by which intravacuolar parasite movement and IFN-γ-induced parasite egression occur.