Intracellular localization of the Epstein-Barr virus BFRF1 gene product in lymphoid cell lines and oral hairy leukoplakia lesions

A novel protein encoded by the BFRF1 gene of the Epstein-Barr virus was identified recently [Farina et al. (2000) J Virol 74:3235-3244], which is antigenic "in vivo" and expressed early in the viral replicative cycle. In the present study, its subcellular localization was examined in greater detail comparing Epstein-Barr virus (EBV) induced producing and nonproducing cell lines by immunofluorescence: in 12-0-tetradecanoyl phorbol-13-acetate (TPA)-induced Raji and B95-8 cells, as well as in anti-IgG-stimulated Akata cells, the protein appeared to be localized over the cell nuclear membrane. A similar nuclear membrane localization was observed in epithelial cells of oral hairy leukoplakia, a pathological manifestation of permissive EBV infection. In contrast, upon transfection of BFRF1 in the EBV-negative Burkitt's lymphoma cell line DG75, the protein was localized predominantly over the plasma membrane. The membrane localization was abolished when DG75 cells were transfected with a C-terminal deletion mutant of BFRF1 lacking the transmembrane domain. Because induced Raji cells do not produce virus, the above observations indicate that the nuclear membrane localization is not associated with viral production, but requires the expression of EBV genes, and suggest that additional proteins, expressed early during viral lytic infection, might be necessary to target the protein to the nuclear membrane. Immunogold electron microscopy on ultrathin cryosections of induced B95-8 cells showed that BFRF1 on the nuclear membranes was concentrated over multilayered domains representing areas of active viral replication or at the sites of viral budding, suggesting that BFRF1 is involved in the process of viral assembly.     

Lymphocyte calcium signaling involves dihydropyridine-sensitive L-type calcium channels: facts and controversies

Calcium influx into lymphocytes is essential for activation, differentiation, and effector functions. While several channel- and receptor-types contribute to calcium influx, voltage-gated calcium channels (VGCC) mediate a well-characterized calcium influx pathway that is most exclusively identified in excitable cells. The role of L-type VGCCs, which belong to high-voltage activated calcium channels and are defined as dihydropyridine (DHP) receptors in excitable cells, is well documented. Interestingly, while lymphocytes do not range in the excitable cell category, the modulatory role of DHP agonists and antagonists and the identification of L-type VGCC-related molecules in B and T lymphocytes, mainly in Th2 cells, suggest these proteins are involved in the calcium response of these cells. Because the identity and the regulation of DHP receptors/channels in lymphocytes is far from being solved, we will discuss the challenging issues of demonstrating a role of L-type VGCCs in nonexcitable cells and the arguments supporting their role in lymphocytes. We will comment on the limitation of the use of DHP agonists and antagonists to ascertain a specific involvement of L-type VGCCs in lymphocyte calcium signaling. Finally, we will provide new clues on the interest of a potential use of DHP antagonists in Th2-cell-mediated pathology.     

Genome-Wide Study of Pseudomonas aeruginosa Outer Membrane Protein Immunogenicity Using Self-Assembling Protein Microarrays

Pseudomonas aeruginosa is responsible for potentially life-threatening infections in individuals with compromised defense mechanisms and those with cystic fibrosis. P. aeruginosa infection is notable for the appearance of a humoral response to some known antigens, such as flagellin C, elastase, alkaline protease, and others. Although a number of immunogenic proteins are known, no effective vaccine has been approved yet. Here, we report a comprehensive study of all 262 outer membrane and exported P. aeruginosa PAO1 proteins by a modified protein microarray methodology called the nucleic acid-programmable protein array. From this study, it was possible to identify 12 proteins that trigger an adaptive immune response in cystic fibrosis and acutely infected patients, providing valuable information about which bacterial proteins are actually recognized by the immune system in vivo during the natural course of infection. The differential detections of these proteins in patients and controls proved to be statistically significant (P < 0.01). The study provides a list of potential candidates for the improvement of serological diagnostics and the development of vaccines.Pseudomonas aeruginosa is a gram-negative bacterium represented by different strains. PAO1 is the most representative one, and it contains one chromosome comprising 5,570 open reading frames (ORFs) (21). Ubiquitous in the environment, it rarely causes respiratory tract infections in healthy individuals but causes life-threatening lung infections in cystic fibrosis (CF) patients (24, 27), linking it directly to the disease''s poor prognosis (38).CF is a hereditary disease affecting 30,000 people in the United States alone, and it results from the impaired function of the cystic fibrosis transmembrane conductance regulator-encoded chloride channel. This defect leads to the accumulation of nutrient-rich mucus inside the respiratory tract, which is readily colonized by bacteria and difficult for immune cells and antibiotics to penetrate (13). Although these patients are generally colonized early by other pathogens, such as Staphylococcus aureus and Haemophilus influenzae, by the age of 18, 85% have chronic pseudomonal lung infections (23).P. aeruginosa is also associated with nosocomial infections, especially in the immunocompromised (6, 10). Multidrug-resistant strains in intensive care units as well as outside the hospital setting have been described, making the development of alternate treatment strategies a necessity (9, 29). CF patients and other at-risk individuals would benefit from a functional vaccine, but none has been approved for use so far (8, 16).In the late 1970s, evidence about proteins acting as important virulence factors in Pseudomonas aeruginosa infection called attention to exotoxin A (protein PA1148), alkaline protease (protein PA1248), and elastase (protein PA3724) (18, 43). Currently, diagnostic serology for P. aeruginosa is mostly based on these three proteins and has a sensitivity of 80% in chronic infections (36). This test is much less sensitive at the onset of infection, when serology alone does not detect pseudomonal infection in more than 43% of patients. The discovery of new antigens to complement the current panel of antigens would increase diagnostic sensitivity in all infection phases.Pseudomonas presents different antigens on the surface depending on the microenvironment it colonizes, the mucoid phenotype being a hallmark for lung infections (5, 35). Which proteins are present on the pathogen''s surface in each microenvironment has not yet been fully characterized; however, this information would be invaluable for diagnostics development and reverse vaccinology studies.Early studies executed in the 1980s probed Western blots made from gel electrophoresis-fractionated P. aeruginosa extracts with serum to reveal reactive bands. Some of these reactive bands were identified to be specific outer membrane proteins (OMPs), such as OMPs E, H2, and I, as well as porin F, but due to technical limitations, not all bands could be identified (15, 20). Notably, all of these proteins, as well as the secreted proteins alkaline protease and elastase from the earlier studies, are externally displayed proteins, confirming the importance of this class of proteins in triggering an immune response, as they are readily seen by the immune system. Therefore, it would be useful to execute a systematic comprehensive study to analyze all predicted OMPs, both known and hypothetical, in an unbiased screening experiment.These also represent good vaccine candidates based on data from phase I and II clinical trials using two P. aeruginosa OMPs in vaccines (8). A phase III trial of the external surface flagellin protein also showed promising results with previously uninfected CF patients (7). However, testing proteins as candidate antigens ordinarily requires antigen expression and purification, which are notoriously difficult for membrane proteins, making a comprehensive study of all P. aeruginosa OMPs with traditional methods impossible, particularly because the P. aeruginosa genome is very GC rich, which reduces the yield of protein in common expression systems.Protein microarrays provide one mechanism for executing an unbiased screen. Recent studies demonstrate immune responses from plague patients against a Yersinia pestis protein microarray containing 144 virulence related factors, 14 of which were detected by all the patients tested (25). Similar protein microarray studies have been done for selected antigens from Francisella tularensis (39), Borrelia burgdorferi (44), Coxiella burnetti (2), severe acute respiratory syndrome coronavirus (45), and others. In some cases, difficulty in producing proteins has been encountered. In the C. burnetti study, ∼500 proteins could not be studied (∼25%) due to difficulties in producing some individual proteins for array spotting. The high-throughput nature of microarrays allows easy execution of comparative studies of the humoral response mounted against different pathogens. In a recent study, Keasey and collaborators demonstrated that by probing a Yersinia pestis-specific protein microarray with serum from animals infected with other gram-negative bacteria, it was possible to find shared cross-reactive proteins (common to several pathogens), fingerprint proteins (common to two or more bacteria), and signature proteins (specific to each pathogen) (17).However, the traditional protein microarray printing method, which relies upon printing purified proteins, may encounter limitations. Chief among these is the difficulty in producing and purifying a broad range of proteins in order to obtain both adequate and consistent protein yields, resulting in an excessively large dynamic range of protein yields displayed on the array. This is particularly relevant in circumstances in which the targets are either hydrophobic or large.To address these issues, we previously reported the development of a novel method for displaying proteins on protein microarrays, called the nucleic acid-programmable protein array (NAPPA), which is particularly advantageous for membrane proteins. In contrast to the traditional method for producing protein microarrays, NAPPA entails printing the cDNAs that encode the proteins that are “self-assembled” at the time of the experiment by transcribing and translating proteins in situ on the array surface (33). The cDNAs are situated in an expression vector such that they produce proteins with a carboxyl-terminal fusion tag that both allows protein capture at the surface and confirms full-length translation. This unique approach obviates the need to express and purify any protein, avoids protein aggregation by immediate capture on the slide surface, and ensures that the proteins displayed have been produced freshly at the time of assay.NAPPAs display a uniform amount of protein in each feature (34) and work well for detecting humoral immune responses in a micro-enzyme-linked immunosorbent assay (micro-ELISA) format (1, 32). NAPPA has been demonstrated to express and display membrane proteins with an efficiency that exceeds 90% (34), perhaps by avoiding cell toxicity and protein aggregation. Based on the observation that NAPPA-immobilized proteins interact with other proteins as expected (33), we reasoned that many relevant epitopes should be displayed and that NAPPA could be used to test candidate membrane antigens in P. aeruginosa.Using bioinformatics tools based on amino acid composition, homology to known OMPs, and finding transmembrane ß-barrels, we identified 266 gene products among all of the 5,570 ORFs in PAO1 that were likely to reside in the outer membrane, are part of exposed surface organelles, or are secreted into the surrounding media. These proteins were represented on the surface of the NAPPAs, and patient sera were used to screen for their antigenicity.     

Noise correlations and SNR in phased‐array MRS

The acquisition of magnetic resonance spectroscopy (MRS) signals by multiple receiver coils can improve the signal‐to‐noise ratio (SNR) or alternatively can reduce the scan time maintaining a reliable SNR. However, using phased array coils in MRS studies requires efficient data processing and data combination techniques in order to exploit the sensitivity improvement of the phased array coil acquisition method. This paper describes a novel method for the combination of MRS signals acquired by phased array coils, even in presence of correlated noise between the acquisition channels. In fact, although it has been shown that electric and magnetic coupling mechanisms produce correlated noise in the coils, previous algorithms developed for MRS data combination have ignored this effect. The proposed approach takes advantage of a noise decorrelation stage to maximize the SNR of the combined spectra. In particular Principal Component Analysis (PCA) was exploited to project the acquired spectra in a subspace where the noise vectors are orthogonal. In this subspace the SNR weighting method will provide the optimal overall SNR. Performance evaluation of the proposed method is carried out on simulated ¹H‐MRS signals and experimental results are obtained on phantom ¹H‐MR spectra using a commercially available 8‐element phased array coil. Noise correlations between elements were generally low due to the optimal coil design, leading to a fair SNR gain (about 0.5%) in the center of the field of view (FOV). A greater SNR improvement was found in the peripheral FOV regions. Copyright © 2009 John Wiley & Sons, Ltd.     

Direct correlation between human herpesvirus-8 seroprevalence and classic Kaposi's sarcoma incidence in Northern Sardinia

The human herpesvirus-8 (HHV-8) has been associated with the development of Kaposi's sarcoma. A high incidence of classic Kaposi's sarcoma has been described in Sardinia, an island West of Italy's mainland. Different seroepidemiological analyses have reported that prevalence of HHV-8 infection varies worldwide: a high HHV-8 seroprevalence has been shown in Italy. The present survey was carried out to evaluate the correlation between HHV-8 infection and classic Kaposi's sarcoma incidence in northern Sardinia. Blood samples were collected from 226 healthy donors born and resident in five different areas of North Sardinia. Seroprevalence to HHV-8 was determined searching antibodies to viral lytic proteins by immunofluorescence in sera diluted at 1:10. Classic Kaposi's sarcoma incidence data spanning a period of 23 years were examined in the areas studied. The present screening revealed that seroprevalence was 35%, within a range of 15.3-46.3% in the five areas, although it should be considered that the seroprevalence to HHV-8 can be established more accurately by the combined use of different assays. Age emerged as an important risk factor. Indeed, subjects aged > 50 years showed a higher seroprevalence to HHV-8 as compared with younger individuals. A strong direct correlation between HHV-8 prevalence and classic Kaposi's sarcoma incidence has been also observed. The wide diffusion of HHV-8 in Sardinia appears to represent an important factor in the high incidence of classic Kaposi's sarcoma reported in the island. However, additional co-factors, such as age, sex, genetic traits, or viral strain pathogenicity, are likely to play a role in the development of the disease.     

New technological developments in the clinical imaging of atherosclerotic plaque

Direct visualization of the composition of the atherosclerotic plaque during its natural history and after therapeutic intervention may be helpful in detecting lesions with high risk of acute events and in understanding progression and regression of the disease. A wide variety of invasive and non-invasive imaging techniques is available to detect clue aspects of atherosclerosis from the early stage to the clinical evidence appearance. We will firstly review the ongoing technological and clinical research on both invasive and non-invasive techniques. Afterward, we will discuss in detail the use of high-resolution, multi-contrast magnetic resonance imaging for non-invasive imaging of the plaque and its characterization in terms of its various components (i.e., thickness, lipid, fibrous, calcium, or thrombus). Finally, we will describe the potential of quantitative analysis in describing of plaque constituents with improved reproducibility.     

Immunogenicity and safety in newborns of a new recombinant hepatitis B vaccine containing the S and pre-S2 antigens.

A study to evaluate the safety and immunogenicity of a recombinant hepatitis B vaccine containing the S and pre-S2 antigens (GenHevac B Pasteur) was conducted in healthy newborn infants. All infants received 20 micrograms of vaccine within 24 h of birth and at 1 and 2 months with a booster injection at month 12. The vaccine was administered alone in 19 infants born to low risk mothers, i.e. surface antigen (HBsAg)-negative and antibody to the core antigen (Anti-HBc)-positive mothers. The vaccine was administered in combination with 100 IU hepatitis B immune globulin (HBIg) at birth and 1 month in 18 infants born to high risk mothers, i.e. HBsAg positive mothers. In the group not receiving HBIg, the anti-HBs seroconversion rate at the 10 mIU ml-1 threshold was 50% 1 month after the first injection. In both groups, the anti-HBs seroconversion rates were 100% 1 month after the third injection and greater than 85% 1 month after the second injection. After the booster injection greater than 90% of the infants had an anti-HBs titre greater than 1000 mIU ml-1 which will probably provide them with adequate protection for several years. The kinetics of the anti-pre-S2 response was similar to that of the anti-HBs response and 100% of infants in both groups had seroconverted 1 month after the second injection of the vaccine. The side effects were scarce, all mild and transient.(ABSTRACT TRUNCATED AT 250 WORDS)     

T-type calcium channels are inhibited by fluoxetine and its metabolite norfluoxetine

Fluoxetine, a widely used antidepressant that primarily acts as a selective serotonin reuptake inhibitor, also inhibits various neuronal ion channels. Using the whole-cell patch-clamp technique, we have examined the effects of fluoxetine and norfluoxetine, its major active metabolite, on cloned low-voltage-activated T-type calcium channels (T channels) expressed in tsA 201 cells. Fluoxetine inhibited the three T channels Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3 in a concentration-dependent manner (IC(50) = 14, 16, and 30 microM, respectively). Norfluoxetine was a more potent inhibitor than fluoxetine, especially on the Ca(V)3.3 T current (IC(50) = 5 microM). The fluoxetine block of T channels was voltage-dependent because it was significantly enhanced for T channels in the inactivated state. Fluoxetine caused a hyperpolarizing shift in steady-state inactivation, with a slower rate of recovery from the inactivated state. These results indicated a tighter binding of fluoxetine to the inactivated state than to the resting state of T channels, suggesting a more potent inhibition of T channels at physiological resting membrane potential. Indeed, fluoxetine and norfluoxetine at 1 microM strongly inhibited cloned T currents (approximately 50 and approximately 75%, respectively) in action potential clamp experiments performed with firing activities of thalamocortical relay neurons. Altogether, these data demonstrate that clinically relevant concentrations of fluoxetine exert a voltage-dependent block of T channels that may contribute to this antidepressant's pharmacological effects.     

Family caregiving for AIDS patients in the Democratic Republic of Congo

We conducted a qualitative study of women who were caregivers for HIV/AIDS-affected spouses in Bumbu Zone, Kinshasa, Democratic Republic of Congo in 2003. Twelve caregiving women, six home-based care workers and five key informants were interviewed via focus group discussions. Most women reported huge problems in providing care to their spouses due to psychological, social and economic factors. The secrecy around HIV/AIDS issues and care was a significant theme in the findings. The self-reported health status of the caregivers indicated poor health.     

Efficacy of two one-week rabeprazole/levofloxacin-based triple therapies for Helicobacter pylori infection

BACKGROUND: One-week low-dose proton pump inhibitor-based triple therapies have usually proved to be effective treatments for Helicobacter pylori infection. AIM: To investigate the eradication efficacy, safety profile and patient compliance of two triple therapies containing a standard dose of rabeprazole and a new fluoroquinolone, levofloxacin. METHODS: One hundred patients referred to us for gastroscopy, who were H. pylori-positive, were consecutively recruited in a prospective, open-label study. The enrolled patients were randomised to receive a seven-day course of rabeprazole 20 mg o.d. plus levofloxacin 500 mg o.d. and either amoxycillin 1 g b.d. (RLA group) or tinidazole 500 mg b.d. (RLT group). Their H. pylori status was assessed by means of histology and rapid urease test at entry, and by 13C-urea breath test 8 weeks after the end of treatment. RESULTS: All 100 enrolled patients completed the study. Forty-six of 50 patients treated with RLA (both PP and ITT analysis: 92%; 95% CI: 81-98%) and 45 of 50 with RLT (both PP and ITT analysis: 90%: 95% CI: 78-97%), became H. pylori-negative. Slight or mild side-effects occurred in 4 (8%) patients of the RLA group and in 5 (10%) of the RLT group. CONCLUSIONS: This study demonstrates the efficacy of two 1-week rabeprazole-based triple therapies including levofloxacin to eradicate H. pylori. These regimens prove to be safe, well-tolerated, and achieved good eradication rates. Levofloxacin may be an effective alternative to clarithromycin in triple therapy regimens.