Respiratory syncytial virus activates epidermal growth factor receptor to suppress interferon regulatory factor 1-dependent interferon-lambda and antiviral defense in airway epithelium.

Respiratory syncytial virus (RSV) persists as a significant human pathogen that continues to contribute to morbidity and mortality. In children, RSV is the leading cause of lower respiratory tract infections, and in adults RSV causes pneumonia and contributes to exacerbations of chronic lung diseases. RSV induces airway epithelial inflammation by activation of the epidermal growth factor receptor (EGFR), a tyrosine kinase receptor. Recently, EGFR inhibition was shown to decrease RSV infection, but the mechanism(s) for this effect are not known. Interferon (IFN) signaling is critical for innate antiviral responses, and recent experiments have implicated IFN-λ (lambda), a type III IFN, as the most significant IFN for mucosal antiviral immune responses to RSV infection. However, a role for RSV-induced EGFR activation to suppress airway epithelial antiviral immunity has not been explored. Here, we show that RSV-induced EGFR activation suppresses IFN regulatory factor (IRF) 1-induced IFN-λ production and increased viral infection, and we implicate RSV F protein to mediate this effect. EGFR inhibition, during viral infection, augmented IRF1, IFN-λ, and decreased RSV titers. These results suggest a mechanism for EGFR inhibition to suppress RSV by activation of endogenous epithelial antiviral defenses, which may be a potential target for novel therapeutics.

A soluble inorganic pyrophosphatase from the cattle tick Rhipicephalus microplus capable of hydrolysing polyphosphates.

Polyphosphates have been found in all cell types examined to date and play diverse roles depending on the cell type. In eukaryotic organisms, polyphosphates have been investigated mainly in mammalian cells, and only a few studies have addressed arthropods. Pyrophosphatases have been shown to regulate polyphosphate metabolism. However, these studies were restricted to trypanosomatids. Here we focus on the tick Rhipicephalus microplus, a haematophagous ectoparasite that is highly harmful to cattle. We produced a recombinant R. microplus pyrophosphatase (rRmPPase) with the aim of investigating its kinetic parameters using polyphosphates as substrate. Molecular docking assays of RmPPase with polyphosphates were also carried out. The kinetic and Hill coefficient parameters indicated that rRmPPase has a greater affinity, higher catalytic efficiency and increased cooperativity for sodium phosphate glass type 15 (polyP15 ) than for sodium tripolyphosphate (polyP3 ). Through molecular docking, we found that polyP3 binds close to the Mg2+ atoms in the catalytic region of the protein, participating in their coordination network, whereas polyP15 interactions involve negatively charged phosphate groups and basic amino acid residues, such as Lys56, Arg58 and Lys193; polyP15 has a more favourable theoretical binding affinity than polyP3 , thus supporting the kinetic data. This study shows, for the first time in arthropods, a pyrophosphatase with polyphosphatase activity, suggesting its participation in polyphosphate metabolism.

Matrix protein CCN1 induced by bacterial DNA and CpG ODN limits lung inflammation and contributes to innate immune homeostasis.

To defend against pulmonary infections, lung epithelial cells are equipped with complex innate immunity closely linked to inflammation. Dysregulated innate immunity/inflammation leads to self-perpetuating lung injury. The CpG motif in bacterial DNA is one of the factors involved in bacterial infection-associated inflammation. Bacterial DNA and synthetic CpG oligonucleotide (ODN) induced CCN1 secretion from lung epithelial cells, functioning as a potential "braking" signal to prevent uncontrolled inflammatory responses. CpG ODN-induced endoplasmic reticulum (ER) stress resulted in Src-Y527 phosphorylation (pY527) and Src/CCN1 vWF domain dissociation. Src-Y527 activated caveolin-1 (cav-1) phosphorylation at Y14 and then modulated CCN1 secretion via pCav-1 interaction with the CCN1 IGFbp domain. Functionally, secreted CCN1 promoted anti-inflammatory cytokine interleukin (IL)-10 release from epithelial cells via integrin αVß6-PKC, and this subsequently suppressed tumor necrosis factor (TNF)-α, macrophage inflammatory protein 2 (MIP-2)-2 secretion and neutrophil infiltration in the lungs. Collectively, bacterial DNA/CpG ODN-stimulated CCN1 secretion via the BiP/GRP78-Src(Y527)-JNK-Cav-1(Y14) pathway and CpG-induced CCN1 conferred anti-inflammatory roles. Our studies suggested a novel paradigm by which the lung epithelium maintains innate immune homeostasis after bacterial infection.

The effect of an anti-HLA-B27 immune response on CTL recognition of Chlamydia.

The interplay between triggering bacteria and HLA-B27 in the pathogenesis of the spondyloarthropathies remains one of the most active areas of investigation in the rheumatic diseases. This has proved difficult to study systematically in the clinical setting, and in this study we utilized a rat model to address the influence that B27-related immunity may have on the process of generating anti-Chlamydia immunity. When splenocytes from HLA-B27 DNA-immunized Lewis (LEW) animals received restimulation in vitro with Chlamydia-treated cells from B27-transgenic LEW rats, we observed that in addition to the expected CTL recognition of HLA-B27, there was also anti-Chlamydia CTL killing of Chlamydia-sensitized syngeneic fibroblast targets. This was not seen when responding cells in vitro were naive LEW splenocytes. To confirm the existence of CTLs recognizing both HLA-B27 and Chlamydia, LEW rats were immunized with B27-transgenic LEW cells, instead of the B27 DNA construct. Splenocytes from the immune rats were restimulated in vitro with Chlamydia-treated B27-transgenic LEW cells. In this instance, the CTLs retained the allele-specific recognition of HLA-B27, as well as recognition of Chlamydia-sensitized syngeneic fibroblasts. Thus, if there is prior expansion of an immune response against HLA-B27, then the resulting splenocytes demonstrate a reduced threshold for generating a primary anti-Chlamydia CTL response. These studies implicate a dynamic interrelationship between recognition of HLA-B27 and Chlamydia trachomatis. The results may have implications for deciphering the cellular basis of Chlamydia-induced reactive arthritis.

Creating HIV-1 reverse transcriptase cytotoxic T lymphocyte target structures by HLA-A2 heavy chain modifications.

Vigorous HIV-1-specific CD8(+) cytotoxic T lymphocyte (CTL) responses play an important role in the control of HIV-1 replication and the induction of a strong, broadly cross-reactive CTL response remains an important goal of HIV vaccine development. It is known that the display of high levels of class I MHC-viral peptide complexes at the cell surface of target cells is necessary to elicit a strong CTL response. We now report two strategies to enhance the presentation of defined HIV-1 epitope-specific CTL target structures, by incorporating subdominant HIV-1 reverse transcriptase (RT) CTL epitope sequences into the human class I MHC molecule HLA-A2. We show that either incorporation of HIV-1 CTL epitopes into the signal sequence of HLA or tethering of epitopes to the HLA-A2 heavy chain provide simple ways to create effective CTL target structures that can be recognized and lysed by human HLA-A2-restricted RT-specific CD8(+) CTL. Moreover, cells expressing these epitope-containing HLA-A2 constructs stimulated the generation of primary epitope-specific CTL in vitro. These strategies offer new options in the design of plasmid DNA-based vaccines or immunotherapeutics for the induction of CTL responses against subdominant HIV-1 epitopes.

Anti-major histocompatibility complex antibody responses in macaques via intradermal DNA immunizations.

In simian immunodeficiency virus (SIV) models, immunization of macaques with uninfected human cells or human major histocompatibility complex (MHC) proteins can induce xenogeneic immune responses which can protect the animals from subsequent SIV challenges. These studies suggest that the induction of anti-MHC immune responses can be a viable vaccine strategy against human immunodeficiency virus type 1 (HIV-1). We have previously shown in mouse studies that DNA immunization with class I and class II MHC-encoding plasmids can elicit both xenogeneic and allogeneic antibody responses against conformationally intact MHC molecules (Vaccine 17 (1999) 2479-92). Here we take these observations one step closer to human applications and report that intradermal needle immunizations of non-human primates with plasmid DNA encoding human MHC alleles can safely elicit xenogeneic anti-MHC antibody responses. Moreover, injecting macaques with DNA encoding a specific macaque allogeneic MHC induced anti-allogeneic MHC antibodies production. These studies show that DNA immunization with MHC-encoding vectors can indeed be used to induce specific anti-human xenogeneic, as well as anti-macaque allogeneic MHC immunity in non-human primates. This strategy could thus be used to mobilize anti-MHC antibody response which may be useful as part of an anti-HIV-1 vaccination approach.

Xenogeneic and allogeneic anti-MHC immune responses induced by plasmid DNA immunization.

Major histocompatibility complex (MHC) proteins are known to be incorporated into the human immunodeficiency virus (HIV-1) envelope as the virion buds from the host cell surface. Studies using simian immunodeficiency virus (SIV) infection of macaques have demonstrated that immunization with uninfected human cells or purified HLA proteins can provide protection from challenge with live SIV when it is grown in human cells expressing the same MHC alleles. Thus the induction of anti-MHC immune responses represents an important option to consider with respect to vaccine design for SIV and HIV. Here we examine plasmid DNA immunization strategies as an alternative to cellular or protein immunogens for the induction of xenogeneic and allogeneic immune responses in C57BL/6 mice and in an HLA transgenic mouse model system, respectively. We compared the immunogenicity of HLA-A2- and HLA-B27-expressing splenocytes with the corresponding plasmid DNA immunogens. Results from the transgenic mouse experiments indicate that plasmid DNA immunization with both class I and class II MHC-encoding vectors can elicit antibody responses recognizing conformationally intact MHC molecules. Our data also show that immunization with class I MHC-encoding DNA immunogens can elicit cytotoxic T-lymphocyte responses, demonstrating the potential to mobilize both antibody and cell-mediated anti-MHC immune responses in the context of this approach to HIV-1 vaccine design.

Epitope-specific cytotoxic T lymphocyte induction by minigene DNA immunization.

Plasmid DNA vaccines encoding full-length antigen often induce both potent antibody and cytotoxic T lymphocyte (CTL) responses. Here, we examine strategies to exclusively elicit epitope-specific CTL responses using DNA constructs expressing a minimal class I MHC-restricted epitope of the nucleoprotein (NP) of influenza virus. The effects of the addition of an ER leader sequence or cytokine combination on minigene-induced CTL responses in vivo were assessed following both delivery by needle injection into skeletal muscle and by gene gun bombardment into skin epidermis. Our data indicate that the leader sequence enhanced the magnitude of the CTL responses, whereas co-injection of the cytokine genes IL-12 and GM-CSF had a minimal effect. An antibody response against NP was not observed in any of the mice receiving the minigene constructs.

[Migrant populations at the Hôpital de l'Enfance of Lausanne (HEL): evaluation inquiry, taking care and contribution of cultural mediators-translators]. / Les populations migrantes à l'Hôpital de l'Enfance de Lausanne (HEL): état des lieux, prise en charge et apport des traducteurs-médiateurs culturels.

The number of patients belonging to migrant populations who consult Lausanne's Hôpital de l'Enfance (HEL) has increased massively in an exponential manner. HEL is a facility dedicated to children with a vocation of public health and training in a university setting and it has to make a point of developing an adequate and pertinent health care system for these populations. In order to do this, a study was undertaken in the form of a prospective survey including over a thousand ambulatory patients. Administrative data (origin, date of migration, nature of the permit and legal situation), social data (home language, professional situation, number of siblings), medical (diagnosis) and psychosomatic data (sleep, enuresis) was recorded as well as data testing the level of understanding between carrier and patient. The study allowed us to define priorities of intervention: introduction of a service of translators--cultural mediators trained by the association "Appartenances" (as well as on going follow-up and assessment of this service), training of health care workers in the fields of cultural mediation and the different aspects of migrant population medicine and, finally, the creation of a steering group within the Institution. Having allowed rapid and spectacular improvement in the quality of the service provided by the HEL, this process is also included in the will to improve health care given to migrant populations at local and national levels in accordance with the priorities defined by the WHO in this domain. It is this experience which is reported in this paper.

Microbial keratitis resulting in loss of the eye.

BACKGROUND AND OBJECTIVES: The purpose of this study was to determine the clinical characteristics of infected corneal ulcers resulting in loss of the eye. PATIENTS AND METHODS: The authors conducted a retrospective study of all cases requiring evisceration or enucleation due to microbial keratitis at Wills Eye Hospital between January 1, 1989, and December 31, 1995. Medical records were reviewed to determine the past medical and ophthalmic history, duration of symptoms and treatment prior to referral, and the size of the ulcer at time of presentation. Treatment, culture results, and clinical course were also analyzed. RESULTS: During the study period, 1.8% (17 of 965) of the patients with corneal ulcers admitted to Wills Eye Hospital underwent evisceration or enucleation for microbial keratitis. The median age of the patients was 67 years (+/- 20.1 years). A majority of the patients (82%, 14 of 17) had a history of preexisting ocular disease resulting in poor visual acuity. The median duration of symptoms prior to presentation to Wills Eye Hospital was 11.4 days (+/- 13.9 days). The average size of the corneal infiltrate was 40.8 mm2 (+/- 38.7 mm2). The most common pathogens were Pseudomonas (7 cases) and Streptococcus (3 cases). Patients required evisceration (14 cases) or enucleation (3 cases) due to uncontrolled infection. CONCLUSIONS: Microbial keratitis resulting in loss of the eye occurred typically in patients who were elderly with preexisting poor visual acuity, who presented with severe infections due to virulent organisms or delayed treatment.

Chalazion-induced hyperopia as a cause of decreased vision.

This article presents three cases of decreased vision due to acquired hyperopia, which were caused by a chalazion of the upper eyelid. Through manifest refraction and computerized corneal topographic analysis, acquired hyperopia associated with central corneal flattening was revealed. These findings were responsible for the blurred vision that was reversed by chalazion resolution or removal. Although not usually considered a risk factor for refractive disorders other than astigmatism, chalazia of the upper eyelid can present as a decrease in vision associated with reversible central corneal flattening and acquired hyperopia.

Neural fraud detection in credit card operations.

This paper presents an online system for fraud detection of credit card operations based on a neural classifier. Since it is installed in a transactional hub for operation distribution, and not on a card-issuing institution, it acts solely on the information of the operation to be rated and of its immediate previous history, and not on historic databases of past cardholder activities. Among the main characteristics of credit card traffic are the great imbalance between proper and fraudulent operations, and a great degree of mixing between both. To ensure proper model construction, a nonlinear version of Fisher's discriminant analysis, which adequately separates a good proportion of fraudulent operations away from other closer to normal traffic, has been used. The system is fully operational and currently handles more than 12 million operations per year with very satisfactory results.

Metodologia de avaliaçäo cardiológica do atleta normal e cardiopata / Methodology for cardiac evaluation of normal athletes and with heart disease

O exame cardiológico do atleta tem, entre seus principais objetivos, a detecçäo de alguma cardiopatia desconhecida, que poderia levar a problemas futuros, com a manutençäo da prática esportiva. O cardiologista deve estar a par das alteraçöes que podem ser encontradas no "coraçäo do atleta", para identificar anormalidades da propedêutica potencialmente importantes, bem como conhecer como as alteraçöes habituais regridem quando o indivíduo abandona a atividade esportiva. Säo discutidos os exames mínis que deve ser realizados na avaliaçäo do atleta, tendo em vista a dificuldade de obtençäo das condiçöes tecnológicas ideais. Entretanto, säo abordados também exames mais sofisticados, que seräo realizados de acordo com a necessidade de cada caso.

The effect of vitamin E on erythrocyte hemolysis and lipid peroxidation in newborn premature infants.

The biochemical effect of vitamin E supplementation to mothers with threatened premature delivery and to premature infants after birth has been studied. Although a weak correlation was found between maternal and cord blood vitamin E levels at birth, cord blood levels were not significantly higher in the infants from supplemented mothers than those from unsupplemented mothers. Furthermore, maternal vitamin E treatment did not prevent either erythrocyte hemolysis or lipid peroxide formation in the premature infants after birth. On the other hand, intramuscular vitamin E to infants after birth produced a marked biochemical effect, with both zero erythrocyte hemolysis and low lipid peroxide formation when serum vitamin E increased above 2 mg/100 ml. We conclude that intramuscular vitamin E immediately after birth is necessary to achieve a biochemical effect of vitamin E in the early neonatal period. (No cases of retrolental fibroplasia occurred in the present study.)

Mixed (lichenoid and macular) cutaneous amyloidoses.

Between October 1972 and Paril 1974 a total of four patients suffering from lichen amyloidosus were admitted to the wards of the Department of Dermatology, University Hospital, Valladolid. All these patients also showed a macular amyloidosis associated with the lichenoid eruption. The authors emphasize that macular amyloidosis is quite commonly found as an associated and closely related condition in lichen amyloidosus.