NS1 protein secretion during the acute phase of West Nile virus infection

The West Nile virus (WNV) nonstructural protein NS1 is a protein of unknown function that is found within, associated with, and secreted from infected cells. We systematically investigated the kinetics of NS1 secretion in vitro and in vivo to determine the potential use of this protein as a diagnostic marker and to analyze NS1 secretion in relation to the infection cycle. A sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for detection of WNW NS1 (polyclonal-ACE) was developed, as well as a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). The 4G4-ACE detected native NS1 antigens at high sensitivity, whereas the polyclonal-ACE had a higher specificity for recombinant forms of the protein. Applying these assays we found that only a small fraction of intracellular NS1 is secreted and that secretion of NS1 in tissue culture is delayed compared to the release of virus particles. In experimentally infected hamsters, NS1 was detected in the serum between days 3 and 8 postinfection, peaking on day 5, the day prior to the onset of clinical disease; immunoglobulin M (IgM) antibodies were detected at low levels on day 5 postinfection. Although real-time PCR gave the earliest indication of infection (day 1), the diagnostic performance of the 4G4-ACE was comparable to that of real-time PCR during the time period when NS1 was secreted. Moreover, the 4G4-ACE was found to be superior in performance to both the IgM and plaque assays during this time period, suggesting that NS1 is a viable early diagnostic marker of WNV infection.

Pattern and correlates of inpatient admission during the initial acute phase of first-episode psychosis

Objectives: The first aim of this study was to examine the rate, pattern and correlates of inpatient admission during the first 3 months of treatment for first-episode psychosis (FEP). The second aim was to determine whether the pattern of inpatient admission during this period was associated with remission of psychotic symptoms or inpatient service use at 15-month follow-up. Method: One hundred and four consecutive patients with FEP at a specialist treatment service were approached to participate in a follow-up study. Patients were grouped on the basis of the pattern of inpatient admission (none, one, or multiple) during the first 3 months of treatment. Clinical ratings at baseline and 3-month follow-up, and ratings of remission of psychotic symptoms at 3 and 15-month follow-up, were available for two-thirds of the patients. Inpatient data for the 15-month follow-up period were derived from an electronic database for most patients (n = 98). Results: Eighty (76.9%) of the 104 patients were admitted to an inpatient unit during the first 3 months of treatment. Fifty-nine (56.7%) patients had a single admission and 21 (20.2%) had multiple admissions. At baseline, inpatient admission was associated with a diagnosis of affective psychosis and more severe behavioural and functional disturbance but not positive psychotic symptoms. Multiple admissions were associated with risks to self or others at baseline and 3-month follow-up, and lack of remission of positive symptoms at 3 and 15-month follow-up. There was no association between the pattern of inpatient admission during the initial 3-month period and inpatient service use during the following 12-month period. Conclusions: The substantial proportion of young patients with FEP admitted to hospital emphasizes the need for youth-friendly treatment environments and practices. Although patients with multiple admissions during the initial treatment period are less likely to achieve remission, these patients are no more likely to establish a pattern of revolving-door hospitalizations compared with other patients.

An antigen capture enzyme-linked immunosorbent assay reveals high levels of the dengue virus protein NS1 in the sera of infected patients

We describe the development of a capture enzyme-linked immunosorbent assay for the detection of the dengue virus nonstructural protein NS1. The assay employs rabbit polyclonal and monoclonal antibodies as the capture and detection antibodies, respectively. Immunoaffinity-purified NS1 derived from dengue 2 virus-infected cells was used as a standard to establish a detection sensitivity of approximately 4 ng/ml for an assay employing monoclonal antibodies recognizing a dengue 2 serotype-specific epitope. A number of serotype cross-reactive monoclonal antibodies were also shown to be suitable probes for the detection of NS1 expressed by the remaining three dengue virus serotypes. Examination of clinical samples demonstrated that the assay was able to detect NS1 with minimal interference from serum components at the test dilutions routinely used, suggesting that it could form the basis of a useful additional diagnostic test for dengue virus infection. Furthermore, quantitation of NS1 levels in patient sera may prove to be a valuable surrogate marker for viremia. Surprisingly high levels of NS1, as much as 15 mu g/ml, were found in acute-phase sera taken hom some of the patients experiencing serologically confirmed dengue 2 virus secondary infections but was not detected in the convalescent sera of these patients. In contrast, NS1 could not be detected in either acute-phase or convalescent serum samples taken from patients with serologically confirmed primary infection. The presence of high levels of secreted NS1 in the sera of patients experiencing secondary dengue virus infections, and in the context of an anamnestic antibody response, suggests that NS1 may contribute significantly to the formation of the circulating immune complexes that are suspected to play an important role in the pathogenesis of severe dengue disease.

Equine laminitis: cleavage of laminin 5 associated with basement membrane dysadhesion

Reasons for performing study: The key lesion of laminitis is separation at the hoof lamellar dermal-epidermal interface. For this to happen the structural and adhesion proteins of the basement membrane zone must be altered. Which proteins and how damage to them leads to the lamellar separation of laminitis is unknown. Objectives: To investigate lamellar hemidesmosome and cytoskeleton damage and basement membrane dysadhesion using light microscopy (LM) and immunofluorescence microscopy (IFM). Methods: Cryostat sections of lamellar tissues from 2 control and 6 Standardbred horses with oligofructose induced laminitis were studied using LM and IFM. Plectin, integrin alpha(6) and BP230 antibody was used to label hemidesmosome intracellular plaque proteins and anti-BP180 and anti-laminin 5 (L5) was used to label anchoring filament (AF) proteins. Cytoskeleton intermediate filaments were labelled using anti-cytokeratin 14. The primary antibodies of selected sections were double labelled to show protein co-localisation. Results: Laminitis caused reduction of transmembrane integrin alpha(6), the AF proteins BP180 and L5,and failure of co-localisation of BP180 and L5. Proteins of the inner hemidesmosomal plaque, plectin and BP230, were unaffected. Conclusions: Loss of co-localisation of L5 and BP180 suggests that, during the acute phase of laminitis, L5 is cleaved and therefore, the AFs connecting the epidermis to the dermis, fail. Without a full complement of AFs separation at the lamellar dermo-epidermal junction occurs. Potential relevance: Suppressing or inhibiting metalloproteinase activity may prevent L5 cleavage and therefore the lamellar dermo-epidermal separation of laminitis.

Circadian blood pressure variation after acute stroke

We aimed to characterise the patterns of circadian blood pressure (BP) variation after acute stroke and determine whether any relationship exists between these patterns and stroke outcome. BP was recorded manually every 4 h for 48 h following acute stroke. Patients were classified according to the percentage fall in mean systolic BP (SBP) at night compared to during the day as: dippers (fall >= 10-= 20%); non-dippers (>= 0-

Particle formation during soy protein precipitation

The current success of soy foods is driving soy ingredient manufacturers to develop innovative products for food manufacturers. One such innovation is separating the soy proteins glycinin and beta-conglycinin to take advantage of their individual functional and nutritional properties. Precipitation by acidification is a low-cost method for separating these two proteins. Separation is achieved by preferentially precipitating glycinin at pH ~ 6 while beta-conglycinin remains in solution. Understanding the particle formation during protein precipitation is important as it can influence the efficiency of the precipitation process as well as subsequent downstream processes such as the particle-liquid separation step, usually achieved by centrifugation. Most of the previous soy protein precipitation studies are limited to precipitation at pH 4 as this is the pH range most commonly used in the commercial manufacturing of soy protein isolates. To date, there have been no studies on the particle formation during precipitation at pH > 5.Precipitation of soy protein is generally thought to occur by the rapid formation of primary particles in the size range of 0.1 - 0.3 microns followed by aggregation of these particles via collision to aggregates of size about 1 - 50 microns. The formation of the primary particles occurs on a time scale much shorter than that of the overall precipitation process (Nelson and Glatz, 1985). This study shows that precipitation of soy protein is indeed rapid. At high pH levels, binary liquid-liquid separation occurs forming a protein-rich heavy phase. The protein-rich phase appears as droplets which can be coalesced to form a uniform bulk layer under centrifugation forces. Upon lowering the pH level by the addition of acid, further protein is precipitated as amorphous material which binds the droplets together to form aggregates of amorphous precipitates. Liquid-liquid separation has been observed in many protein solutions but this phenomenon has only scarcely been reported in the literature for soy proteins. It presents an exciting opportunity for an innovative product. Features of the liquid-phase protein such as protein yield and purity will be characterized.

Contemporaneous fluctuations in T cell responses to persistent herpes virus infections

The classical paradigm for T cell dynamics suggests that the resolution of a primary acute virus infection is followed by the generation of a long-lived pool of memory T cells that is thought to be highly stable. Very limited alteration in this repertoire is expected until the immune system is re-challenged by reactivation of latent viruses or by cross-reactive pathogens. Contradicting this view, we show here that the T cell repertoire specific for two different latent herpes viruses in the peripheral blood displayed significant contemporaneous co-fluctuations of virus-specific CD8(+) T cells. The coordinated responses to two different viruses suggest that the fluctuations within the T cell repertoire may be driven by sub-clinical viral reactivation or a more generalized 'bystander' effect. The later contention was supported by the observation that, while absolute number of CD3(+) T cells and their subsets and also the cell surface phenotype of antigen-specific T cells remained relatively constant, a loss of CD62L expression in the total CD8(+) T cell population was coincident with the expansion of tetramer-positive virus-specific T cells. This study demonstrates that the dynamic process of T cell expansion and contractions in persistent viral infections is not limited to the acute phase of infection, but also continues during the latent phase of infection.

Six-month outcome for dysphagia following traumatic brain injury: Radiological assessment

The coexistence of a swallowing impairment, or dysphagia, can severely impact upon the medical condition and recovery of a child with traumatic brain injury (TBI; Logemann, Pepe, & Mackay, 1994). Despite this fact, there is limited data that provide evidence of the progression or outcome of dysphagia in the pediatric population post-TBI (Rowe, 1999). The present study aimed to (1) provide a prospective radiologically based profile of swallowing outcome and (2) determine the clinical significance of any persistent physiological swallowing deficits by investigating the presence/absence of any coexistent respiratory complications. Seven children with moderate/severe TBI were evaluated via an initial videofluoroscopic swallowing assessment (VFSS) at an average of 24.1 days postinjury, during the acute phase of management. A follow-up VFSS was conducted at an average of 7 months, 3 weeks postinjury. The physiological impairment, swallowing safety, swallowing efficiency, and functional swallowing outcomes of the acute phase post-TBI were compared with reassessment results at 6 months post-TBI. The presence/absence of lower respiratory tract infection/respiratory complications in the past 6 months postinjury were recorded.VFSS revealed a number of residual physiological oropharyngeal swallowing impairments and reduced swallowing efficiency. However, all participants presented with clinically safe and functional swallowing outcomes at 6 months post-TBI, with no recent history of respiratory complication. This study indicates good functional swallowing and respiratory outcomes for patients at 6-months post-TBI despite the presence of persistent physiological swallowing impairment.

Physiological consequences of early-life insult

The most commonly observed severe lung injuries in early life are the respiratory distress syndrome in premature infants and the acute respiratory distress syndrome in children. Both diseases are characterised by alveolar instability, fluid filled airspace and some degree of airway obstruction. In the acute phase, collapsed alveoli can be reopened with positive end-expiratory pressure and lung recruitment. New insight into the physiology of lung recruitment suggests that the shape of the pressure–volume curve is defined by the change in rate of alveolar opening and closing. Reduced lung volumes and severe ventilation maldistribution are found in the acute phase but may persist during childhood. Any severe lung injury in this early phase of life can cause significant structural and functional damage to the developing lung. Follow-up studies of children with chronic lung disease have shown that the functional abnormalities will improve but may still be present in later childhood.

Acute cadmium chloride administration induces hepatic and renal CYP2A5 mRNA, protein and activity in the mouse: involvement of transcription factor NRF2

Modulation of the cytochrome P450 (CYP) monooxygenase system by cadmium was investigated in male, adult DBA/2J mice treated with a single dose (16 mumol/kg body weight, i.p.) of cadmium chloride (CdCl2). Total CYP content of liver and kidney microsomes decreased maximally (56% and 85%, respectively) 24 and 18 h, respectively, after CdCl2 treatment. Progressive increases of hepatic coumarin 7-hydroxylase (COH) activity; indicative of CYP2A5 activity, relative to the total CYP content were seen at 8 h (2-fold), 12 h (3-fold), 18 h (12-fold), and 24 h (15-fold). Similar changes were seen in the kidney. Liver and kidney CYP2A5 mRNA levels increased maximally 12 and 4 h after treatment and decreased to almost half 6 h later. In contrast, kidney and liver CYP2A5 protein levels increased maximally at 18 and 24 h. The CYP2A5 mRNA levels in the kidney and liver increased after Cd treatment in Nrf2 +/+ but not in Nrf2 -/- mouse. This study demonstrates that hepatic and kidney CYP2A5 is upregulated by cadmium with a somewhat faster response in the kidney than the liver. The strong upregulation of the CYP2A5 both at mRNA and enzyme activity levels, with a simultaneous decrease in the total CYP concentration suggest an unusual mode of regulation of CYP2A5 in response to cadmium exposure, amongst the CYP enzymes. The observed decrease in the mRNA but not in protein levels after maximal induction may suggest involvement of post-trancriptional mechanisms in the regulation. Upregulation of CYP2A5 by cadmium in the Nrf2 +/+ mice but not in the Nrf2 -/- mice indicates a role for this transcription factor in the regulation. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

Phase 1 study of HPV16-specific immunotherapy with E6E7 fusion protein and ISCOMATRIX (TM) adjuvant in women with cervical intraepithelial neoplasia

Purpose: Persistent infection of cervical epithelium with high risk human papillomavirus (HPV) results in cervical intraepithelial neoplasia (CIN) from which squamous cancer of the cervix can arise. A study was undertaken to evaluate the safety and immunogenicity of an HPV 16 immunotherapeutic consisting of a mixture of HPV16 E6E7 fusion protein and ISCOMATRIX(TM) adjuvant (HPV16 Immunotherapeutic) for patients with CIN. Experimental design: Patients with CIN (n = 3 1) were recruited to a randomised blinded placebo controlled dose ranging study of immunotherapy. Results: Immunotherapy was well tolerated. Immunised subjects developed HPV16 E6E7 specific immunity. Antibody, delayed type hypersensitivity, in vitro cytokine release, and CD8 T cell responses to E6 and E7 proteins were each significantly greater in the immunised subjects than in placebo recipients. Loss of HPV16 DNA from the cervix was observed in some vaccine and placebo recipients. Conclusions : The HPV16 Immunotherapeutic comprising HPV16E6E7 fusion protein and ISCOMATRIX(TM) adjuvant is safe and induces vaccine antigen specific cell mediated immunity. (C) 2004 Elsevier Ltd. All rights reserved.

Caveolins in the repair phase of acute renal failure after oxidative stress

Ischaemia-reperfusion and toxic injury are leading causes of acute renal failure (ARF). Both of these injury initiators use secondary mediators of damage in oxygen-derived free radicals. Several recent publications about ischaemia-reperfusion and toxin-induced ARF have indicated that plasma membrane structures called caveolae, and their proteins, the caveolins, are potential participants in protecting or repairing renal tissues. Caveolae and caveolins have previously been ascribed many functions, a number of which may mediate cell death or survival of injured renal cells. This review proposes possible pathophysiological mechanisms by which altered caveolin-1 expression and localization may affect renal cell survival following oxidative stress.