Antibodies with specificity to native gp120 and neutralization activity against primary human immunodeficiency virus type 1 isolates elicited by immunization with oligomeric gp160.

Current human immunodeficiency virus type 1 (HIV-1) envelope vaccine candidates elicit high antibody binding titers with neutralizing activity against T-cell line-adapted but not primary HIV-1 isolates. Serum antibodies from these human vaccine recipients were also found to be preferentially directed to linear epitopes within gp120 that are poorly exposed on native gp120. Systemic immunization of rabbits with an affinity-purified oligomeric gp160 protein formulated with either Alhydrogel or monophosphoryl lipid A-containing adjuvants resulted in the induction of high-titered serum antibodies that preferentially bound epitopes exposed on native forms of gp120 and gp160, recognized a restricted number of linear epitopes, efficiently bound heterologous strains of monomeric gp120 and cell surface-expressed oligomeric gp120/gp41, and neutralized several strains of T-cell line-adapted HIV-1. Additionally, those immune sera with the highest oligomeric gp160 antibody binding titers had neutralizing activity against some primary HIV-1 isolates, using phytohemagglutinin-stimulated peripheral blood mononuclear cell targets. Induction of an antibody response preferentially reactive with natively folded gp120/gp160 was dependent on the tertiary structure of the HIV-1 envelope immunogen as well as its adjuvant formulation, route of administration, and number of immunizations administered. These studies demonstrate the capacity of a soluble HIV-1 envelope glycoprotein vaccine to elicit an antibody response capable of neutralizing primary HIV-1 isolates.     

Urinary C-type natriuretic peptide excretion: a potential novel biomarker for renal fibrosis during aging

Renal aging is characterized by structural changes in the kidney including fibrosis, which contributes to the increased risk of kidney and cardiac failure in the elderly. Studies involving healthy kidney donors demonstrated subclinical age-related nephropathy on renal biopsy that was not detected by standard diagnostic tests. Thus there is a high-priority need for novel noninvasive biomarkers to detect the presence of preclinical age-associated renal structural and functional changes. C-type natriuretic peptide (CNP) possesses renoprotective properties and is present in the kidney; however, its modulation during aging remains undefined. We assessed circulating and urinary CNP in a Fischer rat model of experimental aging and also determined renal structural and functional adaptations to the aging process. Histological and electron microscopic analysis demonstrated significant renal fibrosis, glomerular basement membrane thickening, and mesangial matrix expansion with aging. While plasma CNP levels progressively declined with aging, urinary CNP excretion increased, along with the ratio of urinary to plasma CNP, which preceded significant elevations in proteinuria and blood pressure. Also, CNP immunoreactivity was increased in the distal and proximal tubules in both the aging rat and aging human kidneys. Our findings provide evidence that urinary CNP and its ratio to plasma CNP may represent a novel biomarker for early age-mediated renal structural alterations, particularly fibrosis. Thus urinary CNP could potentially aid in identifying subjects with preclinical structural changes before the onset of symptoms and disease, allowing for the initiation of strategies designed to prevent the progression of chronic kidney disease particularly in the aging population.     

Species specificity of iron delivery in hybridomas

Summary Studies with Human x Human (HxH), Human x Mouse (HxM), and Mouse x Mouse (MxM) hybridomas have enabled us to define specific factors that affect hybridoma growth in a species-specific manner. Three transferrins and three lipophilic iron chelates have been tested for their ability to support hybridoma proliferation and antibody production. The results of these studies demonstrate that HxH hybridomas do not respond to bovine transferrin a⁺ concentrations up to 100 μg/ml and are approximately 100-fold less responsive to mouse transferrin than to human transferrin. HxM and MxM hybridomas respond equally to human or mouse transferrin but are 100-fold less sensitive to bovine transferrin. An antibody to the human transferrin receptor inhibited the growth-promoting activity of human or mouse transferrin on HxH hybridomas but was ineffective on HxM hybridomas. This semonstrated the functionality of the human transferrin receptor in HxH hybridomas and that human, mouse, and bovine transferrin were interacting through the mouse transferrin receptor in HxM hybridomas. HxH and HxM hybridomas respond similarly to three different iron chelates exhibiting 80 to 110% of the growth response to human transferrin. MxM hybridomas fail to respond to the iron chelates at similar concentrations, suggesting that the human genome present in the other hybridoma species confers a unique ability for utilizing iron when delivered in this form.     

Assessment of lake sturgeon (Acipenser fulvescens) recruitment in a regulated spawning tributary of Rainy Lake,Ontario

Continued study of the relationship between lake sturgeon (Acipenser fulvescens) recruitment and hydroelectric dams and operations, in a variety of river systems and habitat types is needed to improve the ability to predict and monitor impacts of the hydroelectric industry on this species. Herein, we present results of a juvenile lake sturgeon study aimed at addressing concerns over an inferred lack of recruitment resulting from spawning downstream of a hydroelectric generating station (HGS). Two years of sampling (2015 and 2016) were conducted in five sections of a 41 km long reach of the Seine River, Ontario, a lake sturgeon spawning tributary of Rainy Lake. Using an established gillnetting method, deepwater habitat was targeted to capture juvenile lake sturgeon to assess relative abundance, recruitment (cohort strength), and growth. Deepwater habitat, defined as water depths >6 m in this system, comprised only 2.1% of the wetted area in this study area. Within these habitats, a total of 331 lake sturgeon capture events were observed over the 2-years study period. The majority of the lake sturgeon catch (85%) was comprised of age-0 to age-5 individuals (both sampling years combined). Although inter-annual variation in cohort strength was apparent, each cohort between 2006 and 2016 was represented. The spatial distribution of cohorts varied among river reaches with younger individuals (age-0 and age-1) occupying reaches proximal to the Sturgeon Falls HGS, and larger, older individuals (age-2 to age-5) occupying reaches further downstream. The rarity of age-6+ individuals can likely be explained by ongoing downstream redistribution of juveniles over time, out of the Seine River and into Rainy Lake. Growth of juvenile lake sturgeon captured in the Seine River was above average relative to conspecifics from other rivers in the Hudson Bay drainage. Unfortunately, baseline data sets required to facilitate comparisons of contemporary (post-construction Sturgeon Falls HGS) versus historical (i.e. pre- Sturgeon Falls HGS) lake sturgeon recruitment, or to evaluate the influence of the Seine River Water Management Plan (2004) on lake sturgeon recruitment, are lacking. However, juvenile Lake Sturgeon are more abundant in this system than what had been surmised based on recent studies which implemented random sampling. Results indicate that juvenile lake sturgeon may reside in spawning tributaries for several years (age-0 to age-5) prior to seeking alternate habitats and highlights the value of targeted sampling (i.e. by depth) along the flow axis of rivers downstream of spawning areas when assessing lake sturgeon recruitment patterns.     

Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and antitumor effects

We recently demonstrated that Venezuelan equine encephalitis virus-based replicon particle (VRPs) encoding tumor antigens could break tolerance in the immunomodulatory environment of advanced cancer. We hypothesized that local injection of VRP-expressing interleukin-12 (IL-12) at the site of injections of VRP-based cancer vaccines would enhance the tumor-antigen-specific T cell and antibody responses and antitumor efficacy. Mice were immunized with VRP encoding the human tumor-associated antigen, carcinoembryonic antigen (CEA) (VRP-CEA(6D)), and VRP-IL-12 was also administered at the same site or at a distant location. CEA-specific T cell and antibody responses were measured. To determine antitumor activity, mice were implanted with MC38-CEA-2 cells and immunized with VRP-CEA with and without VRP-IL-12, and tumor growth and mouse survival were measured. VRP-IL-12 greatly enhanced CEA-specific T cell and antibody responses when combined with VRP-CEA(6D) vaccination. VRP-IL-12 was superior to IL-12 protein at enhancing immune responses. Vaccination with VRP-CEA(6D) plus VRP-IL-12 was superior to VRP-CEA(6D) or VRP-IL-12 alone in inducing antitumor activity and prolonging survival in tumor-bearing mice. Importantly, local injection of VRP-IL-12 at the VRP-CEA(6D) injection site provided more potent activation of CEA-specific immune responses than that of VRP-IL-12 injected at a distant site from the VRP-CEA injections. Together, this study shows that VRP-IL-12 enhances vaccination with VRP-CEA(6D) and was more effective at activating CEA-specific T cell responses when locally expressed at the vaccine site. Clinical trials evaluating the adjuvant effect of VRP-IL-12 at enhancing the immunogenicity of cancer vaccines are warranted.     

Treating mites in a bat colony: a case study

Since many bats used in research are wild-caught, dealing with mite infestation can present a unique challenge to the animal care staff. The authors tested the efficacy of fipronil spray for eliminating mites from a bat colony.     

Attachment of Escherichia coli O157:H7 to the surfaces and internal structures of apples as detected by confocal scanning laser microscopy

Confocal scanning laser microscopy (CSLM) was used to demonstrate the attachment of Escherichia coli O157:H7 transformed with a plasmid encoding for green fluorescent protein (GFP) to the surface and within the internal structures of nonwaxed Red Delicious cv. apples. Apples at 2 or 25 degrees C were inoculated with an E. coli O157:H7 cell suspension at 2 or 25 degrees C. The effect of a negative temperature differential (cold inoculum, warm apple), a positive differential (warm inoculum, cold apple), and no differential (warm inoculum, warm apple), in combination with a pressure differential (atmospheric versus 10,130 Pa), on the attachment and infiltration of cells was determined. CSLM stereo images of external surfaces of apples subjected to all combinations of test parameters showed preferential cellular attachment to discontinuities in the waxy cuticle on the surface and to damaged tissue surrounding puncture wounds, where the pathogen was observed at depths up to 70 microm below the skin surface. Attachment to lenticels was sporadic but was occasionally observed at depths of up to 40 microm. Infiltration through the floral tube and attachment to seeds, cartilaginous pericarp, and internal trichomes were observed in all apples examined, regardless of temperature differential during inoculation. The pressure differential had no effect on infiltration or attachment of E. coli O157:H7. Image analysis to count cells at various depths within tissues was used to quantitatively compare the extent of infiltration into various apple structures as well as the effects of the temperature differential. Puncture wounds harbored greater numbers of the pathogen at greater depths than did other sites examined. Attachment or infiltration of cells was greater on the intact skin and in lenticels, russet areas, and the floral tube of apples inoculated under a negative temperature differential compared to those inoculated under no temperature differential. The results suggest that E. coli O157:H7 attached to internal core structures or within tissues of apples may evade decontamination treatments. Interventions designed to deliver disinfectants to these locations or to remove viable cells of E. coli O157:H7 and other pathogens from apples by other means need to be developed and validated.     

Activation of EphA2 kinase suppresses integrin function and causes focal-adhesion-kinase dephosphorylation

Interactions between receptor tyrosine kinases of the Eph family and their ligands, ephrins, are implicated in establishment of organ boundaries and repulsive guidance of cell migration during development, but the mechanisms by which this is achieved are unclear. Here we show that activation of endogenous EphA2 kinase induces an inactive conformation of integrins and inhibits cell spreading, migration and integrin-mediated adhesion. Moreover, EphA2 is constitutively associated with focal-adhesion kinase (FAK) in resting cells. Within one minute after stimulation of EphA2 with its ligand, ephrin-A1, the protein tyrosine phosphatase SHP2 is recruited to EphA2; this is followed by dephosphorylation of FAK and paxillin, and dissociation of the FAK-EphA2 complex. We conclude that Eph kinases mediate some of their functions by negatively regulating integrins and FAK.     

Whole animal and gill tissue oxygen uptake in the Eastern oyster, Crassostrea virginica: Effects of hypoxia, hypercapnia, air exposure, and infection with the protozoan parasite Perkinsus marinus(1)

The Eastern oyster, Crassostrea virginica, lives in shallow coastal waters and experiences many different environmental extremes including hypoxia, hypercapnia and air exposure and many oysters are infected with the protozoan parasite Perkinsus marinus. The effects of these conditions on oyster metabolism, as measured by oxygen uptake, were investigated. Mild hypercapnia had no effect on the ability of oysters to regulate oxygen uptake in hypoxic water, as measured by the B2 coefficient of oxygen regulation. The average B2 was -0.060x10(-3) (+/-0.01x10(-3) S.E.M.; n=20; low and high CO(2) treatments combined) in oysters uninfected with P. marinus and -0.056x10(-3) (+/-0.01x10(-3) S.E.M.; n=16; low and high CO(2) treatments combined) in infected oysters. There was no significant effect of light to moderate infections of P. marinus on oxygen regulation. Nor did the presence of P. marinus have an effect on the rate of oxygen uptake of whole animals in well-aerated water. In well-aerated conditions, oxygen uptake was significantly reduced by moderate hypercapnia in oysters when data from uninfected and infected oysters were combined. Mean oxygen uptake of infected oysters under hypercapnia (pCO(2)=6-8 Torr; pH 7) was 9.10 μmol O(2) g ww(-1) h(-1) +/-0.62 S.E.M. (n=9), significantly different from oxygen uptake under normocapnia (pCO(2) 相似文献