Role of endothelial nitric oxide synthase in endothelial activation: insights from eNOS knockout endothelial cells

The objective of this study was to determine whether absence of endothelial nitric oxide synthase (eNOS) affects the expression of cell surface adhesion molecules in endothelial cells. Murine lung endothelial cells (MLECs) were prepared by immunomagnetic bead selection from wild-type and eNOS knockout mice. Wild-type cells expressed eNOS, but eNOS knockout cells did not. Expression of neuronal NOS and inducible NOS was not detectable in cells of either genotype. Upon stimulation, confluent wild-type MLECs produced significant amounts of NO compared with N-monomethyl-L-arginine-treated wild-type cells. eNOS knockout and wild-type cells showed no difference in the expression of E-selectin, P-selectin, intracellular adhesion molecule-1, and vascular cell adhesion molecule-1 as measured by flow cytometry on the surface of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31)-positive cells. Both eNOS knockout and wild-type cells displayed the characteristics of resting endothelium. Adhesion studies in a parallel plate laminar flow chamber showed no difference in leukocyte-endothelial cell interactions between the two genotypes. Cytokine treatment induced endothelial cell adhesion molecule expression and increased leukocyte-endothelial cell interactions in both genotypes. We conclude that in resting murine endothelial cells, absence of endothelial production of NO by itself does not initiate endothelial cell activation or promote leukocyte-endothelial cell interactions. We propose that eNOS derived NO does not chronically suppress endothelial cell activation in an autocrine fashion but serves to counterbalance signals that mediate activation. vascular biology; atherosclerosis; mouse models     

Real-Time, label-free monitoring of tumor antigen and serum antibody interactions

Conventional techniques for the detection of biomolecular interactions can be limited by the need for exogenous labels, time- and labor-intensive protocols, as well as by poor sensitivity levels. A refractometer instrument has been reconfigured to detect biomolecular interactions through changes in surface plasmon resonance (SPR). The binding kinetics and affinity values of anti-NY-ESO-1 monoclonal antibody, ES121, to the cancer-testis antigen NY-ESO-1 were determined according to the surface heterogeneity model and resulted in K(D) values of 1.3x10(-9) and 2.1x10(-10) M. The reconfigured instrument was then used to measure the interaction between tumor antigens and serum antibodies against these antigens in preselected cancer patient sera samples. The tumor antigens assayed included NY-ESO-1, SSX2 and p53, all used as recombinant proteins containing polyhistidine tags. These results demonstrated that the instrument is capable of detecting the binding of serum antibodies from cancer patient sera to immobilized tumor antigens, consistent with those observed previously in ELISA-based experiments. These results demonstrate the potential of SPR technology for the rapid diagnosis and monitoring immune responses.     

Preferential expression of a plant cystatin at nematode feeding sites confers resistance to Meloidogyne incognita and Globodera pallida

The expression patterns of three promoters preferentially active in the roots of Arabidopsis thaliana have been investigated in transgenic potato plants in response to plant parasitic nematode infection. Promoter regions from the three genes, TUB-1, ARSK1 and RPL16A were linked to the GUS reporter gene and histochemical staining was used to localize expression in potato roots in response to infection with both the potato cyst nematode, Globodera pallida and the root-knot nematode, Meloidogyne incognita. All three promoters directed GUS expression chiefly in root tissue and were strongly up-regulated in the galls induced by feeding M. incognita. Less activity was associated with the syncytial feeding cells of the cyst nematode, although the ARSK1 promoter was highly active in the syncytia of G. pallida infecting soil grown plants. Transgenic potato lines that expressed the cystatin OcIDeltaD86 under the control of the three promoters were evaluated for resistance against Globodera sp. in a field trial and against M. incognita in containment. Resistance to Globodera of 70 +/- 4% was achieved with the best line using the ARSK1 promoter with no associated yield penalty. The highest level of partial resistance achieved against M. incognita was 67 +/- 9% using the TUB-1 promoter. In both cases this was comparable to the level of resistance achieved using the constitutive cauliflower mosaic virus 35S (CaMV35S) promoter. The results establish the potential for limiting transgene expression in crop plants whilst maintaining efficacy of the nematode defence.     

Satisfaction (not) guaranteed: re-evaluating the use of animal models of type 1 diabetes

Without a doubt, rodent models have been instrumental in describing pathways that lead to pancreatic beta-cell destruction, evaluating potential causes of type 1 diabetes and providing proof-of-principle for the potential of immune-based interventions. However, despite more than two decades of productive research, we are still yet to define an initiating autoantigen for the human disease, to determine the precise mechanisms of beta-cell destruction in humans and to design interventions that prevent or cure type 1 diabetes. In this Perspective article, we propose that a major philosophical change would benefit this field, a proposition that is based on evaluation of situations in which rodent models have provided useful guidance and in which they have led to disappointments.     

Rapid population decline in red knots: fitness consequences of decreased refuelling rates and late arrival in Delaware Bay

Most populations of migrant shorebirds around the world are in serious decline, suggesting that vital condition-dependent rates such as fecundity and annual survival are being affected globally. A striking example is the red knot (Calidris canutus rufa) population wintering in Tierra del Fuego, which undertakes marathon 30,000 km hemispheric migrations annually. In spring, migrant birds forage voraciously on horseshoe crab eggs in Delaware Bay in the eastern USA before departing to breed in Arctic polar deserts. From 1997 to 2002 an increasing proportion of knots failed to reach threshold departure masses of 180-200 g, possibly because of later arrival in the Bay and food shortage from concurrent over-harvesting of crabs. Reduced nutrient storage, especially in late-arriving birds, possibly combined with reduced sizes of intestine and liver during refuelling, had severe fitness consequences for adult survival and recruitment of young in 2000-2002. From 1997 to 2002 known survivors in Delaware Bay were heavier at initial capture than birds never seen again, annual survival of adults decreased by 37% between May 2000 and May 2001, and the number of second-year birds in wintering flocks declined by 47%. Population size in Tierra del Fuego declined alarmingly from 51,000 to 27,000 in 2000-2002, seriously threatening the viability of this subspecies. Demographic modelling predicts imminent endangerment and an increased risk of extinction of the subspecies without urgent risk-averse management.     

Protists decrease in size linearly with temperature: ca. 2.5% degrees C(-1)

An inverse relationship between organism size and rearing temperature is widely observed in ectotherms ('the temperature-size rule', TSR). This has rarely been quantified for related taxa, and its applicability to protists also required testing. Here, we quantify the relationship between temperature and mean cell volume within the protists by a meta-analysis of published data covering marine, brackish water and freshwater autotrophs and heterotrophs. In each of 44 datasets, a linear relationship between temperature and size could not be rejected, and a negative trend was found in 32 cases (20 gave significant negative regressions, p < 0.05). By combining 65 datasets, we revealed, for each 1 degrees C increase, a cell-size reduction of 2.5% (95% CI of 1.7-3.3%) of the volume observed at 15 degrees C. The value did not differ across taxa (amoebae, ciliates, diatoms, dinoflagellates, flagellates), habitats, modes of nutrition or combinations of these. The data are consistent with two hypotheses that are capable of explaining the TSR in ectotherms generally: (i) resource, especially respiratory gas, limitation; and (ii) fitness gains from dividing earlier as population growth increases. Using the above relationship we show how changes in cell numbers with temperature can be estimated from changes in biomass and vice versa; ignoring this relationship would produce a systematic error.     

Systemic overexpression of IL-10 induces CD4+CD25+ cell populations in vivo and ameliorates type 1 diabetes in nonobese diabetic mice in a dose-dependent fashion

Early systemic treatment of nonobese diabetic mice with high doses of recombinant adeno-associated virus (rAAV) vector expressing murine IL-10 prevents type 1 diabetes. To determine the therapeutic parameters and immunological mechanisms underlying this observation, female nonobese diabetic mice at 4, 8, and 12 wk of age were given a single i.m. injection of rAAV-murine IL-10 (10(4), 10(6), 10(8), and 10(9) infectious units (IU)), rAAV-vector expressing truncated murine IL-10 fragment (10(9) IU), or saline. Transduction with rAAV-IL-10 at 10(9) IU completely prevented diabetes in all animals injected at all time points, including, surprisingly, 12-wk-old animals. Treatment with 10(8) IU provided no protection in the 12-wk-old injected mice, partial prevention in 8-wk-old mice, and full protection in all animals injected at 4 wk of age. All other treatment groups developed diabetes at a similar rate. The rAAV-IL-10 therapy attenuated pancreatic insulitis, decreased MHC II expression on CD11b+ cells, increased the population of CD11b+ cells, and modulated insulin autoantibody production. Interestingly, rAAV-IL-10 therapy dramatically increased the percentage of CD4+CD25+ regulatory T cells. Adoptive transfer studies suggest that rAAV-IL-10 treatment alters the capacity of splenocytes to impart type 1 diabetes in recipient animals. This study indicates the potential for immunomodulatory gene therapy to prevent autoimmune diseases, including type 1 diabetes, and implicates IL-10 as a molecule capable of increasing the percentages of regulatory cells in vivo.     

Interaction of glycoprotein H of human herpesvirus 6 with the cellular receptor CD46

Human herpesvirus 6 (HHV-6) employs the complement regulator CD46 (membrane cofactor protein) as a receptor for fusion and entry into target cells. Like other known herpesviruses, HHV-6 encodes multiple glycoproteins, several of which have been implicated in the entry process. In this report, we present evidence that glycoprotein H (gH) is the viral component responsible for binding to CD46. Antibodies to CD46 co-immunoprecipitated an approximately 110-kDa protein band specifically associated with HHV-6-infected cells. This protein was identified as gH by selective depletion with an anti-gH monoclonal antibody, as well as by immunoblot analysis with a rabbit hyperimmune serum directed against a gH synthetic peptide. In reciprocal experiments, a monoclonal antibody against HHV-6 gH was found to co-immunoprecipitate CD46. Studies using monoclonal antibodies directed against specific CD46 domains, as well as engineered constructs lacking defined CD46 regions, demonstrated a close correspondence between the CD46 domains involved in the interaction with gH and those previously shown to be critical for HHV-6 fusion (i.e. short consensus repeats 2 and 3).     

Radiation-induced cell inactivation can increase the cancer risk

Radiation can inactivate cells that are replaced by dividing neighboring cells. If cells on the way to malignancy can fill the deficit faster than healthy cells, their number increases. A major part of the radon-induced lung cancers in the Colorado miners can be explained by a moderate increase in the replacement probability.     

Impact responses of the flexed human knee using a deformable impact interface

Blunt impact trauma to the patellofemoral joint during car accidents, sporting activities, and falls can produce a range of injuries to the knee joint, including gross bone fracture, soft tissue injury, and/or microinjuries to bone and soft tissue. Currently, the only well-established knee injury criterion applies to knee impacts suffered during car accidents. This criterion is based solely on the peak impact load delivered to seated cadavers having a single knee flexion angle. More recent studies, however, suggest that the injury potential, its location, and the characteristics of the damage are also a function of knee flexion angle and the stiffness of the impacting structure. For example, at low flexion angles, fractures of the distal patella are common with a rigid impact interface, while at high flexion angles splitting of the femoral condyles is more evident. Low stiffness impact surfaces have been previously shown to distribute impact loads over the anterior surface of the patella to help mitigate gross and microscopic injuries in the 90 deg flexed knee. The objective of the current study was to determine if a deformable impact interface would just as effectively mitigate gross and microscopic injuries to the knee at various flexion angles. Paired experiments were conducted on contralateral knees of 18 human cadavers at three flexion angles (60, 90, 120 deg). One knee was subjected to a fracture level impact experiment with a rigid impactor, and the opposite knee was impacted with a deformable interface (3.3 MPa crush strength honeycomb material) to the same load. This (deformable) impact interface was effective at mitigating gross bone fractures at approximately 5 kN at all flexion angles, but the frequency of split fracture of the femoral condyles may not have been significantly reduced at 120 deg flexion. On the other hand, this deformable interface was not effective in mitigating microscopic injuries observed for all knee flexion angles. These new data, in concert with the existing literature, suggest the chosen impact interface was not optimal for knee injury protection in that fracture and other minor injuries were still produced. For example, in 18 cadavers a total of 20 gross fractures and 20 subfracture injuries were produced with a rigid interface and 5 gross fractures and 21 subfracture injuries with the deformable interface selected for the current study. Additional studies will be needed to optimize the knee impact interface for protection against gross and microscopic injuries to the knee.