Early determinants of atherosclerosis in paediatric systemic lupus erythematosus.

OBJECTIVES: To assess traditional and non-traditional cardiovascular risk factors and to determine the prevalence and correlates of early vascular markers of atherosclerosis in paediatric systemic lupus erythematosus (pSLE). METHODS: Fifty-four adolescents with pSLE had cardiovascular risk factor assessment, disease activity and vascular testing including carotid intima-media thickness (CIMT), flow-mediated dilatation (FMD), arterial stiffness measures, and myocardial perfusion studies. RESULTS: The traditional risk factors of hypertension, elevated triglycerides, apolipoprotein B, haemoglobin A1c and insulin levels and non-traditional risk factors of elevated homocysteine and fibrinogen were present (all p<0.001). Some arterial stiffness measures, central pulse wave velocity and characteristic impedance were elevated (p<0.001), but CIMT, FMD and myocardial perfusion were normal. Cumulative prednisone dose correlated with total cholesterol (r=0.5790, p<0.001) and elevated LDL-C (r=0.4488, p=0.0012). Hydroxychloroquine treatment correlated negatively with total cholesterol (r=-0.4867, p=0.0002), LDL-C (r=-0.4805, p=0.0002) and apolipoprotein B (r=-0.4443, p=0.0011). In multivariate analysis LDL-C correlated with cumulative prednisone dose and negatively with hydroxychloroquine treatment (R2=0.40, p<0.001). CONCLUSIONS: An increased burden of traditional and non-traditional risk factors and early evidence of insulin resistance and increased central arterial stiffness were present in paediatric SLE. Disease-specific and therapy-related factors are likely modifying these cardiovascular risk profiles warranting prospective longitudinal studies.

Transitions in insect respiratory patterns are controlled by changes in metabolic rate.

We examined the respiratory patterns of Rhodnius prolixus and Gromphadorhina portentosa as metabolic rates varied with temperature to determine whether insects transition from discontinuous (DGC), cyclical and continuous respiration as a response to increasing aerobic demand. Using flow through respirometry we: (1) determined the effects of temperature on metabolic rate; (2) objectively defined periods of spiracular closure; (3) observed whether there was a correlation between metabolic rate and length of spiracular closure. At low temperatures both species exhibit lengthy periods of spiracular closure reflecting a discontinuous respiratory pattern. As metabolic rate increased, periods of spiracular closure decreased and insects displayed a more cyclical pattern of respiration. As metabolic rates increased even further under the highest experimental temperatures, periods of spiracular closure decreased even more and a continuous respiratory pattern was employed by both species. Our results suggest that the three described respiratory patterns in insects are not distinct but are instead a continuum of respiratory responses driven by the metabolic demand experienced by the insect.

Metabolic rate controls respiratory pattern in insects.

The majority of scientific papers on the subject of respiratory patterns in insects have dealt with the discontinuous gas-exchange cycle (DGC). The DGC is characterized by the release of bursts of CO(2) from the insect, followed by extended periods of spiracular closure. Several hypotheses have been put forward to explain the evolutionary origin and physiological function of this unusual respiratory pattern. We expand upon one of these (the oxidative damage hypothesis) to explain not only the occurrence of the DGC but also the mechanistic basis for the transition to two other well-characterized respiratory patterns: the cyclic pattern and the continuous pattern. We propose that the specific pattern employed by the insect at any given time is a function of the amount of oxygen contained in the insect at the time of spiracular closure and the aerobic metabolic rate of the insect. Examples of each type of pattern are shown using the insect Rhodnius prolixus. In addition, contrary to the expectations deriving from the hygric hypothesis, it is demonstrated that the DGC does not cease in Rhodnius in humid air.

Electroless metallization onto pulsed plasma deposited poly(4-vinylpyridine) surfaces.

Pulsed plasma-chemical deposition of poly(4-vinylpyridine) is found to be a highly effective way of functionalizing solid surfaces with pyridine ring centers. These surfaces can be metallized via complexation to Pd2+ ions from solution, followed by autocatalytic electroless deposition of either copper or nickel films.

Rewritable DNA microarrays.

Thiol-terminated single-stranded deoxyribonucleic acids (ssDNA) can be immobilized onto pulsed plasma deposited poly(allylmercaptan) surfaces via disulfide bridge chemistry and are found to readily undergo nucleic acid hybridization. Unlike other methods for oligonucleotide attachment to solid surfaces, this approach is shown to be independent of substrate material or geometry, and amenable to highly efficient rewriting.

Malarial infection in Aedes aegypti : effects on feeding, fecundity and metabolic rate.

We have examined metabolic rate, lipid and carbohydrate of female Aedes aegypti during 10 days following a malaria-infected bloodmeal. In parallel, we determined bloodmeal size, portions retained and diuresed, and subsequent fecundity. We found that mosquitoes obtained identical masses of blood when feeding on an infected or control host. However, infected mosquitoes lost more mass during diuresis and retained a smaller mass. Infection led to a significant reduction in fecundity, the extent of which could not be explained by the difference in post-diuresis bloodmeal mass alone. We found no differences in lipid or carbohydrate content between infected and control mosquitoes during the 10 days post-infection, although infected mosquitoes had a lower body mass than controls. Metabolic rates were not different between groups, except during blood digestion, where the metabolic rate was lower in infected mosquitoes. These results suggest that infection by malaria does not lead to an increase in metabolic rate during the phases of midgut invasion and sporogony. However, infection does have a measurable effect on fecundity and subsequent body mass of the infected females.

Water acquisition and partitioning in Drosophila melanogaster: effects of selection for desiccation-resistance.

We examined physiological features related to water balance in five replicate populations of Drosophila melanogaster that have undergone selection for enhanced resistance to desiccation (D populations) and in five replicate control (C) populations. Adult D flies contain 34 % more water than the control flies. We examined two hypotheses for increased water acquisition in the D flies: (i) that they accumulate more water early in development and (ii) that they have a reduced post-eclosion diuretic water loss. We found no evidence of differential water or dry mass acquisition between the C and D populations prior to adulthood. We also found no evidence of differential post-eclosion diuresis, i.e. both C and D groups showed insignificant changes in water volume in the 4 h period immediately after eclosion. In addition, we quantified water content in the intra- and extracellular compartments of the C and D populations and were able to identify the hemolymph as the primary storage site of the 'extra' water carried by the desiccation-resistant flies. We estimated that 68 % of the increased water volume observed in the D flies was contained in the hemolymph. Desiccation-resistance was strongly correlated with hemolymph volume and only weakly with intracellular water volume. Survival during desiccation was also strongly related to the carbohydrate content of the D flies. It has been presumed that the D flies accumulate carbohydrate primarily as intracellular glycogen, which would result in a significant increase in intracellular water volume. We found that carbohydrate content was weakly correlated with intracellular water volume and more strongly with hemolymph volume. The carbohydrate pool in the D flies may, therefore, be contained in the extracellular compartment as well as in cells. These results are suggestive of the importance of modifications in hemolymph volume and hemolymph solute concentrations in the evolution of enhanced desiccation-tolerance in populations of Drosophila melanogaster.

Sodium and chloride regulation in freshwater and osmoconforming larvae of Culex mosquitoes.

In this study, we examined aspects of Na(+) and Cl(-) regulation in mosquito larvae of the genus Culex, a group that includes species that tolerate high salinity as well as other forms that are restricted to fresh water. When the euryhaline osmoconformer C. tarsalis was acutely transferred from 30 % to 50 % sea water, the patterns of hemolymph Na(+) and Cl(-) regulation were similar. The underlying regulatory mechanisms for these two ions have very different characteristics. In C. tarsalis, Na(+) efflux was significantly elevated compared with the rates measured in the freshwater-restricted C. quinquefasciatus, while Cl(-) influx was relatively lower. The modulation of Na(+) efflux and Cl(-) influx allowed C. tarsalis to avoid a potential salt load and ionic disturbance in the hemolymph during an acute increase ( )in salinity. The observed adjustment of NaCl regulation departs from that determined for other euryhaline organisms and is integral to the osmoconforming response. At the other extreme of the salinity spectrum, we observed that C. tarsalis faces difficulties in ion regulation in habitats with low NaCl levels because of its inability to reduce ion efflux and adjust ion absorption rates to maintain hemolymph ion balance. In contrast, C. quinquefasciatus exhibited a reduced ion efflux and the ability to upregulate Na(+) uptake, traits necessary to extend its lower salinity limit.

Postponed aging and desiccation resistance in Drosophila melanogaster.

Studies with the fruit fly, Drosophila melanogaster, have repeatedly shown that selection for postponed reproduction leads to increases in mean life span and increased stress resistance; including increased resistance to desiccation, starvation and ethanol vapors. We show that desiccation resistance declines with age in both short- and long-lived flies suggesting that desiccation resistance may serve as a useful biomarker for aging-related declines in physiological performance. We examined the physical basis of desiccation resistance in five replicate populations selected for postponed reproduction and five replicate control populations. The variables examined were water content, rates of water loss during desiccation, and water content at time of death due to desiccation. In the absence of desiccation stress, both the flies exhibiting postponed senescence and their controls maintained constant water content throughout their lifetimes. In the presence of desiccation stress, the short-lived flies showed significantly higher rates of water loss at all ages than did the long-lived flies. Flies from the two treatments did not differ in water content at death. Our results indicate that water loss rates are the major determinant of desiccation resistance. Water loss rates are under genetic control and covary with age in populations with genetically-determined postponed senescence.

The physiology of salinity tolerance in larvae of two species of Culex mosquitoes: the role of compatible solutes.

We investigated the physiological basis for differences in salinity tolerance ranges in mosquito larvae of the genus Culex. We examined the response of larvae of C. quinquefasciatus, a freshwater obligate, and C. tarsalis, a euryhaline osmoconformer, following transfer from fresh water to 34% sea water. Hemolymph Na(+) and Cl(-) levels increased similarly in both species, indicating that ion regulation does not differ under these conditions. C. quinquefasciatus responded to increased environmental salinity with increased hemolymph levels of serine, but suffered a significant reduction in levels of trehalose. C. tarsalis responded to increased environmental salinity with increased hemolymph levels of both proline and trehalose. When C. tarsalis larvae were held in 64% sea water, which C. quinquefasciatus larvae cannot tolerate, hemolymph proline and trehalose were accumulated approximately 50-fold and twofold, respectively, relative to freshwater values. We found that proline serves as both an intra- and extracellular compatible solute in C. tarsalis, the first such circumstance documented in an animal in response to increased environmental salinity. Analyses of the acute responses of the two species to an increase in salinity (from 30% to 50% sea water) indicate that larvae of C. tarsalis are able to volume-regulate via drinking and to attenuate increases in hemolymph NaCl concentration using unknown mechanisms during large, rapid increases in salinity.

Regulation of compatible solute accumulation in larvae of the mosquito Culex tarsalis: osmolarity versus salinity.

In this study, we demonstrate that two of the osmolytes utilized in the osmoconforming strategy of larval Culex tarsalis are regulated by two fundamentally different signals. When the external osmolality was increased using salinity (sea salts), hemolymph NaCl, proline and trehalose concentrations increased significantly. When sorbitol was used to increase the external osmolality without an elevation in salt concentration, hemolymph NaCl and proline concentrations decreased, whereas hemolymph trehalose concentration increased. The results suggest that proline accumulation was cued by increases in salinity, whereas trehalose levels followed increases in osmolality. Interestingly, we found that C. tarsalis larvae accumulated the exogenous sorbitol in the hemolymph in an osmoconforming manner. We conducted further studies in which changes in hemolymph NaCl concentrations were manipulated using changes in environmental salinity. The results suggested that hemolymph proline accumulation was cued by the proximal signal of hemolymph NaCl levels. Regardless of which solute (sea salts, sorbitol or mixtures thereof) was used to raise the external osmolality, trehalose accumulation tracked the increase in total osmolality of the medium. These findings indicate that the synthesis and accumulation of these two osmolytes are regulated by two independent signals.

Using laboratory selection for desiccation resistance to examine the relationship between respiratory pattern and water loss in insects.

We conducted concurrent measurements of rates of CO2 and H2O release from individual fruit flies Drosophila melanogaster taken from populations subjected to three different selective regimes: (1) populations selected for resistance to desiccation (D flies); (2) populations maintained as their controls (C flies); and (3) the ancestral populations of the D and C populations (O flies). In the D flies, water loss rates were significantly reduced, the standard error of the regression (SER) of the CO2 release pattern measured over the survival period of the flies was increased, and the ratio of CO2 loss to H2O loss (VCO2/VH2O) was increased. Correlations across all 15 populations from the three selection treatments indicate that survival time was negatively correlated with water loss rate, positively correlated with the SER of CO2 release and positively correlated with the VCO2/VH2O ratio. We did not, however, find a significant correlation between the SER of CO2 release and rates of water loss or the VCO2/VH2O ratio.

The effect of respiratory pattern on water loss in desiccation-resistant Drosophila melanogaster.

We measured CO2 and H2O release from individual fruit flies from five populations of Drosophila melanogaster selected for resistance to desiccation (D flies). Our previous work found that these flies survive for an extended period in dry air, have an increase in the peak height and frequency of CO2 release, as measured by the standard error of a linear regression (SER) of CO2 release for the entire survival period, and have reduced water loss rates (VH2O) compared with their control or ancestor populations. In the present study, we examined the following respiratory characteristics: VCO2, VH2O, the SER of CO2 release and the ratio of VCO2 to VH2O in the D flies. Correlations between these characters were calculated in order to determine the effect of respiratory pattern on water loss. We found that, within the D flies, neither periodic release of CO2 nor an increased SER for CO2 release was associated with reduced water loss. In addition, an increased SER was positively correlated with both an increased water loss rate and a decreased survival time. Therefore, although selection for desiccation resistance leads to both an increased SER and a decreased rate of water loss in the D flies, the increased SER does not significantly reduce respiratory water loss.

Metabolic reserves and evolved stress resistance in Drosophila melanogaster.

We have examined starvation and desiccation resistance in 43 outbred populations of Drosophila melanogaster that have diverged from a common ancestral population as a result of a variety of defined selection protocols. The populations differ up to 8.5-fold in desiccation resistance and up to 10-fold in starvation resistance. We used these populations to search for evolved physiological changes that might explain the differences in stress resistance. We examined two hypotheses for increased stress resistance that had been proposed previously in the literature: (1) that increments in starvation resistance are principally the result of differential lipid accumulation, and (2) that changes in glycogen accumulation play a role in evolved increases in resistance to desiccation stress. By quantifying desiccation resistance, starvation resistance, lipid content, and carbohydrate content in each of our populations of flies, we were able to demonstrate strong correlations between the capacity of the flies to resist starvation and the quantity of lipid or carbohydrate that the flies had stored. The strongest correlation (R2 = 0.99) was observed when the total energy content of both the lipid and carbohydrate stores was regressed against starvation resistance. These results demonstrate that the flies responded to selection for starvation resistance through a genetically determined increase in both lipid and carbohydrate storage. Similar analyses of the correlation between lipid storage or total energy storage and desiccation resistance revealed no significant correlations. Carbohydrate storage was significantly correlated with desiccation resistance in female but not in male flies. These results suggest that different forms of stress are resisted with distinct physiological mechanisms and that the evolutionary response of the flies to stress selection is specific to the stress imposed.

An analysis of resource allocation in response to dietary yeast in Drosophila melanogaster.

Drosophila melanogaster exhibit an increase in fecundity and a decrease in length of life and starvation resistance when the diet is enriched through the addition of live yeast. It has been proposed that this represents a necessary energetic trade-off between reproductive and somatic functions. We examined the metabolic aspects of this trade-off. We measured egg production, dry wt, somatic lipid and carbohydrate storage, and metabolic rate in response to changing amounts of live dietary yeast. These variables were measured in five replicate populations selected for postponed aging and five replicate short lived control populations. We find that all ten populations show an overall increase in egg production and decrease in the amount of stored metabolites in the presence of increasing amounts of yeast. All populations increase metabolic rate in response to increasing amounts of live dietary yeast. The energetics of this phenomenon suggest that increased egg production results from increased acquisition of nutrients available in yeast with only a small redirection of resources from storage to egg production.

CO2 release patterns in Drosophila melanogaster: the effect of selection for desiccation resistance.

We used laboratory natural selection on insects as a means of investigating the role of patterns of gas exchange in desiccation resistance. We used 15 populations of Drosophila melanogaster: five selected for desiccation resistance, five control populations and five ancestral populations. Using flow-through respirometry, we found that D. melanogaster from all populations produced irregular peaks of CO2 release. To quantify the height and frequency of these peaks, we used the standard error of a linear regression (SER) through the recordings of CO2 release. The values for the SER were significantly larger in the populations selected for desiccation resistance than in the control and ancestral populations. Occasionally, highly periodic peaks of CO2 release were observed in the desiccation-resistant populations only. Maximum SER was found to be strongly correlated with survival time in dry air among selection treatments, but not among individuals within a population. Access to dietary water resulted in lower SER values. These data demonstrate that gas exchange is physiologically controlled in Drosophila melanogaster and that the pattern of gas exchange can change under selection. The relationship between these CO2 release patterns and classic discontinuous ventilation is discussed.

Lectin binding to extracellularly melanized microfilariae of Brugia malayi from the hemocoel of Anopheles quadrimaculatus.

Binding patterns of fluorescein isothiocyanate (FITC)- and gold-conjugated lectins to extracellularly melanized sheathed and exsheathed microfilariae of subperiodic Brugia malayi, isolated from and in situ in the abdominal hemocoel of Anopheles quadrimaculatus 72-hr postinfection, were examined. Five FITC-conjugated lectins [Helix pomatia agglutinin (HPA), Arachis hypogaea (peanut agglutinin-PNA), Triticum vulgaris (wheat germ agglutinin-WGA), Lens culinaris (lentil-LCH), and Concanavalin A (Con A)] with specificities for different carbohydrate moieties were tested for binding to isolated melanized microfilariae and observed with transmitted light and fluorescence microscopy. All five FITC-lectins bound strongly to the acellular material accompanying the melanin deposits on the surface of isolated melanized microfilariae. Significant inhibition of FITC-lectin binding occurred when lectins were preincubated with their complementary carbohydrates before testing. H. pomatia agglutinin binding was totally inhibited by N-acetyl-D-glucosamine and N-acetyl-D-galactosamine. Other lectins were partially inhibited, such as PNA by galactose and lactose; WGA by N-acetylneuraminic acid; LCH by N-acetyl-D-glucosamine, mannose, glucose, and methyl alpha-D-mannopyranoside; and Con A by mannose and methyl alpha-D-mannopyranoside. Three gold-conjugated lectins (HPA, PNA, and Con A), examined by using transmission electron microscopy, bound to the outer surface of the acellular material associated with the melanin deposits on isolated melanized microfilarial sheaths and melanized microfilariae and to the remnants of lysed hemocytes found in the proximity of the melanized deposits. Con A in the presence of gold-labeled horseradish peroxidase, examined by using transmission electron microscopy, showed random binding within the melanized capsule formed around the microfilarial sheath in situ. These results indicate that the acellular material accompanying melanin deposits on melanized microfilarial sheaths and sheathed and exsheathed microfilariae contain several glycoconjugates with exposed carbohydrate moieties and are possibly glycoproteins. These glycoproteins could be the by-products of the activation of the prophenoloxidase by the microfilariae.

Comparative study of hemolymph phenoloxidase activity in Aedes aegypti and Anopheles quadrimaculatus and its role in encapsulation of Brugia malayi microfilariae.

Hemolymph phenoloxidase activity of sugar-fed and blood-fed females of Anopheles quadrimaculatus and Aedes aegypti showed similar characteristics. Phenoloxidase was present as an inactive proenzyme in both mosquito species and was partially activated during collection of the hemolymph. In both mosquito species, phenoloxidase activity was modulated by different buffers and activated phenoloxidase did not need Ca2+. Enzymatic activity was higher in the hemocytes than in the plasma in both mosquito species. Trypsin, laminarin, and blood-feeding on uninfected and Brugia malayi-infected jirds enhanced hemolymph phenoloxidase activity in both mosquito species. The appearance of hemolymph phenoloxidase activity was inhibited by p-nitrophenyl p'-guanidinobenzoate HCl, soybean trypsin inhibitor, ethylenediaminetetraacetic acid, diethyldithiocarbamic acid, saturated 1-phenyl-2-thiourea and reduced glutathione, but not by benzamidine in A. quadrimaculatus. The appearance of hemolymph phenoloxidase activity was inhibited by benzamidine, diethyldithiocarbamic acid, saturated 1-phenyl-2-thiourea, reduced glutathione, p-nitrophenyl p'-guanidinobenzoate and soybean trypsin inhibitor, but not by ethylenediamine-tetraacetic acid in A. aegypti. It is suggested that in both mosquito species, blood-feeding and migration of sheathed microfilariae in the homocoel activated the prophenoloxidase in the hemolymph and caused the encapsulation and melanization of microfilarial sheaths and microfilariae of B. malayi.

Ultrastructural comparison of extracellular and intracellular encapsulation of Brugia malayi in Anopheles quadrimaculatus.

Ultrastructural aspects of extracellular humoral encapsulation of microfilariae of Brugia malayi in the hemocoel of Anopheles quadrimaculatus were compared with those of intracellular encapsulation of first-stage larvae (L1) of the same parasite species, in the thoracic muscle cells of the same species of mosquito. The results showed that extracellular humoral encapsulation of microfilarial sheaths, and sheathed and exsheathed microfilariae, in the hemocoel of mosquitoes occurs around the parasite within the first 6 hr postingestion, apparently without initial participation of hemocytes. Hemocytes and their remnants were observed near the parasite during the first 6 hr postingestion. Within the next 24 hr, hemocytes attach to the initial humoral capsule. By contrast, intracellular encapsulation of L1S is initiated by the accumulation of a dense cytoplasmic layer derived from the infected thoracic muscle cell. Melanin deposits accumulate in this layer adjacent to the parasite cuticle, again without visible participation of hemocytes.

Intracellular development of subperiodic Brugia malayi influenced by mosquito thoracic muscle cells.

The in vitro development of 1-day-old intracellularly lodged larvae of Brugia malayi cultured in infected excised thoraces of selected susceptible and refractory strains of Aedes aegypti and Anopheles quadrimaculatus was compared with larvae reared in vivo. In susceptible mosquitoes, both in vitro and in vivo, larvae developed normally and abnormally. In refractory mosquitoes this pattern of both normal and abnormal development was also observed, except that comparatively fewer larvae developed to the infective third-stage larvae (L3) in vitro than in vivo and that more first-stage larvae (L1) were intracellularly melanized in vivo than in vitro. These studies indicate that factors in the thoracic muscle cells of the mosquito greatly affect the development of B. malayi microfilariae to L3. Intracellular melanization of L1 in An. quadrimaculatus, previously demonstrated in vivo, rarely occurred in vitro. These studies therefore suggest that refractoriness and melanization of B. malayi larvae in the thoraces of An. quadrimaculatus are controlled by two different and separate mechanisms.