Amplification of cytokine-specific ELISAs increases the sensitivity of detection to 5-20 picograms per milliliter.

The ability to detect a protein is always limited to the sensitivity of the assays available. Progress in improving the sensitivity of protein detection will allow a more complete understanding of biological systems. Of particular interest to the field of immunology is the ability to characterize an immune response based upon the pattern of cytokines that are released in response to antigen. A Th1 response is characterized by the presence of IL-2, IL-12, TNF and IFN-gamma, whereas a Th2 response is characterized by IL-4, IL-5, IL-6 and IL-10. Often, these cytokines are present in in vitro-derived culture supernatants at extremely low concentrations and are therefore very difficult to detect. Although a number of improvements have been made to the sensitivity of the relevant detection assays, the most successful assays involve the presence of the cells being cultured thereby limiting the number of tests per culture to one. Here we describe an enhanced ELISA protocol where the sensitivity is equivalent or better than corresponding cell-based assays. This protocol will permit the sensitive measurement of multiple cytokines per single culture supernatant.

Fibrinolytic and haemostatic responses to desamino-D-arginine vasopressin (DDAVP) administered by intravenous and subcutaneous routes in healthy subjects.

DDAVP was administered at 0.4 microgram kg-1 intravenous (i.v.) and subcutaneous (s.c.) routes to 6 healthy subjects in a double blind crossover study. Both study treatments were well tolerated. Flushing occurred after both treatments but was more prominent after i.v. than after s.c. DDAVP. Mild transient local discomfort at the s.c. injection site occurred in 5 of 6 subjects. The mean peak factor VIII (FVIII) response was 369% and 247% of baseline after i.v. and s.c. DDAVP respectively and the maximum increase in FVIII occurred earlier with the i.v. route. Changes in FVIII antigen (FVIII:Ag) and von Willebrand factor antigen (vWF:Ag) were also monitored. Tissue-type plasminogen activator (t-PA) activity measured by a chromogenic assay employing soluble fibrin had a median peak value of 2.9 IU ml-1 at 20 min after i.v. and of 1.9 IU ml-1 at 60 min after s.c. DDAVP. t-PA antigen was also measured so that the specific activity of circulating t-PA could be determined. Preinjection median values of 14,650 and 13,700 IU mg-1 increased to peak median values of 236,200 IU mg-1 at 20 min after i.v. and 202,400 IU mg-1 at 60 min after s.c. DDAVP. Plasminogen activator inhibitor (PAI) activity fell following DDAVP and became undetectable in some subjects during the sampling period. The ratio of maximum fibrinolytic response was similar to the ratio of maximum haemostatic responses obtained by two routes of injection. Our results indicate that s.c. DDAVP might successfully replace i.v. DDAVP in several applications such as confirmation of haemostatic or fibrinolytic responsiveness in patient groups; for obtaining FVIII enriched plasma; as well as its obvious potential usefulness in home treatment of haemophilia A and von Willebrand's disease.

Spline solution to terminal zero acceleration problems in biomechanical data.

An augmented cubic spline function was evaluated as an alternative solution to the end-point problem which frequently arises in the smoothing and differentiation of biomechanical position-time data. The usual procedures of natural cubic spline functions and digital filtering have the undesirable effect of forcing the accelerations to zero at the ends of the data set. The proposed augmented cubic spline procedure does not have this characteristic but rather requires the curve to pass through a single extra point at each end of the data set. Using two different criteria with large acceleration magnitudes near the end of the data set, we have compared the effectiveness of all three methods in terms of the total curve (root mean square error) and in terms of the last few points (percent and algebraic error). In both experiments the augumented cubic spline procedure was found to be superior to both digital filtering and natural cubic spline functions. It was concluded that this technique could be used for smoothing and differentiating biomechanical data in instances where the underlying function is unknown and the accelerations at the end points of the data set are suspected of being non-zero.

Quantification of intersegmental reactions during rapid swing motion.

A simple mathematical model was developed to quantify the non-muscular reactions between two adjacent segments undergoing rapid free-segment motion. Newtonian equations of motion were used to simulate the trajectories of the two segments using the input parameters of linear acceleration and muscular moment acting only about the proximal end of the proximal link. The intersegmental muscular force and moment were mathematically considered to be zero, and no constraints were placed on the motion of the two segments. The motion of the thigh and shank during the swing phase of running was simulated by the model and compared to the real motion obtained from cinematographic records. The simulation demonstrated that proximal thigh motion has a significant effect on the distal shank motion. Depending on the initial conditions, intersegmental reactions alone can produce large increases in distal segment speed. The role of the knee musculature in preventing these reactions during the swing phase of running was inferred.

A study of tomite structure and metamorphosis in Dendrocometes paradoxus stein (suctoria, ciliophora) using silver staining and scanning electron microscopy.

The organisation of the ciliary bands in the tomite of Dendrocometes paradoxus has been examined using silver staining and scanning electron microscopy (SEM). Stages in the metamorphosis of the tomite have also been examined using SEM. The tomite has a flattened ventral surface and a convex dorsal surface. The posterior end of the tomite is always marked by a small papilla, the remnant of the last point of attachment to either the trophont, or the residual body when the whole trophont transforms into a tomite. The ciliary bands around the tomite have an uneven distribution. On the right hand side of the body and posteriorly there are three bands of cilia. On the left hand side of the body and anteriorly there are four bands of cilia. The number of cilia in the rows that make up the bands also varies. Posteriorly and to the right hand, side the bands are much wider than elsewhere on the body. The ciliary bands are located around the base of the dorsal convex surface. A series of pore-like structures is seen on the right hand side of the flattened ventral surface. During metamorphosis the positions of the ciliary bands are clearly seen even when the feeding tentacles start to emerge.

A three-dimensional, six-segment chain analysis of forceful overarm throwing.

A three-dimensional, six-segment model was applied to the pitching motion of three professional pitchers to analyze the kinematics and kinetics of the hips, upper trunk, humerus and forearm plus hand of both the upper limbs. Subjects were filmed at 250 frames per second. An inverse dynamics approach and angular momentum principle with respect to the proximal endpoint of a rigid segment were employed in the analysis. Results showed considerable similarities between subjects in the kinetic control of trunk rotation about the spine's longitudinal axis, but variability in the control of trunk lean both to the side and forward. The kinetics of the throwing shoulder and elbow joint were comparable between subjects, but the contribution of the non-throwing upper limb was minimal and variable. The upper trunk rotators played a key role in accelerating the ball to an early, low velocity near stride foot contact. After a brief pause they resumed acting strongly in a positive direction, though not enough to prevent trunk angular velocity slowing, as the musculature of the arm applied a load at the throwing shoulder. The interaction moment from the proximal segments assisted the forearm extensor in slowing flexion and producing rapid elbow extension near ball release. The temporal onset of muscular torques was not in a strictly successive proximal-to-distal sequence.

Relationship between doses of prostaglandin F2alpha and stages of the breeding season for synchronization of estrus and ovulation in ewes.

Two experiments were conducted to compare estrous response to three doses (8, 16 and 24 mg) of prostaglandin F2alpha (PGF2alpha) administered by intramuscular injection to ewes between day 6 and 12 of the estrous cycle (Experiment I) and to ewes on unknown days of the estrous cycle during four different stages of the breeding season (Experiment II). In experiment I, a total of 41 ewes were treated with PGF2alpha. The injection of 24 mg PGF2alpha resulted in a higher proportion of ewes exhibiting estrus (13/14, 92.9%) within 5 days after treatment as compared to the other two doses (2/12 and 10/15, for 8 and 16 mg PGF2alpha, respectively). However, there was no significant difference for the proportion between 16 mg and 24 mg PGF2alpha. In experiment II, PGF2alpha was given to ewes on the 3rd of February (early breeding season), the 28th of February (mid-early breeding season), the 10th of April (mid breeding season) and the 27th of May (late breeding season). These was a significant difference for the proportion of ewes exhibiting estrus between the early breeding season and the other three seasons (P<0.05) but not for ewes ovulating. Throughout the breeding season, 16 mg PGF2alpha appeared to be slightly better than the other two doses (8 and 24 mg) although there was no overall difference in the estrous responses to treatment among the three doses. However, a significant difference in the proportion of ewes ovulating was found among the three doses of PGF2alpha (P<0.05). Especially, 16 mg PGF2alpha was significantly superior to 8 mg (P<0.01) and 24 mg (P<0.05). It was considered that there was a complicated relationship between the doses of PGF2alpha and the stages of the breeding season for induction of estrus and ovulation in the ewe.

Stress-dependent and gender-specific neuroregulatory roles of the apelin receptor in the hypothalamic-pituitary-adrenal axis response to acute stress.

The neuropeptide apelin is expressed in hypothalamic paraventricular and supraoptic nuclei and mediates its effects via activation of the apelin receptor (APJ). Evidence suggests a role for apelin and APJ in mediating the neuroendocrine response to stress. To understand the physiological role of APJ in regulation of the hypothalamic-pituitary-adrenal (HPA) axis, we measured ACTH and corticosterone (CORT) plasma levels in male and female mice lacking APJ (APJ knockout, APJ KO) and in wild-type controls, in response to a variety of acute stressors. Exposure to mild restraint, systemic injection of lipopolysaccharide (LPS), insulin-induced hypoglycaemia and forced swim (FS) stressors, elevated plasma ACTH and CORT levels in wild-type mice. Acute mild restraint significantly increased plasma ACTH and CORT to a similar level in APJ KO mice as in wild-type mice. However, an intact APJ was required for a conventional ACTH, but not CORT, response to LPS administration in male mice and to insulin-induced hypoglycaemia in male and female mice. In contrast, APJ KO mice displayed an impaired CORT response to acute FS stress, regardless of gender. These data indicate that APJ has a role in regulation of the HPA axis response to some acute stressors and has a gender-specific function in peripheral immune activation of the HPA axis.

The vasopressin V1b receptor modulates plasma corticosterone responses to dehydration-induced stress.

Vasopressin V1b receptor knockout (V1b⁻/⁻) mice were used to investigate a putative role for the V1b receptor (V1bR) in fluid regulation and in the hypothalamic-neurohypophysial system (HNS) and hypothalamic-pituitary-adrenal (HPA) axis responses to osmotic stress induced by water deprivation (WD). Male wild-type and V1b⁻/⁻ mice were housed in metabolic cages to allow determination of water intake and urine volume and osmolality. When provided with food and water ad lib., spontaneous urine volume and urine osmolality did not differ between genotypes. Similarly, WD for 24 h caused comparable decreases in urine volume and increases in urine osmolality irrespective of genotype. WD resulted in an increase in plasma corticosterone concentration in wild-type animals; however, this WD-induced increase in plasma corticosterone was significantly attenuated in V1b⁻/⁻ mice. Comparable increases in neuronal activation, indicated by increased c-fos mRNA expression, and in vasopressin mRNA expression occurred in both the supraoptic nucleus and paraventricular nucleus (PVN) of wild-type and V1b⁻/⁻ mice following WD; however, the WD-induced decrease in corticotrophin-releasing hormone mRNA expression seen in the PVN of wild-type mice was not observed in the PVN of V1b⁻/⁻ mice. These data suggest that, although the vasopressin V1bR is not required for normal HNS function, it is necessary for a full HPA-axis response to the osmotic stress of WD.

Stimulus-specific neuroendocrine responses to osmotic challenges in apelin receptor knockout mice.

The expression of the novel peptide apelin and its receptor APJ within specific regions of the brain, in particular the magnocellular neurones of the hypothalamus and the circumventricular organs, has implicated the apelinergic system in mechanisms controlling fluid homeostasis. In addition, apelin and APJ are considered to be involved in controlling arginine vasopressin (AVP) secretion into the circulation and release within the hypothalamic-neurohypophysial system. To clarify the role of APJ during regulation of fluid homeostasis, we compared the effects of osmotic stimulation on the urinary concentrating capacities and central nervous system responses of salt-loaded (SL) and water-deprived (WD) female APJ knockout (APJ(-/-)) mice and wild-type controls. SL resulted in a significantly increased urine volume in APJ(-/-) mice compared to wild-type controls, whereas WD in APJ(-/-) mice failed to reduce urine volume as seen in wild-type controls. AVP transcripts in the supraoptic and paraventricular nuclei and plasma AVP concentrations were significantly attenuated in SL APJ(-/-) mice compared to SL wild-type, but increased comparably in wild-type and APJ(-/-) mice after WD. Analysis of c-fos mRNA expression in the median preoptic nucleus and subfornical organ in response to either WD or SL showed attenuated expression in APJ(-/-) compared to wild-type mice. These findings further implicate the apelinergic system in mechanisms controlling fluid homeostasis, particularly at a neuroendocrine level, and suggest stimulus-specific involvement in vasopressinergic activity.

Neighborhood social environments and the distribution of low birthweight in Chicago.

OBJECTIVES: This study examined the socioeconomic precursors of disparities in maternal health by measuring the associations of nine neighborhood-level indicators of social phenomena with low infant birthweight. METHODS: Vital records and census data for the Chicago metropolitan area in 1990 were merged (n = 112,327); a logistic regression model predicting low birthweight was estimated by backward elimination. RESULTS: With individual-level variables held constant, six neighborhood-level indicators predicted low birthweight, together contributing to a variation in rate of 5.5%. Community economic hardship and housing costs were positively associated with low birthweight, while community socioeconomic status, crowded housing, and high percentages of young and African-American residents were negatively associated with low birthweight. CONCLUSIONS: Maternal health inequalities should be explored in the context of historical segregation, social stratification, the dynamics of social support, and resource sharing among communities. Several community characteristics associated with poverty are negatively associated with low birthweight. The traditional focus on individual risk factors for low birthweight limits our understanding.

The in vitro interaction of dexmedetomidine with human liver microsomal cytochrome P4502D6 (CYP2D6).

The effect of dexmedetomidine DEX on cytochrome P4502D6 (CYP2D6)-dependent dextromethorphan O-demethylase (DEXTROase) activity was studied using native human liver microsomes. DEX (0.01-4.0 microM inhibited DEXTROase activity (IC50 = 1.8 +/- 0.25 microM; mean +/- SD; N = 5 livers) and was less potent than quinidine (QND), prototypical and clinically relevant CYP2D6 inhibitor (IC50 = 0.22 +/- 0.02 microM; mean Ki = 0.07 microM). Similar results were obtained with human B-lymphoblast microsomes containing cDNA-expressed CYP2D6 (DEX, IC50 = 2.2 microM; QND, IC50 0.15 microM). Formal kinetic analyses indicated that DEX was a reversible mixed (competitive/noncompetitive) inhibitor of DEXTROase activity in human liver microsomes, where Kies > Ki and alpha > 1 (Ki = 0.4 +/- 0.2 microM; Kies = 2.3 +/- 0.9 microM; alpha = 8.1 +/- 6.8; N = 3 livers). In addition, DEX elicited a Type IIb difference spectrum (lambda max approximately 436 nm; lambda min approximately 414 nm) when added to cDNA-expressed CYP2D6 under aerobic (oxidized) conditions. These data indicated that DEX was able to bind reversibly to the heme (ferric) iron of CYP2D6. It is postulated that binding occurs via the 4(5)-substituted imidazole moiety. In this instance, binding was characterized by a spectral dissociation constant (Ks) of 0.4 microM that was identical to the Ki obtained with native human liver microsomes.

Lower extremity forces and torques during systematic variation of non-weight bearing motion.

Component and resultant external forces and torques at ankle, knee, and hip joints of unrestrained lower extremity motion were computed as resultant limb velocities were systematically changed. Cinematographic and modeling techniques were used to analyze 45 trials of five skilled soccer players who performed slow, medium and fast velocity kicks. A model of the lower extremity was incorporated, with kinematic data and segment mass estimates, into computer programs to quantify the mechanical variables. Monotonic increases in magnitudes of all kinetic elements occurred as the velocity of kicking was increased. Significant differences were also observed in the maximum values of the net parallel and perpendicular external force actions, and resultant torques at the joints with changes in limb velocity.