Interleukin (IL) 31 induces in cynomolgus monkeys a rapid and intense itch response that can be inhibited by an IL-31 neutralizing antibody.

BACKGROUND: Overexpression or administration of interleukin 31 (IL-31) has been shown to induce a profound itch response in mice and dogs. The chronic pruritus observed in mouse IL-31 transgenic mice results in the development of skin lesions and alopecia through excoriation from excessive scratching, a condition similar to that observed in patients with atopic dermatitis (AD). OBJECTIVE: To test whether IL-31 induces pruritus in non-human primates and, if so, whether treatment with an anti-IL-31 neutralizing monoclonal antibody (mAb) can block the response. METHODS: A series of studies was conducted in cynomolgus monkeys to evaluate the itch response to recombinant cynomolgus IL-31 (cIL-31) administration. Three routes of cIL-31 administration (intravenous, intradermal, and subcutaneous) were evaluated. Subcutaneous treatment with a humanized anti-human IL-31 mAb cross-reactive to cIL-31 was subsequently tested for its ability to block the response to intradermal cIL-31 administration. RESULTS: Each route of cIL-31 delivery elicited a scratching response immediately after cIL-31 administration and lasted at least 3 h. Treatment with the IL-31 mAb inhibited the cIL-31-mediated scratching response in a dose-dependent manner. CONCLUSION: These results demonstrate that an IL-31 mAb can inhibit IL-31-mediated pruritus in vivo, and could be an effective therapy for pruritic skin conditions like AD where IL-31 upregulation may play a role.

Platelet-derived growth factor C (PDGF-C), a novel growth factor that binds to PDGF alpha and beta receptor.

We have characterized platelet-derived growth factor (PDGF) C, a novel growth factor belonging to the PDGF family. PDGF-C is a multidomain protein with the N-terminal region homologous to the extracellular CUB domain of neuropilin-1, and the C-terminal region consists of a growth factor domain (GFD) with homology to vascular endothelial growth factor (25%) and PDGF A-chain (23%). A serum-sensitive cleavage site between the two domains allows release of the GFD from the CUB domain. Competition binding and immunoprecipitation studies on cells bearing both PDGF alpha and beta receptors reveal a high affinity binding of recombinant GFD (PDGF-CC) to PDGF receptor-alpha homodimers and PDGF receptor-alpha/beta heterodimers. PDGF-CC exhibits greater mitogenic potency than PDGF-AA and comparable or greater mitogenic activity than PDGF-AB and PDGF-BB on several mesenchymal cell types. Analysis of PDGF-CC in vivo in a diabetic mouse model of delayed wound healing showed that PDGF-CC significantly enhanced repair of a full-thickness skin excision. Together, these studies describe a third member of the PDGF family (PDGF-C) as a potent mitogen for cells of mesenchymal origin in in vitro and in vivo systems with a binding pattern similar to PDGF-AB.

Endothelial adenosine transporter characterization in perfused guinea pig hearts.

Adenosine (Ado), a smooth muscle vasodilator and modulator of cardiac function, is taken up by many cell types via a saturable transporter, blockable by dipyridamole. To quantitate the influences of endothelial cells in governing the blood-tissue exchange of Ado and its concentration in the interstitial fluid, one must define the permeability-surface area products (PS) for Ado via passive transport through interendothelial gaps [PS(g)(Ado)] and across the endothelial cell luminal membrane (PS(ecl)) in their normal in vivo setting. With the use of the multiple-indicator dilution (MID) technique in Krebs-Ringer perfused, isolated guinea pig hearts (preserving endothelial myocyte geometry) and by separating Ado metabolites by HPLC, we found permeability-surface area products for an extracellular solute, sucrose, via passive transport through interendothelial gaps [PS(g)(Suc)] to be 1.9 +/- 0.6 ml. g(-1). min(-1) (n = 16 MID curves in 4 hearts) and took PS(g)(Ado) to be 1. 2 times PS(g)(Suc). MID curves were obtained with background nontracer Ado concentrations up to 800 micrometer, partially saturating the transporter and reducing its effective PS(ecl) for Ado. The estimated maximum value for PS(ecl) in the absence of background adenosine was 1.1 +/- 0.1 ml. g(-1). min(-1) [maximum rate of transporter conformational change to move the substrate from one side of the membrane to the other (maximal velocity; V(max)) times surface area of 125 +/- 11 nmol. g(-1). min(-1)], and the Michaelis-Menten constant (K(m)) was 114 +/- 12 microM, where +/- indicates 95% confidence limits. Physiologically, only high Ado release with hypoxia or ischemia will partially saturate the transporter.

Cardiac endothelial transport and metabolism of adenosine and inosine.

The influence of transmembrane flux limitations on cellular metabolism of purine nucleosides was assessed in whole organ studies. Transcapillary transport of the purine nucleosides adenosine (Ado) and inosine (Ino) via paracellular diffusion through interendothelial clefts in parallel with carrier-mediated transendothelial fluxes was studied in isolated, Krebs-Henseleit-perfused rabbit and guinea pig hearts. After injection into coronary inflow, multiple-indicator dilution curves were obtained from coronary outflow for 90 s for 131I-labeled albumin (intravascular reference tracer), [3H]arabinofuranosyl hypoxanthine (AraH; extracellular reference tracer and nonreactive adenosine analog), and either [14C]Ado or [14C]Ino. Ado or Ino was separated from their degradative products, hypoxanthine, xanthine, and uric acid, in each outflow sample by HPLC and radioisotope counting. Ado and Ino, but not AraH, permeate the luminal membrane of endothelial cells via a saturable transporter with permeability-surface area product PS(ecl) and also diffuse passively through interendothelial clefts with the same conductance (PSg) as AraH. These parallel conductances were estimated via fitting with an axially distributed, multi-pathway, four-region blood-tissue exchange model. PSg for AraH were approximately 4 and 2.5 ml. g(-1). min(-1) in rabbits and guinea pigs, respectively. In contrast, transplasmalemmal conductances (endothelial PS(ecl)) were approximately 0.2 ml. g(-1). min(-1) for both Ado and Ino in rabbit hearts but approximately 2 ml. g(-1). min(-1) in guinea pig hearts, an order of magnitude different. Purine nucleoside metabolism also differs between guinea pig and rabbit cardiac endothelium. In guinea pig heart, 50% of the tracer Ado bolus was retained, 35% was washed out as Ado, and 15% was lost as effluent metabolites; 25% of Ino was retained, 50% washed out, and 25% was lost as metabolites. In rabbit heart, 45% of Ado was retained and 5% lost as metabolites, and 7% of Ino was retained and 3% lost as metabolites. We conclude that endothelial transport of Ado and Ino is a prime determinant of their metabolic fates: where transport rates are high, metabolic transformation is high.

Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice.

Correction of the obese state induced by genetic leptin deficiency reduces elevated levels of both blood glucose and hypothalamic neuropeptide Y (NPY) mRNA in ob/ob mice. To determine whether these responses are due to a specific action of leptin or to the reversal of the obese state, we investigated the specificity of the effect of systemic leptin administration to ob/ob mice (n = 8) on levels of plasma glucose and insulin and on hypothalamic expression of NPY mRNA. Saline-treated controls were either fed ad libitum (n = 8) or pair-fed to the intake of the leptin-treated group (n = 8) to control for changes of food intake induced by leptin. The specificity of the effect of leptin was further assessed by 1) measuring NPY gene expression in db/db mice (n = 6) that are resistant to leptin, 2) measuring NPY gene expression in brain areas outside the hypothalamus, and 3) measuring the effect of leptin administration on hypothalamic expression of corticotropin-releasing hormone (CRH) mRNA. Five daily intraperitoneal injections of recombinant mouse leptin (150 micrograms) in ob/ob mice lowered food intake by 56% (P < 0.05), body weight by 4.1% (P < 0.05), and levels of NPY mRNA in the hypothalamic arcuate nucleus by 42.3% (P < 0.05) as compared with saline-treated controls. Pair-feeding of ob/ob mice to the intake of leptin-treated animals produced equivalent weight loss, but did not alter expression of NPY mRNA in the arcuate nucleus. Leptin administration was also without effect on food intake, body weight, or NPY mRNA levels in the arcuate nucleus of db/db mice. In ob/ob mice, leptin did not alter NPY mRNA levels in cerebral cortex or hippocampus or the expression of CRH mRNA in the hypothalamic paraventricular nucleus (PVN). Leptin administration to ob/ob mice also markedly reduced serum glucose (8.3 +/- 1.2 vs. 24.5 +/- 3.8 mmol/l; P < 0.01) and insulin levels (7,263 +/- 1,309 vs. 3,150 +/- 780 pmol/l), but was ineffective in db/db mice. Pair-fed mice experienced reductions of glucose and insulin levels that were < 60% of the reduction induced by leptin. The results suggest that in ob/ob mice, systemic administration of leptin inhibits NPY gene overexpression through a specific action in the arcuate nucleus and exerts a hypoglycemic action that is partly independent of its weight-reducing effects. Furthermore, both effects occur before reversal of the obesity syndrome. Defective leptin signaling due to either leptin deficiency (in ob/ob mice) or leptin resistance (in db/db mice) therefore leads directly to hyperglycemia and the overexpression of hypothalamic NPY that is implicated in the pathogenesis of the obesity syndrome.

Recombinant ob protein reduces feeding and body weight in the ob/ob mouse.

To determine whether the product of the recently cloned ob gene functions as an adipose-related satiety factor, recombinant murine ob protein was administered intraperitoneally to ob/ob mice. Monomeric ob protein given as single morning injections to groups of three animals at seven doses ranging from 5 to 100 micrograms reduced 24-h chow consumption in a dose-dependent manner from values of 81 +/- 6.8% of control (10-micrograms dose, P = 0.04) to 29 +/- 7.7% of control (100-micrograms dose, P < 0.0001). Daily injections of 80 micrograms of ob protein into six ob/ob mice for 2 wk led to an 11 +/- 1.6% decrease in body weight (P = 0.0009) and suppressed feeding to 26 +/- 4.9% of baseline (P < 0.0001), with significant reduction of serum insulin and glucose levels. The effect of recombinant ob protein on feeding was not augmented by cofactors secreted by adipose tissue, nor did exposure of adipose tissue to ob protein affect intracellular ob mRNA levels. Posttranslational modification of ob protein was not required for activity; however, addition of a hexahistidine tag to the amino terminus of the mature ob protein resulted in prolonged suppression of feeding after injection into ob/ob mice. These results demonstrate a direct effect of the ob protein to suppress feeding in the ob/ob mouse and suggest that this molecule plays a critical role in regulating total body fat content.

Triple-label beta liquid scintillation counting.

The detection of radioactive compounds by liquid scintillation has revolutionized modern biology, yet few investigators make full use of the power of this technique. Even though multiple isotope counting is considerably more difficult than single isotope counting, many experimental designs would benefit from using more than one isotope. The development of accurate isotope counting techniques enabling the simultaneous use of three beta-emitting tracers has facilitated studies in our laboratory using the multiple tracer indicator dilution technique for assessing rates of transmembrane transport and cellular metabolism. The details of sample preparation, and of stabilizing the liquid scintillation spectra of the tracers, are critical to obtaining good accuracy. Reproducibility is enhanced by obtaining detailed efficiency/quench curves for each particular set of tracers and solvent media. The numerical methods for multiple-isotope quantitation depend on avoiding error propagation (inherent to successive subtraction techniques) by using matrix inversion. Experimental data obtained from triple-label beta counting illustrate reproducibility and good accuracy even when the relative amounts of different tracers in samples of protein/electrolyte solutions, plasma, and blood are changed.

Capillary endothelial transport of uric acid in guinea pig heart.

Much of the adenosine formed in the heart is degraded by endothelial enzymes to uric acid, which is exported across the coronary capillary endothelial cell membrane before renal excretion. Because previous experiments suggested that cell permeability for uric acid is either very high (similar to water) or very low, multiple indicator-dilution experiments were carried out to distinguish between the two possibilities. An intravascular reference tracer, 131I-labeled albumin, and an extracellular reference tracer, L-[3H]glucose, were injected together with [14C]uric acid as a bolus into the coronary inflow, while fractionating the venous outflow for 90 s. Recovery of injected uric acid averaged 99.0 +/- 2.9% (mean +/- SD, n = 12) that of L-glucose. Peak capillary extraction of L-glucose and uric acid averaged 0.38 +/- 0.032 and 0.42 +/- 0.035 (P less than 0.005) compared with albumin. Except at the peaks, the dilution curves for [14C]uric acid and L-[3H]glucose coincided closely, indicating that little uric acid was transported into cells. The dilution curves were analyzed using an axially distributed, multipathway, four region mathematical model, to estimate membrane permeability-surface area (PS) products. Since the endothelial cell PS for uric acid was low (0.12 +/- 0.09 ml.g-1.min-1), approximately 3% of the PS reported for adenosine, the possibility of flow-limited exchange for uric acid is ruled out. To estimate steady-state endothelial concentrations of uric acid in vivo, equations were developed describing electrochemical potential gradients for dissociated and undissociated forms of a weak acid. Despite endothelial production, intracellular concentrations that are lower than outside are expected because the negative membrane potential and lower cellular pH assist uric acid efflux.