Treating cat allergy with monoclonal IgG antibodies that bind allergen and prevent IgE engagement.

Acute allergic symptoms are caused by allergen-induced crosslinking of allergen-specific immunoglobulin E (IgE) bound to Fc-epsilon receptors on effector cells. Desensitization with allergen-specific immunotherapy (SIT) has been used for over a century, but the dominant protective mechanism remains unclear. One consistent observation is increased allergen-specific IgG, thought to competitively block allergen binding to IgE. Here we show that the blocking potency of the IgG response to Cat-SIT is heterogeneous. Next, using two potent, pre-selected allergen-blocking monoclonal IgG antibodies against the immunodominant cat allergen Fel d 1, we demonstrate that increasing the IgG/IgE ratio reduces the allergic response in mice and in cat-allergic patients: a single dose of blocking IgG reduces clinical symptoms in response to nasal provocation (ANCOVA, p = 0.0003), with a magnitude observed at day 8 similar to that reported with years of conventional SIT. This study suggests that simply augmenting the blocking IgG/IgE ratio may reverse allergy.

Glucagon orchestrates stress-induced hyperglycaemia.

Hyperglycaemia is commonly observed on admission and during hospitalization for medical illness, traumatic injury, burn and surgical intervention. This transient hyperglycaemia is referred to as stress-induced hyperglycaemia (SIH) and frequently occurs in individuals without a history of diabetes. SIH has many of the same underlying hormonal disturbances as diabetes mellitus, specifically absolute or relative insulin deficiency and glucagon excess. SIH has the added features of elevated blood levels of catecholamines and cortisol, which are not typically present in people with diabetes who are not acutely ill. The seriousness of SIH is highlighted by its greater morbidity and mortality rates compared with those of hospitalized patients with normal glucose levels, and this increased risk is particularly high in those without pre-existing diabetes. Insulin is the treatment standard for SIH, but new therapies that reduce glucose variability and hypoglycaemia are desired. In the present review, we focus on the key role of glucagon in SIH and discuss the potential use of glucagon receptor blockers and glucagon-like peptide-1 receptor agonists in SIH to achieve target glucose control.

Hyperalgesia, synovitis and multiple biomarkers of inflammation are suppressed by interleukin 1 inhibition in a novel animal model of gouty arthritis.

BACKGROUND: Monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystal-induced interleukin 1 beta (IL1beta) release contributes to inflammation in subcutaneous air pouch and peritoneal models of acute gout and pseudogout. However, consequences of IL1 inhibition have not been explored in more clinically relevant models of crystal-induced arthritis. OBJECTIVE: To develop a novel mouse model of acute gouty ankle arthritis and use it to assess the effects of genetic deletion of IL1 receptor type (IL1R1) and of exogenous mIL1 Trap (a high-affinity blocker of mouse IL1alpha and IL1beta) on pain, synovitis and systemic inflammatory biomarkers. METHODS: MSU crystals were injected into the mouse ankle joint and pain and ankle swelling were measured over 4 days. The effects of IL1 inhibition were determined in this model, and in the comparator models of crystal-induced peritonitis and subcutaneous air pouch inflammation. RESULTS: Both IL1R1-null mice and mice pretreated with mIL1 Trap showed reduced neutrophil influx in MSU and CPPD crystal-induced peritonitis and air pouch models (p<0.05). In the ankle joint model, both IL1R1 knockout mice and pretreatment with mIL1 Trap were associated with significant reductions in MSU crystal-induced elevations in hyperalgesia, inflammation, serum amyloid A and the levels of multiple inflammatory cytokines and chemokines (p<0.05). Additionally, it was found that administration of mIL1 Trap after MSU crystal injection reduced established hyperalgesia and ankle swelling. CONCLUSIONS: IL1 inhibition both prevented and relieved pain and ankle joint inflammation in response to intra-articular MSU crystals in mice. Results suggested that IL1 Trap has the potential to both prevent and treat gouty arthritis.

Expression of BMP-7 and USAG-1 (a BMP antagonist) in kidney development and injury.

Once developed, end-stage renal disease cannot be reversed by any current therapy. Bone morphogenetic protein-7 (BMP-7), however, is a possible treatment for reversing end-stage renal disease. Previously, we showed that the BMP antagonist uterine sensitization-associated gene-1 (USAG-1, also known as ectodin and sclerostin domain-containing 1) negatively regulates the renoprotective action of BMP-7. Here, we show that the ratio between USAG-1 and BMP-7 expression increased dramatically in the later stage of kidney development, with USAG-1 expression overlapping BMP-7 only in differentiated distal tubules. Examination of USAG-1 expression in developing kidney indicated that a mosaic of proximal and distal tubule marker-positive cells reside side by side in the immature nephron. This suggests that each cell controls its own fate for becoming a proximal or distal tubule cell. In kidney injury models, the ratio of USAG-1 to BMP-7 expression decreased with kidney damage but increased after subsequent kidney regeneration. Our study suggests that USAG-1 expression in a kidney biopsy could be useful in predicting outcome.

Delta-like ligand 4 (Dll4) is induced by VEGF as a negative regulator of angiogenic sprouting.

Genetic deletion studies have shown that haploinsufficiency of Delta-like ligand (Dll) 4, a transmembrane ligand for the Notch family of receptors, results in major vascular defects and embryonic lethality. To better define the role of Dll4 during vascular growth and differentiation, we selected the postnatal retina as a model because its vasculature develops shortly after birth in a highly stereotypic manner, during which time it is accessible to experimental manipulation. We report that Dll4 expression is dynamically regulated by VEGF in the retinal vasculature, where it is most prominently expressed at the leading front of actively growing vessels. Deletion of a single Dll4 allele or pharmacologic inhibition of Dll4/Notch signaling by intraocular administration of either soluble Dll4-Fc or a blocking antibody against Dll4 all produced the same set of characteristic abnormalities in the developing retinal vasculature, most notably enhanced angiogenic sprouting and increased endothelial cell proliferation, resulting in the formation of a denser and more highly interconnected superficial capillary plexus. In a model of ischemic retinopathy, Dll4 blockade also enhanced angiogenic sprouting and regrowth of lost retinal vessels while suppressing ectopic pathological neovascularization. Our data demonstrate that Dll4 is induced by VEGF as a negative feedback regulator and acts to prevent overexuberant angiogenic sprouting, promoting the timely formation of a well differentiated vascular network.

VEGF trap as a novel antiangiogenic treatment currently in clinical trials for cancer and eye diseases, and VelociGene- based discovery of the next generation of angiogenesis targets.

The concept that tumors can be controlled by directly targeting their vascular supply has finally come of age, because clinical trials using a humanized monoclonal antibody that blocks VEGF have demonstrated exciting efficacy in cancer patients, as well as in vascular eye diseases that can lead to blindness. However, data suggest that these current regimens may not provide complete VEGF inhibition and, thus, that the maximum therapeutic potential of VEGF blockade has not yet been achieved. We describe the status of a very potent and high-affinity VEGF blocker, termed the VEGF Trap, that may provide the opportunity to maximize the potential of VEGF blockade in cancer as well as in vascular eye diseases. We also describe use of the VEGF Trap as a research tool, when coupled to high-throughput mouse genetics approaches such as VelociGene that can be exploited in strategies to discover and validate the next generation of angiogenesis targets.

Ciliary neurotrophic factor improves diabetic parameters and hepatic steatosis and increases basal metabolic rate in db/db mice.

Obesity plays a central role in the development of insulin resistance and type 2 diabetes. We therefore examined the effects of a modified form of ciliary neurotrophic factor [Axokine, which is hereafter referred to as ciliary neurotrophic factor (CNTF)Ax15], which uses a leptin-like mechanism to reduce body weight, in the db/db murine model of type 2 diabetes. In previous studies, weight loss produced by CNTF treatment could largely be attributed to its effects on food intake. In contrast, CNTFAx15 treatment of db/db mice caused significantly greater weight loss and marked improvements in diabetic parameters (e.g., levels of glucose, insulin, triglyceride, cholesterol, and nonesterified free fatty acids) than could be accounted for by reduced caloric intake alone. These beneficial effects, above and beyond those seen in animals controlled for either food restriction or body weight, correlated with the ability of CNTFAx15 to increase metabolic rate and energy expenditure and reduce hepatic steatosis while enhancing hepatic responsiveness to insulin. The hepatic effects were linked to rapid alterations in hepatic gene expression, most notably reduced expression of stearoyl-CoA desaturase 1, a rate-limiting enzyme in the synthesis of complex lipids that is also markedly suppressed by leptin in ob/ob mice. These observations further link the mechanisms of CNTF and leptin action, and they suggest important, beneficial effects for CNTF in diabetes that may be distinct from its ability to decrease food intake; instead, these effects may be more related to its influence on energy expenditure and hepatic gene expression.

Activation of the hypothalamic arcuate nucleus predicts the anorectic actions of ciliary neurotrophic factor and leptin in intact and gold thioglucose-lesioned mice.

Similar to leptin, ciliary neurotrophic factor (CNTF) suppresses appetite and selectively reduces body fat in leptin-deficient ob/ob mice. To assess the relative importance of specific regions of the hypothalamus in mediating these effects, we administered a CNTF analogue (CNTFAx15) or leptin to mice made obese by administration of gold thioglucose (GTG), which destroys a well-defined portion of the medial basal hypothalamus. CNTFAx15 treatment reduced appetite and body weight in obese GTG-lesioned C57BL/6 mice, whereas leptin failed to effect similar changes regardless of whether treatment was initiated before or after the lesioned mice had become obese. Because leptin does not reduce food intake or body weight in most forms of obesity (a condition termed 'leptin resistance'), we also investigated the actions of leptin in GTG-lesioned leptin-deficient (ob/ob) mice. By contrast to C57BL/6 mice, leptin treatment reduced food intake and body weight in GTG-lesioned ob/ob mice, although the effect was attenuated. To further compare the neural substrates mediating the anorectic actions of leptin and CNTF, we determined the patterns of neurone activation induced by these proteins in the hypothalamus of intact and GTG-lesioned mice by staining for phosphorylated signal transducer and activator of transcription 3 (pSTAT3). CNTFAx15 stimulated robust pSTAT3 signalling in neurones of the medial arcuate nucleus in both intact and lesioned C57BL/6 and ob/ob mice. Leptin administration stimulated pSTAT3 signalling in only a few neurones of the medial arcuate nucleus in intact or lesioned C57BL/6 mice, but elicited a robust response in intact or lesioned ob/ob mice. By contrast to CNTFAx15, leptin treatment also resulted in prominent activation of STAT3 in several areas of the hypothalamus outside the medial arcuate nucleus. This leptin-induced pSTAT3 signal was at least as prominent in intact and GTG-lesioned C57BL/6 mice as it was in ob/ob mice, and thus was not correlated with appetite suppression or weight loss. These results indicate that the medial arcuate nucleus is a key mediator of appetite suppression and weight loss produced by CNTF and leptin, whereas GTG-vulnerable regions play a role only in leptin-induced weight loss. Other regions of hypothalamus in which pSTAT3 signal is induced by leptin may regulate energy metabolism through mechanisms other than appetite reduction.

Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways.

Skeletal muscle is composed of multinucleated fibres, formed after the differentiation and fusion of myoblast precursors. Skeletal muscle atrophy and hypertrophy refer to changes in the diameter of these pre-existing muscle fibres. The prevention of atrophy would provide an obvious clinical benefit; insulin-like growth factor 1 (IGF-1) is a promising anti-atrophy agent because of its ability to promote hypertrophy. However, the signalling pathways by which IGF-1 promotes hypertrophy remain unclear, with roles suggested for both the calcineurin/NFAT (nuclear factor of activated T cells) pathway and the PtdIns-3-OH kinase (PI(3)K)/Akt pathway. Here we employ a battery of approaches to examine these pathways during the hypertrophic response of cultured myotubes to IGF-1. We report that Akt promotes hypertrophy by activating downstream signalling pathways previously implicated in activating protein synthesis: the pathways downstream of mammalian target of rapamycin (mTOR) and the pathway activated by phosphorylating and thereby inhibiting glycogen synthase kinase 3 (GSK3). In contrast, in addition to demonstrating that calcineurin does not mediate IGF-1-induced hypertrophy, we show that IGF-1 unexpectedly acts via Akt to antagonize calcineurin signalling during myotube hypertrophy.

Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo.

Skeletal muscles adapt to changes in their workload by regulating fibre size by unknown mechanisms. The roles of two signalling pathways implicated in muscle hypertrophy on the basis of findings in vitro, Akt/mTOR (mammalian target of rapamycin) and calcineurin/NFAT (nuclear factor of activated T cells), were investigated in several models of skeletal muscle hypertrophy and atrophy in vivo. The Akt/mTOR pathway was upregulated during hypertrophy and downregulated during muscle atrophy. Furthermore, rapamycin, a selective blocker of mTOR, blocked hypertrophy in all models tested, without causing atrophy in control muscles. In contrast, the calcineurin pathway was not activated during hypertrophy in vivo, and inhibitors of calcineurin, cyclosporin A and FK506 did not blunt hypertrophy. Finally, genetic activation of the Akt/mTOR pathway was sufficient to cause hypertrophy and prevent atrophy in vivo, whereas genetic blockade of this pathway blocked hypertrophy in vivo. We conclude that the activation of the Akt/mTOR pathway and its downstream targets, p70S6K and PHAS-1/4E-BP1, is requisitely involved in regulating skeletal muscle fibre size, and that activation of the Akt/mTOR pathway can oppose muscle atrophy induced by disuse.

Identification of ubiquitin ligases required for skeletal muscle atrophy.

Skeletal muscle adapts to decreases in activity and load by undergoing atrophy. To identify candidate molecular mediators of muscle atrophy, we performed transcript profiling. Although many genes were up-regulated in a single rat model of atrophy, only a small subset was universal in all atrophy models. Two of these genes encode ubiquitin ligases: Muscle RING Finger 1 (MuRF1), and a gene we designate Muscle Atrophy F-box (MAFbx), the latter being a member of the SCF family of E3 ubiquitin ligases. Overexpression of MAFbx in myotubes produced atrophy, whereas mice deficient in either MAFbx or MuRF1 were found to be resistant to atrophy. These proteins are potential drug targets for the treatment of muscle atrophy.

Stromal cells expressing ephrin-B2 promote the growth and sprouting of ephrin-B2(+) endothelial cells.

Ephrin-B2 is a transmembrane ligand that is specifically expressed on arterial endothelial cells (ECs) and surrounding cells and interacts with multiple EphB class receptors. Conversely, EphB4, a specific receptor for ephrin-B2, is expressed on venous ECs, and both ephrin-B2 and EphB4 play essential roles in vascular development. The bidirectional signals between EphB4 and ephrin-B2 are thought to be specific for the interaction between arteries and veins and to regulate cell mixing and the making of particular boundaries. However, the molecular mechanism during vasculogenesis and angiogenesis remains unclear. Manipulative functional studies were performed on these proteins in an endothelial cell system. Using in vitro stromal cells (OP9 cells) and a paraaortic splanchnopleura (P-Sp) coculture system, these studies found that the stromal cells expressing ephrin-B2 promoted vascular network formation and ephrin-B2(+) EC proliferation and that they also induced the recruitment and proliferation of alpha-smooth muscle actin (alpha-SMA)-positive cells. Stromal cells expressing EphB4 inhibited vascular network formation, ephrin-B2(+) EC proliferation, and alpha-SMA(+) cell recruitment and proliferation. Thus, these data suggest that ephrin-B2 and EphB4 mediate reciprocal interactions between arterial and venous ECs and surrounding cells to form each characteristic vessel. (Blood. 2001;98:1028-1037)

VEGF-initiated blood-retinal barrier breakdown in early diabetes.

PURPOSE: The objectives of this study were to (1) determine whether endogenous vascular endothelial growth factor (VEGF) triggers diabetic blood-retinal barrier breakdown, and (2) identify the site as well as phenotype of the hyperpermeable diabetic retinal vessels. METHODS: Retinal VEGF mRNA levels were quantified in 1-week diabetic rats using the RNase protection assay. VEGF bioactivity was blocked via the systemic administration of a highly specific VEGF-neutralizing soluble Flt/F(c) construct (VEGF TrapA(40)). An inactive IL6 receptor/F(c) construct (IL6R Trap) was used as an isotype control. Blood-retinal barrier breakdown was quantified using the Evans blue technique and was spatially localized with fluorescent microspheres. RESULTS: Retinal VEGF mRNA levels in 1-week diabetic animals were 3.2-fold higher than in nondiabetic controls (P < 0.0001). Similarly, retinal vascular permeability in 8-day diabetic animals was 1.8-fold higher than in normal nondiabetic controls (P < 0.05). Diabetes-induced blood-retinal barrier breakdown was dose-dependently inhibited with VEGF TrapA(40), with 25 mg/kg producing complete inhibition of the diabetes-induced increases (P < 0.05). Blood-retinal barrier breakdown in diabetic animals treated with solvent alone or IL6R Trap did not differ significantly from untreated diabetic animals (P > 0.05). Spatially, early blood-retinal barrier breakdown was localized to the retinal venules and capillaries of the superficial retinal vasculature. CONCLUSIONS: Early blood-retinal barrier breakdown in experimental diabetes is VEGF dependent and is restricted, in part, to the venules and capillaries of the superficial inner retinal vasculature. VEGF inhibition should prove a useful therapeutic approach in the treatment of early diabetic blood-retinal barrier breakdown.

Excessive tumor-elaborated VEGF and its neutralization define a lethal paraneoplastic syndrome.

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen and key regulator of both physiologic and pathologic (e.g., tumor) angiogenesis. In the course of studies designed to assess the ability of constitutive VEGF to block tumor regression in an inducible RAS melanoma model, mice implanted with VEGF-expressing tumors sustained high morbidity and mortality that were out of proportion to the tumor burden. Documented elevated serum levels of VEGF were associated with a lethal hepatic syndrome characterized by massive sinusoidal dilation and endothelial cell proliferation and apoptosis. Systemic levels of VEGF correlated with the severity of liver pathology and overall clinical compromise. A striking reversal of VEGF-induced liver pathology and prolonged survival were achieved by surgical excision of VEGF-secreting tumor or by systemic administration of a potent VEGF antagonist (VEGF-TRAP(R1R2)), thus defining a paraneoplastic syndrome caused by excessive VEGF activity. Moreover, this VEGF-induced syndrome resembles peliosis hepatis, a rare human condition that is encountered in the setting of advanced malignancies, high-dose androgen therapy, and Bartonella henselae infection. Thus, our findings in the mouse have suggested an etiologic role for VEGF in this disease and may lead to diagnostic and therapeutic options for this debilitating condition in humans.

Ephrin-B3 is the midline barrier that prevents corticospinal tract axons from recrossing, allowing for unilateral motor control.

Growing axons follow highly stereotypical pathways, guided by a variety of attractive and repulsive cues, before establishing specific connections with distant targets. A particularly well-known example that illustrates the complexity of axonal migration pathways involves the axonal projections of motor neurons located in the motor cortex. These projections take a complex route during which they first cross the midline, then form the corticospinal tract, and ultimately connect with motor neurons in the contralateral side of the spinal cord. These obligatory contralateral connections account for why one side of the brain controls movement on the opposing side of the body. The netrins and slits provide well-known midline signals that regulate axonal crossings at the midline. Herein we report that a member of the ephrin family, ephrin-B3, also plays a key role at the midline to regulate axonal crossing. In particular, we show that ephrin-B3 acts as the midline barrier that prevents corticospinal tract projections from recrossing when they enter the spinal gray matter. We report that in ephrin-B3(-/-) mice, corticospinal tract projections freely recross in the spinal gray matter, such that the motor cortex on one side of the brain now provides bilateral input to the spinal cord. This neuroanatomical abnormality in ephrin-B3(-/-) mice correlates with loss of unilateral motor control, yielding mice that simultaneously move their right and left limbs and thus have a peculiar hopping gait quite unlike the alternate step gait displayed by normal mice. The corticospinal and walking defects in ephrin-B3(-/-) mice resemble those recently reported for mice lacking the EphA4 receptor, which binds ephrin-B3 as well as other ephrins, suggesting that the binding of EphA4-bearing axonal processes to ephrin-B3 at the midline provides the repulsive signal that prevents corticospinal tract projections from recrossing the midline in the developing spinal cord.

Ciliary neurotrophic factor activates leptin-like pathways and reduces body fat, without cachexia or rebound weight gain, even in leptin-resistant obesity.

Ciliary Neurotrophic Factor (CNTF) was first characterized as a trophic factor for motor neurons in the ciliary ganglion and spinal cord, leading to its evaluation in humans suffering from motor neuron disease. In these trials, CNTF caused unexpected and substantial weight loss, raising concerns that it might produce cachectic-like effects. Countering this possibility was the suggestion that CNTF was working via a leptin-like mechanism to cause weight loss, based on the findings that CNTF acts via receptors that are not only related to leptin receptors, but also similarly distributed within hypothalamic nuclei involved in feeding. However, although CNTF mimics the ability of leptin to cause fat loss in mice that are obese because of genetic deficiency of leptin (ob/ob mice), CNTF is also effective in diet-induced obesity models that are more representative of human obesity, and which are resistant to leptin. This discordance again raised the possibility that CNTF might be acting via nonleptin pathways, perhaps more analogous to those activated by cachectic cytokines. Arguing strongly against this possibility, we now show that CNTF can activate hypothalamic leptin-like pathways in diet-induced obesity models unresponsive to leptin, that CNTF improves prediabetic parameters in these models, and that CNTF acts very differently than the prototypical cachectic cytokine, IL-1. Further analyses of hypothalamic signaling reveals that CNTF can suppress food intake without triggering hunger signals or associated stress responses that are otherwise associated with food deprivation; thus, unlike forced dieting, cessation of CNTF treatment does not result in binge overeating and immediate rebound weight gain.

Biological action of angiopoietin-2 in a fibrin matrix model of angiogenesis is associated with activation of Tie2.

The endothelial cell (EC) specific tyrosine kinase receptor, Tie2, interacts with at least two ligands, angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2). Ang1 stimulates Tie2 receptor autophosphorylation, while Ang2 has been reported to inhibit Ang1-induced Tie2 receptor autophosphorylation. We studied the effects of Ang1 and Ang2 in an in vitro model of angiogenesis. Human ECs (HUVEC), cultured on 3-D fibrin matrices, were treated with conditioned media (CM) from stably transfected cells expressing human Ang1 or Ang2, or with purified recombinant proteins. EC tube formation was measured as a differentiation index (DI), calculated as the ratio of total tube length over residual of EC monolayer. CM from Ang1 overexpressing A10 SMC or HEK293T cells induced profound HUVEC differentiation, resulting in the formation of extensive capillary-like tubes within 48 h (DI: 24.58+/-5.91 and 19.13+/-7.86, respectively) vs. control (DI: 2.73+/-1.68 and 2.15+/-1.45, respectively, both P<0.001). Interestingly, CM from two independent cell lines overexpressing Ang2 also produced a significant increase in EC differentiation (DI: 9.22+/-3.00 and 9.72+/-4.84, both P<0.005 vs. control) although the degree of angiogenesis was significantly less then that seen with Ang1. Addition of Ang1* (a genetically engineered variant of naturally occurring Ang1) or Ang2 also resulted in dose dependent increases in DI, which were blocked by an excess of soluble Tie2 receptor (20 microg/ml). Both Ang1* and Ang2 induced modest increases in [3H]thymidine incorporation into HUVECs (20 and 26%, respectively), which were inhibited by excess soluble Tie2. Although Ang2 was unable to induce significant Tie2 receptor phosphorylation during a 5-min exposure, a 24-h pretreatment with Ang2, followed by brief re-exposure, produced Tie2 phosphorylation in HUVEC comparable to that produced by Ang1*. These results demonstrate for the first time that Ang2 may have a direct role in stimulating Tie2 receptor signaling and inducing in vitro angiogenesis. Our findings suggest that the physiological role of Ang2 is more complex than previously recognized: acting alternately to promote or blunt Tie2 receptor signaling in endothelial cells, depending on local conditions.