Dupilumab in adolescents with uncontrolled moderate-to-severe atopic dermatitis: results from a phase IIa open-label trial and subsequent phase III open-label extension.

BACKGROUND: Dupilumab (monoclonal antibody inhibiting IL-4/IL-13 signalling) is approved for use in adolescents aged ≥ 12 years with inadequately controlled moderate-to-severe atopic dermatitis (AD). Dupilumab significantly improved AD signs/symptoms in a 16-week, randomised, placebo-controlled phase III trial in adolescents (NCT03054428). OBJECTIVES: To characterize the pharmacokinetics of dupilumab, and long-term safety and efficacy in adolescents. METHODS: This was a global, multicentre, phase IIa, open-label, ascending-dose, sequential cohort study with a phase III open-label extension (OLE) in adolescents with moderate-to-severe AD. In the phase IIa study, patients received one dupilumab dose (2 mg kg-1 or 4 mg kg-1 ) and 8 weeks of pharmacokinetic sampling. Thereafter, patients received the same dose weekly for 4 weeks, with 8-week safety follow-up. Patients then enrolled in the OLE, continuing 2 mg kg-1 or 4 mg kg-1 dupilumab weekly. Primary end points were dupilumab concentration-time profile and incidence of treatment-emergent adverse events (TEAEs). Secondary outcomes included Eczema Area and Severity Index (EASI). RESULTS: Forty adolescents received dupilumab in the phase IIa study; 36 enrolled in the OLE. Dupilumab showed nonlinear, target-mediated pharmacokinetics. Mean ± SD trough dupilumab concentrations in serum at week 48 (OLE) were 74 ± 19 mg L-1 and 161 ± 60 mg L-1 for 2 mg kg-1 and 4 mg kg-1 , respectively. Dupilumab was well tolerated over 52 weeks; the most common TEAEs were nasopharyngitis (week 52: 41% [2 mg kg-1 ], 47% [4 mg kg-1 ]) and AD exacerbation (29%, 42%). After one dupilumab dose in the phase IIa study, EASI improved from baseline to week 2 [mean ± SD reduction -34% ± 20% (2 mg kg-1 ) and -51% ± 29% (4 mg kg-1 )]. With continuing treatment, EASI scores improved further [week 52: -85% ± 12% (2 mg kg-1 ) and -84% ± 20% (4 mg kg-1 )]. CONCLUSIONS: In adolescents with moderate-to-severe AD, dupilumab's pharmacokinetic profile was similar to that in adults. These 52-week safety and efficacy data support long-term use of dupilumab in this patient population. What's already known about this topic? Adolescents with moderate-to-severe atopic dermatitis (AD) have high unmet medical need, with significant disease burden and limited treatment options. Dupilumab (monoclonal antibody against interleukin-4 receptor α) is approved for the treatment of adolescents with moderate-to-severe AD who are inadequately responsive to standard of care (U.S.A.) or candidates for systemic therapy (European Union). A 16-week, randomized, placebo-controlled phase III trial in adolescents demonstrated significant improvements in AD signs/symptoms with an acceptable safety profile. What does this study add? These studies demonstrate the long-term safety and efficacy of dupilumab in adolescents with moderate-to-severe AD for up to 52 weeks of treatment, thus extending and reinforcing the findings from the 16-week dupilumab phase III trial. The data from these studies also support the use of dupilumab in combination with current standard of care (topical corticosteroids), which was not evaluated in the 16-week phase III monotherapy trial.

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.

Bedside diagnosis of mitochondrial dysfunction after malignant middle cerebral artery infarction.

BACKGROUND: The study explores whether the cerebral biochemical pattern in patients treated with hemicraniectomy after large middle cerebral artery infarcts reflects ongoing ischemia or non-ischemic mitochondrial dysfunction. METHODS: The study includes 44 patients treated with decompressive hemicraniectomy (DCH) due to malignant middle cerebral artery infarctions. Chemical variables related to energy metabolism obtained by microdialysis were analyzed in the infarcted tissue and in the contralateral hemisphere from the time of DCH until 96 h after DCH. RESULTS: Reperfusion of the infarcted tissue was documented in a previous report. Cerebral lactate/pyruvate ratio (L/P) and lactate were significantly elevated in the infarcted tissue compared to the non-infarcted hemisphere (p < 0.05). From 12 to 96 h after DCH the pyruvate level was significantly higher in the infarcted tissue than in the non-infarcted hemisphere (p < 0.05). CONCLUSION: After a prolonged period of ischemia and subsequent reperfusion, cerebral tissue shows signs of protracted mitochondrial dysfunction, characterized by a marked increase in cerebral lactate level with a normal or increased cerebral pyruvate level resulting in an increased LP-ratio. This biochemical pattern contrasts to cerebral ischemia, which is characterized by a marked decrease in cerebral pyruvate. The study supports the hypothesis that it is possible to diagnose cerebral mitochondrial dysfunction and to separate it from cerebral ischemia by microdialysis and bed-side biochemical analysis.

Recirculation usually precedes malignant edema in middle cerebral artery infarcts.

OBJECTIVES: In patients with large middle cerebral artery (MCA) infarcts, maximum brain swelling leading to cerebral herniation and death usually occurs 2-5 days after onset of stroke. The study aimed at exploring the pattern of compounds related to cerebral energy metabolism in infarcted brain tissue. METHODS: Forty-four patients with malignant MCA infarcts were included after decision to perform decompressive hemicraniectomy (DHC). Cerebral energy metabolism was in all patients monitored bedside by 1-3 microdialysis catheters inserted into the infarcted hemisphere during DHC. In 29 of the patients, one microdialysis catheter was also placed in the non-infarcted hemisphere. MCA blood-flow velocity was monitored bilaterally by transcranial Doppler ultrasound. RESULTS: The interstitial glucose levels were in both sides within normal limits throughout the monitoring period. Mean lactate/pyruvate (LP) ratio was very high in infarcted tissue immediately after DHC. The ratio slowly decreased but did not reach normal level during the study period. In the infarcted hemisphere, MCA blood-flow velocities increased from approximately 42 cm/s 1 day prior to DHC (nine of nine patients) to approximately 60 cm/s at day 4. CONCLUSIONS: Normal interstitial glucose level in the infarcted hemisphere in combination with substantial MCA blood-flow velocities bilaterally even before DHC was performed indicates that malignant brain swelling usually commences when the embolus/thrombosis has been largely resolved and recirculation of the infarcted area has started. The protracted increase of the LP ratio in infarcted tissue might indicate mitochondrial dysfunction.

Comparative study of MBR and activated sludge in the treatment of paper mill wastewater.

The study was based on a full scale activated sludge plant (AS) compared to a parallel operated pilot membrane bioreactor (MBR) with flat sheets membranes. Both systems received their influent from an anaerobic bioreactor treating paper mill wastewater. MBR produced an effluent of much better quality than AS in terms of suspended solids, containing 1 mg/L or less in 80% of the monitoring time, while the AS effluent contained 12 mg/L. This could save the necessity of further treatment by filtration in the case of MBR. Other effluent quality parameters, such as organic matter (COD and BOD), phosphorus and ammonia nitrogen, did not indicate substantial differences between AS and MBR. Calcium carbonate scaling and formation of a bacterial layer on the membrane caused severe flux reduction. The membrane blockage because of scaling and biofouling proved to be very serious, therefore, it required proper and more complicated maintenance than the AS system. This study leads to the conclusion that in the case of paper mill wastewater, after anaerobic biotreatment, if there is no need for excellent effluent quality in terms of suspended solids, the replacement of the AS by the MBR would not be strongly justified, mainly because of maintenance cost.

Advanced treatment by anaerobic process followed by aerobic membrane bioreactor for effluent reuse in paper mill industry.

The operation of an activated sludge process at a paper mill (AIPM) in Hedera, Israel, was often characterized by disturbances. As part of a research and development project, a study on new biological treatment was initiated. The study included the operation of three pilot units: a. anaerobic treatment by upflow anaerobic sludge blanket (UASB); b. aerobic treatment by two pilot units including activated sludge and membrane bioreactor (MBR), which have been operated in parallel for comparison reasons. The pilot plant working on anaerobic treatment performed COD reduction from 2,365 to 755 mg/L, expressed as average values. Based on the pilot study, a full scale anaerobic treatment system has been erected. During a period of 100 days, after achieving steady state, the MBR system provided steady operation performance, while the activated sludge produced effluent characterized by oscillatory qualities. The following results, based on average values, indicate much lower suspended solids concentrations in the MBR effluent, 2.5 mg/L, as compared to 25 mg/L in the activated sludge. The ability to develop and maintain a concentration of over 11,000 mg/L of mixed liquor volatile suspended solids in the MBR enabled an intensive bioprocess at relatively high cell residence time. This study demonstrates that the anaerobic process, followed by aerobic MBR can provide effluent of high quality which can be considered for economic reuse in the paper mill industry.

Membrane bioreactors for final treatment of wastewater.

The full-scale existing treatment plant in a paper mill in Hedera, Israel, includes equalization, solids separation by either straining or by dissolved air flotation and biological treatment by activated sludge. The operation of the existing biological process is often characterized by disturbances, mainly bad settling, voluminous bioflocs, followed by wash-out of the biosolids. This paper summarizes the results obtained in a study based on a pilot plant including a membrane biological reactor (MBR) compared to the "conventional" activated sludge process in the aerobic treatment of the effluent obtained from an anaerobic reactor. During the pilot operation period (about 90 days after achieving steady state) the MBR system provided steady operation performance, while the activated sludge produced effluent characterized by oscillatory values. The results are based on average values and indicate much lower levels of suspended solids in the MBR effluent, 2.5 mg/L, as compared to 37 mg/L in the activated sludge. As a result, the total organic mater content was also substantially lower in the MBR effluent, 129 vs 204 mg/L as COD, and 7.1 vs 83 mg/L as BOD. The MBR enabled better nitrification. The ability to develop and maintain a concentration of over 11,000 mg/L of mixed liquor volatile suspended solids in the MBR bioreactor enabled an intensive bioprocess at relatively high cell residence time. As a result the biosolids which had to be removed as excess sludge were characterized by relatively low volatile/total suspended solids ratio, around 0.78. This could facilitate and lower the cost of biosolids treatment and handling. The results of this comparative study indicate that in the case of MBR there will be no need for further treatment, while after activated sludge additional filtration will be required. The study leads to the conclusion that MBR will be the best technology for aerobic treatment of the anaerobic effluent of the paper mill.

Bedside biochemical monitoring of the penumbra zone surrounding an evacuated acute subdural haematoma.

We describe a penumbra zone with increased biochemical vulnerability in cerebral cortex underlying an evacuated acute subdural haematoma. Two microdialysis catheters were placed in this zone and one catheter was placed in the opposite, less injured hemisphere. The microdialysis perfusates were analysed bedside for glucose, pyruvate, lactate, glutamate, and glycerol. In the penumbra zone, but not in the opposite hemisphere, energy metabolism was seriously disturbed with signs of cell membrane degradation. During an adverse event (decrease in haemoglobin level, systemic blood pressure and cerebral perfusion pressure) the perturbation of energy metabolism increased in this zone. Energy metabolism recovered and the signs of cell membrane degradation disappeared after normalization of the physiological parameters. We use the term biochemical penumbra zone to describe an area with signs of energy failure and cell membrane degradation, which has a capacity to regain a normal metabolic pattern but also an increased vulnerability to secondary insults.

Intracerebral microdialysis and bedside biochemical analysis in patients with fatal traumatic brain lesions.

BACKGROUND: Microdialysis with bedside biochemical analysis was used to monitor cerebral biochemical alterations that precede and accompany increase in intracranial pressure (ICP), resulting in a complete cessation of cerebral blood flow. METHODS: Seven patients, who died due to an untreatable increase in ICP, were included. The patients originate from a large, consecutive series of severely head injured patients (n: 95) monitored with intracerebral microdialysis (perfusion rate 0.3 microl/min). One microdialysis catheter was inserted via a separate burr hole frontally to that used for the intraventricular catheter ("better" position) and one catheter was inserted into cerebral cortex surrounding an evacuated focal contusion or underlying an evacuated haematoma ("worse" position). Biochemical analyses of glucose, lactate, glycerol, urea, glutamate, and pyruvate were performed at the bedside. All samples were frozen for subsequent HPLC (high-performance liquid chromatography) analyses of amino acids and ions. RESULTS: Decreases in glucose and pyruvate and increases in lactate, glycerol, glutamate, and lactate/pyruvate (la/py) ratio characterized cerebral ischaemia. The measured markers give information regarding substrate availability (glucose), redox state of the tissue (la/py ratio), degradation of glycerophospholipids in cell membranes (glycerol), and extracellular concentration of excitatory amino acids (glutamate). In the "worse" position biochemical deterioration occurred before the increase in ICP. In the "better" position biochemical deterioration was usually observed after the increase in ICP. CONCLUSION: Changes of cerebral energy metabolism that accompany cerebral ischaemia follow a certain pattern and may be detected at the bedside by intracerebral microdialysis before the secondary damage causes an increase in ICP.

Brain energy metabolism during controlled reduction of cerebral perfusion pressure in severe head injuries.

OBJECTIVE: To study cerebral biochemical markers with intracerebral microdialysis and bedside analysis in patients with severe head injuries treated with a controlled reduction of cerebral perfusion pressure (CPP). DESIGN: Prospective observational study. SETTING: Neurological intensive care unit in a university hospital. PATIENTS: A consecutive series of 48 patients with severe head injuries and intracranial pressure (ICP) above 20 mmHg after conventional treatment. INTERVENTIONS: Reduction of CPP was attained with i. v. infusion of beta1-antagonist (metoprolol) and an alpha2-agonist (clonidine). One microdialysis catheter was inserted via a burr hole frontally to that used for the intraventricular catheter ("better" position). In 27 patients one or more catheters were inserted into cerebral cortex surrounding an evacuated focal contusion or underlying an evacuated haematoma ("worse" position). Perfusion rate was 0.3 microl/min and samples were taken every 30 or 60 min. The levels of glucose, pyruvate, lactate, glycerol and glutamate were analysed and displayed bedside. RESULTS: After initiation of treatment mean CPP decreased from 73 to 62 mmHg. During the first 96 h CPP was less than 60 mmHg and less than 50 mmHg during 30% and 8% of the time, respectively. The treatment was associated with a gradual normalisation of all biochemical markers in the "better" as well as the "worse" catheter position. CONCLUSION: The study shows that pharmacological decrease in CPP according to the "Lund concept" is associated with a normalisation of cerebral metabolism. The study also indicates that intracerebral microdialysis can be used for evaluation of new treatment strategies.

Screw placement in transoral atlantoaxial plate systems: an anatomical study.

OBJECT: The placement of an anterior atlantoaxial plate after transoral odontoid resection has been described by Harms. Recently, the authors of biomechanical and clinical studies have shown that this procedure, especially in combination with posterior wiring, is a good alternative to established, isolated posterior atlantoaxial fixation techniques. Reports on the anatomy of the atlas and axis primarily focus on the posterior surgical approach. Scarce research regarding the quantitative anatomy of the anterior aspect of C-1 and C-2 has been reported. This study was undertaken to measure relevant dimensions of C-1 and C-2 and their relation to the anterior transoral approach. The aim of the study was to determine "safe zones" for screw placement in anterior atlantoaxial plate fixation. METHOD: Fifty human dry C-1 and C-2 vertebrae were obtained for direct anatomical, radiographic, and computerized tomography (CT) measurements. Thirty-two linear and four angular parameters were evaluated. All measurements were made using a digital caliper, ruler, or goniometer. Anatomical measurements were correlated with radiographic (anteroposterior, lateral, and craniocaudal) and CT (0.5-mm-slice thickness) measurements of the corresponding vertebrae. Additionally, bone mineral density (BMD) measurements of C-1 and C-2 were obtained in 20 patients. A safe zone for anterior screw placement in an atlas of bilateral trapezoid shape could be characterized. The average medial and lateral height of the trapezoid was 4.1 +/- 1.01 mm (range 1.4-6.7 mm) and 12.9 +/- 1.73 mm (range 8.7-17.4 mm), respectively. The distance between the sagittal plane and the medial and lateral walls of the trapezoid was 10.2 +/- 1.42 mm (range 8.9-12.8 mm) and 23.5 +/- 2.98 mm (range 21.7-30.7 mm), respectively. The average depth of the lateral masses was 22.3 +/- 2.04 mm (range 17.0-26.7 mm) in the sagittal plane. The average BMD in the safe zone of C-1 was 0.89 +/- 0.11 g/cm3 (range 0.75-1.01 g/cm3). Bone mineral density measurements at C-2 revealed a spheroid zone of low density at the basis of the dens (0.68 +/- 0.09 g/cm3). In contrast, high zones of BMD were found near the articular surfaces (C1-2: 0.97 +/- 0.11 g/cm3; C2-3: 0.94 +/- 0.12 g/cm3). The safe zone for anterior axis screw placement was V-shaped, limited cranially by a zone of low bone density and laterally by the vertebral artery groove. Correlations between radiographic and anatomical measurements were generally good (r2 = 0.78-0.95), but they were higher between CT and anatomical measurements (r2 = 0.86-0.99). CONCLUSIONS: A quantitative understanding of the anterior anatomy of C-1 and C-2 is necessary when considering anterior atlantoaxial plate fixation after transoral odontoid resection. In this study the authors defined safe zones for anterior atlas and axis screw placement. The anterior atlantoaxial plate, as originally described by Harms, only partially respects these safe zones.

Incidence of intracerebral and subarachnoid haemorrhage in southern Sweden.

OBJECTIVES: Spontaneous intracranial haemorrhage-that is, mainly subarachnoid haemorrhage (SAH) and primary intracerebral haemorrhage (PICH)-constitutes an important part of all strokes. As previous epidemiological studies have demonstrated highly variable incidence rates, we conducted a large prospective investigation of all haemorrhagic strokes during a 1 year period. METHODS: Twelve hospitals serving a defined population of 1.14 million in southern Sweden registered all cases with spontaneous intracranial haemorrhage, including those found dead outside hospitals, during 1996. All patients were examined with CT of the brain or underwent necropsy. Incidence rates adjusted to the Swedish population for age and sex, as well as location of haematoma and prevalence of risk factors were calculated. RESULTS: A total of 106 patients with SAH and 341 patients with PICH were identified. The annual incidence/100 000 was 10.0 (6.4 for men and 13.5 for women) for SAH and 28.4 (32.2 for men and 24.7 for women) for PICH when adjusted to the Swedish population. Subarachnoid haemorrhage affected twice as many women as men. The incidence of both types of haemorrhage increased with advancing age, but in particular, this was the case for supratentorial PICH. Lobar haematomas were the most common (51.6%) type of PICH. Among patients with PICH, 37% had hypertension, 41% other vascular disease, and 12% were on oral anticoagulation. Among patients with SAH, 28% had hypertension and 18% vascular disease before the haemorrhage but no one was on treatment with oral anticoagulation. CONCLUSIONS: The incidence of PICH was high, especially for the older age groups. PICH was, on average, three times as common as SAH. The study underscores the importance of PICH and SAH as significant stroke subgroups.

Intracerebral microdialysis in clinical practice: baseline values for chemical markers during wakefulness, anesthesia, and neurosurgery.

OBJECTIVE: The study was undertaken to measure baseline values for chemical markers in human subjects during wakefulness, anesthesia, and neurosurgery, using intracerebral microdialysis. METHODS: Microdialysis catheters were inserted into normal posterior frontal cerebral cortex in nine patients who were undergoing surgery to treat benign lesions of the posterior fossa. The perfusion rate was 1.0 microl/min during anesthesia/neurosurgery and the early postoperative course and 0.3 microl/min during the later course. Bedside biochemical analyses of glucose, pyruvate, lactate, glycerol, glutamate, and urea were performed before, during, and after neurosurgery. After the bedside analyses, all samples were frozen for subsequent high-performance liquid chromatographic analyses of amino acids. RESULTS: The following baseline values were obtained during wakefulness (perfusion rate, 0.3 microl/min): glucose, 1.7+/-0.9 mmol/L; lactate, 2.9+/-0.9 mmol/L; pyruvate, 166+/-47 micromol/L; lactate/pyruvate ratio, 23+/-4; glycerol, 82+/-44 micromol/L; glutamate, 16+/-16 mmol/L; urea, 4.4+/-1.7 mmol/L. Marked increases in the levels of all chemical markers were observed at the beginning and end of anesthesia/surgery. CONCLUSION: The study provides human baseline levels for biochemical markers that can presently be measured at the bedside during neurointensive care. In addition, some changes that occurred under varying physiological conditions are described.

Essential requirement of BMPs-2/4 for both osteoblast and osteoclast formation in murine bone marrow cultures from adult mice: antagonism by noggin.

Bone morphogenetic proteins (BMPs) have been heretofore implicated in the induction of osteoblast differentiation from uncommitted progenitors during embryonic skeletogenesis and fracture healing. We have tested the hypothesis that BMPs are also involved in the osteoblastogenesis that takes place in the bone marrow in postnatal life. To do this, we took advantage of the properties of noggin, a recently discovered protein that binds BMP-2 and -4 and blocks their action. Addition of human recombinant noggin to bone marrow cell cultures from normal adult mice inhibited both osteoblast and osteoclast formation; these effects were reversed by exogenous BMP-2. Consistent with these findings, BMP-2 and -4 and BMP-2/4 receptor transcripts and proteins were detected in these primary cultures, in a bone marrow-derived stromal/osteoblastic cell line, as well as in murine adult whole bone; noggin expression was also documented in all these preparations. Moreover, addition of antinoggin antibody caused an increase in osteoblast progenitor formation. These findings suggest that BMP-2 and -4 are expressed in the bone marrow in postnatal life and serve to maintain the continuous supply of osteoblasts and osteoclasts; and that, in fact, BMP-2/4-induced commitment to the osteoblastic lineage is a prerequisite for osteoclast development. Hence, BMPs, perhaps in balance with noggin and possibly other antagonists, may provide the tonic baseline control of the rate of bone remodeling on which other inputs (e.g., hormonal, biomechanical, etc.) operate.

Expression and function of Gdf-5 during digit skeletogenesis in the embryonic chick leg bud.

Bone morphogenetic proteins (BMPs) constitute a large family of secreted signals involved in the formation of the skeleton but the specific function of each member of this family remains elusive. GDF-5 is a member of the BMP family which has been implicated in several skeletogenic events including the induction and growth of the appendicular cartilages, the determination of joint forming regions, and the establishment of tendons. Here, we have studied the function of GDF-5 in digit skeletogenesis by analyzing the effects of its local administration in the developing autopod of embryonic chick and the regulation of its pattern of gene expression by other signals involved in digit morphogenesis. As reported in the mouse, the gdf-5 gene exhibits a precise distribution in the joint-forming regions of the developing chicken digital rays. GDF-5 beads implanted at the tip of the digits promote intense cartilage growth and fail to induce morphological or molecular signs of joint formation. Furthermore, GDF-5 beads implanted in the interdigits inhibit the formation of joints in the adjacent digits. These data suggest that the role of GDF-5 in joint formation is the control of growth and differentiation of the cartilage of the epiphyseal regions of the phalanges rather than accounting for the differentiation of the sinovial joint tissues. The interdigital mesoderm in spite of its potential to form ectopic digits with their tendinous apparatus failed to form either ectopic cartilages or ectopic tendons after the implantation of GDF-5 beads in the stages preceding cell death. At difference with other BMPs, GDF-5 exhibited only a weak cell death promoting effect. The BMP antagonist Noggin binds to GDF-5 and is able to inhibit all the observed effects of this growth factor in vivo. Potential interactions of GDF-5 with other signals involved in digits morphogenesis were also explored. BMP-7 regulates negatively the expression of gdf-5 gene in the joint forming regions and local treatment with Noggin induces the ectopic expression of gdf-5 in the interdigital mesoderm. Retroviral-induced misexpression of Indian or Sonic Hedgehog genes in the developing digits leads to the formation of digits without joints in which gdf-5 expression occurs throughout the entire perichondrial surface. In conclusion, this study indicates that GDF-5 is a signal regulated by other BMPs which controls the growth and differentiation of the epiphyses of the digital cartilages acting in close relationship with Hedgehog signaling.

Biochemical and biophysical characterization of refolded Drosophila DPP, a homolog of bone morphogenetic proteins 2 and 4.

The mature C-terminal signaling domain of the Drosophila Decapentaplegic proprotein (DPP) can be efficiently refolded from chaotrope-solubilized inclusion bodies with the aid of a membrane protein-solubilizing detergent, high concentrations (0.75-2 M) of NaCl, and low temperatures (5-15 degreesC). The disulfide-linked homodimeric product contains N-terminal heparin-binding sites that were utilized as intrinsic affinity tags to obtain a highly enriched preparation in one chromatographic step. A subsequent C4 reverse phase high pressure liquid chromatography step provides high purity, salt-free protein that is amenable to biophysical and structural studies at a yield of approximately 3 mg/liter of bacterial culture. The dimeric protein is correctly folded as determined by electrophoretic, spectroscopic, chemical, and proteolytic analyses. Refolded DPP is also bioactive as shown by induction of chondrogenesis in embryonic chick limb bud cells and by high affinity binding to Noggin, an antagonist of bone morphogenetic protein signaling. In contrast to bone morphogenetic proteins extracted from demineralized bone or overexpressed in cell culture, the refolded Escherichia coli-expressed protein is not glycosylated at a conserved N-linked site and is therefore homogeneous. The C-terminal domain dimer is more hydrophobic and thus less soluble than its unfolded or partially folded forms, necessitating highly solubilizing conditions for recovery after folding in vitro. Hence solubilization of the mature ligand may be one of the principal roles of the large (250-400 amino acids) N-terminal prodomains of transforming growth factor-beta superfamily members, shown to act as intramolecular chaperones in vivo.

Increased expression of CNTF receptor alpha in denervated human skeletal muscle.

The functional receptor for ciliary neurotrophic factor (CNTF) is comprised of a CNTF binding entity termed CNTF receptor alpha (CNTFRalpha), and 2 signaling molecules called LIF receptor beta and gp130. CNTFRalpha can be released from the cell surface; the soluble form can confer CNTF responsiveness to cells. CNTFRalpha has recently been localized to several nonneuronal cell types including rat skeletal muscle fibers. In this study we examined the expression pattern of CNTFRalpha in normal, denervated and dystrophic human muscle. In muscle biopsies from 12 normal subjects, 16 cases of neurogenic muscular atrophy, 4 cases of Duchenne muscular dystrophy, and 4 cases of limb girdle dystrophy, CNTFRalpha mRNA levels were determined by Northern blotting. Transcript levels were significantly increased in cases of neurogenic atrophy compared to normal controls and dystrophic muscle. By nonradioactive in situ hybridization, CNTFRalpha transcripts were detected in the sarcoplasm of both normal sized and atrophic muscle fibers. In addition, soluble CNTFRalpha was elevated 4.4-fold in the urine of ALS patients compared to normal adults. These results suggest that the expression of CNTFRalpha in human skeletal muscle fibers is regulated by innervation. This regulation appears to be selective, because CNTFRalpha mRNA was not increased in dystrophic human muscle. Increased CNTFRalpha could confer higher sensitivity to CNTF during neurodegeneration or nerve fiber regeneration.

Enhancing leptin response by preventing SH2-containing phosphatase 2 interaction with Ob receptor.

Leptin is an adipocyte-derived cytokine that regulates food intake and body weight via interaction with its Ob receptor (ObR). Serum leptin levels are chronically elevated in obese humans, suggesting that obesity may be associated with leptin resistance and the inability to generate an adequate ObR response. Evidence suggests that transcriptional activation of target genes by STAT3 (signal transducer and activator of transcription) in the hypothalamus is a critical pathway that mediates leptin's action. Herein we report that activation of ObR induces the tyrosine phosphorylation of the tyrosine phosphatase SH2-containing phosphatase 2 (SHP-2) and demonstrate that Tyr986 within the ObR cytoplasmic domain is essential to mediate phosphorylation of SHP-2 and binding of SHP-2 to ObR. Surprisingly, mutation of Tyr986 to Phe, which abrogates SHP-2 phosphorylation and binding to the receptor, dramatically increases gene induction mediated by STAT3. Our findings indicate that SHP-2 is a negative regulator of STAT3-mediated gene induction after activation of ObR and raise the possibility that blocking the interaction of SHP-2 with ObR could overcome leptin resistance by boosting leptin's weight-reducing effects in obese individuals.

A sweat gland-derived differentiation activity acts through known cytokine signaling pathways.

The sympathetic innervation of sweat glands undergoes a target-induced noradrenergic to cholinergic/peptidergic switch during development. Similar changes are induced in cultured sympathetic neurons by sweat gland cells or by one of the following cytokines: leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), or cardiotrophin-1 (CT-1). None of these is the sweat gland-derived differentiation activity. LIF, CNTF, and CT-1 act through the known receptors LIF receptor beta (LIFRbeta) and gp130 and well defined signaling pathways including receptor phosphorylation and STAT3 activation. Therefore, to determine whether the gland-derived differentiation activity was a member of the LIF/CNTF cytokine family, we tested whether it acted via these same receptors and signal cascades. Blockade of LIFRbeta inhibited the sweat gland differentiation activity in neuron/gland co-cultures, and extracts of gland-containing footpads stimulated tyrosine phosphorylation of LIFRbeta and gp130. An inhibitor (CGX) of molecules that bind the CNTFRalpha, which is required for CNTF signaling, did not affect the gland-derived differentiation activity. Soluble footpad extracts induced the same changes in NBFL neuroblastoma cells as LIF and CNTF, including increased vasoactive intestinal peptide mRNA, STAT3 dimerization, and DNA binding, and stimulation of transcription from the vasoactive intestinal peptide cytokine-responsive element. Thus, the sweat gland-derived differentiation activity uses the same signaling pathway as the neuropoietic cytokines, and is likely to be a family member.