Time kinetics of bone defect healing in response to BMP-2 and GDF-5 characterised by in vivo biomechanics.

This study reports that treatment of osseous defects with different growth factors initiates distinct rates of repair. We developed a new method for monitoring the progression of repair, based upon measuring the in vivo mechanical properties of healing bone. Two different members of the bone morphogenetic protein (BMP) family were chosen to initiate defect healing: BMP-2 to induce osteogenesis, and growth-and-differentiation factor (GDF)-5 to induce chondrogenesis. To evaluate bone healing, BMPs were implanted into stabilised 5 mm bone defects in rat femurs and compared to controls. During the first two weeks, in vivo biomechanical measurements showed similar values regardless of the treatment used. However, 2 weeks after surgery, the rhBMP-2 group had a substantial increase in stiffness, which was supported by the imaging modalities. Although the rhGDF-5 group showed comparable mechanical properties at 6 weeks as the rhBMP-2 group, the temporal development of regenerating tissues appeared different with rhGDF-5, resulting in a smaller callus and delayed tissue mineralisation. Moreover, histology showed the presence of cartilage in the rhGDF-5 group whereas the rhBMP-2 group had no cartilaginous tissue. Therefore, this study shows that rhBMP-2 and rhGDF-5 treated defects, under the same conditions, use distinct rates of bone healing as shown by the tissue mechanical properties. Furthermore, results showed that in vivo biomechanical method is capable of detecting differences in healing rate by means of change in callus stiffness due to tissue mineralisation.

Enforced covalent trimerization increases the activity of the TNF ligand family members TRAIL and CD95L.

Variants of human TRAIL (hTRAIL) and human CD95L (hCD95L), encompassing the TNF homology domain (THD), interact with the corresponding receptors and stimulate CD95 and TRAILR2 signaling after cross-linking. The murine counterparts (mTRAIL, mCD95L) showed no or only low receptor binding and were inactive/poorly active after cross-linking. The stalk region preceding the THD of mCD95L conferred secondary aggregation and restored CD95 activation in the absence of cross-linking. A corresponding variant of mTRAIL, however, was still not able to activate TRAIL death receptors, but gained good activity after cross-linking. Notably, disulfide-bonded fusion proteins of the THD of mTRAIL and mCD95L with a subdomain of the tenascin-C (TNC) oligomerization domain, which still assembled into trimers, efficiently interacted with their cognate cellular receptors and robustly stimulated CD95 and TRAILR2 signaling after secondary cross-linking. Introduction of the TNC domain also further enhanced the activity of THD encompassing variants of hTRAIL and hCD95L. Thus, spatial fixation of the N-terminus of the THD appears necessary in some TNF ligands to ensure proper receptor binding. This points to yet unanticipated functions of the stalk and/or transmembrane region of TNF ligands for the functionality of these molecules and offers a broadly applicable option to generate recombinant soluble ligands of the TNF family with superior activity.

Radiation-induced suppression of the Bmp2 signal transduction pathway in the pluripotent mesenchymal cell line C2C12: an in vitro model for prevention of heterotopic ossification by radiotherapy.

Heterotopic ossification is a common complication after total hip replacement. Clinical studies showed the effectiveness of radiation for prevention of heterotopic ossification. The mechanism of radiotherapy responsible for the reduction of heterotopic ossification is unclear. The purpose of this study was to study an analogue model showing a time- and dose-dependent effect of radiation. Using cells of the defined embryonic mouse cell line C2C12, the influence of ionizing radiation on the Bmp-induced signal cascade leading to osteogenic differentiation was analyzed. Binding of iodinated Bmp2 to the receptors, Smad1 activation, and alkaline phosphatase (ALP) activity were determined in cells with or without irradiation. The cytotoxic effect of radiotherapy was evaluated using viability tests. Radiotherapy reduced formation of the Bmp2/Bmp receptor complex. This effect was dependent on dose. The phosphorylation (activation) of Smad1 decreased after irradiation in a time-dependent manner, whereas the level of total Smads was not influenced by radiotherapy. The ALP activity decreased after radiotherapy. A dose of 7 Gy delivered 6 h before or after incubation with Bmp resulted in about a 30% decrease in ALP activity. No signs of cytotoxic effects were observed within the time window studied using doses of 0 to 20 Gy. The time- and dose-dependent effect of radiotherapy for prevention of heterotopic ossification known from the results of clinical studies has an analogue in the C2C12 cell model. The primary mechanism of radiotherapy seems to be an influence on cellular responsiveness to the Bmp2-induced osteoblastic differentiation. The results suggest a down-regulation of the Bmp2/receptor complex.

Dual role of the IL-12/IFN-gamma axis in the development of autoimmune myocarditis: induction by IL-12 and protection by IFN-gamma.

IL-12 and IFN-gamma positively regulate each other and type 1 inflammatory responses, which are believed to cause tissue damage in autoimmune diseases. We investigated the role of the IL-12/IFN-gamma (Th1) axis in the development of autoimmune myocarditis. IL-12p40-deficient mice on a susceptible background resisted myocarditis. In the absence of IL-12, autospecific CD4(+) T cells proliferated poorly and showed increased Th2 cytokine responses. However, IFN-gamma-deficient mice developed fatal autoimmune disease, and blockade of IL-4R signaling did not confer susceptibility to myocarditis in IL-12p40-deficient mice, demonstrating that IL-12 triggers autoimmunity by a mechanism independent of the effector cytokines IFN-gamma and IL-4. In conclusion, our results suggest that the IL-12/IFN-gamma axis is a double-edged sword for the development of autoimmune myocarditis. Although IL-12 mediates disease by induction/expansion of Th1-type cells, IFN-gamma production from these cells limits disease progression.

Radiation-induced reduction of BMP-induced proteoglycan synthesis in an embryonal mesenchymal tissue equivalent using the chicken "limb bud" test.

PURPOSE: Heterotopic ossification (HO) is a common complication following total hip replacement. Clinical studies showed the effectiveness of irradiation for prevention of heterotopic ossification. The mechanism of radiotherapy responsible for the reduction of heterotopic ossification is unclear. The purpose of this study was to find a suitable cell system, which can reproduce in-vitro data resulting from clinical in-vivo studies. The establishment of such a cell model allows detailed analyses of the mechanism of radiotherapy. METHOD: The chicken limb bud test was used as an in-vitro model. The cells acquired by the limb bud test were irradiated with different doses (0 Gy, 3 Gy, 7 Gy, 10 Gy, 20 Gy). Irradiation was set either 1 hour before, or 1 or 3 days after BMP-2 incubation. The synthesis of proteoglycans (PGS) upon treatment with bone morphogenetic protein (BMP)-2 was measured in cells incubated with BMP-2 for 4 days followed by 35SO4(2-) labeling for 6 hours. Labeled proteoglycans were precipitated using Alcian blue and measured in a raytest radio-TLC analyzer. The incubation with BMP-2 was defined to correlate the in-vivo stimulus meaning the operation. RESULTS: The proteoglycan synthesis was significantly reduced by irradiation 1 hour before or 1 day after BMP-2 incubation, if the dosage was at least 7 Gy. Higher doses than 7 Gy did not lead to lower proteoglycan levels. There was only a trend for a reduction of proteoglycan synthesis by 3 Gy irradiation, but no significant difference compared to the non-irradiated control. An irradiation 3 days after BMP-2 incubation had no effect on proteoglycan. CONCLUSION: A dose and time dependent effect of radiation on BMP-2-induced proteoglycan synthesis was observed. Therefore the results of clinical in-vivo studies were reproduced exactly by the limb bud test. We established an in-vitro cell model to analyze the mechanism of the prevention of heterotopic ossification by radiotherapy on cellular or sub-cellular level.

Upon prolonged allergen exposure IL-4 and IL-4Ralpha knockout mice produce specific IgE leading to anaphylaxis.

BACKGROUND: IL-4 and IL-13 are key regulators in atopic disorders and both signal through the receptor chain IL-4Ralpha. IL-4 and IL-13 are also the only cytokines known to induce class switching to IgE. We sought to compare allergen-specific IgE responses and allergic reactivity of wild-type (wt) mice with IL-4-/- and IL-4Ralpha-/- mice, which lack both IL-4 and IL-13 functions. METHODS: BALB/c wt, IL-4-/- and IL-4Ralpha-/- mice were immunized with ovalbumin intranasally or intraperitoneally and specific antibody titers were measured by ELISA. Bronchoalveolar lavage fluids and lung tissue were analyzed cytologically and histologically. Allergic reactivity was determined by active cutaneous anaphylaxis and anaphylactic shock. RESULTS: wt mice immunized intranasally or intraperitoneally showed high titers of specific IgE 3 and 6 weeks after primary sensitization, resulting in cutaneous anaphylaxis and anaphylactic shock upon challenge. Intranasal sensitization resulted in airway eosinophilia and goblet cell metaplasia. In contrast, IL-4-/- and IL-4Ralpha-/- mice showed no specific IgE after 3 weeks, but produced high titers after 6 weeks. At this time cutaneous anaphylaxis and anaphylactic shock could be induced as in wt mice, but lung pathology was absent. CONCLUSIONS: We conclude that upon long-term allergen exposure, alternative switch mechanisms independent of IL-4 and IL-4Ralpha may induce IgE but not asthma-like lung pathology. This may be relevant for the development of allergic disease, since long-term allergen exposure is a frequent condition during allergic sensitization.

The crystal structure of the BMP-2:BMPR-IA complex and the generation of BMP-2 antagonists.

BACKGROUND: Bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs) belong to the large transforming growth factor-beta (TGF-beta) superfamily of multifunctional cytokines. Signaling of the BMPs requires the binding of the BMP to the BMP cell surface receptors BMPR-IA, BMPR-IB, and BMPR-II. Similar to other cytokines, members of the TGF-beta superfamily exhibit stringent specificity in their ligand-receptor interactions, which may be a reason for the qualitative and quantitative differences in cellular responses. To understand how BMPs and GDFs activate their receptors, it is important to determine structure and binding mechanisms of ligand-receptor complexes. We have used BMP-2 as a key representative of the BMPs to identify the epitopes for type I and type II receptor binding by mutational interaction analyses and have solved the crystal structure of a BMP2:BMPR-IA receptor ectodomain complex. METHODS: To identify amino acid side chains involved in receptor binding, a collection of in vitro mutagenized human BMP-2 variants was prepared and subjected to interaction analyses with use of the receptor ectodomains of BMPR-IA, BMPR-II, and ActR-II immobilized on a biosensor system. The biological activity of the BMP-2 variants was measured by BMP-2 dependent expression of alkaline phosphatase (ALP) in C2C12 cells. For crystallization, a complex of BMP-2 and the ectodomain of BMPR-IA was formed in solution, purified, and crystallized as described(12). RESULTS: The ligand-receptor interaction analysis of the BMP-2 variants identified distinct epitopes for type I and type II receptor binding. Because the structure of TGF-beta-like proteins has been compared with that of an open hand, the binding epitope for the type I receptor was-on the basis of its location-termed "wrist" epitope. The crystal structure of the BMP-2:BMPR-IA ectodomain complex revealed a key feature of the ligand-receptor interaction: a large hydrophobic residue (Phe85) within a hydrophobic patch of BMPR-IA fit into a hydrophobic pocket composed of residues of both BMP-2 monomers. A second epitope identified by alanine mutagenesis scanning was termed the "knuckle" epitope on the basis of its location on the outer side of the "finger" segments of BMP-2. Mutations in either the wrist epitope or the knuckle epitope produced variants with altered biological activities. Variants with antagonistic properties were exclusively generated by mutations in the knuckle epitope of BMP-2. CONCLUSIONS AND CLINICAL RELEVANCE: The identification and characterization of the two receptor binding epitopes in BMP-2 provide new insight into the primary steps of BMP-receptor activation. Because of the structural similarities between members of the TGF-beta superfamily, it can be assumed that the data presented in this work are transferable to other TGF-beta receptor systems. Because of the association with various diseases, the generation of antagonists of other TGF-beta superfamily members might generate potent tools for basic research and therapeutic approaches.

Type III TGF-beta receptor-independent signalling of TGF-beta2 via TbetaRII-B, an alternatively spliced TGF-beta type II receptor.

Transforming growth factor-beta (TGF-beta) signals through membrane-bound serine/threonine kinase receptors, which upon stimulation phosphorylate Smad proteins and thereby trigger their nuclear translocation and transcriptional activity. Although the three mammalian isoforms of TGF-beta are highly homologous at the level of sequence, analysis of their in vivo function by gene knockouts revealed striking differences, suggesting no significant functional redundancy between TGF-beta1, -2 and -3. While signal transduction by TGF-beta1 has been well characterized, receptor binding and activation by the TGF-beta2 isoform is less well understood. Here, we show that TbetaRII-B, an alternatively spliced variant of the TGF-beta type II receptor, is a TGF-beta2 binding receptor, which mediates signalling via the Smad pathway in the absence of any TGF-beta type III receptor (TbetaRIII). L6 cells lacking endogenous TbetaRIII as well as TbetaRII-B do not respond to TGF-beta2. Transfection of these cells with TbetaRII-B restores TGF-beta2 sensitivity. The expression of TbetaRII-B is restricted to cells originating from tissues such as bone where the isoform TGF-beta2 has a predominant role. This reflects the importance of this receptor in TGF-beta isoform-specific signalling.

BMP-2 antagonists emerge from alterations in the low-affinity binding epitope for receptor BMPR-II.

Bone morphogenetic protein-2 (BMP-2) induces bone formation and regeneration in adult vertebrates and regulates important developmental processes in all animals. BMP-2 is a homodimeric cysteine knot protein that, as a member of the transforming growth factor-beta (TGF-beta) superfamily, signals by oligomerizing type I and type II receptor serine-kinases in the cell membrane. The binding epitopes of BMP-2 for BMPR-IA (type I) and BMPR-II or ActR-II (type II) were characterized using BMP-2 mutant proteins for analysis of interactions with receptor ectodomains. A large epitope 1 for high-affinity BMPR-IA binding was detected spanning the interface of the BMP-2 dimer. A smaller epitope 2 for the low-affinity binding of BMPR-II was found to be assembled by determinants of a single monomer. Symmetry-related pairs of the two juxtaposed epitopes occur near the BMP-2 poles. Mutations in both epitopes yielded variants with reduced biological activity in C2C12 cells; however, only epitope 2 variants behaved as antagonists partially or completely inhibiting BMP-2 activity. These findings provide a framework for the molecular description of receptor recognition and activation in the BMP/TGF-beta superfamily.

Bone morphogenetic protein 2 (BMP-2) induces sequential changes of Id gene expression in the breast cancer cell line MCF-7.

Bone morphogenetic proteins (BMPs) are involved in the development of various organs including the mammary gland. They are well-regulated and act in a time-, concentration- and cell-type-specific manner. We found that BMP-2 is expressed in primary breast tumor tissue samples and in breast cancer cell lines. Hybridization of labeled cDNA, obtained from the breast cancer cell line MCF-7, against the Atlas human cDNA expression array revealed differential gene expression depending on BMP-2 treatment. The most prominent changes were observed for the helix-loop-helix proteins Id-1, Id-2 and Id-3. Id-1 expression had increased severalfold after 4 h and was even higher after 24 h. Id-2 and Id-3 were more strongly induced after 4 h and showed no further significant change after 24 h. Analysis of cell-cycle distribution revealed a marked increase of the sub-G1 phase after 48 h in serum-deprived cells. In the presence of BMP-2 no change was observed over 48 h indicating that BMP-2 does not induce apoptosis. In addition, expression of caspase-3 was reduced in BMP-2-treated cells after 24 h. In summary, our results clearly indicate that BMP-2 is a susceptibility factor keeping the cells ready for the integration of various other signals for cell progression.

Improving the refolding yield of interleukin-4 through the optimization of local interactions.

Interleukin-4 (IL-4) is a multifunctional cytokine that plays an important role in the regulation of various immune responses. However, the development of IL-4 or IL-4 variants into potential therapeutic drugs is hindered by the low efficiency of the in vitro refolding process of this protein. In this work, we have investigated the improvement of the refolding yield of IL-4 using two different rational design approaches. The first one is based on the so-called inverse hydrophobic effect and involved the replacement of a solvent exposed, non-conserved, hydrophobic residue (W91) by serine. This led to an increase in stability of 1.4 kcal mol(-1) and shifted the midpoint transition temperature (Tm) from 62 to 70 degrees C. The second approach is based on the stabilization of alpha-helices through the introduction of favorable local interactions. This strategy resulted in the following helix sequence for helix C of IL-4, 68ASAAEANRHKQLIRFLKRLDRNLWGLAG95. The mutant protein was stabilized by 0.5 kcal mol(-1), the Tm shifted to 68 degrees C, and a two-fold increase in the refolding yield was consistently observed. Our results make the large-scale production of IL-4 derivatives economically more viable, suggest that a similar approach can be applied to other related proteins, and may represent a general strategy to improve in vitro refolding yields through the selective optimization of the stability of alpha-helices.

IL-4 enhances proliferation and mediator release in mature human mast cells.

Tissue mast cells (MC) are recognized as key effector cells of immediate-type allergic reactions releasing inflammatory mediators and cytokines on stimulation with antigen, but they also might be involved in IgE-independent inflammatory and tissue repair processes. The mechanism of human MC regulation in tissue is not fully understood. Here, we show that IL-4, in synergy with stem cell factor (SCF), regulates the function of purified human MC isolated from intestinal tissue. Whereas SCF induced only marginal proliferation of MC cultured in vitro up to 4 weeks, addition of IL-4 and SCF strongly increased the proliferation rate. Moreover, IL-4, which by itself had no visible effect on human MC, enhanced the release of histamine, leukotriene C4, and IL-5 in MC triggered by IgE receptor crosslinking. The IL-4 effects occurred in a dose-dependent fashion (ED50 = 100 pg/ml) and could be totally blocked by a competitive IL-4 receptor antagonist. Our data indicate that IL-4 is an important regulator of human MC function and suggest that mature MC retain the capacity to proliferate in a particular tissue environment.

Activation of STAT5 by IL-4 relies on Janus kinase function but not on receptor tyrosine phosphorylation, and can contribute to both cell proliferation and gene regulation.

We have investigated mechanisms and consequences of STAT5 activation through the human IL-4 receptor (IL-4R). By functionally expressing receptor mutants in the murine pro-B cell line Ba/F3, we could show that phosphorylated tyrosine residues within the IL-4R alpha chain are dispensable for IL-4-induced STAT5 activity. However, disruption of a membrane-proximal proline-rich sequence motif ('box1') in either subunit of the bipartite IL-4R abolished not only ligand-induced tyrosine phosphorylation of Janus kinases JAK1 and JAK3, but also IL-4-triggered activation of STAT5 and concomitant cell proliferation. A dominant-negative version of STAT5b, but not of STAT5a, interfered with IL-4-induced DNA synthesis in Ba/F3 cells, suggesting an involvement of STAT5b in the control of cell proliferation through IL-4R. Reporter gene experiments finally showed that transcription from promoters of STAT5 target genes can be specifically induced by challenging cells with IL-4, and that both STAT5a and STAT5b can contribute to IL-4-triggered transcriptional control.

[EHBMP-2. Initial BMP analog with osteoinductive properties]. / EHBMP-2. Erstes BMP-Analog mit osteoinduktiven Eigenschaften.

For the first time a non natural BMP-variant (EHBMP-2) with osteoinductive properties was produced by expression in E. coli through specific mutation of the amino acid sequence. The substitution of 12 N-terminal amino acids by a nonsense sequence results in a neglectible affinity of EHBMP-2 to the extracellular matrix. In vitro EHBMP-2 induces dose-dependent cartilage formation in neonatal muscle tissue. Single intramuscular implantation in mice results in the formation of an ossicle with functional active bone marrow. The size of the ossicle depends on the amount of implanted EHBMP-2 and can significantly be increased by the combination with a collagen carrier. The largest bone formation is observed after injection of EHBMP-2 containing collagen suspensions. In rats a stronger osteoinductive activity can be achieved by coupling of EHBMP-2 to collagen discs than by coupling natural BMP-2 to the same collagen carrier. Critical size defects in rats' mandibular angels can be restored by the combination of granular collagenous bone matrix (ICBM) with EHBMP-2. Further investigations have to show whether the altered pharmacokinetics of EHBMP-2 has advantages regarding its therapeutical use and tissue-engineering.

Crystal structure of human bone morphogenetic protein-2 at 2.7 A resolution.

Homodimeric bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor beta (TGF-beta) superfamily that induces bone formation and regeneration, and determines important steps during early stages of embryonic development in vertebrates and non-vertebrates. BMP-2 can interact with two types of receptor chains, as well as with proteins of the extracellular matrix and several regulatory proteins. We report here the crystal structure of human BMP-2 determined by molecular replacement and refined to an R-value of 24.2 % at 2.7 A resolution. A common scaffold of BMP-2, BMP-7 and the TGF-betas, i.e. the cystine-knot motif and two finger-like double-stranded beta-sheets, can be superimposed with r. m.s. deviations of around 1 A. In contrast to the TGF-betas, the structure of BMP-2 shows differences in the flexibility of the N terminus and the orientation of the central alpha-helix as well as two external loops at the fingertips with respect to the scaffold. This is also known from the BMP-7 model. Small secondary structure elements in the loop regions of BMP-2 and BMP-7 seem to be specific for the respective BMP-subgroup. Two identical helix-finger clefts and two distinct cavities located around the central 2-fold axis of the dimer show characteristic shapes, polarity and surface charges. The possible function of these specific features in the interaction of BMP-2 with its binding partners is discussed.

The interleukin-4 receptor activates STAT5 by a mechanism that relies upon common gamma-chain.

Interleukin (IL)-4 signaling proceeds via cytoplasmic activation of the Janus kinases JAK1 and JAK3 and the signal transducer and activator of transcription STAT6. We show that the IL-4 receptor, like other cytokine receptor systems utilizing the common receptor gamma-chain (gammac), is also connected to a signaling pathway that involves STAT5. Both STAT5a and STAT5b become tyrosine-phosphorylated and acquire specific DNA-binding properties in response to IL-4 receptor stimulation in the murine pro-B cell line Ba/F3. In preactivated human T cells, STAT5 became activated in an IL-4-dependent fashion as assayed by IL-4-induced STAT5 translocation from the cytoplasm to the cell nucleus and by binding to cognate DNA. Moreover, stimulation of preactivated human T cells by IL-4 led to specific transcriptional up-regulation of STAT5 target genes. IL-4 receptor-mediated STAT5 activation is dependent on the presence of gammac and JAK3 within the receptor complex. In COS-7 cells, the JAK/STAT pathway leading from the IL-4 receptor to STAT5-dependent regulation of a reporter gene relied largely on coexpression of JAK3. In Ba/F3 cells, studies on signal transduction evoked by directed specific receptor homo- or heterodimerization revealed that STAT5 activation can be triggered exclusively by IL-4R heterodimers containing gammac.

Inductive properties of recombinant human BMP-2 produced in a bacterial expression system.

Recombinant human BMP-2, produced in E. coli, refolded and concentrated to a purity of more than 98%, has been demonstrated to be biologically active. In vitro, amounts of 0.4 microg BMP-2 or more induced new cartilage formation in 27 out of 47 samples of a neonatal muscle tissue assay, with chondroneogenesis occurring 14 days after a four-hour contact between BMP-2 and the muscle tissue. In vivo, BMP-2 was implanted in the thigh muscle of ICR mice for a period of three weeks. Amounts of 4 microg BMP-2 and more showed heterotopic bone formation in 15 out of 17 samples. When BMP-2 was combined with a collagen carrier, amounts of 0.4 microg protein or more induced heterotopic bone formation in 30 out of 33 samples four weeks after the implantation in the abdominal wall of Sprague-Dawley rats. The results show that the E. coli-derived BMP-2 was active in different assay systems in concentrations equal to those required with mammalian cell-expressed BMP-2. It could also be demonstrated that a single morphogen (BMP-2) is enough to initiate the differentiation process associated with bone induction. The presented bacterial expression system also offers the opportunity to produce large quantities of recombinant BMP-2 for clinical applications.

[Biological activity of E. coli expressed BMP-4]. / Biologische Aktivität von E.-coli-exprimiertem BMP-4.

BMP-4 is physiologically present in low concentrations in human bone matrix. So far the protein has only been produced in small quantities by expression in mammalian cell cultures. In this study we investigated the biological activity of E. coli-expressed BMP-4. In vitro neonatal rat muscle tissue was incubated together with BMP-4 during 4 h, followed by an incubation period of 14 days on cellulose acetate membranes in BMP-free medium. The addition of 0.4 microgram BMP-4 induced cartilage formation in 1/8 samples while 4 micrograms BMP-4 showed chondroneogenesis in 2/10 samples. When the BMP-4 concentration was increased to 40 micrograms, new cartilage formation was seen in 5/7 samples. In vivo BMP-4 was implanted intramuscularly for 3 weeks in ICR mice. Amounts of 10 micrograms rhBMP-4 and more (up to 100 micrograms) constantly induced heterotopic ossicle formation. BMP-4 was also combined with a collagen carrier and implanted for 2 and 4 weeks in the abdominal muscle of SD rats. While 0.4 microgram BMP-4 showed no bone or cartilage formation, the amount of 40 micrograms BMP-4 showed new heterotopic cartilage formation, followed by endochondral ossification in almost all samples. The results prove that E. coli-expressed BMP-4 possesses the same inductive properties as mammalian-cell-expressed BMP-4.

Radiation-induced impairment of bone healing can be overcome by recombinant human bone morphogenetic protein-2.

Radiotherapy of head and neck tumors very often results in impaired healing of craniomaxillofacial bones in the vicinity. Management of radionecrosis of bones after radiotherapy is an important clinical challenge. Bone morphogenetic proteins (BMPs) induce new bone differentiation. The aim of this study is to investigate the potential of BMPs in ameliorating radiation-induced impaired bone repair. Two 3-mm diameter defects were created in the calvaria of rats. The defects were treated with different doses of recombinant human (rh) BMP-2 using collagen type I as a carrier. Irradiation with a single dose of 1,200 rad was performed 2 or 7 days preoperatively. Unirradiated animals served as controls. New bone formation was assessed by quantitation of radiographs of the calvaria and histology on day 21 after surgery. Untreated, unirradiated defects showed a spontaneous osseous regeneration of 90 +/- 7% of the defect area within 21 days. Irradiation of the site (1,200 rad 2 days preoperatively) resulted in a profound decrease in the bone fill of the untreated defect (5 +/- 2%). Recombinant human BMP-2 in soluble collagen type I carrier delivered to the defect resulted in a significant increase of new bone formation (34 +/- 14%, P < 0.01 for 25 micrograms rhBMP-2; 77 +/- 19% for 35 micrograms rhBMP-2, P < 0.01). Type I collagen carrier alone resulted in only 7 +/- 2% healing. In conclusion, radiation-induced impairment of calvarial repair can be overcome by rhBMP-2. Thus, the concept of BMP-2-induced regeneration has potential applications in reconstructive craniomaxillofacial surgery after irradiation.

IL-4 receptor complexes containing or lacking the gamma C chain are inhibited by an overlapping set of antagonistic IL-4 mutant proteins.

IL-4 signals on lymphocytes and other cells through a heterodimeric complex of two cytokine receptor proteins, IL-4R alpha and gamma C. IL-4 variants with mutations in the positions Tyr124, and (less efficiently) Arg121 and Ser125 are deficient in interaction with gamma zero and can be applied as IL-4 antagonists. Some cells respond to IL-4 without using gamma zero presumably by recruiting an alternative subunit into the receptor complex. We have investigated the effects of IL-4 mutant proteins on cells bearing this second type of IL-4 receptor complex. Mutations in the amino acid residues Arg 121 and Tyr124, but not Ser125, completely prevented cellular responses mediated by type 2 IL-4 receptors. Both receptor types are therefore affected by mutations in an overlapping but not identical site of the IL-4 molecule.