Validation of Bone Conversion in Osteoconductive and Osteoinductive Bone Substitutes

Bone reconstruction can be performed with an autogeneic graft from various donor regions. Osteoconductive and osteoinductive bone substitutes originate from substances of diverse chemical and morphological types and can have a synthetic or a biological derivation. Alongside autogeneic bone transplants and allogenic and xenogeneic bone implants, alloplastic bone replacements of synthetic or semi-synthetic origin are being used for defect reconstruction. In an animal model in rabbits five bone substitutes and one autogeneic graft were surgically incorporated into identical bone defects (10times 10 mm in size) in six anatomically defined regions of the skull. With scintigraphic and histological methods, the metabolic dynamics of the bone is examined as it reacts to the transplantation of autogeneic bone or to implanted bone replacement material. The different autogeneic, xenogeneic and alloplastic bone replacement materials can be differentiated according to the functional quality of the new tissue and the dynamics of the bone conversion thus induced. In the comparison of mineralized, osteoconductive bone subsitutes (TCP, HA, calcium carbonate ceramics) with demineralized, osteoinductive implants (DBM new, DBM old) and autogeneic bone grafts, the bone inducing matrices show the largest quantity of new bone formation, making possible a volume-constant reconstruction. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pore membrane and/or filament interacting like protein 1 (POMFIL1) is predominantly expressed in the nervous system and encodes different protein isoforms

We have isolated and characterized a novel differentially spliced gene predominantly expressed in the nervous system, which encodes protein isoforms with significant homology to the alpha-actinin protein superfamily, the Caenorhabditis elegans UNC-53 protein and weak homology to the nuclear membrane protein POM121. Similar to POM121 the primary structures show a hydrophobic region that is likely to form one or more adjacent transmembrane segment(s). Indirect immunofluorescence with antibodies against a synthetic peptide gave staining of the nucleus. Target experiments with EGFP (enhanced green fluorescent protein)-fusion proteins confirmed the nuclear localization. Two further members of this gene family could be isolated. All three pore membrane and/or filament interacting like (POMFIL) genes are differentially expressed in neuronal tumor cell lines. In 40% of tested primary neuroblastomas expression of POMFIL1 is strongly reduced and after brain injury POMFIL1 protein expression is upregulated, indicating that POMFIL1 is involved in the process of neuron growth and regeneration, as well as in neural tumorigenesis.     

Premature chromosome condensation in humans associated with microcephaly and mental retardation: a novel autosomal recessive condition

We report a novel autosomal recessive disorder characterized by premature chromosome condensation in the early G2 phase. It was observed in two siblings, from consanguineous parents, affected with microcephaly, growth retardation, and severe mental retardation. Chromosome analysis showed a high frequency of prophase-like cells (>10%) in lymphocytes, fibroblasts, and lymphoblast cell lines with an otherwise normal karyotype. (3)H-thymidine-pulse labeling and autoradiography showed that, 2 h after the pulse, 28%-35% of the prophases were labeled, compared with 9%-11% in healthy control subjects, indicating that the phenomenon is due to premature chromosome condensation. Flow cytometry studies demonstrate that the entire cell cycle is not prolonged, compared with that in healthy control subjects, and compartment sizes did not differ from those in healthy control subjects. No increased reaction of the cells to X-irradiation or treatments with the clastogens bleomycin and mitomycin C was observed, in contrast to results in the cell-cycle mutants ataxia telangiectasia and Fanconi anemia. The rates of sister chromatid exchanges and the mitotic nondisjunction rates were inconspicuous. Premature entry of cells into mitosis suggests that a gene involved in cell-cycle regulation is mutated in these siblings.     

Genomewide comparison of DNA sequences between humans and chimpanzees

A total of 8,859 DNA sequences encompassing ~1.9 million base pairs of the chimpanzee genome were sequenced and compared to corresponding human DNA sequences. Although the average sequence difference is low (1.24%), the extent of changes is markedly different among sites and types of substitutions. Whereas ~15% of all CpG sites have experienced changes between humans and chimpanzees, owing to a 23-fold excess of transitions and a 7-fold excess of transversions, substitutions at other sites vary in frequency, between 0.1% and 0.5%. If the nucleotide diversity in the common ancestral species of humans and chimpanzees is assumed to have been about fourfold higher than in contemporary humans, all possible comparisons between autosomes and X and Y chromosomes result in estimates of the ratio between male and female mutation rates of ~3. Thus, the relative time spent in the male and female germlines may be a major determinant of the overall accumulation of nucleotide substitutions. However, since the extent of divergence differs significantly among autosomes, additional unknown factors must also influence the accumulation of substitutions in the human genome.     

Thioredoxin-2 but not thioredoxin-1 is a substrate of thioredoxin peroxidase-1 from Drosophila melanogaster: isolation and characterization of a second thioredoxin in D. Melanogaster and evidence for distinct biological functions of Trx-1 and Trx-2

As Drosophila melanogaster does not contain glutathione reductase, the thioredoxin system has a key function for glutathione disulfide reduction in insects (Kanzok, S. M., Fechner, A., Bauer, H., Ulschmid, J. K., Müller, H. M., Botella-Munoz, J., Schneuwly, S., Schirmer, R. H., and Becker, K. (2001) Science 291, 643-646). In view of these unique conditions, the protein systems participating in peroxide metabolism and in redox signaling are of special interest. The genes for a second thioredoxin (DmTrx-2) and a thioredoxin peroxidase (DmTPx-1) were cloned and expressed, and the proteins were characterized. In its disulfide form, the 13-kDa protein thioredoxin-2 is a substrate of thioredoxin reductase-1 (K(m) = 5.2 microm, k(cat) = 14.5 s(-1)) and in its dithiol form, an electron donor for TPx-1 (K(m) = 9 microm, k(cat) = 5.4 s(-1)). DmTrx-2 is capable of reducing glutathione disulfide with a second order rate constant of 170 m(-1) s(-1) at pH 7.4 and 25 degrees C. Western blot analysis indicated that this thioredoxin represents up to 1% of the extractable protein of D. melanogaster Schneider cells or whole fruit flies. Recombinant thioredoxin peroxidase-1 (subunit molecular mass = 23 kDa) was found to be a decameric protein that can efficiently use Trx-2 but not Trx-1 as a reducing substrate. The new electron pathway found in D. melanogaster is also representative for insects that serve as vectors of disease. As a first step we have cloned and functionally expressed the gene that is the orthologue of DmTrx-2 in the malaria mosquito Anopheles gambiae.     

Some new aspects of 17alpha-estradiol metabolism in man

17Alpha-estradiol (1,3,5(10)-estratriene-3,17alpha-diol) together with a tracer dose of the tritium-labeled compound was administered orally and sublingually to male volunteers. The serum concentrations of 17alpha-estradiol (free and liberated by enzymatic hydrolysis) were quantified by GC/MS, and the serum total radioactivity and urinary radioactivity excretion were determined. After oral administration, 17alpha-estradiol was rapidly and intensively conjugated; only tiny quantities of the free steroid (<1% of total) appeared in serum. Sublingual administration resulted in temporary (up to 3 h p.a.) higher serum levels of the free compound. The metabolite patterns obtained by TLC of extracts from serum and urine demonstrated that 17alpha-estradiol is the subject of a poor phase I metabolism in man. A great discrepancy was found in the serum concentrations of 17alpha-estradiol (free + conjugated) determined by GC/MS and the serum radioactivity expressed in 17alpha-estradiol equivalents. By TLC analysis of the steroid conjugates extracted from serum, various 17alpha-estradiol conjugate peaks were found. By enzymatic hydrolysis with beta-glucuronidase/aryl sulfatase from Helix pomatia they were only partially cleaved. Thus, the difference between the serum radioactivity and the 17alpha-estradiol levels determined by GC/MS had to be attributed to an incomplete conjugate hydrolysis. It has been shown with the synthesized 17alpha-estradiol sulfate conjugates that only the 3-sulfate is cleaved by enzymatic hydrolysis, whereas the 17-sulfate group resists enzymatic hydrolysis. The methanolysis procedure (acetyl chloride in MeOH) has proved to be an efficient method for cleaving both the 3-sulfate group and the 17-sulfate group. In contrast to the 17alpha-estradiol conjugates in serum, the urinary conjugates were intensively split by the enzyme preparation. From this, it has to be concluded that the serum conjugates were deconjugated and newly reconjugated before urinary excretion.     

Promotion of skin graft tolerance across MHC barriers by mobilization of dendritic cells in donor hemopoietic cell infusions

Flt3 ligand (FL) dramatically increases the number of immunostimulatory dendritic cells (DC) and their precursors in bone marrow (BM) and secondary lymphoid tissues. Herein we tested the ability of FL-mobilized donor hemopoietic cells to promote induction of skin graft tolerance across full MHC barriers. C57BL/10 (B10; H2(b), IE(-)) mice were given 10(8) spleen cells (SC) from normal or FL-treated, H-2-mismatched B10.D2 (H2(d), IE(+)) donors i.v. on day 0, 200 mg/kg i.p. cyclophosphamide on day 2, and 10(7) T cell-depleted BM cells from B10.D2 mice on day 3. B10.D2 skin grafting was performed on day 14. Indefinite allograft survival (100 days) was induced in recipients of FL-SC, but not in mice given normal SC. Tolerance was associated with blood macrochimerism and was confirmed by second-set skin grafting with donor skin 100 days after the first graft. In tolerant mice, peripheral donor-reactive T cells expressing TCR Vbeta11 were deleted selectively. Immunocompetence of tolerant FL-SC-treated mice was proven by rapid rejection of third-party skin grafts. To our knowledge this is the first report that mobilization of DC in donor cell infusions can be used to induce skin graft tolerance across MHC barriers, accompanied by specific deletion of donor-reactive T cells.     

Synergistic antitumor effect of bispecific CD19 x CD3 and CD19 x CD16 diabodies in a preclinical model of non-Hodgkin's lymphoma

To target NK cells against non-Hodgkin's lymphoma, we constructed a bispecific diabody (BsDb) with reactivity against both human CD19 and FcgammaRIII (CD16). Bacterially produced CD19 x CD16 BsDb specifically interacted with both CD19(+) and CD16(+) cells and exhibited significantly higher apparent affinity and slower dissociation from the tumor cells than from effector cells. It was able to induce specific lysis of tumor cells in the presence of isolated human NK cells or nonfractionated PBLs. The combination of the CD19 x CD16 BsDb with a previously described CD19 x CD3 BsDb and CD28 costimulation significantly increased the lytic potential of human PBLs. Treatment of SCID mice bearing an established Burkitt's lymphoma (5 mm in diameter) with human PBLs, CD19 x CD16 BsDb, CD19 x CD3 BsDb, and anti-CD28 mAb resulted in the complete elimination of tumors in 80% of animals. In contrast, mice receiving human PBLs in combination with either diabody alone showed only partial tumor regression. These data clearly demonstrate the synergistic effect of small recombinant bispecific molecules recruiting different populations of human effector cells to the same tumor target.