Digital measurements: a different approach to evaluate bone formation. A technical report.

In this study a digital measurement technique has been proposed to quantify bone formation on histological images. Two standard parietal defects were created in 30 adult rabbits. The animals were divided into six groups. Four animals of each group were randomly chosen as experimental group in which osteopontin-coated hydroxyapatite (OPN-HA) and hydroxyapatite (HA) were inserted alternatively in created defects. To observe the spontaneous healing process of defects, one animal of each group was used as control animal and these created defects did not receive any implants. The animals were sacrificed after 1, 2, 6, 12, 18 and 30 weeks. The histological sections were magnified (x100) and scanned digitally. The newly formed bone surfaces within the healing area were indicated and quantified by means of Adobe Photoshop 7 software. This measuring technique was found to be reliable and reproducible. The results of this study show no significant differences in bone formation between the OPN-HA and non-coated HA defects, although a significant difference in bone formation was measured at the margins of the defects treated with OPN-HA.

Osteopontin and bone metabolism in healing cranial defects in rabbits.

Non-collagen proteins such as bone sialoprotein and osteopontin (OPN) form 10% of the extracellular bone matrix. In this study, the influence of OPN on bone repair was investigated. Human OPN (Innogenetics) was produced by a recombinant technique and bonded onto the surface of hydroxyapatite (Interpore 200). Thirty rabbits were divided into six equal groups. A circular defect (10mm) was prepared in each parietal bone. In four rabbits of each group the left and right defects were filled with either OPN-coated hydroxyapatite (OPN-HA) or non-coated hydroxyapatite (HA). One sham animal of each group received no implants. The animals were killed after 1, 2, 6, 12, 18 and 30 weeks. The histological sections were scanned and analysed digitally. There were no statistically significant differences in total bone formation between defects filled with OPN-HA and HA. Bone formation at the borders of the healing area was significantly higher in defects filled with OPN-HA than in those filled with HA. Less bone formation was noted in the OPN-HA and HA groups at the centre of the healing area than at the borders of the healing area and the dural area. Although some animals in the sham group showed a high level of bone formation in the dural area, this was not significantly different to that in the dural area of the other groups. There was no sign of infection or tissue rejection of the graft.

Expression and processing of the activin-A/erythroid differentiation factor precursor: a member of the transforming growth factor-beta superfamily.

The biosynthesis and intracellular processing of the polypeptide precursor of the beta A-chain of the fertility hormone inhibin were assessed by infecting a wide spectrum of cell types with a recombinant vaccinia virus. Most cell lines, including follicular granulosa cells, secrete both prohormone and mature hormone as homodimers (activin) composed of disulfide-linked subunits of 54 kDa (proactivin-A) and 14 kDa (activin-A), respectively, and a small amount of prohormone-mature hormone heterodimers. Mature activin is secreted from mouse pituitary cells (AtT-20), while pig kidney cells [PK(15)] secrete mostly proactivin. More prohormone is secreted in the presence of NH4Cl, suggesting that prohormone processing is facilitated by low pH. Proactivin-A is not a ligand for the mannose-6-phosphate/insulin growth factor-II receptor. The recombinant activin stimulates FSH release from pituitary cells and differentiates erythroleukemia cell lines in vitro.

Osteopontin and bone metabolism: a histology and scintigraphy study in rats.

Osteopontin (OPN) is one of the major non-collagen proteins in extracellular bone matrix. To elucidate the function of OPN in bone metabolism, a cellular defect was created in parietal bone and tibia of 12 rats. In Group 1, the left defects were filled with OPN-coated hydroxyapatite (OPN-H). In Group 2, the right defects were filled with non-coated hydroxyapatite (N-H). In both groups, the contra lateral defects were used as control defects. In Group 3, OPN-H was inserted in the left defects and N-H in the right defects. Bone metabolism was measured by (45)Ca and technetium-99m methylene diphosphonate scintigraphy for 4 weeks. Scintigraphy did not show any significant differences in bone metabolism between the defects filled with OPN-H and N-H. A higher bone metabolism was measured between the parietal defects filled with OPN-H or N-H in comparison with the parietal control defects. This difference, however, was not significant and was less for tibia defects. Histological observation (7th week) shows less inflammatory cells at the tibia defects filled with OPN-H compared to the tibia defects filled with N-H. This study did not show any acceleration or inhibition of bone metabolism in parietal or tibia bone in rats, but there is some evidence that OPN might influence inflammatory cells in bone matrix.

A prospective multicenter study of the efficacy and tolerability of cryopreserved allogenic human keratinocytes to treat venous leg ulcers.

Allogeneic human keratinocyte cultures have been used to treat burn wounds, donor sites, and chronic skin ulcers with some success. Cryopreservation of these cultures allows for the production of large standardized batches that are readily available for use. The aim of the study presented in this report was to study effects of cryopreserved cultured allogenic human keratinocytes (CryoCeal) on chronic lower extremity wounds. Parameters were measured to study efficacy, tolerability, pain associated with chronic wounds, and quality of life of patients. Twenty-seven patients with hard-to-heal venous leg ulcers received a maximum of 9 applications of CryoCeal in a prospective, uncontrolled multicenter study lasting 48 weeks. Eleven out of 27 patients (41%; 95% CI: 22%-61%) had complete wound closure within 24 weeks (1 week). The time required for complete wound closure in these 11 patients ranged from 4.1 to 24.9 weeks. Only 1 patient had recurrence of the ulcer at 48 weeks. Local (wound) pain scores decreased from a mean of 2.5 at baseline to 0.9 at week 24. Fifty percent of the patients attained a pain score of 0 after 12 weeks and remained stable at this score until the end of the study. Overall, the patient quality of life was better at week 24, compared to baseline values. The treatment was well tolerated, and wound infection was the most frequently occurring adverse event.

Prevention of renal allograft rejection in primates by blocking the B7/CD28 pathway.

BACKGROUND: There is accumulating evidence that blockade of the costimulatory pathways offers a valid approach for immune suppression after solid organ transplantation. In this study, the efficacy of anti-CD80 and anti-CD86 monoclonal antibodies (mAbs) in combination with cyclosporine (CsA) to prevent renal allograft rejection was tested in non-human primates. METHODS: Rhesus monkeys were transplanted with a partly major histocompatibility complex-matched kidney on day 0. Anti-CD80 and anti-CD86 mAbs were administered intravenously daily for 14 days starting at day - 1. CsA was given intramuscularly for 35 days starting just after transplantation. The kidney function was monitored by determining serum creatinine levels. RESULTS: The combination of anti-CD80 and anti-CD86 mAbs completely abrogated the mixed lymphocyte reaction. Untreated rhesus monkeys rejected the kidney allograft in 5-7 days. Treatment with anti-CD80 plus anti-CD86 mAbs resulted in a significantly prolonged graft survival of 28+ 7 days (P=0.025). There were no clinical signs of side effects or rejection during treatment. Kidney graft rejection started after the antibody therapy was stopped. The anti-mouse antibody response was delayed from day 10 to 30 after the first injection. No difference in graft survival was observed between animals treated with CsA alone or in combination with anti-CD80 and anti-CD86 mAbs. However, treatment with anti-CD80 and anti-CD86 mAbs reduced development of vascular rejection. CONCLUSIONS: In combination, anti-CD80 and antiCD86 mAbs abrogate T-cell proliferation in vitro, delay the anti-mouse antibody response in vivo, and prevent graft rejection and development of graft vascular disease in a preclinical vascularized transplant model in non-human primates.

Follistatins neutralize activin bioactivity by inhibition of activin binding to its type II receptors.

Follistatin is an activin-binding protein, which inhibits activin bioactivity in several biological systems. In the present study it is demonstrated that preincubation of iodinated activin A with follistatin, purified from porcine follicular fluid, completely abolished the binding of activin to activin type IIA, IIB2 and IIB4 receptors, and consequently to activin type IB receptor, transiently transfected in COS cells. Binding of activin A to membrane proteins on the activin-responsive P19 embryonal carcinoma cells was also prevented by this follistatin preparation. The same results were obtained with a carboxy-terminally truncated form of follistatin (FS-288), which is only present in minor amounts in the purified follistatin preparation. Since FS-288 has a high affinity for heparan sulfate proteoglycans on the cell surface, we tested whether membrane-bound FS-288 presents activin A to the different activin receptors, thereby facilitating activin binding. FS-288 did bind to the cell surface of transfected COS cells, but inhibited the binding of activin A to its receptors IIA, IIB2 and IIB4. Furthermore, after addition of FS-288 to K562 erythroleukemia cells, the total binding of activin via cell surface-bound FS-288 was increased, whereas the binding of activin A to activin type II and type I receptors present on these cells was inhibited. These findings reveal that different forms of follistatin can neutralize activin bioactivity by interference with binding of activin to all known activin type II receptors, rather than that they inhibit the binding of the type I receptor to the activin/activin type II receptor complex. In addition, our studies indicate that cell surface-associated follistatin cannot present ligand to signalling receptors.

Apparent Greig cephalopolysyndactyly and sinus node disease.

We present the clinical findings and follow-up data from birth to 10.5 years in a boy with Greig cephalopolysyndactyly who, in addition, presents sinus node disease ("sick sinus syndrome"). The significance of the concurrence of Greig cephalopolysyndactyly syndrome, an autosomal dominant condition mapped at 7p13, and sinus node disease is discussed.

Expression of functional mouse antibodies directed against the tumour marker human placental alkaline phosphatase in non-lymphoid cells.

A mouse hybridoma cell line was isolated which produces monoclonal antibodies (MAbs) of the IgG2b, kappa subtype directed against the tumour-associated marker human placental alkaline phosphatase (hPLAP). The mRNAs coding for the heavy (H) and light (L) chains were cloned as cDNA copies. These genes were then separately inserted into the eukaryotic expression vector pSV23p, under control of the SV40 early promoter. Both genes were introduced with the DEAE-dextran technique in COSI cells, and 72 hr after transfection, 10 ng/ml functional antibodies could be detected in the supernatant of the cells. Permanent CHO cell lines secreting 100 ng/ml functional antibodies were established upon transfection of CHO (dhfr-) cells with the plasmids containing the H and L cDNAs and the plasmid pAdD26SVp-(A)-3 carrying the mouse dihydrofolate reductase (dhfr) gene. A plasmid construction in which we inserted a stop codon-containing sequence behind the hinge region of the H-chain cDNA sequence yielded immuno-competent F(ab')2 molecules upon transfection of COS or CHO cells. Our results indicate that not only lymphoid cells, but also non-lymphoid cells, are capable of synthesis and assembly of immunoglobulin chains that are immunologically fully competent.

Expression in non-lymphoid cells of mouse recombinant immunoglobulin directed against the tumour marker human placental alkaline phosphatase.

From a mouse hybridoma cell line secreting a monoclonal antibody directed against the tumour marker human placental alkaline phosphatase, mRNA coding for the H and L chains of this antibody was isolated and cloned as cDNA. Sequence analysis of the H and L chain cDNAs confirmed the IgG2b,kappa subtype previously established. Recloning the H and L chain cDNA information into SV40-based vectors enabled us to obtain expression of functional immunoglobulin upon cotransfection into COS or CHO dhfr- cells. This illustrates that non-lymphoid cells also have the capacity to assemble active immunoglobulins.

Interstitial deletion of the short arm of chromosome 2 in a moderately mentally retarded boy without gross clinical stigmata.

This report describes a moderately mentally retarded boy in whom there is interstitial deletion of the short arm of chromosome 2 (del(2)(p11p21)) associated with the presence in all cells of a small acentric fragment.

[Aglossia-adactylia with jejunal atresia]. / Aglossie-adactylie avec atresie jejunale.

Case report of a newborn with the aglossy-adactyly syndrome associated with complete jejunal atresia. Review of the literature does not allow any conclusion concerning the etiology of the observed malformations. The jejunal atresia, present in this case is probably coincidental. Attention is drawn on the possibility of prenatal diagnosis of obstruction of the digestive tract by ultrasound whenever pregnancy is complicated by hydramnios.

Use of monoclonal antibodies to detect human placental alkaline phosphatase.

Convenient, sensitive, and specific solid-phase immunoassays involving monoclonal antibody are described for the determination of human placental alkaline phosphatase (hPLAP). An endogenous enzyme immunoassay combined the specificity of the immunological and the enzymatic reactions. Alternatively, a solid-phase "sandwich" radioimmunoassay involving immobilized polyclonal rabbit anti-hPLAP in combination with iodinated monoclonal antibody provided some additional advantages. Both tests can be used to detect hPLAP from various sources, e.g., in human sera during pregnancy or as a tumor marker. The radioimmunoassay detected an increase in hPLAP at nine weeks of gestation. We discuss the use of monoclonal antibodies for the differentiation of different alkaline phosphatase isoenzyme types by electrophoresis on starch gel.

The occurrence of human placental alkaline phosphatase (PLAP) in extracts of normal, benign and malignant tissues of the female genital tract.

In addition to its presence in extracts of carcinomatous tissues from the vulva, endometrium and ovary, PLAP can also be found in tissue extracts of benign conditions of the endometrium and myometrium. We detected slightly elevated levels of PLAP in patients with myoma, raised levels in 2/2 patients with endometrial polyps and high values in 2/2 patients with glandulocystic hyperplasia. Moreover, normal endometrium and fragments of normal Fallopian tube also contained fairly high amounts of endogenous PLAP. These results have important implications with regard to the possible use of PLAP as a tumour marker or of anti-PLAP monoclonal antibodies in radioimmunolocalization or radioimmunotherapy.

Prolonged skin graft survival by administration of anti-CD80 monoclonal antibody with cyclosporin A.

Costimulation via the B7/CD28 pathway is an important signal for the activation of T cells. Maximal inhibition of T-cell activation and the induction of alloantigen-specific nonresponsiveness in vitro was achieved using anti-CD80 monoclonal antibody (mAb) in combination with cyclosporin A (CsA). Based on this knowledge, the efficacy of the prophylactic treatment of anti-CD80 mAb and CsA on allogeneic skin graft survival was tested in a preclinical rhesus monkey model. No side effects have been observed. Administration of anti-CD80 mAb resulted in high mAb serum levels that decreased to undetectable values around day 7. At the same time, the anti-mouse antibody response started to develop. The anti-CD80 mAb bound to peripheral blood mononuclear cells and was detectable in lymph node and grafted skin during the treatment period. The skin graft survival time of untreated or suboptimally CsA-treated rhesus monkeys was 10 days. Treatment with CsA (blood levels of 100-160 ng/ml) in combination with anti-CD80 mAb (0.5 mg/kg) resulted in a significantly increased skin graft survival time to 14 days. Eventually, skin grafts in all rhesus monkeys were rejected, which coincided with an increase in helper and cytotoxic T-cell frequency and induction of an antibody response directed against the donor antigens. Therefore, treatment of anti-CD80 mAb in combination with CsA has significant immunosuppressive potency, but was unable to induce donor-specific nonresponsiveness in skin graft recipients.

Screening of sera and tumor extracts of cancer patients using a monoclonal antibody directed against human placental alkaline phosphatase.

A sensitive endogenous enzyme immunoassay involving an anti-human placental alkaline phosphatase (PLAP) monoclonal antibody was used in the screening of sera and tumor extracts of patients with various types of cancer. In sera of breast cancer patients an incidence of 5.2% was recorded. This value rose to 43% when tumor extracts were analyzed. For lung and bronchial cancers we found 11.2% seropositive patients. On several occasions a good correlation was observed between the PLAP determinations and the histopathological staging of tumor tissue.

Identification of two amino acids in activin A that are important for biological activity and binding to the activin type II receptors.

Activins are members of the transforming growth factor-beta family of growth and differentiation factors. In this paper, we report the results of a structure-function analysis of activin A. The primary targets for directed mutagenesis were charged, individual amino acids located in accessible domains of the protein, concentrating on those that differ from transforming growth factor-beta2, the x-ray crystal structure of which is known. Based on the activities of the recombinant activin mutants in two bioassays, 4 out of 39 mutant proteins (D27K, K102A, K102E, and K102R) produced in a vaccinia virus system were selected for further investigation. After production in insect cells and purification of these four mutants to homogeneity, they were studied in bioassays and in cross-linking experiments involving transfected receptor combinations. Mutant D27K has a 2-fold higher specific bio-activity and binding affinity to an ActRIIA/ALK-4 activin receptor complex than wild type activin, whereas mutant K102E had no detectable biological activity and did not bind to any of the activin receptors. Mutant K102R and wild type activin bound to all the activin receptor combinations tested and were equipotent in bioassays. Our results with the Lys-102 mutants indicate that the positive charge of amino acid 102 is important for biological activity and type II receptor binding of activins.

Truncated activin type II receptors inhibit bioactivity by the formation of heteromeric complexes with activin type I. receptors.

Truncated activin type II receptors have been reported to inhibit activin receptor signaling in Xenopus embryos, although the mechanism of action for this effect has not been fully understood. In the present study we demonstrate that in P19 embryonal carcinoma cells both the induction of the activin responsive 3TP-lux reporter construct and the inhibition of retinoic acid-induced neuronal differentiation by activin are blocked by expression of a truncated activin receptor. To reveal the mechanism of action of truncated activin receptors, the interaction between different activin receptors has been investigated upon coexpression in COS cells followed by cross-linking of 125I-activin A and subsequent immunoprecipitation. Complexes between a truncated activin type IIA receptor and activin type IA and type IB receptors can be formed, as demonstrated by coimmunoprecipitation of these type I receptors with the truncated activin type IIA receptor. Other type I receptors known as ALK-1 and ALK-6 also coimmunoprecipitate with the truncated type IIA receptor, whereas ALK-3 and ALK-5 do not. Furthermore, the activin type IIB2 receptor does not coimmunoprecipitate with the truncated type IIA receptor, but decreases activin binding to the truncated type IIA receptor. In double immunoprecipitation experiments with cell lysates from COS cells, in which full-length activin type IIA and type IIB2 receptors were cotransfected, no interaction between these receptors was found. In contrast, homomeric complexes of full-length activin type IIA receptors were detected. These results implicate that truncated activin receptors can interfere with activin signaling by interacting with activin type I receptors. Additionally, truncated activin type IIB2 receptors might also interfere with type IIA receptor signaling by decreasing activin binding to the type IIA receptor and therefore might be more potent in inhibiting activin signal transduction. Furthermore, our data indicate that truncated type IIA receptors can interact with other type I receptors and as such might inhibit signal transduction by type I receptors other than activin type IA and type IB receptors.