The FGFRL1 receptor is shed from cell membranes, binds fibroblast growth factors (FGFs), and antagonizes FGF signaling in Xenopus embryos

FGFRL1 (fibroblast growth factor receptor like 1) is the fifth and most recently discovered member of the fibroblast growth factor receptor (FGFR) family. With up to 50% amino acid similarity, its extracellular domain closely resembles that of the four conventional FGFRs. Its intracellular domain, however, lacks the split tyrosine kinase domain needed for FGF-mediated signal transduction. During embryogenesis of the mouse, FGFRL1 is essential for the development of parts of the skeleton, the diaphragm muscle, the heart, and the metanephric kidney. Since its discovery, it has been hypothesized that FGFRL1 might act as a decoy receptor for FGF ligands. Here we present several lines of evidence that support this notion. We demonstrate that the FGFRL1 ectodomain is shed from the cell membrane of differentiating C2C12 myoblasts and from HEK293 cells by an as yet unidentified protease, which cuts the receptor in the membrane-proximal region. As determined by ligand dot blot analysis, cell-based binding assays, and surface plasmon resonance analysis, the soluble FGFRL1 ectodomain as well as the membrane-bound receptor are capable of binding to some FGF ligands with high affinity, including FGF2, FGF3, FGF4, FGF8, FGF10, and FGF22. We furthermore show that ectopic expression of FGFRL1 in Xenopus embryos antagonizes FGFR signaling during early development. Taken together, our data provide strong evidence that FGFRL1 is indeed a decoy receptor for FGFs.

Detection of gelatinolytic activity in developing basement membranes of the mouse embryo head by combining sensitive in situ zymography with immunolabeling

Genetic evidence indicates that the major gelatinases MMP-2 and MMP-9 are involved in mammalian craniofacial development. Since these matrix metalloproteinases are secreted as proenzymes that require activation, their tissue distribution does not necessarily reflect the sites of enzymatic activity. Information regarding the spatial and temporal expression of gelatinolytic activity in the head of the mammalian embryo is sparse. Sensitive in situ zymography with dye-quenched gelatin (DQ-gelatin) has been introduced recently; gelatinolytic activity results in a local increase in fluorescence. Using frontal sections of wild-type mouse embryo heads from embryonic day 14.5-15.5, we optimized and validated a simple double-labeling in situ technique for combining DQ-gelatin zymography with immunofluorescence staining. MMP inhibitors were tested to confirm the specificity of the reaction in situ, and results were compared to standard SDS-gel zymography of tissue extracts. Double-labeling was used to show the spatial relationship in situ between gelatinolytic activity and immunostaining for gelatinases MMP-2 and MMP-9, collagenase 3 (MMP-13) and MT1-MMP (MMP-14), a major activator of pro-gelatinases. Strong gelatinolytic activity, which partially overlapped with MMP proteins, was confirmed for Meckel's cartilage and developing mandibular bone. In addition, we combined in situ zymography with immunostaining for extracellular matrix proteins that are potential gelatinase substrates. Interestingly, gelatinolytic activity colocalized precisely with laminin-positive basement membranes at specific sites around growing epithelia in the developing mouse head, such as the ducts of salivary glands or the epithelial fold between tongue and lower jaw region. Thus, this sensitive method allows to associate, with high spatial resolution, gelatinolytic activity with epithelial morphogenesis in the embryo.

[Guidance of interventions in subintimal recanalization and fenestration of dissection membranes using a novel dual-lumen intravascular ultrasound catheter]

PURPOSE: To evaluate the feasibility and effectiveness of IVUS-guided puncture for gaining controlled target lumen reentry in subintimal recanalization of chronic iliac/femoral artery occlusions and in fenestration of aortic dissections. MATERIALS AND METHODS: Between 5/2004 and 12/2005 12 consecutive patients (7 male, 5 female; mean age 64.6 +/- 12.0 years) with chronic critical limb ischemia and ischemic complications of aortic dissection were treated using the Pioneer catheter. This 6.2-F dual-lumen catheter combines a 20-MHz IVUS transducer with a pre-shaped extendable, hollow 24-gauge nitinol needle. This coaxial needle allows real-time IVUS-guided puncture of the target lumen and after successful reentry a 0.014" guidewire may be advanced through the needle into the target lumen. 7 patients were treated for aortic dissection and 5 patients (with failed previous attempts at subintimal recanalization) for chronic arterial occlusion. Patients with aortic dissection (5 type A dissections, 2 type B dissections) had developed renal ischemia (n = 2), renal and mesenteric ischemia (n = 2), or low extremity ischemia (n = 3). Patients with chronic arterial occlusions (2 common iliac artery occlusions, 3 superficial femoral artery occlusions) experienced ischemic rest pain (n = 4), and a non-healing foot ulcer (n = 1). RESULTS: The technical success rate using the Pioneer catheter was 100%. The recanalization/fenestration time was 37 +/- 12 min. Procedure-related complications did not occur. In 10 cases a significant improvement of clinical symptoms was evident. One patient with aortic dissection and ischemic paraplegia required subsequent surgical intervention. One patient had persistent ischemic rest pain despite successful recanalization of a superficial femoral artery occlusion. CONCLUSION: The Pioneer catheter is a reliable device which may be helpful for achieving target lumen reentry in subintimal recanalization of chronic occlusions and in fenestration of aortic dissections.

The effect of rhBMP-2 around endosseous implants with and without membranes in the canine model

BACKGROUND: Bone morphogenetic protein (BMP) is a potent differentiating agent for cells of the osteoblastic lineage. It has been used in the oral cavity under a variety of indications and with different carriers. However, the optimal carrier for each indication is not known. This study examined a synthetic bioabsorbable carrier for BMP used in osseous defects around dental implants in the canine mandible. METHODS: Twelve canines had their mandibular four premolars and first molar teeth extracted bilaterally. After 5 months, four implants were placed with standardized circumferential defects around the coronal 4 mm of each implant. One-half of the defects received a polylactide/glycolide (PLGA) polymer carrier with or without recombinant human BMP-2 (rhBMP-2), and the other half received a collagen carrier with or without rhBMP-2. Additionally, one-half of the implants were covered with a non-resorbable (expanded polytetrafluoroethylene [ePTFE]) membrane to exclude soft tissues. Animals were sacrificed either 4 or 12 weeks later. Histomorphometric analysis included the percentage of new bone contact with the implant, the area of new bone, and the percentage of defect fill. This article describes results with the PLGA carrier. RESULTS: All implants demonstrated clinical and radiographic success with the amount of new bone formed dependent on the time and presence/absence of rhBMP-2 and presence/absence of a membrane. The percentage of bone-to-implant contact was greater with rhBMP-2, and after 12 weeks of healing, there was approximately one-third of the implant contacting bone in the defect site. After 4 weeks, the presence of a membrane appeared to slow new bone area formation. The percentage of fill in membrane-treated sites with rhBMP-2 rose from 24% fill to 42% after 4 and 12 weeks, respectively. Without rhBMP-2, the percentage of fill was 14% rising to 36% fill, respectively. CONCLUSIONS: After 4 weeks, the rhBMP-2-treated sites had a significantly higher percentage of contact, more new bone area, and higher percentage of defect fill than the sites without rhBMP-2. After 12 weeks, there was no significant difference in sites with or without rhBMP-2 regarding percentage of contact, new bone area, or percentage of defect fill. In regard to these three outcomes, comparing the results with this carrier to the results reported earlier with a collagen carrier in this study, only the area of new bone was significantly different with the collagen carrier resulting in greater bone than the PLGA carrier. Thus, the PLGA carrier for rhBMP-2 significantly stimulated bone formation around dental implants in this model after 1 month but not after 3 months of healing. The use of this growth factor and carrier combination appears to stimulate early bone healing events around the implants but not quite to the same degree as a collagen carrier.

Effect of two different bioabsorbable collagen membranes on guided bone regeneration: a comparative histomorphometric study in the dog mandible

BACKGROUND: This study compared bone regeneration following guided bone regeneration with two bioabsorbable collagen membranes in saddle-type bone defects in dog mandibles. METHODS: Three standardized defects were created, filled with bone chips and deproteinized bovine bone mineral (DBBM), and covered by three different methods: control = no membrane; test 1 = collagen membrane; and test 2 = cross-linked collagen membrane (CCM). Each side of the mandible was allocated to one of two healing periods (8 or 16 weeks). The histomorphometric analysis assessed the percentage of bone, soft tissue, and DBBM in the regenerate; the absolute area in square millimeters of the bone regenerate; and the distance in millimeters from the bottom of the defect to the highest point of the regenerate. RESULTS: In the 8-week healing group, two dehiscences occurred with CCM. After 8 weeks, all treatment modalities showed no significant differences in the percentage of bone regenerate. After 16 weeks, the percentage of bone had increased for all treatment modalities without significant differences. For all groups, the defect fill height increased between weeks 8 and 16. The CCM group showed a statistically significant (P = 0.0202) increase over time and the highest value of all treatment modalities after 16 weeks of healing, CONCLUSIONS: The CCM showed a limited beneficial effect on bone regeneration in membrane-protected defects in dog mandibles when healing was uneventful. The observed premature membrane exposures resulted in severely compromised amounts of bone regenerate. This increased complication rate with CCM requires a more detailed preclinical and clinical examination before any clinical recommendations can be made.

Effect of two bioabsorbable barrier membranes on bone regeneration of standardized defects in calvarial bone: a comparative histomorphometric study in pigs

BACKGROUND: The effect of two different bioabsorbable collagen membranes on bone regeneration was assessed in standardized, membrane-protected calvarial defects in pigs. METHODS: Two standardized defect types (6 x 6 x 6 mm and 9 x 9 x 9 mm) were produced in the calvaria of pigs: empty defects without a membrane (group 1; eight defects per size); defects filled with deproteinized bovine bone mineral (DBBM) without a membrane (group 2; eight defects per size); defects filled with DBBM and covered by a collagen membrane (group 3; eight defects per size); and defects filled with DBBM and covered by a cross-linked collagen membrane (CCM) (group 4; eight defects per size). Sacrifice took place 16 weeks after surgery, and the following parameters were analyzed: descriptive histology; semiquantitative histology (SQH), assessing bone regeneration in the whole defect area; and histomorphometric analysis of the percentage of bone and DBBM in the regenerated area at three different depth levels of the defect. RESULTS: Using SQH, both membrane types resulted in significantly better bone regeneration compared to groups 1 and 2, irrespective of the defect size (P <0.005), with no difference between the two membranes. In the histomorphometric analysis, the layer immediately below the surface exhibited a significantly higher percentage of bone in groups 3 (27%) and 4 (36%) versus the two other groups for the 9 x 9 x 9-mm defects. No such differences were apparent for the 6 x 6 x 6-mm defects or the other two depth levels (bottom and middle layer) for either defect size. CONCLUSIONS: The two collagen membranes tested significantly enhanced bone regeneration, especially in the superficial level of the calvarial bone defects. The prototype CCM did not provide any further advantage in the present animal model.

Differential expression and activity of 11beta-hydroxysteroid dehydrogenase in human placenta and fetal membranes from pregnancies with intrauterine growth restriction

OBJECTIVES: To study the expression and the function of the 11beta-hydroxysteroid dehydrogenase enzyme 1 (11beta-HSD1) and 2 (11beta-HSD2) in placenta and the fetal membranes from pregnancies with intrauterine growth restriction (IUGR) and from controls. METHODS: Amnion, chorion, decidua and cotyledon were separated from placenta; mRNA was analyzed by TaqMan real-time technology and proteins by Western blot; enzyme activities were measured by the conversion of 3H-cortisol to 3H-cortisone and vice versa. RESULTS: Predominant mRNA expression (p < 0.001) was found for 11beta-HSD1 in chorion and for 11beta-HSD2 in decidua and cotyledon. In pregnancies with IUGR, 11beta-HSD1 was upregulated in chorion (mean DeltaCt 11beta-HSD:18S mRNA 193.5 vs. 103.0 in controls respectively, p < 0.05) and 11beta-HSD2 was downregulated in decidua (mean DeltaCt 11beta-HSD2:18S mRNA 0.18 vs. 15.88 in controls respectively, p < 0.05). 11beta-HSD1 protein levels were reduced in amnion and 11beta-HSD1 and 11beta-HSD2 oxidase activity in decidua and cotyledon were reduced from pregnancies with IUGR. CONCLUSION: Reduced synthesis or activity of 11beta-HSD1 or 2 in cases of IUGR is shown in some but not in all tissues. The local mRNA expression of 11beta-HSD1 in chorion may reflect a mechanism on the post-transcriptional gene regulation to stimulate the formation of cortisone in IUGR. To provoke increasing activity with oxidase stimulators could be a future therapy in cases of IUGR.

Complement Factor P in choroidal neovascular membranes of patients with age-related macular degeneration

To evaluate whether complement Factor P (properdin) was present in surgically removed choroidal neovascular membranes of patients with age-related macular degeneration (AMD) and to investigate whether associated pre- and postoperative clinical characteristics can be correlated.

GABARAP deficiency modulates expression of NaPi-IIa in renal brush-border membranes

Renal reabsorption of inorganic phosphate (P(i)) is mainly mediated by the Na(+)-dependent P(i)-cotransporter NaPi-IIa that is expressed in the brush-border membrane (BBM) of renal proximal tubules. Regulation and apical expression of NaPi-IIa are known to depend on a network of interacting proteins. Most of the interacting partners identified so far associate with the COOH-terminal PDZ-binding motif (TRL) of NaPi-IIa. In this study GABA(A) receptor-associated protein (GABARAP) was identified as a novel interacting partner of NaPi-IIa applying a membrane yeast-two-hybrid system (MYTH 2.0) to screen a mouse kidney library with the TRL-truncated cotransporter as bait. GABARAP mRNA and protein are present in renal tubules, and the interaction of NaPi-IIa and GABARAP was confirmed by using glutathione S-transferase pulldowns from BBM and coimmunoprecipitations from transfected HEK293 cells. Amino acids 36-68 of GABARAP were identified as the determinant for the described interaction. The in vivo effects of this interaction were studied in a murine model. GABARAP(-/-) mice have reduced urinary excretion of P(i), higher Na(+)-dependent (32)P(i) uptake in BBM vesicles, and increased expression of NaPi-IIa in renal BBM compared with GABARAP(+/+) mice. The expression of Na(+)/H(+) exchanger regulatory factor (NHERF)1, an important scaffold for the apical expression of NaPi-IIa, is also increased in GABARAP(-/-) mice. The absence of GABARAP does not interfere with the regulation of the cotransporter by either parathyroid hormone or acute changes of dietary P(i) content.

Probing the function of ionotropic and G protein-coupled receptors in surface-confined membranes

This article reports on recent electrical and optical techniques for investigating cellular signaling reactions in artificial and native membranes immobilized on solid supports. The first part describes the formation of planar artificial lipid bilayers on gold electrodes, which reveal giga-ohm electrical resistance and the insertion and characterization of ionotropic receptors therein. These membranes are suited to record a few or even single ion channels by impedance spectroscopy. Such tethered membranes on planar arrays of microelectrodes offer mechanically robust, long-lasting measuring devices to probe the influence of different chemistries on biologically important ionotropic receptors and therefore will have a future impact to probe the function of channel proteins in basic science and in biosensor applications. In a second part, we present complementary approaches to form inside-out native membrane sheets that are immobilized on micrometer-sized beads or across submicrometer-sized holes machined in a planar support. Because the native membrane sheets are plasma membranes detached from live cells, these approaches offer a unique possibility to investigate cellular signaling processes, such as those mediated by ionotropic or G protein-coupled receptors, with original composition of lipids and proteins.

Increased periodontal inflammation in women with preterm premature rupture of membranes

OBJECTIVES To evaluate possible differences in periodontal inflammatory, microbiological and clinical parameters between women with preterm premature rupture of membranes (PPROM) and controls with uncomplicated pregnancies. MATERIALS AND METHODS Fifty-six women (32 test (PPROM) and 24 controls (uncomplicated pregnancies)) were examined at three time-points (T1: gestational weeks 20-35, T2: within 48 h after parturition, T3: 4-6 weeks after parturition). The examinations included assessment of the Periodontal Screening Index, collection of gingival crevicular fluid (GCF) and subgingival as well as vaginal bacterial sampling. RESULTS Periodontal inflammation was found to be higher in the test compared with the control group (p < 0.05) and decreased over time in both groups (p < 0.05). Microbiological outcomes showed no intergroup differences (p > 0.05) in prevalence of bacteria, but a decrease in subgingival periodontopathogens from T1 to T2 in the test group (p < 0.05) was observed. Interleukin (IL)-1β levels in GCF at T2 were not different between groups (p > 0.05). In women with PPROM, GCF levels of IL-8 (p < 0.05) and C-reactive protein (p < 0.05) were lower and IL-10 levels higher (p < 0.05) compared with controls. CONCLUSIONS Periodontal inflammation is elevated during pregnancy and seems to be more pronounced in women with PPROM. CLINICAL RELEVANCE The findings of the present study revealed an association between periodontal inflammation and PPROM, thus emphasizing the importance of optimizing self-performed oral hygiene in pregnant women.

Treatment of Retained Fetal Membranes: Comparison of the Postpartum Period after Routine Treatment or Routine Treatment Including an Additional Phytotherapeutic Substance in Dairy Cattle in Switzerland

Background: The therapy of retained fetal membranes (RFM) is a controversial subject. In Switzerland, intrauterine antibiotics are routinely administered although their effect on fertility parameters is questionable. The objective of this study was to compare the post-partal period after a routine treatment of RFM in 2 groups: one group received a placebo additionally (A), whereas the other group received a phytotherapeutic substance (lime bark) (B) additionally. The routine treatment of RFM included an attempt to manually remove the fetal membranes (for a maximum of 5 min), intramuscular administration of oxytetracycline and intrauterine treatment with tetracycline. In case of an elevated rectal temperature (>39.0°C), an additional non-steroidal inflam-matory drug was allowed. Methods: Cows undergoing caesarean section, suffering from prolapse of the uterus, deep cervical or vaginal injuries, hypocalcaemia, and illnesses during the last 14 days before calving were excluded. Cows had to be more than 265 days pregnant. Only cows that were artificially inseminated after RFM were included. Group stratification was done according to the last number on the ear tag (even/uneven) with (n = 50) cows in group A and (n = 55) cows in group B. Results: The number of treatments after the initial treatment of RFM was not significantly different between groups. The median interval from calving to the first insemination was 77 days in group A compared to 82 days in group B (p = 0.72). The number of AI’s until conception was not significantly different between groups. The median number of days open was 89 days in group A compared to 96 days in group B (p = 0.57). The culling rate was not significantly different between groups. Conclusion: There was neither a difference between the groups concerning therapies within the first 50 days after RFM nor concerning the subsequent fertility variables.

Diagnosis and therapy of retained fetal membranes, puerperal metritis and clinical endometritis in cattle: Results of the Online-survey among Swiss practitioners. II. Puerperal metritis and clinical endometritis

The aim of the study was to obtain the diagnostic and therapeutic approach among Swiss practitioners in cows with puerperal metritis and clinical endometritis (part 2). All members of the Association for ruminant health were contacted per email via the newsletter. The survey was completed by 128 veterinarians, partially responded by 140 veterinarians. The following main symptoms of puerperal metritis were stated by the practitioners: purulent vaginal discharge, fever and reduced appetite. A vaginal and rectal examination was performed to diagnose the disease. Usually, an intrauterine treatment with tetracycline or cefapirin was done. Parenteral administration of tetracycline or penicillin was often combined with PGF(2α), NSAIDS or cortisone. Clinical endometritis was also diagnosed by vaginal and rectal examination and the main symptom indicated was purulent vaginal discharge. The therapy consisted of the administration of PGF(2α), uterine infusions predominantly with cefapirin, and rarely with parenteral administration of antibiotics. Further diagnostic tools were not used and normally cows were not rechecked. The success of the therapy of puerperal metritis and clinical endometritis was judged to be satisfactory to excellent.

Diagnosis and therapy of retained fetal membranes, puerperal metritis and clinical endometritis in cattle: Results of the Online-survey among Swiss practitioners. I Retained fetal membranes

The aim of this study was to obtain the diagnostic and therapeutic approach among Swiss practitioners in cows with retained fetal membranes (RFM) (part 1). All members of the Association for ruminant health were contacted per email via the newsletter. The survey was completed by 128 veterinarians, partially responded by 140 veterinarians. The manual removal of the fetal membranes is practiced by 129 of the responding veterinarians. Cows with/without fever are treated usually with intrauterine antibiotics. Cows with RFM with/without fever are most commonly treated parenterally with tetracycline or penicillin. The use of cephalosporins and quinolones in cows with fever is more common than in cows without fever. With the present results of the survey veterinarians should critically question the supposed benefits of the manual removal of the placenta and the use of antibiotics in cows with RFM.