Measurement of the solar gravitational deflection of radio waves using geodetic very-long-baseline interferometry data, 1979-1999.

We used very-long-baseline interferometry (VLBI) to measure the deflection by the Sun of radio waves emanating from distant compact radio sources. This bending is characterized in the parametrized post-Newtonian formalism by gamma, which is unity in general relativity. Using a large geodetic VLBI data set, we obtained gamma=0.9998(3)+/-0.0004(5) (estimated standard error). We found no systematic biases from our analysis of subgroups of data.

Anti-endothelial cell antibodies in Behçet's disease.

OBJECTIVE: Circulating antibodies that bind to human endothelial cells cultured in vitro have been detected in a variety of diseases, including Behçet's disease. In this disorder the reported prevalence of AECA has varied widely. One likely source of variability is the ELISA assay itself, in which differing conditions and reagents have been used in different reports. METHODS: We have re-examined the frequency of AECA in 132 Turkish Behçet's patients and 50 healthy Turkish controls, comparing several different methods of preparing the target endothelial cells. Human umbilical vein endothelial cells (HUVEC) were used either: 1) fresh and non-treated, 2) fixed, or 3) TNF alpha-stimulated. All stages of the procedures were performed at room temperature. RESULTS: In Behçet's patients, using fresh, non-treated HUVEC, 17 of 130 (13.1%) and 9 of 132 (6.8%) sera were positive for IgG- and IgM-AECA, respectively. However, among 50 normal controls, 2 (4.0%) had IgG-positive and 4 (8.0%) had IgM-positive ELISAs under the same conditions. The difference in the frequency of positives between patients and controls was not statistically significant. Fixed HUVEC and TNF alpha-treated HUVEC gave similar results as well. When group means were examined, only the mean for IgG-AECA determined with TNF alpha-stimulated HUVEC reached statistical significance. CONCLUSION: The discrepancy between our data and earlier reports in the literature probably reflects the methodological differences alluded to, and highlights the difficulties in interpreting ELISA assays for AECA.

Role of vitamins in skin care.

Therapeutic (vitamins A and D, and their analogs) and antioxidant (vitamins C, E, and coenzyme Q) vitamins play an increasing role in skin care. Their benefits range from skin conditions such as acne and psoriasis to the protection against environmental insults.

Filamins as integrators of cell mechanics and signalling.

Filamins are large actin-binding proteins that stabilize delicate three-dimensional actin webs and link them to cellular membranes. They integrate cellular architectural and signalling functions and are essential for fetal development and cell locomotion. Here, we describe the history, structure and function of this group of proteins.

Inhibition of melanosome transfer results in skin lightening.

The chemical basis of melanogenesis is well documented, but the mechanism of melanosome transfer and the regulation of pigmentation by keratinocyte-melanocyte interactions are not well understood. Therefore we examined the effects of serine protease inhibitors on skin pigmentation and found that the protease-activated receptor 2, expressed on keratinocytes, may regulate pigmentation via keratinocyte-melanocyte interactions. Here we show that modulation of protease-activated receptor 2 activation affects melanosome transfer into keratinocytes, resulting in changes in pigment production and deposition. SLIGRL, the protease-activated receptor 2 activating peptide, enhanced melanosome ingestion by keratinocytes, thus increasing pigment deposition. RWJ-50353, a serine protease inhibitor, led to reduced pigment deposition in melanocytes and depigmentation. Electron microscopy studies illustrated an accumulation of immature melanosomes inside melanocytes and abnormal dendrite dynamics in RWJ-50353-treated epidermal equivalents. RWJ-50353 induced a visible and dose-dependent skin lightening effect in the dark-skinned Yucatan swine. Examinations by electron microscopy indicated that the in vivo transfer of melanosomes from melanocytes to keratinocytes was affected. Our data suggest that modulation of keratinocyte-melanocyte interactions via the protease-activated receptor 2 pathway affects melanosome transfer. The use of RWJ-50353 to modulate protease-activated receptor 2 activation could lead to a new class of depigmenting agents.

Transfer of nonassisted hatched and hatching human blastocysts after in vitro fertilization.

OBJECTIVE: To determine the feasibility of performing blastocyst transfer 6 days after oocyte insemination. DESIGN: Retrospective clinical study. SETTING: University-based IVF center. PATIENT(S): All cases of IVF over a 1-year span of time (June 1998-1999) in which seven 2PN embryos were available for transfer. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Implantation, pregnancy, and multiple pregnancy rates. RESULT(S): Transfer of blastocysts on days 5 and 6 resulted in implantation rates of 69% and 33% (P=0.0006), clinical pregnancy rates of 89% and 59% (P=0.05), and multiple pregnancy rates of 39% and 10% (P=0.03), respectively. In cases in which blastocysts were spontaneously hatching or hatched on day 6 (9% of embryos), implantation and pregnancy rates were 52% and 80%, respectively. Embryos were successfully frozen in the hatched or hatching state with resultant clinical pregnancies. CONCLUSION(S): Transfer of embryos can be delayed to day 6 after oocyte insemination at which time a small percentage of embryos will hatch. Hatching of embryos by day 6 is a favorable prognostic factor for IVF outcome. Embryos that fail to hatch by day 6 may have a lower implantation potential. Difficulty with hatching embryos sticking to the transfer catheter was not encountered. Furthermore, hatching and hatched embryos can be frozen and with subsequent transfer result in pregnancies.

Using videotaped specimens to test quality control in a computer-assisted semen analysis system.

OBJECTIVE: To determine the feasibility of using semen samples previously recorded on videotape for intralaboratory and interlaboratory quality control of computer-assisted semen analysis (CASA) systems. DESIGN: Blinded, controlled study. SETTING: Pooled semen specimens from two normal human volunteers in an academic research environment. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Semen parameters from a videotape analyzed internally and by four external laboratories. RESULT(S): Preliminary experiments designed to examine intralaboratory variation by repeated analysis of semen samples recorded on videotape revealed some significant differences for every variable examined. When these data were analyzed by using the larger biologic error caused by subsampling, no significant differences were found for any of the variables examined. When either a standard set or the specific laboratories' sets of parameters were used to analyze the same videotaped semen specimen, no statistically significant differences were detected for sperm concentration for motility among the five laboratories after the biological error caused by subsampling was applied to results. CONCLUSION(S): These data strongly suggest that videotaped semen specimens can serve as quality control for intralaboratory and interlaboratory testing of CASA equipment as long as the biologic error caused by subsampling is used to compare results.

The protease-activated receptor 2 regulates pigmentation via keratinocyte-melanocyte interactions.

Close association exists between melanocytes, the pigment melanin-producing cells in the body, and their neighboring keratinocytes. Keratinocytes are the pigment recipients and skin pigmentation is the result of this interaction. While the chemical basis of melanin production (melanogenesis) is well documented, the molecular mechanism of melanosome transfer needs to be elucidated. We are now providing first evidence that the protease-activated receptor 2 (PAR-2) expressed on keratinocytes, but not on melanocytes, is involved in melanosome transfer and therefore may regulate pigmentation. Activation of PAR-2 with trypsin or with the peptide agonist SLIGRL induced pigmentation in both two- and three-dimensional cocultures of keratinocytes and melanocytes, but not in cocultures that were spatially separated, indicating the need for intimate cell-cell contact. Topical application of SLIGRL on human skin transplanted on SCID mice resulted in a visible skin darkening. Histological examination revealed increased deposits of melanin in the keratinocytes. Inhibition of PAR-2 activation by RWJ-50353, a serine protease inhibitor, resulted in depigmentation and changes in expression of melanogenic-specific genes. Keratinocyte-melanocyte contact was essential for this depigmenting effect. Topical application of this inhibitor induced lightening of the dark skin Yucatan swine, which was confirmed by histochemical analysis. The results presented here suggest a novel mechanism for the regulation of pigmentation, mediated by the activation or inhibition of the keratinocyte receptor PAR-2.

Evaluation of endothelial cell migration with a novel in vitro assay system.

In this study we introduce a novel in vitro 'oil-drop' assay system for the measurement of endothelial cell (EC) migration, based on the original concept of the Teflon fence assay (Pratt et al., 1984; Am. J. Pathol. 117: 349-354). An aliquot of 15-20,000 human umbilical vein EC (HUVEC) is pipetted through a layer of mineral oil. The cells readily attach, spread and migrate on the surface of a matrix-coated tissue culture dish as a confluent circular monolayer. Migration is measured as the net increase in the total area covered at 24 hours. We have used this system to quantify EC migration on matrices composed of a mixture of type I collagen and either von Willebrand factor (vWF) or fibronectin (FN) in the presence or absence of tumor necrosis factor alpha (TNFalpha). Plating efficiency on both vWF/collagen and FN/collagen, measured by counting cells after attachment and spreading, is about 80%. With this method, migration on vWF/collagen was about 6.4 mm(2) and 5.3 mm(2) for TNFalpha-treated and untreated HUVEC, respectively. HUVEC migration on FN/collagen was slightly greater - 6.4 mm(2) and 6.5 mm(2) with and without TNFalpha treatment, respectively. During the 24 hour time period, HUVEC numbers increased 30-40% on vWF/collagen, and 60-80% on FN/collagen, with increased proliferation observed with TNF-alpha treatment. EC proliferation could be completely inhibited by 2 mM hydroxyurea. This assay system has proven useful in our studies to quantify cell migration and proliferation.

Glycoprotein Ibalpha can mediate endothelial cell migration on von Willebrand factor-containing substrata.

We have previously shown that, in addition to the vitronectin receptor (VNR, alpha(v)beta(3)), the GP Ib complex can participate in endothelial cell (EC) attachment to von Willebrand Factor (vWF) (D. A. Beacham, M. S. Cruz, and R. I. Handin, 1995, Thromb. Haemostas. 73, 309-317; D. A. Beacham, L.-P. Tran, and S. S. Shapiro, 1997, Blood 89, 4071-4077). In this study we have investigated the functional roles of these vWF receptors in the migration of untreated and TNFalpha-treated EC on vWF, a mixture of vWF and type I collagen, and on vitronectin (VN). In agreement with previous studies (D. I. Leavesley, M. A. Schwartz, M. Rosenfeld, and D. A. Cheresh, 1993, J. Cell Biol. 121, 163-170), the migration of untreated and TNFalpha-treated EC on VN was dependent entirely on the VNR. Migration of untreated EC on vWF was inhibited 10-15% by recombinant vWF-A1, the GP Ibalpha-binding domain on vWF which abrogates the platelet GP Ibalpha-vWF interaction. In contrast, migration of TNFalpha-treated EC on vWF was inhibited 50-60% by vWF-A1 or the anti-GP Ibalpha mAb AS-7 but only 20% by the anti-VNR mAb LM609. On a mixed vWF-collagen substratum, vWF-A1 inhibited untreated EC migration by 45%, and TNFalpha-treated EC migration by 75%. The possible role of EC proliferation was eliminated, since hydroxyurea completely inhibited EC proliferation without reducing migration significantly. The anti-GP Ibalpha mAb Ib1 inhibited EC migration by 50%, but reduced proliferation by only 15%. Taken together, our data demonstrate that EC migration on vWF-containing substrata involves the GP Ib complex as well as the VNR and raises the possibility that the VNR and GP Ib act cooperatively in supporting EC migration.

Von Willebrand factor receptor GPIb alpha is expressed by human factor XIIIa-positive dermal dendrocytes and is upregulated by mast cell degranulation.

GPIb alpha, a glycoprotein component of the GPIb-IX-V complex, serves as a platelet membrane receptor that mediates adhesion to von Willebrand factor normally present in the vascular subendothelium. Recent data have demonstrated that GPIb alpha is not restricted to platelets, but is also expressed by endothelium in vitro. In this study, we describe the expression and distribution of GPIb alpha in normal adult and neonatal human skin. GPIb alpha is present, as detected by immunohistochemistry, on endothelial cells and on highly dendritic cells localized within the perivascular space, dermal-epidermal junction, and reticular dermis. By dual-labeling immunofluorescence and confocal microscopy, GPIb alpha-positive cells within the dermal interstitium are demonstrated to represent factor XIIIa-positive dermal dendrocytes. In organ cultures of neonatal human foreskin, mast cell degranulation induced by either substance P or compound 48/80 resulted in transiently increased GPIb alpha expression by dermal dendrocytes. Because the GPIb-IX-V complex plays a part in regulating hemostasis and may be important for cellular interactions with extracellular matrix molecules, these data provide additional insight into the potential function of FXIIIa-positive dermal dendrocytes in skin remodeling and repair.

Arterial shear stress stimulates surface expression of the endothelial glycoprotein Ib complex.

Exposure to shear stress has been shown to alter the expression of a number of surface components of cultured endothelial cells (EC). However, relatively few studies have examined the status of human EC surface proteins after prolonged flow, more closely corresponding to the steady state in vivo. Since the promoter region of glycoprotein (Gp) Ib alpha contains several copies of a putative shear stress response element, 5'-GAGACC-3', we investigated the response of cultured human umbilical vein EC (HUVEC) GpIb alpha to shear stress over a 72 h time period. In response to 30 dynes/cm2 of shear stress, total cell content of GpIb alpha protein was markedly increased above static levels at 7 and 24 h, as determined immunohistochemically. Western blot analysis of whole cell lysates after 24, 48, and 72 h of shear treatment demonstrated a 2.4-, 4.1-, and 3.2-fold increase in total GpIb alpha protein, respectively. Cell surface protein expression of GpIb alpha increased 2.5-fold at 7 h, as measured by quantitative immunofluorescence, and remained at that level at 24 h. After 48 h of shear stress, cell surface GpIb alpha, GpIX, and GpV, analyzed by flow cytometric analysis, were further increased over the levels observed at 24 h. The increase in cell surface membrane expression of GPIb alpha at 24, 48, and 72 h was confirmed by immunoprecipitation of biotinylated surface proteins. No upregulation of GpIb alpha was noted after exposure to shear stress of 1-3 dynes/cm2. These observations imply that under steady-state arterial shear conditions endothelial expression of the GpIb complex is significantly greater than observed in static EC cultures, and raise the possibility of a more important role for this complex under flow, rather than static conditions.

Embryonic stem cell lines derived from human blastocysts.

Human blastocyst-derived, pluripotent cell lines are described that have normal karyotypes, express high levels of telomerase activity, and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages. After undifferentiated proliferation in vitro for 4 to 5 months, these cells still maintained the developmental potential to form trophoblast and derivatives of all three embryonic germ layers, including gut epithelium (endoderm); cartilage, bone, smooth muscle, and striated muscle (mesoderm); and neural epithelium, embryonic ganglia, and stratified squamous epithelium (ectoderm). These cell lines should be useful in human developmental biology, drug discovery, and transplantation medicine.

Human beta-filamin is a new protein that interacts with the cytoplasmic tail of glycoprotein Ibalpha.

We have cloned and sequenced a 9.4-kilobase cDNA specifying a new 280-kDa protein interacting with the cytoplasmic tail of glycoprotein (Gp) Ibalpha and showing considerable homology to actin-binding protein 280 (ABP-280) and chicken retinal filamin. We term this protein human beta-filamin. The gene for beta-filamin localizes to chromosome 3p14.3-p21.1. beta-Filamin mRNA expression was observed in many tissues and in cultured human umbilical vein endothelial cells (HUVECs); only minimal expression was detected in platelets and the megakaryocytic cell line CHRF-288. Like ABP-280, beta-filamin contains an NH2-terminal actin-binding domain, a backbone of 24 tandem repeats, and two "hinge" regions. A polyclonal antibody to the unique beta-filamin first hinge sequence identifies a strong 280-kDa band in HUVECs but only a weak band in platelets, and stains normal human endothelial cells in culture and in situ. We have confirmed the interaction of beta-filamin and GpIbalpha in platelet and HUVEC lysates. In addition, using two-hybrid analysis with deletion mutants, we have localized the binding domain for GpIbalpha in beta-filamin to residues 1862-2148, an area homologous to the GpIbalpha binding domain in ABP-280. beta-Filamin is a new member of the filamin family that may have significance for GpIbalpha function in endothelial cells and platelets.

Cardiolipin binding a light chain from lupus-prone mice.

Autoantibodies in systemic lupus erythematosus react with multiple epitopes on highly conserved molecules such as nucleic acids, cytoskeletal proteins, phospholipids, and phospholipid-binding proteins. Analysis of the heavy- and light-chain variable sequences (VH and VL) has shown that a restricted set of V genes gives rise to these autoantibodies. Several monoclonal antibodies were developed from a strain of mouse prone to lupus (F1 male NZW x BXSB). Two of these antibodies, A1.72 and A1.84, reacted directly with cardiolipin and their VH and VL sequences were analyzed. Surprisingly, these two antibodies had identical light-chain variable sequences despite having substantially different heavy-chain variable sequences. This VL sequence, VL 72/84 was 97% identical with the germ-line sequences with only four single nucleotide substitutions. When this VL sequence was shuffled with the VH sequence of other monoclonal antibodies and expressed as single chain variable fragment (scFv) in Escherichia coli, it imparted cardiolipin-binding activity to the hybrids. Furthermore, the VL 72/84 sequence, when expressed alone without any VH sequence, also bound to cardiolipin. The antibodies and their recombinant fragments were immunoaffinity-purified on cardiolipin liposomes. The dissociation constant of the light chain for cardiolipin was similar to the intact molecule (21 +/- 0.01 vs 20 +/- 0.03 nM). These studies demonstrate that the VL sequence alone, in the absence of any other immunoglobulin domains, can mediate cardiolipin binding, raising the possibility that antigen specificity of certain antibodies may exclusively reside in their light-chain sequences.

Lupus anticoagulants and antiphospholipid antibodies.

In recent years, clinical syndromes involving lupus anticoagulants and antiphospholipid antibodies have come into increasing clinical prominence. Since the discovery that most antiphospholipid antibodies require the presence of anionic phospholipid-binding proteins such as B2-glycoprotein I and prothrombin, a large number of studies have attempted to delineate the specificity of these antibodies. Several mechanisms have been proposed to explain the hypercoagulable state associated with these antibodies. This review attempts to summarize these data and the challenges that confront efforts to delineate the pathogenesis of the prothrombotic state associated with the presence of these antibodies.

Human endothelial cells in culture and in vivo express on their surface all four components of the glycoprotein Ib/IX/V complex.

The platelet glycoprotein Ib (GpIb) complex is composed of four polypeptides: the disulfide-linked GpIb alpha and GpIb beta and the noncovalently associated GpIX and GpV. GpIb alpha contains binding sites for von Willebrand factor and for thrombin and mediates platelet adhesion to the subendothelium under conditions of high shear stress. We have previously shown the presence of GpIb alpha and GpIb beta mRNA and protein in cultured human umbilical vein endothelial cells (HUVECs) as well as the presence of GpIb alpha mRNA and protein in tonsillar endothelium. We, therefore, probed ECs for the presence of the other components of the GpIb/IX/V complex. We have identified the presence of GpIX and GpV mRNA in cultured HUVEC monolayers. The sequence of HUVEC GpIX cDNA was identical to the previously published human erythroleukemia (HEL) cell GpIX cDNA sequence. Two species of GpV mRNA, one of 3 kb and one of 4.4 kb, were found in HUVECs, whereas HEL cells displayed only the 4.4-kb species and the megakaryocytic cell line CHRF-288 contained only the 3-kb species. We previously showed that EC GpIb alpha protein is identical in molecular weight to platelet GpIb alpha. HUVEC GpIb beta, in contrast to its platelet counterpart, has a molecular weight of 50 kD and forms a correspondingly larger disulfide-bonded complex with EC GpIb alpha. The molecular weights of GpIX and GpV were 22 and 88 kD, respectively, identical to the corresponding platelet polypeptides. Furthermore, we have identified all four components of the complex in tonsillar vessels. Using flow cytometry, we have established that all four polypeptides of the GpIb/IX/V complex are expressed on the surface membranes of cultured HUVECs and adult aortic ECs. Furthermore, using two-color fluorescence, we have shown that all ECs expressing GpIb alpha also express GpIX and GpV on their surface. The ratio of GpIb alpha:GpIX:GpV is 1:1:0.5, which is identical to the ratio present in platelets. None of the polypeptides of the GpIb complex could be identified on the surface of human smooth muscle cells or lymphocytes. The presence of all members of the GpIb complex in the EC membrane suggests that this complex may play a role in endothelial function in vivo.