Vitronectin regulates the synthesis and localization of urokinase-type plasminogen activator in HT-1080 cells.

The effect of extracellular matrix composition on the location, amount, and activity of cell-associated urokinase-type plasminogen activator was tested using HT-1080 cells adherent to either fibronectin or vitronectin. Specific immunoprecipitation of newly synthesized urokinase indicated that cells adherent to fibronectin synthesized 2-3-fold more urokinase than cells adherent to vitronectin. Complexes of urokinase and plasminogen activator inhibitor type 1 (PAI-1) were detected in cell layers of vitronectin-adherent but not fibronectin-adherent cells. Inhibition of PAI-1 using a neutralizing monoclonal antibody resulted in a 3-fold increase in urokinase enzymatic activity on vitronectin adherent cells. Urokinase activity on fibronectin adherent cells was only slightly increased following PAI-1 neutralization. Examination of both HT-1080 and normal human fibroblast cells by immunofluorescent microscopy localized urokinase-type plasminogen activator to discrete, focal areas underneath cells adherent to vitronectin. Urokinase was not detectable by immunofluorescence on cells adherent to fibronectin. The addition of exogenous prourokinase to locate urokinase receptors on adherent HT-1080 cells indicated that the focal localization of cell-surface urokinase resulted from the clustering of urokinase receptors following adhesion to vitronectin but not fibronectin-coated substrates. These results suggest that vitronectin can contribute to the control of cell-surface plasmin activity by regulating the synthesis of urokinase and directing the localization of urokinase receptors.     

Urokinase potentiates PDGF-induced chemotaxis of human airway smooth muscle cells

We investigated the chemotactic action of PDGF and urokinase on human airway smooth muscle (HASM) cells in culture. Cells were put in collagen-coated transwells with 8-micro m perforations, incubated for 4 h with test compounds, then fixed, stained, and counted as migrated nuclei by microscopy. Cells from all culture conditions showed some basal migration (migration in the absence of stimuli during the assay), but cells preincubated for 24 h in 10% FBS or 20 ng/ml PDGF showed higher basal migration than cells quiesced in 1% FBS. PDGF(BB), PDGF(AA), and PDGF(AB) were all chemotactic when added during the assay. PDGF chemotaxis was blocked by the phosphatidyl 3'-kinase inhibitor LY-294002, the MEK inhibitor U-0126, PGE(2), formoterol, pertussis toxin, and the Rho kinase inhibitor Y-27632. Urokinase alone had no stimulatory effect on migration of quiescent cells but caused a dose-dependent potentiation of chemotaxis toward PDGF. Urokinase also potentiated the elevated basal migration of cells pretreated in 10% FBS or PDGF. This potentiating effect of urokinase appears to be novel. We conclude that PDGF and similar cytokines may be important factors in airway remodeling by redistribution of smooth muscle cells during inflammation and that urokinase may be important in potentiating the response.     

Plasminogen activation by t-PA on the surface of human melanoma cells in the presence of alpha 2-macroglobulin secretion.

Several human melanoma cell lines produced tissue-type plasminogen activator (t-PA), as detected by zymography and immunocapture assay of culture media and cell lysates. Urokinase (u-PA) was found at only less than or equal to 1% the level of t-PA. Acid eluates of the cell surface indicated that the melanoma cells had t-PA bound on their surface, but no u-PA, and also had a very low capacity to bind exogenous u-PA. After incubation of the melanoma cells with 10% plasminogen-depleted fetal calf serum and human plasminogen, bound plasmin activity could be eluted from the cell surface with tranexamic acid, an analogue of lysine. This indicated that plasminogen was activated on the cell surface. The cell-surface plasmin formation was inhibited by an anti-catalytic monoclonal antibody to human t-PA, and not by an anti-catalytic antibody to u-PA. The melanoma cells also synthesized and secreted alpha 2-macroglobulin (alpha 2M), as shown by alpha 2M-specific mRNA in Northern blotting and detection of alpha 2M protein in conditioned cell culture media. The media were found to inhibit u-PA but not t-PA. This inhibition was related to their alpha 2M content, and immunoabsorption of alpha 2M removed the inhibitory activity. These studies suggest that t-PA can bind to the surface of melanoma cells and generate surface-bound plasmin. Because t-PA and cell-bound plasmin are unaffected by alpha 2M, t-PA may, in the case of melanoma cells, serve an analogous function to u-PA in supporting tumor cell invasion.     

Urokinase receptor (uPAR) regulates complement receptor 3 (CR3)-mediated neutrophil phagocytosis

Urokinase receptor (uPAR) associates in cis with complement receptor 3 (CR3). In the present study, we addressed whether this coupling regulates CR3-mediated phagocytosis. CR3-mediated attachment of iC3b-opsonized sheep red blood cells to human neutrophils and internalization of these cells were reduced by removal of cell-bound uPAR by phosphatidylinositol-specific phospholipase C and reconstituted in the presence of soluble uPAR. The attachment and internalization were suppressed in the presence of anti-uPAR polyclonal antibody, proteolytically inactive urokinase and saccharides that disrupt interaction of uPAR with CR3. Thus, uPAR acts as a cofactor for iC3b binding to CR3 and regulates CR3-mediated phagocytosis.     

Development rates of male bovine nuclear transfer embryos derived from adult and fetal cells

This study compared the nuclear transfer (NT) embryo development rates of adult and fetal cells within the same genotype. The adult fibroblast cells were obtained from a 21-yr-old Brahman bull. The fetal cells were derived from a Day 40 NT fetus previously cloned using cells from the Brahman bull. Overall, similar numbers of blastocysts developed from both adult (53 of 190; 28%) and fetal (39 of 140; 28%) donor cells. Improved blastocyst development rates were observed when fetal cells were serum-starved (serum-fed 12% vs. serum-starved 43%; P < 0.01) whereas there was no similar benefit when adult cells were serum-starved (both serum-fed and serum-starved 28%). Day 30 pregnancy rates were similar for blastocysts derived from adult (6 of 26; 23%) or fetal (5 of 32; 16%) cells. Day 90 pregnancy rates were 3 of 26 for adult and 0 of 32 for the fetal cell lines. One viable bull calf derived from a 21-yr-old serum-starved adult skin fibroblast was born in August 1999. In summary, somatic NT embryo development rates were similar whether adult or fetal cells, from the same genotype, were used as donor cells. Serum starvation of these adult donor cells did not improve development rates of NT embryos to blastocyst, but when fetal cells were serum-starved, there was a significant increase in development to blastocyst.     

Urokinase induces adhesion of monocytes to fibrinogen

Urokinase activates adhesion of monocytic U937 cells to fibrinogen-coated surface. This effect is due to urokinase proteolytic activity and does not depend on the urokinase binding to its receptor.     

The fate of fetal Leydig cells during the development of the fetal and postnatal rat testis

The ultrastructure and developmental fate of the fetal generation of Leydig cells of the rat testis was studied from the 17th day of fetal life up to 100 days after birth. The number of fetal Leydig cells per testis was determined by light microscopic morphometric analysis of semithin plastic sections. In fetal testes (days 17-22 postconception), Leydig cells exhibited a characteristic ultrastructure, containing smooth endoplasmic reticulum, many lipid inclusions and glycogen. Testes of 17-day-old fetuses contained about 25 x 10(3) fetal Leydig cells, rapidly increasing to 90 x 10(3) per testis in 21-day-old fetuses. After birth, fetal Leydig cells per testis remained relatively constant up to 2 weeks (80-90 x 10(3) per testis) and were identified by light and electron microscopy which showed their numerous lipid inclusions, their tendency for clustering and their association with interstitial tissue fibroblasts which partly encapsulated the fetal Leydig cells. From 21-100 days after birth, fetal Leydig cell numbers were quite variable with a mean of 45-60 x 10(3) per testis. These results are the first to show that the fetal generation of Leydig cells persist in the adult testis and do not undergo early postnatal degeneration or dedifferentiation into other interstitial cells. The simultaneous occurrence of the fetal Leydig cells and the adult population of Leydig cells indicates that these cells are distinct cell generations which are developmentally unrelated.     

Bovine placental prostaglandin synthesis: principal cell synthesis as modulated by the binucleate cell

Bovine placentomes were collected during late gestation, prepartum, and immediately postpartum. Postpartum tissues were collected prior to fetal placental release. A procedure for separating fetal placental principal cells from fetal binucleate cells (BNC) was developed. Dispersed fetal placental cells (mixed types), principal cells, and BNC were each examined for their ability to produce prostaglandins (PGs) from arachidonic acid (AA) and to metabolize prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) in vitro. Dispersed fetal placental cells obtained prepartum produced predominantly PGs of the E series (PGEs) from AA (p less than 0.05). PGE synthesis predominated (p less than .05) in cells from postpartum tissue if the fetal placental membranes were subsequently retained, whereas synthesis of PGs of the F series (PGFs) predominated (p less than 0.05) if the fetal membranes were subsequently released. Principal cells were the primary source of fetal placental PG synthesis from AA (p less than 0.05). BNC exhibited a lesser ability to synthesize PGs from AA (p less than 0.05), but were able to convert PGF2 alpha to PGE2. Dispersed fetal placental cells exhibited greater ability to convert PGF2 alpha to PGE2 (p less than 0.05) than did the separated cells. These data suggest the function of a two-cell system within the fetal villi such that the BNC modulate the output of principal cell PG synthesis and/or metabolism.     

Urokinase: a chemotactic factor for polymorphonuclear leukocytes in vivo

The effects of injecting urokinase into subdermal air sacs on the back of mice was studied. Urokinase was leukotactic in the concentration range of 2 X 10(-13) to 2 X 10(-15) M. This response was absolutely dependent on the enzyme activity of the serine esterase, but was found to be independent of generation of the chemotactic complement split product C5a. At high doses of urokinase (greater than 2 X 10(-12) M), no cellular infiltration was observed. Injection of 2 X 10(-10) M urokinase i.p. led to the systemic desensitization of mice when challenged in the skin with a lower dose (2 X 10(-14) M) of urokinase. Urokinase desensitization did not alter the ability of mice to respond to the chemical chemotactic factor f-met-leu-phe or to respond to C5a-dependent chemotactic stimuli. Urokinase desensitized mice failed to demonstrate a chemotactic response to nerve growth factor, thrombin, plasmin, or factor X activating enzyme, all of which were chemotactic in non-urokinase pre-treated animals. The results of these studies indicate the presence of three physiologically independent inflammatory pathways in mice: independent of C5 and not influenced by pretreatment with urokinase, independent of C5 and inhibited by pretreatment with urokinase, and dependent on C5 and not influenced by pretreatment with urokinase.     

An in vitro three-dimensional coculture model of cerebral microvascular angiogenesis and differentiation

Summary The microvasculature of the developing brain is plastic and responds differently to the many insults associated with preterm birth. We developed three-dimensional in vitro culture models for the study of the responses of the developing cerebral micro-vasculature. Beagle brain microvascular endothelial cells (BBMEC) were isolated by differential centrifugation from newborn beagle pups on postnatal Day 1 and placed in three-dimensional culture dispersed in a collagen gel. Alternatively, BBMEC were placed in a three-dimensional coculture with neonatal rat forebrain astrocytes. Cultures were analyzed for extracellular matrix components at 1 and 6 d, and total RNA was extracted for Northern analyses. Urokinase plasminogen activator activity was assayed in both mono- and cocultures of the two cell types. Studies of three-dimensional BBMEC/astrocyte cocultures demonstrated progressive tube formation with only low levels of endothelial proliferation. By 6 d in three-dimensional coculture, the BBMEC formed capillarylike tubes with a wrapping of glial processes, and basement membrane protein synthesis was noted. Urokinase plasminogen zymography suggested intercellular signaling by the two cell types. These data suggest that the three-dimensional beagle brain germinal matrix microvascular endothelial cell/neonatal rat astrocyte coculture provides a good model for the investigation of microvascular responses in the developing brain.     

The plasminogen system and cell surfaces: evidence for plasminogen and urokinase receptors on the same cell type

The capacity of cells to interact with the plasminogen activator, urokinase, and the zymogen, plasminogen, was assessed using the promyeloid leukemic U937 cell line and the diploid fetal lung GM1380 fibroblast cell line. Urokinase bound to both cell lines in a time- dependent, specific, and saturable manner (Kd = 0.8-2.0 nM). An active catalytic site was not required for urokinase binding to the cells, and 55,000-mol-wt urokinase was selectively recognized. Plasminogen also bound to the two cell lines in a specific and saturable manner. This interaction occurred with a Kd of 0.8-0.9 microM and was of very high capacity (1.6-3.1 X 10(7) molecules bound/cell). The interaction of plasminogen with both cell types was partially sensitive to trypsinization of the cells and required an unoccupied high affinity lysine-binding site in the ligand. When plasminogen was added to the GM1380 cells, a line with high intrinsic plasminogen activator activity, the bound ligand was comprised of both plasminogen and plasmin. Urokinase, in catalytically active or inactive form, enhanced plasminogen binding to the two cell lines by 1.4-3.3-fold. Plasmin was the predominant form of the bound ligand when active urokinase was added, and preformed plasmin can also bind directly to the cells. Plasmin on the cell surface was also protected from its primary inhibitor, alpha 2-antiplasmin. These results indicate that the two cell lines possess specific binding sites for plasminogen and urokinase, and a family of widely distributed cellular receptors for these components may be considered. Endogenous or exogenous plasminogen activators can generate plasmin on cell surfaces, and such activation may provide a mechanism for arming cell surfaces with the broad proteolytic activity of this enzyme.     

Production of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle

Genetically modified animals have many poten-tial applications in basic research, human medicine and agriculture. Pronuclear DNA microinjection has been almost the only practical means of producing transgenic animals during the last 20 years, but the low efficiency (1%—5%)[1] of this method has actu-ally been the obstacle that hampered its further appli-cation in animal biotechnology. The birth of Dolly[2], the first somatically cloned animal, made it possible to produce transgenic animals b…     

Production of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle

The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear transfer (NT) embryos. Four types of bovine somatic cells, including granulosa cells, fetal fibroblasts, fetal oviduct epithelial cells and fetal ovary epithelial cells, were transfected with a plasmid (pCE-EGFP-Ires-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation. After 14 days selection with 800 μg/mL G418, transgenic cell lines from each type of somatic cells were obtained. Nontransgenic granulosa cells and all 4 types of transgenic somatic cells were used as nuclear donor to produce transgenic embryos by NT. There was no significant difference in development rates to the blastocyst stage for NT embryos from transgenic and nontransgenic granulosa cells (44.6% and 42.8%, respectively), and transfer of NT embryos derived from transgenic and nontransgenic granulosa cells to recipients resulted in similar pregnancy rates on day 90 (19% and 25%, respectively). The development rates to the blastocyst stage of NT embryos were significantly different among different types of transgenic donor cells (P<0.05). Blastocyst rates from fetal oviduct epithelial cell and granulosa cell (49.1% and 44.6%, respectively) were higher than those from fetal fibroblast (32.7%) and fetal ovary epithelial cell (22.5%). These results suggest that (i) genetic manipulation to donor cells has no negative effect on in vitro and early in vivo developmental competence of bovine NT embryos and (ii) granulosa and fetal oviduct epithelial cells can be used to produce transgenic bovine NT embryos more efficiently. In addition, GFP can be used to select transgenic NT embryos as a non-invasive selective marker.     

Naturally occurring antibody in bovine fetal serum: reactivity against homologous and heterologous species erythrocytes*

Agglutinins that reacted with red cells from members of a different species (hetero-agglutinins) were detected in the serum of bovine fetuses beginning with about 5% in the fifth month of gestation, with the frequency of reactive sera increasing to about 50 % near term. The sera were able to agglutinate red cells from an increasing number of species as fetal age increased, but only exceptionally equalled the number agglutinated by sera from the dams. Antibodies that reacted with bovine red cells (isoantibodies) were detected in bovine fetal sera (anti-L agglutinin, anti-K hemolysin). However, no fetal serum was found that agglutinated the red cells of the respective dam. All fetal isoantibodies were inactivated by heat (56°C/30 min) and 2-mercapto-ethanol (2-ME) treatment, whereas some heteroagglutinins in serum from one fetus were resistant to inactivation by these methods. This suggests that most of the fetal antibodies were other than IgG.     

Isolation of alveolar type II cells from fetal rat lung by differential adherence in monolayer culture

Type II alveolar epithelial cells were isolated from fetal rat lung by differential adherence in monolayer culture. The preparation had a high degree of purity, as assessed by phase contrast microscopy and immunocytochemistry. Purity, based on reactivity with specific anti-adult lung serum (SAALS), which recognizes only type II cells, was 91% for cells isolated from 19-day fetal lungs and 79% for cells isolated from 21-day fetal lungs. The lower purity of type II cells in cultures derived from 1-day postnatal rat lungs (51% cells reactive with SAALS) is probably due to a lower tendency of the type II cells from neonatal rats to adhere to culture dishes than of type II cells from fetal rats. Type II cells isolated from 21-day fetal lungs contained a higher percentage phosphatidylglycerol and incorporated [Me-3H]choline faster into phosphatidylcholine (PC) than type II cells isolated from 19-day fetal lungs. Moreover, in cell preparations derived from lungs at fetal day 21, a higher percentage of epithelial cells contained lamellar bodies than in preparations derived from lungs at fetal day 19. The observation of these differences in the stage of maturation indicates that these differences, which are typical features of the original material, are not obliterated by differentiation during the culture. Type II cells isolated according to the present procedure were capable of synthesizing PC with a high percentage of the disaturated species. This method for the isolation of fetal type II cells may be a useful tool in studies concerning surfactant synthesis and its regulation in the fetal lung.     

Immunocytochemical localization of pro gamma MSH, gamma MSH, ACTH and beta endorphin/beta lipotrophin in the fetal sheep pituitary: an ontogenetic study

An immunocytochemical staining technique was used to localize four fragments [pro gamma MSH, gamma MSH, ACTH and beta endorphin/beta lipotrophin (beta endorphin/beta LPH)] of the proopiomelanocortin molecule in both the adult and fetal sheep pituitary. In the adult sheep anterior pituitary each fragment was localized in cells that were darkly stained, stellate and widely distributed throughout the gland. The same cells, identified in three serial sections, stained with anti-pro gamma MSH, anti-ACTH and anti-beta endorphin/beta LPH. In the fetal sheep anterior pituitary all the proopiomelanocortin derived fragments were present at 38 days gestation. Between about 90 and 130 days of gestation both adult type proopiomelanocortin cells (small, stellate) and uniquely fetal cells (large, columnar) were present. Both adult-type and fetal proopiomelanocortin cells were identified in serial sections of the fetal anterior pituitary, stained with anti-pro gamma MSH, anti-ACTH and anti-beta endorphin/beta LPH. The adult intermediate lobe was immunoreactive with anti-pro gamma MSH and anti-beta endorphin/beta LPH but not with anti-gamma MSH or anti-ACTH. The fetal intermediate lobe was immunoreactive with all four antisera from 60 days gestation.     

Ia-positive nonlymphoid cells and T cell development in murine fetal thymus organ cultures: monoclonal anti-Ia antibodies inhibit the development of T cells

A fetal thymus organ culture system has been developed to study the differentiation of murine thymus-derived immunocompetent cells (T cells) such that cell yields can be easily monitored. This system has been used to study the effects of monoclonal anti-I-A antibodies on the growth of T cells. The addition of anti-I-A antibodies, but not anti-H2K monoclonal antibodies, to fetal thymus organ cultures resulted in a decreased yield of lymphoid cells. Anti-I-A-treated cultures did not produce cells that gave an immune response in MLC assays. Anti-I-A antibodies stained a small subpopulation of nonlymphoid cells in untreated cultures by indirect immunofluorescence that were no longer detectable in cultures that had been pretreated with anti-I-A antibody. Culture of fetal thymus lobes at low temperature (20 degrees C) for 1 wk resulted in a decrease in lymphocyte production, as well as a concomitant increase in the frequency of Ia-positive nonlymphoid cells. Co-culture of fetal liver or anti-thy-1 plus complement-treated adult bone marrow with such Ia-positive cell-enriched fetal thymus lobes at 37 degrees C resulted in the production of T cells. Anti-Thy-1.1 or -1.2 staining by indirect immunofluorescence of cells obtained from co-cultures that differed at the Thy-1 locus showed that the T cells produced were derived from the bone marrow or fetal liver. T cell production occurred in both syngeneic and allogeneic cocultures. However, if co-cultures were made by using 14-day gestation fetal thymus instead of fetal liver or bone marrow as donors of T cell precursors, T cell growth was observed only in syngeneic combinations. These results suggest that Ia-positive nonlymphoid cells play a role in the development of T cells in the fetal thymus, and that "thymus processed" T cell progenitors (but not the more immature progenitors in the fetal liver or bone marrow) are self-Ia restricted in their differentiation.     

Serum antibodies to human fetal antigens in patients with systemic lupus erythematosus (SLE)

Serum antibodies to human fetal antigens were measured by a radiolabeled anti-immunoglobulin binding assay by using human fetal fibroblasts (Flow cell line No. 1000) as target cells. High titers of IgG antibody to the fetal cells were found in sera of patients with systemic lupus erythematosus (SLE). The antibody reacted with surface membrane antigens shared by various fetal tissues of human and murine origin but not by adult tissues. The reaction of the SLE antibody to the fetal cells was inhibited by heterologous antiserum to the Flow 1000 cells and antiserum to murine embryonic fibroblasts, but not by antiserum to human alpha-fetoprotein or human fibronectin. Absorption of SLE serum with isolated nuclei did not abolish the reaction indicating that these were not anti-nuclear antibodies. The antibody activity was found to reside in the F(ab')2 fragment. The serum titer of the anti-fetal antibody was higher in SLE patients with active disease than those in clinical remission.     

Fetal progenitor cells naturally transferred through pregnancy participate in inflammation and angiogenesis during wound healing

The phenotype and fate of fetal microchimeric cells transfered into the maternal circulation during pregnancy are not well described. Since progenitors from distal sites mobilize during wound healing, we analyzed the recruitment and plasticity of fetal progenitors into maternal wounds. Wounds were generated on normal and bleomycin-induced fibrotic skin of parous or pregnant wild-type females with fluorescent GFP(+) fetuses. Analyses were performed on skin and blood specimens through PCR, immunohistochemistry, and flow cytometry. Controls consisted of parous and pregnant females without wounds and virgin females with wounds. Fetal cells were detected in all skin specimens of parous mice as long as healing was not achieved. During early stages of wound healing, fetal cells expressed mainly leukocyte markers, while in later phases endothelial markers prevailed. Fetally derived vessels connected to maternal circulation were also found, demonstrating the transfer of fetal endothelial progenitor cells. Wounding mobilized fetal CD34(+)ckit(-) cells into the blood during pregnancy. Most of this population was CD11b(-)VEGFR2(-). Another part was CD11b(+) with a fraction expressing VEGFR2. VEGFa-spiked Matrigel plugs partially mimicked this fetal progenitor recruitment and mobilization into the blood. In summary, fetal cells that mobilize in response to wounding are mainly progenitor cells and participate in angiogenesis and inflammation.     

Female fetal cells in maternal blood: use of DNA polymorphisms to prove origin

The nucleated erythrocyte (NRBC) is one of the target fetal cell types for noninvasive genetic diagnosis using maternal peripheral blood. However, it is now known that pregnancy can stimulate the production of maternal NRBCs. When isolating female gamma-positive NRBCs, fluorescence in situ hybridization (FISH) analysis may show two X chromosome signals per nucleus, and therefore it cannot be conclusively determined whether the isolated cells are fetal or maternal in origin. The purpose of this study was to develop a means of verifying that a female cell is fetal on the basis of polymorphic short tandem repeat markers. Peripheral blood samples were obtained from women who had just undergone termination of pregnancy. Nucleated candidate fetal cells were isolated by flow-sorting using antibody to the gamma-chain of fetal hemoglobin and Hoechst 33342. FISH analysis was performed using X and Y chromosome specific probes. Female gamma-positive cells and leukocytes were micromanipulated separately and subjected to fluorescent polymerase chain reaction amplification of chromosome 21 and/or 18 STR markers (D21S11, D21S1411, D21S1412, and D18S535). In all ten cases analyzed, the gamma-positive female candidate fetal cells were determined to be fetal in origin by the presence of shared and nonshared DNA polymorphisms when compared with maternal leukocytes. These results show that genetic analysis can be performed on all fetal NRBCs, including female fetal cells that cannot be distinguished from maternal cells based on FISH analysis alone.