Human lymphoblastoid cells produce extracellular matrix-degrading enzymes and induce endothelial cell proliferation, migration, morphogenesis, and angiogenesis

Human lymphoproliferative diseases can be hypothesized to invade locally and to metastatize via mechanisms similar to those developed by a variety of solid tumors, i.e., the secretion of extracellular matrix-degrading enzymes and stimulation of angiogenesis. To assess this hypothesis, Namalwa, Raji, and Daudi cell lines (Burkitt’s lymphoma), LIK and SB cell lines (B-cell lymphoblastic leukemia), CEM and Jurkat cell lines (T-cell lymphoblastic leukemia), and U266 cell line (multiple myeloma) were evaluated for their capacity to produce matrix metalloproteinase-2 and -9, and urokinase-type plasminogen activator. These cell lines were also assessed for their ability: (1) to produce the angiogenic basic fibroblast growth factor and vascular endothelial growth factor; (2) to induce an angiogenic phenotype in cultured endothelial cells, represented by cell proliferation, chemotaxis, and morphogensis; (3) to stimulate angiogenesis in different in vivo experimental models. All cell lines expressed the mRNA for one or both metalloproteinases. Namalwa, Raji, LIK, SB, and U266 cells secreted the active form of both metalloproteinases, while Daudi, CEM, and Jurkat cells produced metalloproteinase-2 but not -9. In contrast, urokinase-type plasminogen activator was secreted only by SB cells. While Raji, LIK, SB, CEM, and Jurkat cells secreted both basic fibroblast growth factor and vascular endothelial growth factor, Daudi and U266 cells produced only the former, and Namalwa cells only the latter. Accordingly, the conditioned medium of all cell lines stimulated cell proliferation and/or chemotaxis in cultured endothelial cells, with the exception of that of Namalwa cells which was ineffective. The conditioned medium of CEM and Jurkat cells induced morphogenesis in cultured endothelial cells grown on a reconstituted basement membrane (Matrigel). Lastly, Namalwa, Raji, LIK, SB, U266, CEM, and Jurkat cells induced angiogenesis and mononuclear cell recruitment in the murine Matrigel sponge model and in a chick embryo chorioallantoic membrane assay. The extent of angiogenesis in both models was strictly correlated with the density of the mononuclear cell infiltrate. The results indicate that human lymphoproliferative disease cells possess both local and remote invasive ability via the secretion of matrix-degrading enzymes and the induction of angiogenesis which is fostered by host inflammatory cells and by an intervening ensemble of angiogenic factors.     

Antibodies in guinea-pigs immunized with kidney and lung basement membranes

The cross-reaction of antibodies to tubular basement membrane (TBM) with alveolar basement membrane (ABM) has been studied in guinea-pigs with tubulointerstitial (TI) nephritis. Forty-three of fifty-two Hartley guinea-pigs immunized with rabbit TBM in complete Freund's adjuvant developed TI nephritis. In addition to linear deposits of guinea-pig IgG and C3 in the TBM, thirty-two of the nephritic animals showed linear immune deposits in ABM; twelve of these animals had thickened alveolar septa and increased numbers of polymorphonuclear (PMN) leucocytes in lung tissue. Sera and eluates of kidney and lung from nephritic guinea-pigs reacted strongly with TBM and more weakly with ABM of normal animals. Absorption experiments suggested that antibodies to TBM and ABM were closely related or identical. Only a minority of guinea pigs immunized with TBM showed in vivo binding of IgG to glomerular basement membrane (GBM). Immunization of guinea-pigs with lung-homogenate-induced antibodies binding to TBM and ABM (in approximately 45% of animals) and to GBM (in approximately 30% of animals). Immunization with crude GBM-induced antibodies which reacted preferentially with TBM and ABM. In contrast, collagenase-treated GBM-induced antibodies preferentially reactive with GBM. TI nephritis was induced in both Strain 13 and Strain 2 guinea-pigs, but the nephropathy developed much faster in Strain 13 animals. Immunization with rabbit TBM-induced antibodies reactive with ABM in 25% of Strain 13 guinea-pigs and in 50% of Strain 2 guinea-pigs, respectively.     

Antigenic surface determinants of chicken lymphoid cells. II. Selective in vivo and in vitro activity of anti-bursa and anti-thymus sera.

Appropriately absorbed turkey antisera to antigenic surface determinants of chicken bursa (ABS) or thymus cells (ATS) were assessed for their selective immunosuppressive activy in vitro and in vivo. The intraperitoneal injection of ABS or ATS into 2-3-week-old normal white Leghorn chickens entailed a significant depletion of B or T cells respectively from spleen and peripheral blood, while bursa and thymus themselves remained unaffected. The potential of this 'peripheral serological bursectomy and thymectomy' paralleled that found after the conventional surgical procedures with subsequent sublethal irradiation. The mean survival time of skin allografts from donors of genotype B4B4 onto B8B8 recipients was significantly prolonged by treatment with ATS (29 plus or minus 12 days) as compared to untreated (14 plus or minus 2 days), normal turkey serum (12 plus or minus 3 days) or ABS-injected (13 plus or minus 2 days) groups. This selective suppression of a T cell-dependent immune reaction by ATS was also confirmed in vitro by its inhibitory action on the graft-versus-host reactivity of adult peripheral blood lymphocytes in the chorioallantoic membrane assay, where normal turkey serum and ABS were again ineffective. Thus, ABS or ATS produced in avian species may serve not only to delineate B and T cells in vitro, but can also be used for selective manipulation of immune reactions of the chicken in vivo.     

Glomerulonephritis in NZW kidneys grafted into NZB/W mice.

After left nephrectomy, 3 10-week old NZB/W mice received orthotopic grafts of kidneys from parental NZW mice of the same age. At autopsy conducted at the age of 33-38 weeks, glomerulonephritis of similar extent was noted in the recipients' own and in the grafted kidneys. Also, very similar granular deposits of immunoglobulins and complement were demonstrated in these kidneys. It was concluded that the absence of glomerulonephritis in NZW mice cannot be attributed to the refractoriness of their kidney to this disease.     

Studies on avian spinal cord chimeras. I. Neurological and histological manifestations.

Spinal cord chimeras were produced by replacing a small fragment of neural tube of a 2-day-old White Leghorn chicken embryo with a similar fragment from a Japanese quail embryo. The embryo mortality was 61%, and 72% of hatched birds were 'cripples' and had to be sacrificed within 5 days after hatching. Forty-nine chimeras, 10.9% of the total number of operated embryos, were alive for more than 3 weeks. For at least 17 days after hatching, all birds behaved like normal chicks, and the grey quail-like feathers were the only manifestations of their chimerism. Initial neurological symptoms of unsteady walking and drooping of the wings were noted in all birds except for 1 that died an accidental death before it became sick. Advanced symptoms characterized by paralysis of the legs forcing the bird to lie on its side were noted in 40 birds. The chimeras could be divided into two groups, each consisting of 24 birds. The short-survival (SS) chimeras of the first group became terminally ill and had to be sacrificed within 3 months. The long-survival (LS) chimeras of the second group showed more protracted disease, in that only 16 of them showed symptoms of the advanced disease, and the majority showed partial or complete recovery. Ten of the LS birds were kept alive for more than 8 months. Furthermore, many LS chimeras lost their grey feathers. The hallmarks of neurohistological manifestations were mononuclear cell infiltrates, demyelinization with preservation of axons and scar formation. These lesions were restricted to the quail fragment of the spinal cord except for 2 birds in which distant cellular infiltrates were observed. Direct immunofluorescence tests for chicken IgG were positive in spinal cords of most SS chimeras but only of some LS chimeras.     

Expression of CD44v3 protein in human endothelial cells in vitro and in tumoral microvessels in vivo

The most universal angiogenic cytokines (VEGF, bFGF, HGF) are all heparin-binding proteins, the function of which is dependent on cell surface heparan sulfate proteoglycans (HSPG). Several proteoglycans have been demonstrated in endothelial cells, but only glypican-1 from the cell surface HSPG subfamily was documented at protein level. Here, we show that CD44v3 is expressed in human immortalized endothelial cells [anchorage-dependent human umbilical vein endothelial cells (HUVEC) and anchorage-independent Kaposi sarcoma (KS-Imm)] at mRNA and protein level, but is absent from the primary culture of human brain microvascular endothelial cells. We have shown that CD44v3 has a large cytoplasmic pool in endothelial cells, but a limited surface expression, mainly at filopodia, colocalized with MMP-2. Angiogenic factors like VEGF or bFGF did not affect surface detection of CD44v3 suggesting a constitutive expression. The putative functional role for endothelial cell surface CD44v3 was identified in chemotaxis assay when anti-CD44v3 antibody pretreatment proved to be inhibitory for HUVEC. Furthermore, we provided evidence for the CD44v3 protein expression in human endothelial cells in vivo in peritumoral microvessels of both human melanoma and glottic cancers, suggesting a role for this part-time heparan sulfate proteoglycan in tumor induced angiogenesis.     

Silver Doped Magnesium Ferrite Nanoparticles: Physico-Chemical Characterization and Antibacterial Activity

Spinel phases, with unique and outstanding physical properties, are attracting a great deal of interest in many fields. In particular, MgFe₂O₄, a partially inverted spinel phase, could find applications in medicine thanks to the remarkable antibacterial properties attributed to the generation of reactive oxygen species. In this paper, undoped and Ag-doped MgFe_2-xAg_xO₄ (x = 0.1 and 0.3) nanoparticles were prepared using microwave-assisted combustion and sol–gel methods. X-ray powder diffraction, with Rietveld structural refinements combined with micro-Raman spectroscopy, allowed to determine sample purity and the inversion degree of the spinel, passing from about 0.4 to 0.7 when Ag was introduced as dopant. The results are discussed in view of the antibacterial activity towards Escherichia coli and Staphylococcus aureus, representative strains of Gram-negative and Gram-positive bacteria. The sol–gel particles were more efficient towards the chosen bacteria, possibly thanks to the nanometric sizes of metallic silver, which were well distributed in the powders and in the spinel phase, with respect to microwave ones, that, however, acquired antibacterial activity after thermal treatment, probably due to the nucleation of hematite, itself displaying well-known antibacterial properties and which could synergistically act with silver and spinel.     

Quantitation of urinary somatomedin-C in children with normal and abnormal growth

The renal excretion of radioimmunoassayable somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) was measured in 12-h overnight urine samples obtained from 88 subjects, aged 3-19 yr. The participants included 34 healthy children (group 1), 29 children with idiopathic growth failure and normal GH stimulation tests (group 2), and 25 GH-deficient subjects (group 3). The mean (+/- SEM) urinary Sm-C/IGF-I excretion in group 1 (28.4 +/- 2.1 mU/kg) was significantly greater than that in group 2 (8.1 +/- 1.6 mU/kg) or group 3 (8.6 +/- 1.3 mU/kg). Twenty-two of the 29 subjects in group 2 had urinary Sm-C/IGF-I values less than 8 mU/kg. After the administration of biosynthetic GH to 12 GH-deficient subjects, urinary Sm-C/IGF-I excretion rose from 10.3 +/- 2.3 to 21.4 +/- 4.2 mU/kg within 12 h (P less than 0.05), indicating that renal excretion of Sm-C/IGF-I is GH dependent. One woman with acromegaly had markedly elevated urinary Sm-C/IGF-I excretion (420 mU/kg). The authenticity of urinary Sm-C/IGF-I was confirmed by high pressure liquid chromatography (HPLC). Assay of serial dilutions of urinary Sm-C/IGF-I demonstrated a direct proportionality between concentration and dilution. Although it is not possible to identify whether urinary Sm-C/IGF-I reflects local or generalized synthesis of the peptide, we hypothesize that quantitation of Sm-C/IGF-I in timed urine collections will yield additional information about GH production and action in children with normal and abnormal growth.     

Angiogenic properties of human immunodeficiency virus type 1 Tat protein.

Extracellular human immunodeficiency virus type 1 (HIV-1) Tat protein promotes growth of spindle cells derived from AIDS-associated Kaposi sarcoma (AIDS-KS), an angioproliferative disease very frequent in HIV-1-infected individuals. Normal vascular cells, progenitors of AIDS-KS cells, proliferate in response to Tat after exposure to inflammatory cytokines, whose levels are augmented in HIV-1-infected individuals and in KS lesions. Here we show that Tat also promotes AIDS-KS and normal vascular cells to migrate and to degrade the basement membrane and stimulates endothelial cell morphogenesis on a matrix substrate. These effects are obtained at picomolar concentrations of exogenous Tat and are promoted by the treatment of the cells with the same inflammatory cytokines stimulating expression of the receptors for Tat, the integrins alpha 5 beta 1 and alpha v beta 3. Thus, under specific circumstances, Tat has angiogenic properties. As Tat and its receptors are present in AIDS-KS lesions, these data may explain some of the mechanisms by which Tat can induce angiogenesis and cooperate in the development of AIDS-KS.     

Advanced atherosclerosis is associated with increased medial degeneration in sporadic ascending aortic aneurysms

Objective

The pathogenesis of non-familial, sporadic ascending aortic aneurysms (SAAA) is poorly understood, and the relationship between ascending aortic atherosclerosis and medial degeneration is unclear. We evaluated the prevalence and severity of aortic atherosclerosis and its association with medial degeneration in SAAA.

Methods and results

Atherosclerosis was characterized in ascending aortic tissues collected from 68 SAAA patients (mean age, 62.9 ± 12.0 years) and 15 controls (mean age, 56.6 ± 11.4 years [P = 0.07]) by using a modified American Heart Association classification system. Upon histologic examination, 97% of SAAA patients and 73% of controls showed atherosclerotic changes. Most SAAA samples had intermediate (types 2 and 3, 35%) or advanced atherosclerosis (types ≥ 4; 40%), whereas most control samples showed minimal atherosclerosis (none or type 1, 80%; P < 0.001 after adjusting for age). In a separate analysis, we examined the total incidence and grade distribution of medial degenerative changes among SAAA samples according to atherosclerosis grade. Advanced atherosclerosis was associated with higher grades of smooth muscle cell depletion (P < 0.001), elastic fiber depletion (P = 0.02), elastic fiber fragmentation (P < 0.001), and mucopolysaccharide accumulation (P = 0.04). Aortic diameter was larger in SAAA patients with advanced atherosclerosis than in patients with minimal (P = 0.04) or intermediate atherosclerosis (P = 0.04). Immunostaining showed marked CD3+ T-cell and CD68+ macrophage infiltration, MMP-2 and MMP-9 production, and cryopyrin expression in the medial layer adjacent to atherosclerotic plaque.

Conclusions

SAAA tissues exhibited advanced atherosclerosis that was associated with severe medial degeneration and increased aortic diameter. Our findings suggest a role for atherosclerosis in the progression of sporadic ascending aortic aneurysms.