Traumatic injury induces interleukin-6 production by human astrocytes.

The brain is being evaluated as a de novo source of cytokines. Because recent evidence indicates that interleukin-6 (IL-6) may influence blood-brain barrier function and vascular permeability, we have sought to determine whether mechanical injury can directly induce in situ cerebral IL-6 production. Adult human astrocyte cultures were subjected to mechanical injury by the in vitro method of fluid percussion barotrauma, developed in our laboratory. Serial supernatant samples were collected for 8 h and evaluated for IL-6 activity using a proliferation assay employing the dependent B cell hybridoma cell line, B9. At optimum injury, the IL-6 level became significantly (P < 0.0001, analysis of variance) elevated from baseline 2 h after trauma and continued to increase over the observation period. Our study shows that following mechanical injury human astrocytes produce IL-6, which may contribute to post-traumatic cerebrovascular dysfunction. Elucidating the precise role of intracerebral cytokines is essential to our understanding of the mechanism responsible for post-traumatic cerebrovascular dysfunction.

Inhibition of tumor cell adhesion to lymph nodes by laminin-related peptide and neuraminidase.

BACKGROUND: Adhesion to lymph nodes, rather than growth stimulation, accounted for preferential colonization of lymph nodes by a metastatic B16 melanoma. We investigated these adhesive interactions. METHODS: Four classes of molecules were tested for inhibition of melanoma adhesion to cryostat sections of lymph node. RESULTS: Calcium chelators ethylenediaminetetraacetic acid and ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetra ace tic acid completely inhibited adhesion (50% adhesion, half-maximal inhibition, at 1 to 3 mmol/L). Cytochalasin B, which impairs contractile microfilaments, inhibited adhesion (60% adhesion at .001 mmol/L, 28% at .01 mmol/L). Colchicine, which disaggregates microtubules, had a similar effect (20% at .01 mmol/L, lowest dose tested). Trypsin slightly increased adhesion (125% adhesion at 10 micrograms/ml). Neuraminidase, which removed sialic acid residues, inhibited it (50% adhesion at 5 micrograms/ml). Gly-arg-gly-asp-ser, a peptide with a cell binding sequence of fibronectin, did not consistently inhibit adhesion (69% adhesion at 0.1 mg/ml, 83% adhesion at 1 mg/ml) or substantially differ from gly-arg-gly-glu-ser-pro (59% adhesion at 0.1 mg/ml, 90% adhesion at 1 mg/ml). In contrast, a peptide with a cell binding region of laminin (tyr-ile-gly-ser-arg) inhibited adhesion (50% adhesion at .05 mg/ml). CONCLUSIONS: Tumor cell-lymph node adhesion is a calcium-dependent process, requiring a functional cytoskeleton, that is mediated by both sialic acid moieties and trypsin-resistant, laminin-related, adhesion molecules.

Human astrocyte production of tumour necrosis factor-alpha, interleukin-1 beta, and interleukin-6 following exposure to lipopolysaccharide endotoxin.

The cytokines, tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, and IL-6, have been found in the human central nervous system. Recent studies have demonstrated that murine astrocytes produce these cytokines when induced with lipopolysaccharide endotoxin (LPS). The present study investigates the kinetics of TNF-alpha, IL-1 beta, and IL-6 production by normal adult human astrocytes when exposed to LPS.

Locally increased metastatic efficiency as a reason for preferential metastasis of solid tumors to lymph nodes.

Metastases from solid tumors to lymph nodes do not portend as poor a prognosis as metastases to other sites. The authors wished to determine whether specific subpopulations of cells metastasized to lymph nodes and whether they have different properties than cells metastatic to visceral sites. Repetitive selection for "spontaneous" metastases of a B16 melanoma to either lung or lymph node increased the incidence of lymph node metastases. Cells derived from pulmonary and lymph node metastases were assayed for their ability to adhere to cryostat sections of lung and lymph node and respond to target organ-conditioned media in serum-free conditions. Both cell types were four times more adherent to lymph node than lung, and consistently attached to the hilar and subcapsular sinuses. Attachment of cells derived from pulmonary metastases to either tissue was threefold greater than that of cells derived from nodal metastases. Lung-conditioned media stimulated proliferation of both cell types, and transiently induced differentiated morphology in cells derived from lymph node metastases, but not in cells from pulmonary metastases. Neither effect was found in lymph-node-conditioned medium. These results suggest that cells metastasize to lymph nodes preferentially not because of a specific predilection for lymph node, but because it is an easy site to colonize. Adhesive interactions in the lymph node rather than trophic ones appear to account for this effect. Cells metastatic to lymph node may be less "malignant" than cells metastatic to visceral sites because less has been required for them to succeed as a metastatic focus.

Search for anti-metastatic therapy: effects of phenytoin on B16 melanoma metastasis.

The ability to metastasize requires that tumor cells be able to degrade matrix. Nontoxic compounds that inhibit matrix digestion might be useful as anti-metastatic agents. We have investigated whether phenytoin, a drug commonly used in clinical practice that inhibits the production of collagenase by some cells, inhibits metastases in a standard animal model of metastasis: In vitro, phenytoin inhibited the proliferative response of B16 F10 melanoma cells to serum-containing media (75% inhibition at 25 micrograms/ml) but had no effect on their ability to degrade a type I collagen gel (1-100 micrograms/ml). Treatment of these cells with phenytoin prior to inoculation in vivo did not inhibit tumor growth, implantation in a surgical wound, or incidence of spontaneous metastases from a primary tumor growing in the foot. Pretreatment of mice with phenytoin (15, 40, and 75 mg/kg/day) diminished pulmonary metastases following tail vein injection in a minimal but dose dependent fashion; mean number of pulmonary colonies 4.6 +/- 3.1 (75/mg/kg/day) vs. 10.2 +/- 9.9 (control). However, tumor growth, implantation, and spontaneous metastases were not inhibited by pretreating the mice with the same doses of phenytoin. It is concluded that phenytoin has an insignificant inhibitory effect on tumor growth and metastasis.

Characterization of the antiviral activity constitutively produced by murine conceptuses: absence of placental mRNAs for interferon alpha and beta.

Antiviral activity has been found in conceptus and placental tissues in numerous species, including mice, pigs, sheep, cattle and humans. In sheep and cattle, the antiviral activity is due to an interferon alpha (IFN-alpha), but in other species the nature of the protein(s) responsible for placental activity is unknown. The objectives of this study were to determine if the constitutive antiviral activity associated with the mouse conceptus is produced as early as the peri-implantation period, and to determine if the activity is due to an IFN-alpha or -beta. Conceptus and placental tissue explants released antiviral activity from Day 4 through at least Day 16 of gestation as measured in an agar overlay bioassay employing CHO cells challenged with vesicular stomatitis virus. This activity was neutralized by antiserum against MuIFN-alpha/beta. The same antiserum failed, however, to immunoprecipitate radiolabeled proteins from medium collected from Day 4 blastocysts cultured in the presence of L-[35S]-methionine. S1 nuclease analysis of placental RNA and screening of ectoplacental cone and extraembryonic ectoderm cDNA libraries with MuIFN-alpha and -beta probes failed to detect IFN related mRNAs, even under relatively non-stringent conditions of hybridization. Thus, while antiviral activity is produced by peri-implantation conceptuses in several diverse mammalian species, it does not appear to be due to a conserved type of IFN in all these species.

Correlation between the release of ovine trophoblast protein-1 by the conceptus and the production of polypeptides by the maternal endometrium of ewes.

We studied the biosynthesis of two proteins, p70 (Mr 70,000; pI 4.0) and p15 (Mr 15,000; pI 5.7), by endometrial tissues from ewes between Days 12 and 24 of pregnancy and between Days 12 and 16 of the oestrous cycle to determine whether production of the two was correlated with the period of biosynthesis of ovine trophoblast protein-1 (oTP-1) by the conceptus. We also compared the protein synthetic activities of endometrium from gravid and non-gravid horns of pregnant ewes at Days 14, 16 and 18 in which the conceptus had been confined to one uterine horn. Proteins p70 and p15 were produced maximally between Days 14 and 20 of pregnancy, but synthesis by endometrial cultures from cyclic ewes was low or absent. Furthermore, synthesis of Protein p70 in particular was much greater by the gravid than non-gravid horn of unilaterally pregnant ewes. We conclude that synthesis of Proteins p70 and p15 by the uterus of sheep coincides with the time of oTP-1 production by the conceptus.

Synthesis and processing of ovine trophoblast protein-1 and bovine trophoblast protein-1, conceptus secretory proteins involved in the maternal recognition of pregnancy.

Ovine and bovine trophoblast protein-1 (oTP-1 and bTP-1) are newly discovered proteins produced by embryonic tissues for a limited period in early gestation. They appear to act as agents that prevent regression of the corpus luteum during early pregnancy in the ewe and cow. Ovine TP-1 [mol wt (Mr), 17,000] consists of three or four isoelectric variants (pI 5.4-5.7), whereas bTP-1, which cross-reacts with antiserum to oTP-1, is found as two predominant Mr classes (Mr, 22,000 and 24,000), each with several isoelectric variants (in the pI range 6.3-6.8). Cell-free translation of ovine conceptus mRNA yields pre-oTP-1 also consists of three or four isoelectric variants, assumed to have arisen by translation of multiple mRNA species. Ovine TP-1 is not glycosylated. When bovine conceptus mRNA is translated, a group of four or five isoforms of pre-bTP-1 are generated, each with a Mr of 19,000. In the presence of microsomes the Mr shifts upward to about 21,500. Bovine conceptuses cultured in presence of either [3H]glucosamine or [3H]mannose incorporate label into both size classes of bTP-1 (Mr, 22,000 and 24,000). Culture in presence of [35S]methionine and tunicamycin gave rise to a nonglycosylated form of bTP-1 with an apparent Mr of 18,000. Treatment of [35S]methionine-labeled bTP-1 with either endoglycosidase-H or peptide:N-glycosidase F yielded products with Mr of 17,000 and 16,000, respectively. bTP-1, although functionally and structurally related to oTP-1, appears to be a glycoprotein carrying at least two Asn-linked oligosaccharides. The two Mr classes of bTP-1 arise as a result of differences in either the number or structure of the carbohydrate chains. Like oTP-1, bTP-1 is probably translated from multiple mRNA species.