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Ferroptosis is a form of programmed cell death associated with inflammation, neurodegeneration, and ischemia. Vitamin E (alpha-tocopherol) has been reported to prevent ferroptosis, but the mechanism by which this occurs is controversial. To elucidate the biochemical mechanism of vitamin E activity, we systematically investigated the effects of its major vitamers and metabolites on lipid oxidation and ferroptosis in a striatal cell model. We found that a specific endogenous metabolite of vitamin E, alpha-tocopherol hydroquinone, was a dramatically more potent inhibitor of ferroptosis than its parent compound, and inhibits 15-lipoxygenase via reduction of the enzyme's non-heme iron from its active Fe3+ state to an inactive Fe2+ state. Furthermore, a non-metabolizable isosteric analog of vitamin E which retains antioxidant activity neither inhibited 15-lipoxygenase nor prevented ferroptosis. These results call into question the prevailing model that vitamin E acts predominantly as a non-specific lipophilic antioxidant. We propose that, similar to the other lipophilic vitamins A, D and K, vitamin E is instead a pro-vitamin, with its quinone/hydroquinone metabolites responsible for its anti-ferroptotic cytoprotective activity.

As an onshore, EU, and OECD member state with an extensive double tax treaty network and with its special tax regime for special purpose vehicles (SPVs), Ireland has emerged as a favored location for establishing vehicles to invest in or hold a wide variety of financial assets, including distressed assets. In particular, Ireland has become a domicile of choice for onshore SPVs in Europe, and it is a preferred alternative to the traditional offshore SPV jurisdictions. Irish SPVs have been used for plain-vanilla securitizations, collateralized loan obligations (CLOs) repackaging of receivables, mortgages, loans (both performing and non-performing), less straightforward synthetic transactions, and more unusual securitizations involving infrastructure projects, such as toll roads. They are also used in various other structures, such as aircraft leasing, life settlement issues, and regulated fund structures.

The objective of the described research effort was to identify a novel serotonin and norepinephrine reuptake inhibitor (SNRI) with improved norepinephrine transporter activity and acceptable metabolic stability and exhibiting minimal drug-drug interaction. We describe herein the discovery of a series of 3-substituted pyrrolidines, exemplified by compound 1. Compound 1 is a selective SNRI in vitro and in vivo, has favorable ADME properties, and retains inhibitory activity in the formalin model of pain behavior. Compound 1 thus represents a potential new probe to explore utility of SNRIs in central nervous system disorders, including chronic pain conditions.

Haemophilus influenzae (Hi) type b transformants RM135 and RM133 were previously found to differ in virulence and capsule content [Zwahlen et a!., Microb Pathog 1986; 1: 465-73]. The more virulent strain RM135 produced less capsule than RM133, but was indicated to differ in its lipopolysaccharide (LPS). We have now performed detailed analyses of the respective LPSs and correlate structure, genetics and the original virulence observation. We found that RM133 expressed identical major LPS structures as the parent strain Rd whereas strain RM135 expressed shorter glycoforms. The genetic basis for this difference was found to be the expression of the phase-variable gene lie 2A, which could be shown to be compromised in RM135. The glycosyltransferase Lic2A is involved in the expression of a digalactoside (α-D-Galp-(1→4)-β-D-Galp(l→) and plays a role in chain elongation from the inner-core region of Hi LPS. One consequence of altered oligosaccharide extension, possibly contributing to the heightened virulence of RM135, was the sialylation of the LPSs. RM135 expressed W-acetylneuraminic acid whereas strain RM133 did not. Sialylation of LPS has been shown to be important for resistance of Hi to the killing effect of normal human serum. We thus propose that lie 2A expression can modulate virulence and that the effect of changes in LPS can outweigh that of capsule copy number. © 2010 Academic Journals Inc.

TLR overactivation may lead to end organ damage and serious acute and chronic inflammatory conditions. TLR responses must therefore be tightly regulated to control disease outcomes. We show in this study the ability of the soluble form of TLR2 (sTLR2) to regulate proinflammatory responses, and demonstrate the mechanisms underlying sTLR2 regulatory capacity. Cells overexpressing sTLR2, or stimulated in the presence of the sTLR2 protein, are hyporesponsive to TLR2 ligands. Regulation was TLR2 specific, and affected NF-κB activation, phagocytosis, and superoxide production. Natural sTLR2-depleted serum rendered leukocytes hypersensitive to TLR2-mediated stimulation. Mice administered sTLR2 together with Gram-positive bacteria-derived components showed lower peritoneal levels of the neutrophil (PMN) chemoattractant, keratinocyte-derived chemokine; lower PMN numbers; and a reduction in late apoptotic PMN. Mononuclear cell recruitment remained unaffected, and endogenous peritoneal sTLR2 levels increased. Notably, the capacity of sTLR2 to modulate acute inflammatory parameters did not compromise the ability of mice to clear live Gram-positive bacteria-induced infection. Mechanistically, sTLR2 interfered with TLR2 mobilization to lipid rafts for signaling, acted as a decoy microbial receptor, and disrupted the interaction of TLR2 with its coreceptor, CD14, by associating with CD14. These findings establish sTLR2 as a regulator of TLR2-mediated inflammatory responses, capable of blunting immune responses without abrogating microbial recognition and may inform the design of novel therapeutics against acute and chronic inflammatory conditions.

Systemic inflammation, which results from the massive release of proinflammatory molecules into the circulatory system, is a major risk factor for severe illness, but the precise mechanisms underlying its control are not fully understood. We observed that prostaglandin ESystemic inflammation, which results from the massive release of proinflammatory molecules into the circulatory system, is a major risk factor for severe illness, but the precise mechanisms underlying its control are not fully understood. We observed that prostaglandin E2 (PGE2), through its receptor EP4, is down-regulated in human systemic inflammatory disease. Mice with reduced PGE2 synthesis develop systemic inflammation, associated with translocation of gut bacteria, which can be prevented by treatment with EP4 agonists. Mechanistically, we demonstrate that PGE2-EP4 signaling acts directly on type 3 innate lymphoid cells (ILCs), promoting their homeostasis and driving them to produce interleukin-22 (IL-22). Disruption of the ILC–IL-22 axis impairs PGE2-mediated inhibition of systemic inflammation. Hence, the ILC–IL-22 axis is essential in protecting against gut barrier dysfunction, enabling PGE2-EP4 signaling to impede systemic inflammation.

A series of N-alkyl-N-arylmethylpiperidin-4-amines have been prepared and are demonstrated to be inhibitors of both serotonin and norepinephrine reuptake.

Human HtrA1 belongs to a widely conserved family of serine proteases involved in various aspects of protein quality control and cell fate. Although HtrA1 has been implicated in the pathology of several diseases, its precise biological functions remain to be established. Through identification of potential HtrA1 targets, studies presented herein propose that within the context of arthritis pathology HtrA1 contributes to cartilage degradation. Elevated synovial HtrA1 levels were detected in fluids obtained from rheumatoid and osteoarthritis patients, with synovial fibroblasts identified as a major source of secreted HtrA1. Mass spectrometry analysis of potential HtrA1 substrates within synovial fluids identified fibronectin as a candidate target, and treatment of fibronectin with recombinant HtrA1 led to the generation of fibronectin-degradation products that may be involved in cartilage catabolism. Consistently, treatment of synovial fibroblasts with HtrA1 or HtrA1-generated fibronectin fragments resulted in the specific induction of matrix metalloprotease 1 and matrix metalloprotease 3 expression, suggesting that HtrA1 contributes to the destruction of extracellular matrix through both direct and indirect mechanisms.

Cytokine receptors exist in membrane-bound and soluble forms. They bind their ligands with comparable affinity. Although most soluble receptors are antagonists because they compete with their membrane counterparts for their ligands, some soluble receptors are agonists. In this case, on target cells, the complex of cytokine and soluble cytokine receptor binds to a second receptor subunit and initiates intracellular signal transduction. The soluble receptors of the interleukin-6 (IL-6) family of cytokines--soluble IL-6 receptor (sIL-6R), sIL-11R, and soluble ciliary neurotrophic factor receptor (sCNTFR)--are agonists. In vivo, the IL-6/sIL-6R complex stimulates several types of target cells not stimulated by IL-6 alone, as they do not express the membrane- bound IL-6R. This process has been named transsignaling. We have shown recently that in several chronic inflammatory diseases, such as chronic inflammatory bowl disease, peritonitis, and rheumatoid arthritis, as well as in colon cancer, transsignaling via the sIL-6R complexed to IL-6 is a crucial point in the maintenance of the disease. The mechanism by which the IL-6/sIL-6R complex regulates the inflammatory or neoplastic state is discussed.

A series of benzothienyloxy propylamines have been prepared and are demonstrated to be inhibitors of both serotonin and norepinephrine reuptake.

Free radical damage has the potential to significantly affect the behavior of cells in culture. In this study the effects of antioxidants (superoxide dismutase, catalase, and vitamin E) and lowered oxygen tension (1% oxygen) on primary culture of rat mammary epithelial cells were examined. Rat mammary epithelial cells were dissociated in collagenase with or without the addition of antioxidants and low oxygen tension, then cultured for 10 d in rat-tail collagen gel matrix and fed with Dulbecco's modified Eagle's F12 medium supplemented with various hormones and growth factors. Growth potential of the mammary cells was enhanced when antioxidants and low oxygen tension were used, alone or in combination, during the cell dissociation period. Using antioxidants and low oxygen tension during the culture period failed to improve growth potential regardless whether cells were dissociated in standard conditions or with antioxidants and low oxygen tension. The use of antioxidants and low oxygen tension during the cell dissociation period also reduced the degree of keratinization of the cells after 10 d of culture. Using antioxidants and low oxygen tension during the cell culture period did not further reduce keratinization if antioxidants and low oxygen tension were used during the dissociation period, but were effective in reducing keratinization if cells were dissociated in standard condition. In this system, antioxidants and low oxygen tension reduced lipid peroxidation during the cell dissociation period. An iron chelator, desferal, can also reduce lipid peroxidation and enhance growth when used during cell dissociation, suggesting the enhanced growth potential by the addition of antioxidants and low oxygen to be due to the reduction of lipid peroxidation.

Objective To determine whether interleukin-6 (IL-6) trans-signaling directs the expression of pre-B cell colony-enhancing factor (PBEF) in vitro and in vivo. Methods Complementary DNA from rheumatoid arthritis (RA) synovial fibroblasts treated with IL-6 and soluble IL-6 receptor (sIL-6R) was used to probe a cytokine microarray. PBEF regulation by the IL-6-related cytokines, IL-6, sIL-6R, oncostatin M (OSM), IL-11, and leukemia inhibitory factor (LIF) was determined by reverse transcription-polymerase chain reaction analysis. IL-6-mediated STAT-3 regulation of PBEF was determined using a cell-permeable STAT-3 inhibitor peptide. Antigen-induced arthritis (AIA) was induced in wild-type (IL-6+/+) and IL-6-deficient (IL-6-/-) mice. PBEF and STAT were detected by immunohistochemistry, immunoblotting, and electrophoretic mobility shift assay. Synovial levels of PBEF were quantified by enzyme immunoassay. Results IL-6 trans-signaling regulated PBEF in a STAT-3-dependent manner. In addition, PBEF was regulated by the IL-6-related cytokine OSM, but not IL-11 or LIF. Flow cytometric analysis of the IL-6-related cognate receptors suggested that OSM regulates PBEF via its OSM receptor and not its LIF receptor. The involvement of PBEF in arthritis progression was confirmed in vivo, where induction of AIA resulted in a 4-fold increase in the synovial expression of PBEF. In contrast, little or no change was observed in IL-6-/- mice, in which the inflammatory infiltrate was markedly reduced and synovial STAT-1/3 activity was also impaired. Analysis of human RA synovial tissue confirmed that PBEF immunolocalized in apical synovial membrane cells, endothelial cells, adipocytes, and lymphoid aggregates. Synovial fluid levels of PBEF were significantly higher in RA patients than in osteoarthritis patients. Conclusion Experiments presented herein demonstrate that PBEF is regulated via IL-6 trans-signaling and the IL-6-related cytokine OSM. PBEF is also actively expressed during arthritis. Although these data confirm an involvement of PBEF in disease progression, the consequence of its action remains to be determined.

Human T-cell leukemia virus type-I (HTLV-I) encodes for the viral protein Tax, which is known to significantly disrupt transcriptional control of cytokines, cytokine receptors and other immuno-modulatory proteins in T cells. Specific dysregulation of these factors can alter the course and pathogenesis of infection. Soluble interleukin-6 receptor (sIL-6R) was shown to circulate at elevated levels in HTLV-I-infected patients, and high expressions of IL-6R and sIL-6R by HTLV-I-infected T cells were clinically and experimentally associated with Tax activity. To examine roles of Tax in expression of the IL-6R gene, the JPX-9 cell line was used, which is derived from Jurkat cell line expressing Tax cDNA. Over-expression of Tax enhanced IL-6R expression but not in Tax mutant JPX-9/M cell line. The clinical relevance of these observations was further demonstrated by ELISA using sera obtained from HTLV-I-infected patients. Our results revealed that sIL-6R levels were apparently elevated in HAM/TSP patients who were expressing Tax in their cells, while ATL patients' cells barely expressed Tax. HTLV-I-infected T-cell lines stimulated by IL-6/sIL-6R showed gp130-mediated STAT3 activity. IL-6/sIL-6R enhanced proliferation of HTLV-I-infected T cells in association with activation of STAT3. Consequently, Tax-mediated regulations of IL-6R and sIL-6R observed in HTLV-I-associated disorders may contribute to proliferation of HTLV-I-infected T cells through activation of inducible STAT3, and ultimately affect malignant growth and transformation of T cells by HTLV-I.

In the cell cycle the transition from G2 phase to cell division (M) is strictly controlled by protein phosphorylation-dephosphorylation reactions effected by several protein kinases and phosphatases. Although much indirect and direct evidence point to a key role of protein phosphatase 2A (PP2A) at the G2/M transition, the control of the enzyme activity prior to and after the transition are not fully clarified. Using synchronized HeLa cells we determined the PP2A activity (i.e. the increment sensitive to inhibition by 2nM okadaic acid) in immunoprecipitates obtained with antibodies raised against a conserved peptide sequence (residues 169-182, Ab(169/182)) of the PP2A catalytic subunit (PP2A C). Two different substrates were offered: the phospho-peptide KR(p)TIRR and histone H1 phosphorylated by means of the cyclin-dependent protein kinase p34(cdc2). The results indicate that in HeLa cells the specific activity of PP2A towards both substrates goes through a minimum in late G2 phase and stays low until metaphase. Treatment of G2 cells with TPA (10(-7) M) caused a reactivation of the downregulated PP2A activity within 20 min, i.e. the same time frame within which TPA was shown earlier to block HeLa cells at the transition from G2 to mitosis [Kinzel et al., 1988. Cancer Res. 48, 1759-1762]. Activation of PP2A was also induced by TPA in mitotic cells. The low activity of PP2A in mitotic cells was accompanied by a strong reaction of mitotic PP2A C with anti-P-Tyr antibodies in Western blots, which was reversed by treatment of mitotic cells with TPA. The results suggest that the activity of cellular PP2A requires downregulation for the transition from G2 phase to mitosis. Unscheduled reactivation of PP2A induced by TPA in late G2 phase appears to inhibit the progress into mitosis.

The defining features of the widely conserved HtrA (high temperature requirement) family of serine proteases are the combination of a catalytic protease domain with one or more C-terminal PDZ domains and reversible zymogen activation. Even though HtrAs have previously been implicated in protein quality control and various diseases, including cancer, arthritis, and neuromuscular disorder, the biology of the human family members is not well understood. Our data suggest that HtrA1 is directly involved in the β-amyloid pathway as it degrades various fragments of amyloid precursor protein while an HtrA1 inhibitor causes accumulation of Aβ in astrocyte cell culture supernatants. Furthermore, HtrA1 colocalizes with β-amyloid deposits in human brain samples. Potential implications in Alzheimer's disease are discussed. © 2005 by The National Academy of Sciences of the USA.

Studies in IL-6-deficient (IL-6−/−) mice highlight that IL-6 contributes to arthritis progression. However, the molecular mechanism controlling its activity in vivo remains unclear. Using an experimental arthritis model in IL-6−/− mice, we have established a critical role for the soluble IL-6R in joint inflammation. Although intra-articular administration of IL-6 itself was insufficient to reconstitute arthritis within these mice, a soluble IL-6R-IL-6 fusion protein (HYPER-IL-6) restored disease activity. Histopathological assessment of joint sections demonstrated that HYPER-IL-6 increased arthritis severity and controlled intrasynovial mononuclear leukocyte recruitment through the CC-chemokine CCL2. Activation of synovial fibroblasts by soluble IL-6R and IL-6 emphasized that these cells may represent the source of CCL2 in vivo. Specific blockade of soluble IL-6R signaling in wild-type mice using soluble gp130 ameliorated disease. Consequently, soluble IL-6R-mediated signaling represents a promising therapeutic target for the treatment of rheumatoid arthritis.

OBJECTIVES We examined the temporal changes in the expression of interleukin 1beta (IL-1beta), tumour necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) in the rat antigen-induced arthritis (AIA) model and investigated how their expression was modulated following disease amelioration by liposomally conjugated methotrexate (G-MLV). METHODS On the day of arthritis induction (day 0), rats were treated with a single intra-articular injection of G-MLV, methotrexate (MTX), a dose of lipid equivalent to G-MLV (E-LIPO) or saline. On days 3 and 7 after disease induction, animals from each experimental group were killed. Joint tissue was examined histologically and for mRNA expression (IL-6, IL-1beta and TNF-alpha) using semiquantitative reverse transcription-polymerase chain reaction. RESULTS There was no significant difference (ANOVA) in knee swelling between MTX-, E-MLV- or saline-treated animals from day 0 to day 7. By day 1, G-MLV significantly reduced knee swelling (1.94+/-0.12 mm; P

Studies on the link between cellular signalling and cell cycle control at the G2 checkpoint have shown that, in HeLa cells, epidermal growth factor (EGF) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) rapidly inhibit the G2-M transition by preventing the key component of mitosis-promoting factor (MPF), p34cdc2, from expressing protein kinase activity. The kinase activity of active MPF is not inhibited; rather, the conversion of pre-MPF to MPF, i.e., the activating dephosphorylation of p34cdc2, at tyrosine is rapidly blocked (Barth and Kinzel, 1994, Exp. Cell Res. 212:383-388; 1995, J. Cell. Physiol., 162:44-51). The phosphatase responsible, cdc25-C, is activated by phosphorylation in mitotic cells starting at the G2-M transition in an autocatalytic loop with MPF (Hoffmann et al., 1993, EMBO J. 12:53-63). We now show that, concomitant with the prevention of MPF activation, EGF and TPA induced a reduction of the activity of cdc25-C in synchronized cultures. Furthermore, treatment of mitotic HeLa cells with TPA did not influence the kinase activity of MPF but caused a rapid decrease of the specific enzyme activity of cdc25-C, probably due to dephosphorylation of the enzyme, as indicated by reduced binding of monoclonal MPM-2 antibody specific for phosphoepitopes in M phase. Because of its inability to induce signalling during division, EGF failed to influence the activity of cdc25-C in mitotic cells. The scenario in cells late in G2 that are committed to enter mitosis may be as follows: In those cells where the signalling pathways responding to EGF as well as those responding to TPA are still open, cdc25-C is prevented by dephosphorylation from exceeding the threshold level of activity required to initiate the activation of and the autocatalytic feedback loop with p34cdc2 and to enter mitosis. Therefore, cdc25-C appears to represent part of an interface between cellular signalling and cell cycle control in G2 phase.

This study was aimed at gaining an understanding of metabolic events responsible for the inhibition of cells in G2 phase, a known physiological restriction site in the cell cycle of multicellular organisms. In an earlier study, phosphatidic acid was proposed as an inhibitory mediator in the epidermal growth factor (EGF)-induced inhibition of A431 cells in G2 phase via the phospholipase C pathway [Kaszkin, Richards and Kinzel (1992) Cancer Res. 52, 5627–5634]. We show here that the phorbol ester phorbol 12-myristate 13-acetate (PMA) induces a reversible inhibition of the G2/M transition in A431 cells under conditions of phospholipase D-catalysed phosphatidic acid formation. Such PMA-induced inhibition in G2 phase is largely attenuated in the presence of 1-propanol (but not of 2-propanol). In this case the amount of phosphatidic acid is reduced to almost control levels, and instead phosphatidylpropanol is formed. In the case of EGF-induced activation of a phospholipase D the amount of phosphatidic acid is only slightly decreased in the presence of a primary alcohol. Under these conditions the EGF-induced G2 delay was not affected. The correlation between the formation of phosphatidic acid and the G2 delay induced by PMA, as well as by an exogenous bacterial phospholipase D (from Streptomyces chromofuscus), could be supported by using synchronized cells in order to increase the population of cells in G2 phase. This study indicates that the formation of substantial amounts of phosphatidic acid immediately before entry into mitosis seems to be important for establishing a delay in the cell cycle at the G2/M border by exogenous ligands.

End buds are the growing terminal structures of the ducts in the mammary gland. They are made up of undifferentiated epithelial cells that, under the hormonal milieu of adult and pregnant females, give rise to the ducts and alveoli of the mature gland. The end buds are of interest in understanding the developmental biology of the gland. They are also likely targets for many mammary carcinogens and, therefore, of interest in understanding the biology of mammary cancer. A method was developed for isolating end buds as a pure subpopulation from collagenase and hyaluronidase digested 4-week-old C57BL/Crgl mouse mammary glands. They were cultured within a rat tail collagen gel matrix for 3 weeks and fed with media containing various combinations of sera, hormones and growth-promoting factors. Growth in response to the different media was measured by photographing individual end bud colonies over time using dark field illumination. The growth of the individual colonies was quantitated using a computer-assisted photodensitometry method of determining the colonies' mass. The end buds showed the greatest growth response (greater than 20-fold increase in 3 weeks with a minimum doubling time of 48 h) to medium supplemented with 50% equine serum and cholera toxin or with less serum (15%) and epidermal growth factor (EGF), hormones and cholera toxin. Initially, the colonies grew logarithmically, then slowed as the colony size increased. This was due, in part, to the cessation of growth by the cells in the center. This end bud culture system differs from that of dissociated mouse mammary cells in a number of ways which are discussed.

Normal and neoplastic mammary cells from both human and rodent sources grow in culture in response to a number of hormones and growth factors. However, with the exception of a few human tumor lines, a consistent growth-promoting effect of estrogens on mammary cells has not been observed. Mammary cells can be shown to respond to other hormones and factors, such as PRL, hydrocortisone, and epidermal growth factor. Recent observations suggest that the pH indicator dye phenol red, found in most media, may be masking any exogenous estrogenic effects by acting as a weak estrogen. To test this possibility, we reexamined the effects of estradiol (E2) and the antiestrogen keoxifene on the growth of normal human, mouse, and rat mammary cells in the absence of phenol red. Primary cultures of these mammary cells were grown within a rat tail collagen gel matrix in a serum-free medium made up of Ham's F-12 and Dulbecco's Modified Eagles' medium (1:1) with and without phenol red. The medium was supplemented with various hormones and growth factors. These supplements were selected for each cell type to produce a variety of conditions from nongrowing to rapidly growing. The effects of E2 (10(-10)-10(-8) M, keoxifene (10(-9)-10(-6) M), and phenol red on growth under these various conditions were examined. Phenol red had no effect on growth, and its absence did not restore a response to E2. Keoxifene, in the presence or absence of E2, also had no effect on growth. Although E2 had no effect on growth, it was able to induce a 150% increase in the progesterone receptor levels in normal mouse mammary cells in culture, indicating that the cells retain their capacity to respond to E2. This work supports the idea that the effect of estrogens on growth in vivo may be mediated through some other factor(s).

Lens culinaris lectin (LCL) immobilized on large 2B-Sepharose beads has been used to investigate the lectin-binding capacity of cell surfaces for the separation of cells by affinity chromatography. Immobilized LCL induced the tissue culture cells studied (HeLa, SV3T3) to bind to the agarose beads. This binding could be prevented by the addition of hapten sugars such as methyl-α d -mannopyranoside (MαMP) and methyl-α d -glucopyranoside (MαGP) according to the affinity of these sugars to LCL in a concentration-dependent manner. The cell-bead linkages were sufficiently strong to resist any appreciable mechanical breakage. The binding of the cells occurred so fast that any release could be started before the cells interacted unspecifically with the beads. Immobilized LCL released most of the cells upon addition of the competing sugar MαMP in a concentration-dependent manner under physiological conditions. The cells retained viability during the separation procedure as demonstrated by subsequent growth in culture. The difficulties so far observed with procedures involving columns have been overcome by a batch technique for controlled cell binding and release. For rapid separation of free from bound cells a gauze of defined pore size is introduced. Methodological problems such as agarose concentration, cell stickiness, lectin amount, and mechanical stability of the bead-cell complex are discussed.

The tumor promoter TPA 2 (12-O-tetradecanoylphorbol-13-acetate) has been shown to exhibit a radiomimetic activity on the cell cycle of HeLa cells ( V. Kinzel, J. Richards, and M. Stohr (1980) Science 210, 429). The response includes a delay of cells in G2 phase. The relation between TPA-induced release of arachidonic acid (AA) and the inhibition in G2 phase was studied. Exogenous AA (>10−4 M; in presence of 10% serum) is shown to delay HeLa cells in G2 and to enhance the effectiveness of TPA in this respect. The inhibition of the TPA-induced AA liberation by fluocinolone acetonide, however, does not influence the TPA-effected G2 delay. The diacylglycerols 1,2-dioctanoyl-glycerol and 1-oleoyl-2-acetyl-glycerol delay HeLa cells in G2 but without major stimulation of AA liberation. On the basis of the data it is concluded that AA released from HeLa cells due to the action of TPA is not involved in the TPA-induced delay of cells in G2 phase.

Mammary glands are enzymatically dissociated and the resulting tissue digest enriched for epithelial cells by isopycnic banding on a density gradient of Percoll. The cells are embedded within a rat tail collagen gel matrix and fed with the appropriate medium. Growth and differentiation are superior in such a system when compared to culture on plastic, using identical media.

End buds from 4- to 5-week-old rat mammary glands were isolated and cultured within a rat tail tendon collagen gel matrix. Media containing equine serum or porcine serum and cholera toxin promoted growth, but not the production of casein or thioesterase II, nor did they induce a state of differentiation as assessed by cell ultrastructure. Medium supplemented with only 5% porcine serum, insulin and cholera toxin did not support growth or differentiation. However, when prolactin, estradiol, progesterone and hydrocortisone were added to this medium, growth was stimulated greatly and a differentiated state was induced as assessed by the production of casein and thioesterase and by the appearance of a highly secretory ultrastructure.

The growth of normal and neoplastic mouse mammary cells was compared in primary cultures on plastic, on rat tail collagen gels and in rat tail collagen gels. Cells on plastic grew for the first few days, then stopped with only a 1- to 3-fold increase in cell number after 2 weeks in culture. Cells grown on or in collagen gels grew continuously over the 2-week culture period with up to 10-fold increase in cell number for cultures on collagen gels and a 20-fold increase for cells embedded in collagen gels. The difference in growth rates between cells grown in collagen gel and those grown on collagen gels was due, in part, to the three-dimensional growth of the colonies in collagen gel their two-dimensional growth on collagen gel. Cells grown on and in collagen gel can produce an electron-dense basal lamina-like structure which is associated with collagen IV and laminin as judged by immunofluorescence. Cells grown on plastic do not form this structure. Cis-OH-proline blocks the production of collagen and inhibits the growth of the cultured cells indicating collagen production to be involved in growth. Rat tail collagen gels are a superior substratum for the growth of mouse mammary cells and this may be related to the cells' ability to form a collagen IV-containing basal lamina-like structure.

Sustained growth of normal mouse mammary epithelial cells in primary culture, leading to an increase in cell number, in response to growth factors [epidermal growth factor (EGF) and fibroblast growth factor (FGF)] or cholera toxin has been achieved by embedding the cells inside collagen cells. Inclusion of agents known to increase the level of cellular cAMP have been found to be favorable for mammary epithelial cell proliferation. Cholera toxin is by far the best of all of the agents tested (prostaglandins E1 and E2, isoproterenol, theophylline, and dibutyryl cAMP). When growth factors (EGF or FGF) are added with cholera toxin, a synergistic effect resulting in a response much greater than with either of them alone is seen. This synergism was best seen in normal mammary epithelial cells from nonpregnant mice. The extent of this synergistic effect was found to be less in normal cells from pregnant mice, suggesting that these cells may be less responsive to EGF during pregnancy. Tumor cells were found to be rather inconsistent in their responses to EGF and cholera toxin, ranging from a minimal response, similar to that of normal cells from pregnant animals, to a maximal response, similar to that of normal cells from nonpregnant animals.

The hormone-responsive mammary tumor 13762NF of the F344 rat was cultured in a collagen gel matrix with the use of a serum-free medium supplemented with hormones and epidermal growth factor (EGF). Hydrocortisone (F) had the greatest effect on cell growth. EGF had no growth-promoting activity when used alone, but it had a significant effect when used with F. There was also a population of cells responsive to progesterone (P) and prolactin (PRL). P synergized with EGF as well as with PRL to promote growth. 17 beta-Estradiol alone or in combination with other hormones had no growth-promoting activity. Receptor levels in the tumor were high for glucocorticoids, intermediate for P and EGF, and low for estrogens. Metastasis in the lung and lymph node showed the same basic hormonal responses as the parent tumor. Cultured cells produced tumors with the same histopathology as the parent tumor when transplanted back into female rats; they had the same receptor levels and when placed back in culture showed a growth response to the same set of hormones. The tumor cells formed colonies that were spherical, which was different from the branching structures formed by normal mammary cells in collagen gel.

When asynchronous and synchronous HeLa cells were incubated with small doses (10(-7) M) of tumor promoter 12 O-tetradecanoylphorbol-13-acetate (TPA), a variety of transient alterations in the replication cycle were detected within 24 hours by the use of independent methods. Especially, a delayed passage through the S phase and influences on the G2 phase resemble x-ray irradiation effects on cell cultures. None of these alterations was observed with the hyperplasiogenic but nonpromoting 4-O-methyl-TPA.

Methods for the primary culture of rat mammary epithelial cells and the response of these cells to various hormones and culture parameters are described. In addition, the techniques for subsequent retransplantation of cultured cells into cleared fat pads of isogenic hosts as a test for normalcy of cultured tissue are detailed. A characteristic and reproducible course of the culture was followed for 2 weeks, after which rat mammary epithelial cells ceased to proliferate and were eventually overgrown by fibroblast-like cells. During the initial 2 weeks in culture, mammary cells and contaminating fibroblast-like cells were responsive to polypeptide and steroid hormones and the percentage of proliferating mammary cells could be enhanced with insulin, prolactin, estradiol, progesterone, and hydrocortisone.

The importance of hormones in the growth, morphogenesis, differentiation and neoplastic transformation of the mammary gland is well recognized due to a large number of experimentsin vivo (for review see 1). However, the precise nature of hormonal involvement is not clearly understood because of the difficulties in analyzing complex in vivo interactions. The role of growth factors in any of these processes is, perhaps, even less well understood, due in part to the complexity ofin vivo interactions and in part to the general lack of knowledge of the role of growth factors in anyin vivo process. The recent advances relating some viral oncogene products to growth factor receptor variants or growth factor sub-units makes it important to understand more fully the role of these molecules in the processes listed above. Culture systems of various types have been used to try and address these questions and are, theoretically, of great advantage since they allow observations and manipulations of cells and tissues in isolation, where experimental variables can be controlled or minimized. The goal of these investigations is to be able to reproduce in culture a system that mimics the in vivo physiological and pathological phemomena of interest. No individual system completely meets this goal, but recent advances allow a better simulation of some of the processes that occurin vivo. Unfortunately, even those systems which closely mimic the in vivo situation in terms of growth and differentiation cannot yet mimic the process of neoplastic transformation in culture.

The effects of phorbol esters on mammary epithelial cells from BALB/cfC3H/Crgl "midpregnant mice" (i.e., mice at the midterm of pregnancy) and from mammary adenocarcinomas (also from BALB/cfC3H/Crgl mice) grown in a collagen gel matrix were studied. 12-O-Tetradecanoylphorbol 13-acetate (TPA), when added to the media, caused an increased proliferation of both normal and cancerous mammary epithelial cells in a dose-dependent manner. The degree of enhancement of proliferation by TPA ranged from no increase in cell number at 3% swine serum (SW) concentration to two to three times the number of cells in the control cultures when 10 or 25% SW was used. Optimal growth was obtained with a TPA concentration of 0.1 or 1.0 microgram/ml. Increasing the SW concentration (3, 5, 10, or 25%) enhanced the proliferative effect of TPA. Cholera toxin (0.01 microgram/ml) enhanced the proliferative effect of TPA on normal cells but had a variable effect on tumor cells. The addition of TPA also resulted in a morphologic change in the epithelial colonies from midpregnant mice and from mammary tumors and caused them to assume a fibroblastic appearance. The addition of 4 alpha-phorbol or 4 alpha-phorbol 12,13-didecanoate to mammary epithelial cultures had no proliferative or morphologic effect. The results demonstrate that TPA has a growth-promoting effect on normal and cancerous mouse mammary epithelial cells.

Cytokine control of the synovial infiltrate is a central process in the development of inflammatory arthritis. In this study, we combine genetic approaches and intervention strategies to describe a fundamental requirement for IL-6-mediated STAT3 signaling in orchestrating the inflammatory infiltrate in monoarticular and systemic models of experimental arthritis. STAT3 activation via the common gp130 signal-transducing receptor for all IL-6-related cytokines led to increased retention of neutrophils and T cells within the inflamed synovium, which included STAT3-regulated IL-17A-secreting T cells. Control of leukocyte infiltration was reliant upon IL-6 signaling via its soluble receptor (termed IL-6 trans signaling), as evidenced by selective blockade of this alternative IL-6 signaling pathway using an engineered variant of soluble gp130 (sgp130Fc). This therapeutic intervention led to substantial clinical improvement in mice with emerging or established incidence of systemic arthritis. These data illustrate that IL-6 control of STAT3 is critical for regulating the synovial infiltrate in inflammatory arthritis, and suggest that selective inhibition of IL-6 trans signaling may provide a more refined intervention strategy for blocking IL-6-driven proarthritic activities.