Preferential alkaline phosphatase isoenzyme induction by sodium butyrate

SW-620, a continuous cell line derived from a poorly differentiated human colon carcinoma, produces two alkaline phosphatases. Under basal conditions the heat-stable, term-placental is the major isoenzyme and the heat-labile, liver/bone/kidney form represents a minor component. Exposing SW-620 cells to sodium butyrate causes induction of increased levels of activity accompanied by a striking shift in isoenzyme distribution not observed heretofore. The activity increase is accounted for entirely by augmentation of the liver/bone/kidney isoenzyme, with the term-placental form not being affected. Two other known alkaline phosphatase inducers, prednisolone and hyperosmolality, do not influence specific activity and isoenzyme distribution. The preferential induction of the liver/bone/kidney form of alkaline phosphatase in SW-620 cells may reflect a butyrate-elicited expression of a more differentiated state.     

Characterization of human foetal intestinal alkaline phosphatase. Comparison with the isoenzymes from the adult intestine and human tumour cell lines.

The molecular structure of human foetal intestinal alkaline phosphatase was defined by high-resolution two-dimensional polyacrylamide-gel electrophoresis and amino acid inhibition studies. Comparison was made with the adult form of intestinal alkaline phosphatase, as well as with alkaline phosphatases isolated from cultured foetal amnion cells (FL) and a human tumour cell line (KB). Two non-identical subunits were isolated from the foetal intestinal isoenzyme, one having same molecular weight and isoelectric point as placental alkaline phosphatase, and the other corresponding to a glycosylated subunit of the adult intestinal enzyme. The FL-cell and KB-cell alkaline phosphatases were also found to contain two subunits similar to those of the foetal intestinal isoenzyme. Characterization of neuraminidase digests of the non-placental subunit showed it to be indistinguishable from the subunits of the adult intestinal isoenzyme. This implies that no new phosphatase structural gene is involved in the transition from the expression of foetal to adult intestinal alkaline phosphatase, but that the molecular changes involve suppression of the placental subunit and loss of neuraminic acid from the non-placental subunit. Enzyme-inhibition studies demonstrated an intermediate response to the inhibitors tested for the foetal intestinal, FL-cell and KB-cell isoenzymes when compared with the placental, adult intestinal and liver forms. This result is consistent with the mixed-subunit structure observed for the former set of isoenzymes. In summary, this study has defined the molecular subunit structure of the foetal intestinal form of alkaline phosphatase and has demonstrated its expression in a human tumour cell line.     

The acid phosphatases of Thermoascus crustaceus, a thermophilic fungus

Thermoascus crustaceus, a filamentous, thermophilic ascomycete with pathogenic potential was cultured on Sabouraud's liquid medium at temperatures from 27 to 47 degrees C for periods up to 7 days. Growth rate and yield were optimal at 37 degrees C. Morphological changes were confined to the cell walls, the thickness being greatest at 47 degrees C, which were also more resistant to mechanical disruption. Significant amounts of acid phosphatase (EC 3.1.3.2) activity occurred in the spent media of all cultures but were greatest at 37 degrees C. The proportions of acid phosphatase activity which were operationally defined as soluble or bound were also documented; the optimum pH for acid phosphatase activity in all fractions was 5.0. Extracts were subjected to polyacrylamide gel electrophoresis under non-denaturing conditions and the gels were stained for acid phosphatase activity. This revealed four electrophoretically distinct acid phosphatases which had different susceptibilities to inhibition by fluoride, phosphate, or tartrate. Effects of growth temperature, or phosphate supplement in the culture medium, on the acid phosphatase isoenzyme pattern were judged to be minor. Cytochemistry at the electron microscope level indicated acid phosphatase activity on the surface, in the periplasmic space, and in the cytoplasm, but no trends with regard to growth conditions. A substantial temperature range can be tolerated by this species but it is concluded that neither the general shape of the cells nor the acid phosphatase isoenzyme pattern changes substantially; this contrasts with previously documented differences for this class of enzyme in dimorphic Sporotrix schenckii.     

Generation of monoclonal antibodies to prostatic acid phosphatase isoenzyme 2 and application in solid-phase enzyme immunoassay

Monoclonal antibodies specific to prostatic acid phosphatase (PAP) isoenzyme 2 were generated by using an improved hybridoma technique. After three subcutaneous immunizations and three intravenous boosters, cell fusion experiments were performed. The hybrid cells were first cultured in a semisolid medium containing methylcellulose and later transferred to a liquid medium for further subculture. Out of a total of 600 colonies recovered after two cell fusion experiments, 13 were shown to exhibit affinity to PAP isoenzyme 2 by radioimmunoassay. Nine hybrid cell lines which showed high affinity and specificity were established for further evaluation. Their immunoglobulin subclass was determined to be immunoglobulin G. The association constants between PAP isoenzyme 2 and each monoclonal antibody were determined by titration curve in radioimmunoassay (RIA). Three of them (PAP 1, PAP 03, and PAP 019) were shown to be over 1 X 10(9) M-1. From the results of a matrix cross-matching procedure, a pair of antibodies (PAP 03 and PAP 1) reacting with discrete antigenic determinants were identified for preparing a solid phase sandwich enzyme immunoassay (EIA) kit. The designed EIA procedure could be performed within 40 min in a one-stage incubation protocol. The assay time was shorter than that of other commercial RIA or EIA kits, and the sensitivity was 0.4 ng/ml which was comparable to that of RIA kits. The EIA kit was shown not to cross-react with human thyroid stimulating hormone, alpha-fetoprotein, carcinoembryonic antigen, and acid phosphatases derived from tissues other than prostate. Therefore, this design was a simple and rapid method with high sensitivity and specificity for determining PAP isoenzyme 2 in human serum.     

Cadmium-induced synthesis of metallothioneins in human lymphocytes and monocytes

Cd2+-binding proteins of peripheral blood lymphocytes and monocytes have not well been characterized so far, although they are expected to be a clue for understanding Cd2+ toxicity in those immune competent cells. We separated a family of Cd2+-binding proteins from Cd2+-exposed human peripheral blood lymphocytes by gel filtration chromatography, and characterized them by SDS-gel electrophoresis. The proteins showed electrophoretic behaviours closely similar to metallothioneins (MTs) of HeLa cells derived from human cervical carcinoma. The proteins were also found in Cd2+-exposed monocytes, and were inducible by Cd2+ in both lymphocytes and monocytes. Anti-MT serum specifically precipitated these proteins, which were thus identified as MTs. These results suggest that the two classes of the cells involved in the immune system possess a protective mechanism against Cd2+ through MTs. A variety of human lymphoid cell lines derived from both T and B cells were also found to have capacity to synthesize MTs in response to Cd2+.     

Alkaline phosphatase and an acid arylamidase as marker enzymes for normal and transformed WI-38 cells

A survey of eleven enzyme activity levels in normal and SV40 transformed (VA-13) WI-38 cells revealed that the transformed cell enzymes differed by a quantitative and qualitative change of alkaline phosphatase and a quantitative loss of an arylamidase. Alkaline phosphatase activity was found to be elevated in the transformed cells at confluency but not in log phase cultures. This elevated activity was heat stable, L-homoarginine resistant and L-phenylalanine sensitive and is probably the term placental isoenzyme. In nontransformed WI-38 cells, the alkaline phosphatase was heat labile, L-homoarginine sensitive and L-phenylalanine resistant and so is probably the liver isoenzyme. While the arylamidase activity from both normal and transformed WI-38 cells had identical pH optima and Km values, the activity was approximately 20 times higher in confluent WI-38 cells than in confluent VA-13 cells. Cytochemical staining techniques for both activities are described that permit identification of fluorescent product within the cells, analysis of activity levels, and separation of cells with high and low activities. Mixtures of WI-38 cells and VA-13 cells separated by flow cytometry on the basis of arylamidase activity were subsequently evaluated for alkaline phosphatase isoenzyme and found to have been simultaneously separated into heat labile and heat stable samples.     

Biochemical and immunological relationships of prostatic and leukocytic acid phosphatases and their subcellular localization

Human acid phosphatases from various sources were purified by a new rapid procedure including chromatofocusing and gel permeation chromatography on an FPLC system. Isoenzymes 2a and 4 (Mr 105,000 and 76,000, respectively) are the prevalent acid phosphatases in prostate, leukocytes and bone marrow. Additional forms are proteolytic products formed during isolation procedure. The source of elevated acid phosphatase in blood serum in metastasizing prostatic carcinoma can be either prostate itself, the tumour or, more likely, bone marrow cells destroyed during the invasion by the tumour cells. Distribution of isoenzymes is not organ specific. It was found using an antiserum against isoenzyme 4 that immunoreactivity is confined to the specific secretory granules both of prostatic epithelium and leukocytes.     

T-leukemia cell lines CCRF-CEM, HPB-ALL, JM and MOLT-4: changes in isoenzyme profiles during induction of differentiation

Biochemical analysis has been used to monitor the induction of differentiation in cultured human T-leukemia cell lines (CCRF-CEM, HPB-ALL, JM and MOLT-4) by the phorbolester 12-0-tetradecanoylphorbol 13-acetate (TPA). The isoenzymes of carboxylic esterase, acid phosphatase, hexosaminidase and lactate dehydrogenase were separated by isoelectric focusing on horizontal thin-layer polyacrylamide gels and stained by histo-cytochemical methods. TPA inhibited the proliferative activity in all four cell lines and led to aggregation of cells seen as floating clusters. TPA induced an increase in number and staining intensity of isoenzymes of all four enzymes in the cell lines studied. This corresponds to an induced isoenzymatic maturation as the progressive increase in number and staining intensity of the isoenzymes parallels the differentiation along the T-cell pathway. However, regardless of the initial stage of arrested differentiation, the cell lines could be induced only to differentiate to a certain more mature stage, but could not be triggered to differentiate terminally with regard to expression of isoenzyme patterns.     

Deletion map of the Escherichia coli K-12 dnaB gene

Summary Acid phosphatase isoenzymes of Chlamydomonas reinhardii were investigated by isoelectric focusing in polyacrylamide gel systems. In this paper we describe in detail an original method for isoelectric focusing of acid phosphatases extracted from wildtype and acid phosphatase-lacking mutant algae, obtained from Laboratoire de Génetique of University of Liège. Three isoenzymes can be separated from the buffer-soluble components of these cells. An additional isoenzyme type can be visualized using the nonionic detergent NP40 as solubilizer. We conclude that these four isoenzymes are releated to the structural gene of the soluble constitutive acid phosphatase, which was shown by their appearance in P ₂ and their total absence in mutant P _a. The pl values of soluble constitutive acid phosphatase isoenzymes range between pH 5.2 and 6.2. As a result of treatment with NP40 the extracts from both wild-type and mutant lines contain two additional active phosphatase forms which can be characterized by their high heat resistance and low pI values. These enzymes are fully active using either -naphthyl phosphate or different acetate esters as substrates.     

Acid phosphatases in germinal and somatic cells of the testes

Four forms of acid phosphatase have been found in the testicular tissue of many mammalian species, but their exact cellular site has remained obscure. In this work, acid phosphatases have been studied in different reproductive organs of the male rat, in somatic cell lines derived by cloning from both rat and mouse testes, in primary cultures of rat Sertoli cells, and in isolated spermatogenic cells of the mouse. Among the reproductive organs, preputial glands show the highest specific activities with p-nitrophenyl phosphate as substrate, followed by the testicular tissue and the different regions of the epididymis. By contrast to that in other tissues, testicular activity with p-nitrophenyl phosphate is not influenced by tartrate and is activated markedly by cobalt (Co2+). Among the somatic cell lines, the highest hydrolysis rates are obtained with naphthyl substrates in the epithelial (TR-1) and myoid (TR-M) cell lines and marginally lower rates in the Leydig (TM3) and Sertoli (TM4) cell lines. With thymolphthalein phosphate, the latter two cell lines show very low activity. These activities are not influenced by different hormones and growth factors in the culture medium. The most marked Co2+-activated reaction with p-nitrophenyl phosphate is found in advanced stages of germinal cells and residual bodies. Primary cultures of Sertoli cells, prepared from rats 10 to 30 days of age, show a slight decrease in acid phosphatase levels; however, the activities are not influenced markedly by addition of follicle-stimulating hormone (FSH) and/or testosterone to the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localisation of High Acid Phosphotyrosine Phosphatase Activity in Afferent Arterioles and Glomeruli of Human Kidney

Summary Endothelial cells contain a variety of specific protein tyrosine phosphatases and an acid phosphatase differing from other known phosphatases. The highest activity of this acid phosphatase with artificial or unspecific substrates is present in the afferent arterioles and glomeruli of human kidney, and the activity is inhibited by nephrotoxic fluoride concentrations, suggesting that it plays a role in circulatory regulation. Here the activity was characterised with physiological substrates. An incubation mixture containing phosphotyrosine or phosphoserine was stable at pH 5 when phosphate-precipitating lead was chelated with tartrate. The activities were studied in frozen sections. Only phosphotyrosine was hydrolysed by some cells. High activity of tartrate-resistant phosphotyrosine phosphatase was present in lymphocytes, endothelial cells of afferent arterioles, and glomerular mesangial cells of kidney, decidual cells, and alveolar macrophages. In lymphocytes the activity was fluoride-resistant and vanadate-sensitive, in other cells fluoride- and vanadate-sensitive. In decidual cells and alveolar macrophages, the activity is due to specific osteoclastic/macrophagic tartrate-resistant acid phosphatase, in lymphocytes to specific protein tyrosine phosphatases, and in endothelial and mesangial cells to a protein tyrosine phosphatase-like acid phosphatase. The results suggest that in endothelial cells of the afferent arterioles, mesangial cells, and lymphocytes the cellular activities are regulated by high constitutive phosphotyrosine phosphatase activity and this may be related to the exceptional cyclosporin A sensitivity of these cells.     

Inhibition of T cell antigen receptor signaling by VHR-related MKPX (VHX), a new dual specificity phosphatase related to VH1 related (VHR).

A cDNA encoding a novel, human, dual-specific protein phosphatase was identified in the Incyte data base. The open reading frame predicted a protein of 184 amino acids related to the Vaccinia virus VH1 and human VH1-related (VHR) phosphatases. Expression VHR-related MKPX (VHX) was highest in thymus, but also detectable in monocytes and lymphocytes. A VHX-specific antiserum detected a protein with an apparent molecular mass of 19 kDa in many cells, including T lymphocytes and monocytes. VHX expression was not induced by T cell activation, but decreased somewhat at later time points. In vitro, VHX dephosphorylated the Erk2 mitogen-activated protein kinase with faster kinetics than did VHR, which is thought to be specific for Erk1 and 2. When expressed in Jurkat T cells, VHX had the capacity to suppress T cell antigen receptor-induced activation of Erk2 and of an NFAT/AP-1 luciferase reporter, but not an NF-kappaB reporter. Thus, VHX is a new member of the VH1/VHR group of small dual-specific phosphatases that act in mitogen-activated protein kinase signaling pathways.     

Functional effects of a monoclonal antibody directed against a distinct epitope on 4F2 molecular complex in human peripheral blood mononuclear cell activation.

The 4F2 antigenic complex is expressed on most human cell lines in culture, on monocytes and activated lymphocytes, but not on resting T and B lymphocytes. Monoclonal antibody (mAb) CB43 recognizes an epitope of the 4F2 heterodimer either located on the light chain or dependent on the conformation of the molecule. The binding of CB43 mAb to peripheral blood mononuclear cells (PBMC) induced a dose-dependent comitogenic effect in the presence of submitogenic concentrations of anti-CD3 mAb. Significant amounts of interleukin (IL)-1 beta but not IL-2 or interferon-gamma were released in the supernatant. Pretreatment of monocytes with CB43 mAb increased the phytohemagglutinin-induced T lymphocyte proliferation. However, CB43 mAb did not exert agonistic effects on activated T lymphocytes. Depletion of CB43+ cells from PBMC decreased the proliferation and generation of cytotoxic effector cells induced by a mannoprotein (MP) derived from Candida albicans cell wall but not by recombinant IL-2. Furthermore, depletion of CB43+ cells from PBMC preactivated with MP or rIL-2 led to a significant decrease in their cytotoxic activity. CB43 mAb did not inhibit the growth of cell lines nor the proliferation of T cells. Thus CB43 mAb identifies a distinct functional epitope on the 4F2 molecular complex and might be useful in further studying the role of this molecule in cellular activation.     

Alkaline phosphatase phenotypes in tumour and non-tumour cell lines: not an invariable marker for neoplastic transformation

The cytochemical localisation and presumed isoenzyme type (based on selective inhibition experiments) of alkaline phosphatase in 5 cell lines derived frrom normal human, rat, mouse and hamster tissues, 6 human lymphoblastoid lines and 6 human and mouse tumour-derived cell lines are described. Enzyme activity varied between the cell lines. An isoenzyme inhibited by L-phenylalanine was present in 3 normal lines, 3 lymphoblastoid lines and 2 tumour lines. The presence of this isoenzyme cannot be used as a marker of neoplastic transformation.     

Analysis of isoenzyme patterns of acid phosphatase in acute leukemias

The status of acid phosphatase isoenzymes was evaluated in cells of patients with acute lymphocytic leukemias or lymphomas by analytical isoelectric focusing in polyacrylamide gels (IEF) on horizontal thin-layer slabs. The isoenzyme patterns were correlated with routine immunological cell surface markers and the relationship of enzyme activity to specific immunological subclasses of ALL is discussed. By isoelectric focusing up to five isoenzyme groups (I-V) containing several isoenzyme were observed. No leukemia specific or additional isoenzyme could be demonstrated. This biochemical characterization showed a marked heterogeneity within two major immunologic subgroups indicating that various differentiation stages of cell maturation could be involved in cALL and T-ALL. According to their degree of maturation along T-cell differentiation axis the leukemic cells displayed no enzyme activity, weak isoenzyme bands or the incomplete or complete isoenzyme pattern seen with normal lymphocytes from human tonsils which were used as controls. The investigation of specific enzymatic patterns can lead to a further definition of subsets of acute leukemias and give insight into lymphopoietic differentiation.     

Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH4 rat pituitary cells.

Okadaic acid, a selective inhibitor of serine/threonine protein phosphatases, was utilized to investigate the requirement for phosphatases in cell cycle progression of GH4 rat pituitary cells. Okadaic acid inhibited GH4 cell proliferation in a concentration-dependent manner with a half-maximal inhibition (IC50) of approximately 5 nM. Treatment of GH4 cells with 10 nM okadaic acid resulted in a 40-60% decrease in phosphatase activity and an increase in the proportion of phosphorylated retinoblastoma (RB) protein. Cell cycle analysis indicated that okadaic acid increased the percentage of cells in G2-M, decreased proportionally the percentage of cells in G1 phase, and had little effect on the percentage of cells in S-phase. The absence of a change in the proportion of S-phase cells indicates that G1-specific phosphatases responsible for dephosphorylation of RB protein were not inhibited by 10 mM okadaic acid. Mitotic index revealed that 10 nM okadaic acid decreased proliferation of GH4 cells specifically by slowing the progression through mitosis. Immunostaining with anti-tubulin demonstrated that 10 nM okadaic acid-treated mitotic cells contained mitotic spindles; however, the spindle apparatus in these cells frequently contained multiple poles. These results suggest that the organization of spindle microtubules during prometaphase requires a protein phosphatase that is sensitive to nanomolar concentrations of okadaic acid. Chromosomes in 10 nM okadaic acid-treated cells appear to be attached to spindle microtubules and the nuclear envelope is absent. The effects of okadaic acid on the spindle differ from those elicited by the calcium channel blocker, nimodipine, indicating that this okadaic acid sensitive phosphatase is not part of the calcium signalling events which participate in mitotic progression.     

Differential spontaneous killing of human and murine tumour cell lines by leucocyte subpopulations from carp peripheral blood leucocytes

Cell populations from carp (Cyprinus carpio L.) peripheral blood leucocytes (PBLs) were examined for nonspecific cytotoxicities. By using monoclonal antibodies (MAbs) against carp thrombocytes (TCL-HB8) and both neutrophils and monocytes (TCL-BE8), PBLs with a density of 1.08 g ml-1 were separated into three fractions: thrombocytes, a mixture of neutrophils and monocytes, and other cells (mainly lymphocytes), and the separated cells were tested for cytotoxic activities against mammalian tumour cell lines (K562, HeLa, P815 and Yac-1 cell). Consequently, the mixture of neutrophils and monocytes exhibited cytolysis against these target cells, whereas the lymphocyte-rich and thrombocyte fractions did not show any cytolysis. To isolate only neutrophils, which do not contain monocytes, the MAb (TCL-BE8) positive cells from PBLs with a density of 1.08-1.09 g ml-1 were separated. Pure isolated neutrophils showed cytotoxic activities against K562 cells, but not P815 cells. Furthermore, analysis of the cytolytic mechanisms indicated that killing of these cells depended on H2O2 or HOCl. These results suggest that both neutrophils and monocytes are effectors for nonspecific cytotoxicity in carp PBLs, and neutrophils may be distinct from monocytes in their reactivity in cytolysis, including target cell selectivity and/or target cell sensitivity, and the cytolytic pathway. In carp, cytotoxicity of target cells can be mediated by several populations of their leucocytes which have cytotoxic capacities with various recognition and cytolytic mechanisms.     

Evidence that SHIP-1 contributes to phosphatidylinositol 3,4,5-trisphosphate metabolism in T lymphocytes and can regulate novel phosphoinositide 3-kinase effectors

The leukemic T cell line Jurkat is deficient in protein expression of the lipid phosphatases Src homology 2 domain containing inositol polyphosphate phosphatase (SHIP) and phosphatase and tensin homolog deleted on chromosome ten (PTEN). We examined whether the lack of expression of SHIP-1 and PTEN is shared by other leukemic T cell lines and PBLs. Analysis of a range of cell lines and PBLs revealed that unlike Jurkat cells, two other well-characterized T cell lines, namely CEM and MOLT-4 cells, expressed the 5'-phosphatase SHIP at the protein level. However, the 3-phosphatase PTEN was not expressed by CEM or MOLT-4 cells or Jurkat cells. The HUT78 cell line and PBLs expressed both SHIP and PTEN. Jurkat cells exhibited high basal levels of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3); the lipid substrate for both SHIP and PTEN) as well as saturated protein kinase B (PKB) phosphorylation. Lower levels of PI(3,4,5)P(3) and higher levels of phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) as well as unsaturated constitutive phosphorylation of PKB were observed in CEM and MOLT-4 cells compared with Jurkat cells. In PBLs and HUT78 cells which express both PTEN and SHIP-1, there was no constitutive PI(3,4,5)P(3) or PKB phosphorylation, and receptor stimuli were able to elicit robust phosphorylation of PKB. Expression of a constitutively active SHIP-1 protein in Jurkat cells was sufficient to reduce both constitutive PKB membrane localization and PKB phosphorylation. Together, these data indicate important differences between T leukemic cells as well as PBLs, regarding expression of key lipid phosphatases. This study provides the first evidence that SHIP-1 can influence the constitutive levels of PI(3,4,5)P(3) and the activity of downstream phosphoinositide 3-kinase effectors in T lymphocytes.     

Cell surface tumor necrosis factor (TNF) accounts for monocyte- and lymphocyte-mediated killing of TNF-resistant target cells

WEHI164 cells are susceptible to cytotoxicity by soluble recombinant or monocyte-derived TNF alpha, as well as to cell-mediated cytotoxicity by monocytes or lymphocytes. In contrast, K562 cells are resistant to lysis by soluble recombinant or natural TNF alpha, but are killed by monocyte or lymphocyte effector cells. Cell-mediated cytotoxicity against both target cell lines is enhanced by treatment of monocyte effector cells with recombinant interferon gamma or lymphocyte effector cells with interleukin-2. However, treatment of monocytes with LPS, or of lymphocytes with PHA, although inducing secretion of soluble TNF alpha in the medium, does not increase cell-mediated cytotoxicity. Anti-TNF alpha neutralizing antibodies partially inhibit monocyte- as well as lymphocyte-mediated cytotoxicity against WEHI164 and K562 cells. Formaldehyde-fixed effector cells are cytotoxic to both target cell lines. Cytotoxicity by fixed effector cells can be inhibited by anti-TNF alpha antibodies. The extent of cell-mediated cytotoxicity induced by treatment of effector cells with stimulators prior to fixation corresponds to the expression of TNF on monocyte membranes, but not to the titers of secreted TNF. The data suggest that membrane-associated TNF alpha may be a mechanism of human monocyte- as well as lymphocyte-mediated cytotoxicity, regardless of whether the target cells are sensitive or insensitive to soluble TNF.