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Human leukocyte antigen G (HLA-G) molecules are crucial for the maternal tolerance against the fetus during pregnancy. Thus, the presence of soluble HLA-G (sHLA-G) in embryo cultures is thought to be correlated to a successful pregnancy after assisted reproductive techniques (ART). Here, we established a rapid detection assay based on Luminex technology, which can be integrated into ART proceedings, allowing sHLA-G quantification in sample volumes of only 10 microl within 1.5 hours. Using this method, sHLA-G levels of 526 single-embryo cultures, 47 two-embryo cultures, and 15 three-embryo cultures were analyzed corresponding to 313 ART cycles. In 117 embryo cultures, sHLA-G was detectable. In single-embryo cultures, the sHLA-G levels were positively correlated to embryo quality (p = 0.048, r = 0.20, n = 100). The presence of sHLA-G in embryo cultures was significantly (p < 0.0001) associated with clinical pregnancy after intracytoplasmatic sperm injections (ICSI), especially in couples with male factor infertility, but not after in vitro fertilization (IVF) or in couples with female infertility. Importantly, in sHLA-G negative embryos, the abortion rate was increased threefold (p = 0.04). In conclusion, the results obtained by our novel method support strongly the diagnostic relevance of sHLA-G for predicting pregnancy outcome after ART. The ultimate conditions for this prediction have to be further investigated in a multicenter study.

Antibacterial proteins realize effective immunological mechanisms against invading pathogens. Some of them exert hemolytic and agglutinating properties. Here, we analyzed two hemolysins isolated from cell lysate (CL(39) and CL(41)) and three hemolytic proteins isolated from coelomic fluid (H(1), H(2) and H(3)) of the annelid Eisenia fetida using mass spectrometry and bioinformatics. We demonstrated the identity of CL(39,41) with fetidin and lysenin; these have been described earlier. H(1-3) share sequence components with fetidin but they seem to be glycosylated as shown for H(1). The results help to resolve a long debate concerning nomenclature and identity of these hemolytic proteins. They support: (1). the concept that the hemolytic proteins originate from chloragocytes; (2). their origin to some extent from large coelomocytes; and (3). the view that they are secreted into CF.

The natural ligands of the S100 EF hand proteins S100A8 and A9 [myeloid-related proteins 8 and 14] have long been searched for in order to further the understanding of the role of the S100A8/A9-expressing monocyte subpopulation in progressing inflammatory processes. We demonstrate that S100A8, S100A9 and the S100A8/A9 heterodimeric complex bind to human dermal microvascular endothelial cell line (HMEC)-1 with an increasing binding capacity progressing from S100A8 < or = S100A9 < or = S100A8/A9. Similar results were obtained in the apolipoprotein E knockout mouse model, where preferably recombinant S100A9 but no S100A8 bound to the endothelium of the aorta ascendens. The binding of the S100A8/A9 heterodimer complex to activated HMEC-1 is specific as demonstrated by a dose-responding and satiable binding curve and the competition of FITC-labeled versus unlabeled protein. The protein character of the binding site was proven by treatment with trypsin. S100A8/A9 binding to HMEC-1 is inducible by lipopolysaccharide and tumor necrosis factor-alpha, and in the presence of calcium. A 163-kDa protein was isolated from a cell lysate of activated HMEC-1 cells using an affinity-chromatography protocol. The endothelial cell-associated ligand proteins isolated by the use of the S100A9 monomer and the S100A8/A9 dimer were subjected to mass spectrometry for protein identification. Clearly, alpha(2)-macroglobulin was identified as a binding partner for the S100A9 monomer, whereas no protein could be identified from the database for the ligand of the S100A8/A9 dimer.

This study was undertaken to investigate the antitumor effect of liposomal hexadecylphosphocholine (L-HPC), a synthetic phospholipid encapsulated into multilamellar vesicles (MLV). The effect of these liposomes was tested in an orthotopic nude mouse model using the human mammary carcinomas MDA-MB 435 and 231. The main interest of the investigation was to study whether activated macrophages are substantially involved in the tumor growth inhibition mechanism. The growth of both MDA-MB 435 and 231 tumors in the mammary fat pad was significantly inhibited by a 14-day intraperitoneal therapy with L-HPC. The remaining tumors were shown to be heavily infiltrated with macrophages. In vitro studies of mPEM demonstrated a significant induction of macrophage-mediated tumor cytotoxicity (MMCTX) against the 2 cell lines by L-HPC. The L-HPC-mediated activation mechanism was characterized to be IL-6 and TNFalpha dependent but rather independent of IL-1alpha and nitric oxide (NO). NMA, a specific inhibitor of NO production, did not inhibit L-HPC-induced MMCTX. Furthermore, L-HPC was shown to upregulate the matrixmetalloproteinases MMP-9 and MMP-2 secretion into the supernatant. Considering cytokine release and production of collagenases, the L-HPC-induced macrophage activation cascade is assumed to be comparable with that of classical activators such as lipopolysaccharide (LPS) and interferon (IFN) gamma. As far as NO production is considered, the L-HPC activation mechanism differs from that caused by LPS and IFN gamma., (Copyright 2001 Wiley-Liss, Inc.)

Earthworm (Eisenia fetida) coelomic fluid contains several leukocytes (coelomocytes): basophils, acidophils and neutrophils as well as chloragocytes. Small coelomocytes and coelomocyte lysate are cytotoxic for the tumor cell target K562. The expression of a lytic factor was investigated by immunocytochemistry using light and transmission electron microscopy. A rat-anti-mouse-perforin-mAb labeled mainly small coelomocytes (nearly 20%) as visualized by light microscopy. TEM analysis using immunogold showed a homogenous labeling in the cytoplasm of small coelomocytes. The highest number of immunogold particles was estimated in coelomocytes with many small cytoplasmic granules. Coelomocytes with large lysosomal granules were also labeled but less intensely. No antibody binding was observed for chloragocytes either in light or electron microscopy. This suggests that the perforin-like activity is associated with only one cell type and that chloragocytes are responsible for other lytic activities. MALDI-MS revealed calreticulin usually associated with perforin in mammalian cells that mediate lysis (e.g. NK, CTL). Together, results strongly suggest the presence of putative perforin in earthworms. This in turn supports the hypothesis that perforin is a conserved component important in immune defense during evolution.

We determined whether the expression of matrix metalloproteinases (MMP) and tissue inhibitors of MMPs (TIMP) in murine macrophages is regulated by the novel synthetic bacterial lipopeptide JBT 3002. Multilamellar liposomes (MLV) encapsulating JBT 3002 (MLV-JBT 3002) stimulated the production of 72-kDa and 92-kDa (gelatinase A and B) type IV collagenase and inhibited the production of murine metalloelastase (MME) in a dose-dependent manner in murine peritoneal macrophages. MLV-JBT 3002 also induced production of TIMP-1. MLV-JBT 3002 did not induce collagenase production in tumor cells. Priming murine macrophages with interferon-gamma (IFN-gamma) inhibited JBT 3002-stimulated production of both MMP-9 and MMP-2 and further inhibited production of MME by a mechanism involving nitric oxide (NO). This conclusion is based on data showing that IFN-gamma failed to inhibit production of MMP in the presence of L-methyl arginine or in macrophages from inducible nitric oxide synthase knockout mice. These data suggest that JBT 3002 differentially regulates the production of various MMPs and TIMP in macrophages.

We describe an electrophysiological method for evaluating the intrinsic invasive potency of tumour cells using renal cells as an in vitro assay system. A high-resistance clone of Madin-Darby canine kidney cells (MDCK-C7) was grown to confluency in a filter cup. Transepithelial electrical resistance across the MDCK-C7 monolayer was measured in a commercially available electrode chamber. After a transepithelial electrical resistance of about 4,000 omega cm2 had been reached, human melanoma or pancreatic carcinoma cells were co-cultivated with the MDCK-C7 monolayer. Both carcinoma cell lines induced resistance breakdown measured after 24 h or later depending on seeding density and cell type. Seeding carcinoma cells on the basolateral surface of MDCK-C7 cells caused a similar decrease in transepithelial resistance of the MDCK-C7 monolayer. Resistance breakdown indicates opening of tight junctions prior to tumour cell invasion. In conclusion, the high-resistance MDCK-C7 cell clone could serve as a valuable biological assay system to determine electrically the metastatic potency of tumour cells in vitro.

We determined whether the implantation of human pancreatic cancer cells into the pancreas of nude mice can be used to select variants with increasing metastatic potential. COLO 357 line fast-growing cells were injected into the spleen or pancreas of nude mice. Hepatic metastases were harvested, and tumor cells were reinjected into the spleen or pancreas. This cycle was repeated several times to yield cell lines L3.6sl (spleen to liver) and L3.6pl (pancreas to liver). The variant cells produced significantly higher incidence and number of lymph node and liver metastases than the parental cells. Their increased metastatic potential was associated with increased expression (mRNA and protein) of the proangiogenic molecules basic fibroblast growth factor, vascular endothelial growth factor, and interleukin-8. The metastatic cells also exhibited increased motility and invasiveness, which were associated with increased expression of collagenase type IV (MMP-9) and decreased expression of E-cadherin. Collectively, the data show that the orthotopic implantation of human pancreatic cancer cells in nude mice is a relevant model with which to study the biology of pancreatic cancer metastasis and to select variant cell lines with enhanced metastatic potential.

We determined whether the IFN-beta gene can be used to suppress angiogenesis, tumor growth, and metastasis of human prostate cancer cells growing in the prostate of nude mice. Highly metastatic PC-3M human prostate cancer cells were engineered to constitutively produce murine IFN-beta subsequent to infection with a retroviral vector containing murine IFN-beta cDNA. Parental (PC-3M-P), control vector-transduced (PC-3M-Neo), and IFN-beta-transduced (PC-3M-IFN-beta) cells were injected into the prostate (orthotopic) or subcutis (ectopic) of nude mice. PC-3M-P and PC-3M-Neo cells produced rapidly growing tumors and regional lymph node metastases, whereas PC-3M-IFN-beta cells did not. PC-3M-IFN-beta cells also suppressed the tumorigenicity of bystander nontransduced prostate cancer cells. PC-3M-IFN-beta cells produced small tumors (3-5 mm in diameter) in nude mice treated with anti-asialo GM1 antibodies and in severe combined immunodeficient/Beige mice. Immunohistochemical staining revealed that PC-3M-IFN-beta tumors were homogeneously infiltrated by macrophages, whereas control tumors contained fewer macrophages at their periphery. Most tumor cells in the control tumors were stained positive by an antibody to proliferative cell nuclear antigen; very few were positively stained by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling. In sharp contrast, PC-3M-IFN-beta tumors contained fewer proliferative cell nuclear antigen-positive cells and many terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling-positive cells. Staining with antibody against CD31 showed that control tumors contained more blood vessels than PC-3M-IFN-beta tumors. PC-3M-IFN-beta cells were more sensitive to lysis mediated by natural killer cells in vitro or to cytostasis mediated by macrophages than control transduced cells. Conditioned medium from PC-3M-IFN-beta cells augmented splenic cell-mediated cytolysis to control tumor cells, which could be neutralized by antibody against IFN-beta. Collectively, the data suggest that the suppression of tumorigenicity and metastasis of PC-3M-IFN-beta cells is due to inhibition of angiogenesis and activation of host effector cells.

The hallmark of developing inflammatory lesions is the excess migration of recruited phagocytes together with the enhanced cell surface expression of adhesion molecules. Recent investigations give evidence that the two myeloid-related proteins MRP8 (S100A8) and MRP14 (S100A9), which are abundant in activated or recruited phagocytes, may have a modulatory role in inflammatory responses. S100A9 displays a regulatory role in the transendothelial migration of human monocytes, and the secreted S100A8/A9 complex may serve as a transport protein to move arachidonic acid to its target cells., (Copyright 2000 S. Karger AG, Basel.)

We studied activation to the tumoricidal state of murine peritoneal macrophages by liposomes containing a new synthetic analogue, JBT3002, of a lipoprotein from the outer wall of a gram-negative bacterium. The liposomes containing JBT3002 or CGP31362 were superior to liposomes containing muramyl tripeptide phosphatidylethanolamine (MTP-PE) for tumoricidal activation in three ways. First, efficient macrophage activation required lower concentrations of JBT3002 or CGP31362 than MTP-PE. Second, macrophage activation by JBT3002 was less dependent on priming by interferon-gamma. Third, MLV-JBT3002 activated tumoricidal properties in both lipopolysaccharide (LPS)-responsive and LPS-nonresponsive macrophages. The activation of tumoricidal properties by MLV-JBP3002 depended on protein tyrosine kinase (PTK) activity associated with phosphorylation of tyrosine. The major mechanism for tumoricidal activity in macrophages incubated with MLV-JBT3002 was due to increased activity of inducible nitric oxide synthase (iNOS) and, hence, production of nitric oxide (NO). We base this conclusion on the results of several experiments. First, MLV-JBT3002 was not directly toxic to tumor target cells. Second, the specific iNOS inhibitor NG-monomethyl-L-arginine abrogated tumor cell lysis by MLV-JBT3002-treated macrophages. Third, macrophages from iNOS knockout mice did not lyse tumor cells, even after incubation with high concentrations of MLV-JBT3002. These data suggest that liposomes containing the synthetic bacterial lipopeptide JBT3002 are potent activators of macrophage tumoricidal properties.

The entry of microorganisms into the body induces inflammatory processes. During this process a sequence of cellular, humoral, non-specific and specific actions are evoked to combat the infection. Macrophages and granulocytes, which are developed from a common progenitor cell, are the cellular components of the specific and non-specific immunoreaction. MRP14 (Macrophage migration inhibitory related protein) and MRP8, two S-100 proteins contained in high concentrations in these cells are obviously essential for adhesion and migration of monocytes and granulocytes. To investigate the transcriptional regulation of these genes we cotransfected constructs expressing CAT under control of the MRP14 promoter and expression constructs of C/EBP alpha and v-myb, two transcription factors involved in myeloid/monocytic differentiation. Transfection with C/EBP alpha revealed a massive enhancement of the MRP14 promoter in both, HL 60 cells (granulocytic differentiated) and L132 fibroblasts. In contrast, v-myb reduces MRP14 promoter activity. Northern blot analysis of L132 cells transfected with the C/EBP alpha expression vector demonstrate that C/EBP alpha is sufficient to enhance MRP14 expression in the context of the whole genome.

We investigated the expression of cytokine genes and tumoricidal properties in human blood monocytes in response to a new synthetic immunomodulating lipopeptide, JBT3002. Incubation of peripheral blood monocytes with free-form JBT3002 or JBT3002 encapsulated in multilamellar phospholipid vesicles (liposomes, MLV-JBT3002) induced tumoricidal properties in a dose-dependent manner. Both MLV-JBT3002 and free-form JBT3002 induced production of tumor necrosis factor alpha, interleukin-1beta, and interleukin-6 in a dose-dependent manner with similar kinetics. Treatment of monocytes with interferon-gamma did not significantly alter the expression of cytokine genes but increased the expression of cytokines induced by MLV-JBT3002 and free-form JBT3002. In contrast to monocyte activation by lipopolysaccharide (LPS), activation by JBT3002 was independent of serum and was not inhibited by CD14-neutralizing antibody. Incubation of monocytes with JBT3002 induced a rapid increase in tyrosine phosphorylation of proteins with apparent molecular masses of 42 and 38 kDa, a migration band shift of c-Jun NH2-terminal kinase 1 (JNK1), and activation of extracellular signaling regulated kinases. Consistent with its effect on cytokine expression, stimulation of these intracellular signaling pathways by JBT3002 was not inhibited in serum-free conditions. Collectively, the data indicate that the synthetic lipopeptide JBT3002 is a potent monocyte activator that modulates monocyte function by mechanisms similar to LPS but by a distinct receptor.

In this study, we endeavored to determine the effectiveness of interferon beta (IFNbeta) gene therapy against highly metastatic murine UV-2237m fibrosarcoma cells. UV-2237m cells were engineered to produce murine IFNbeta constitutively following infection by a retroviral vector harboring the murine IFNbeta gene. Parental (UV-2237m-P), control-vector-transduced (UV-2237m-Neo), and IFNbeta-transduced (UV-2237m-IFNbeta) cells were injected subcutaneously (s.c.) or intravenously (i.v.) into syngeneic mice. Parental and control-transduced cells produced rapidly growing tumors, whereas IFNbeta-transduced cells did not. The tumorigenicity of IFNbeta-sensitive or -resistant parental cells was significantly suppressed when they were injected s.c. together with IFNbeta-transduced cells. The IFNbeta-transduced cells did not inhibit growth of parental cells injected s.c. at a distant site. UV-2237m-IFNbeta cells produced s.c. tumors in nude, SCID/Beige, and natural killer(NK)-cell-compromised syngeneic mice. The IFNbeta-transduced cells were more sensitive to in vitro splenic cell-mediated lysis than were the parental or control-transduced cells. Pretreatment of C3H/HeN mice with the NK-cell-selective antiserum (anti-asialoGM1) partially abrogated the cytotoxic activity of the cells. Cytotoxic activity was not observed in mixed culture of UV-2237m-IFNbeta cells and splenic cells from SCID/Beige mice. Significant cytotoxicity against UV-2237m-IFNbeta cells was mediated by macrophages activated by either IFNgamma, lipopolysaccharide, or a combination of both. Our data led us to conclude that the constitutive expression of IFNbeta can suppress tumorigenicity and metastasis of UV-2237m cells, which is due, in part, to activation of host effector cells.

Multilamellar liposomes from different types of alkylphosphocholines (APC), which had been shown to activate macrophages, were prepared in the presence or absence of polyethylene glycol and the uptake of these multilamellar vesicles (MLV) by J 774A. 1 macrophages was examined. Using l-hydroxypyrene-3,6,8-trisulfonic acid (HPTS), a pH sensitive, highly fluorescent dye, it is possible to microscopically differentiate between liposomes adhered to the outer cell membrane and liposomes endocytosed by macrophages (Finkelstein and Weissmann 1978; Straubinger et al. 1983). In order to quantify the intracellular liposome-associated HPTS, fluorescence of cell lysates was measured at +/-390. We found that phagocytosis of APC-MLV by macrophages is phospholipid type (DCP, TPC, HPC, EPC) and size dependent. Total liposome uptake increased at 37 degrees C for all types of liposomes, plateauing at 4-6 hours, whereas very little liposome attachment could be measured at 4 degrees C. MLV liposomes are incorporated into J 774A.1 macrophages more rapidly and effectively than SUV. DPPE- or DSPE-PEG was used to produce sterical stabilization of the liposomes. The addition of these lipids led to an almost complete inhibition of liposomal uptake by the macrophages over a time period of 24 hours, independent of the PEG-lipid type and -acyl chain length used for liposome preparation. The hypothesis that PEG-like lipids can be powerful tools to prevent APC-MLV from being taken up by the RES and rapidly eliminated from circulation is supported by the present data.

The present study was undertaken to investigate the effect of JT3002, a new synthetic analogue of a lipoprotein from the outer wall of a gram-negative bacterium on the production of cytokines by mouse peritoneal macrophages. Multilamellar liposomes containing different concentrations of JT3002 induced production of the inflammatory cytokines tumor necrosis factor-alpha, interleukin-1 alpha, and interleukin-6 by macrophages in dose- and time-dependent manners. The presence of interferon-gamma enhanced production of tumor necrosis factor-alpha by macrophages exposed to lower concentrations of JT3002 and induced the release of nitric oxide, a potent cytolytic molecule of activated macrophages. Unlike lipopolysaccharide, JT3002 activated macrophages independently of serum, but like lipopolysaccharide, it required protein tyrosine kinase.

The aim of the study was to investigate for the first time the preparation, physical properties and macrophage activating effect of sterically stabilized liposomes made from hexadecylphosphocholine (HPC, Miltefosine) using different poly(ethylene glycol) lipids for coating. We could demonstrate that it is possible to prepare different liposomal vesicle types (MLV, SUV and LUVET) without any problem and with a high stability in buffer (release of hydrophilic marker was < 5% after half a year) and in plasma (t1/2 up to several days). The preparation method, including size of polycarbon membrane filter used for the preparation of LUVETs had the main influence on vesicle size and size distribution. The addition of a charged lipid like DCP and different amounts of PEG-lipid up to 10% had no effect on size and stability of PEG-LUVETs. A comparison of activating potency of PEG-HPC-vesicles with commonly used HPC-liposomes was performed with mouse peritoneal macrophages. HPC-liposomes induced a clear release of NO and TNF from mouse peritoneal macrophages especially in a synergistical action with LPS. On the contrary the effect of PEG-liposomes was similar to control cells after a combined activation in vivo/in vitro. The reduced interaction of these liposomes with the MPS was also demonstrated by an unchanged carbon ink uptake after treatment of mice (i.p.) with liposomes prepared with and without PEG-lipid. PEG-HPC-liposomes combine the advantages of HPC, liposomes and PEG-coating, resulting in a promising preparation for treatment of mammary cancers.

Oligospecific antisera against the hemolytic and hemagglutinating compounds of the coelomic fluid (CF) of the earthworm species Eisenia fetida, Lumbricus terrestris and Aporrectodea caliginosa were prepared. The proteins were bound to the membranes of erythrocytes and injected into rabbits. By the use of these antisera the following results were obtained: 1) The antisera RaL T and RaAC reached a titer of 1:64,000, the detection limit of RaEF was 1:512,000. RaEF was demonstrated to be oligospecific only against three hemolytic proteins by Western blotting. 2) RaEF is able to inhibit the biological activity not only of hemolysins (E. fetida, A. caliginosa) but also of hemagglutinins (L. terrestris, L. rubellus, D. rubidus). 3) Complex carbohydrates, but not simple sugar compounds, are able to inhibit hemolytic and agglutinating activities in the CF of the investigated species. Fet. and alpha 1ac. GP were demonstrated to be strong inhibitors both of the hemolytic and of the hemagglutinating activity, whereas N-acetylglucosamine was only able to inhibit the hemagglutinating activity. 4) The investigated compounds were shown to crossreact in ELISA experiments. 5) By the use of Western blotting, the crossreacting molecules in the CF of the investigated lumbricid species were identified.

Hexadecylphosphocholine (HPC) has been investigated intensively for its cancerostatic properties. One explanation for the mechanism of action of HPC assumes that it plays a role in stimulation of the immune system. In particular, its potency to activate macrophages has already been recognised for different lyso- and ether lipids. Important steps in the cascade for developing cytotoxic effects of macrophages on tumor cells are the release of nitric oxide radicals (NO) and/or tumor necrosis factor (TNF). The aim of our study was to examine the role of HPC as primer and/or trigger for macrophage activation to cytotoxicity. In our experiments we used HPC in free (micellar) or liposomal form in different primer/trigger combinations with lipopolysaccharide (LPS). A weak change in morphology was revealed by electron microscopy, if macrophages were harvested from mice previously treated with HPC or HPC multilamellar vesicles. This observation was quantified by the measurement of NO, TNF and cytotoxic activity of the peritoneal macrophages. A specific release of NO was induced by the combination of in vivo treatment with liposomal HPC and subsequent stimulation by LPS in vitro. This process started only after 12 h of in vitro incubation of macrophages with the endotoxin. The release of TNF was dependent of the primer/trigger combination used. A moderate priming effect was obtained with HPC in liposomal form independently of the trigger. On the other hand, liposomes as priming agents were found to induce a dramatic increase in TNF release after in vitro coculture with the trigger LPS. The high release of NO and TNF is accompanied by only a weak increase in tumor cytostasis. The best results were once more found with macrophages primed with liposomal HPC and then triggered with LPS.

The human histiocytic cell line U 937, which expresses a number of monocyte markers and properties, was investigated with regard to its ability to be activated for NO and tumor necrosis factor (TNF) release after treatment with alkylphosphocholines (APC) and APC liposomes. Using APC multilamellar vesicles (MLV) a clear dose-dependent increase of NO production could be demonstrated for U 937 cells, whereas the corresponding soluble substances had no effect. The time course of NO release was characterised by a peak between 2 h and 12 h and a strong decrease after 24 h. LPS caused no NO release nor the production of TNF in U 937 cells. The simultaneous incubation of the cells with lipopolysaccharide and APC or APC-MLV, led to a strong increase in TNF production. Closer investigation of the time sequence of this synergistic effect demonstrated that cells, that had first been treated with hexadecylphosphocholine (HPC)-MLV and 4 h later with lipopolysaccharide secreted significantly more TNF into the supernatants than in the experiment where both substances were added simultaneously. From these results it was concluded that APC-MLV are possibly able to act as a primer in the process of lipopolysaccharide mediated TNF induction. Furthermore, a positive influence of phorbol 12-myristate 13-acetate (PMA) on the ability of U 937 cells to produce TNF following a treatment with HPC or HPC-MLV could be observed. PMA-pretreated cells were shown to release much more TNF compared to control cells, which led to the supposition that the immunomodifying activity of APC becomes effective only in more highly differentiated cell types.