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The growth of a panel of 22 different human tumor, leukemia, and lymphoma cell lines was examined in a human tumor cloning assay in agar or methylcellulose and a tritiated thymidine uptake assay. The cultures were performed in the absence or presence of increasing concentrations (0.5-500 ng/ml) of nerve growth factor (NGF). The growth of 17 of the 22 cell lines was not significantly and reproducibly affected by NGF. There was minor (1.2-fold) but reproducible stimulation of clonal growth in one glioblastoma cell line (86-HG-39) by NGF, but in this cell line NGF induced no growth modulation in a tritiated thymidine uptake assay. However, clonal growth of another glioblastoma cell line (87-HG-31) and all three lung cancer cell lines tested (HTB 119, HTB 120, CCL 185) could be stimulated up to 3-fold by NGF with a dose-response relationship for the growth factor. Growth stimulation by NGF could be completely reversed by neutralizing anti-NGF antibody and by the tyrosine kinase inhibitor genistein. Evaluation of secondary plating efficiency revealed the stimulation of colony formation as representing self-renewal and not terminal differentiation. Reverse transcriptase-PCR experiments in the five responding cell lines showed expression of both low-affinity NGF receptor (glycoprotein 75) and c-trk transcripts on the mRNA level. Of the five responding cell lines, only 86-HG-39, the cell line with the lowest responsiveness, revealed low-affinity NGF receptor on the protein level; the other four cell lines with high responsiveness, including the three lung cancer cell lines, expressed no low-affinity NGF receptor as shown by fluorescence-activated cell sorter analysis and immunoprecipitation using the ME 20.4 antibody. Immunoprecipitation using anti-trk antibodies was negative in all five responding cell lines. However, binding studies with iodinated NGF showed only low-affinity binding on the 86-HG-39 cell line and only high-affinity binding on the high-responder cell lines CCL 185 and 87-HG-31. In summary, our data suggest that NGF can be operative in stimulation of clonal growth of malignant tumor cells. High-affinity but not low-affinity binding sites mediate signal transduction for clonal growth and signaling involves tyrosine kinase activity.

Neuroendocrine tumors of the gastroenteropancreatic system represent a group of tumors with various diagnostic problems. Especially detection of primary tumor lesions is often difficult. Endoscopic ultrasonography is a relatively new imaging procedure localizing insulinomas preoperatively in about 90% of cases. Thus, previously used invasive preoperative imaging methods are usually unnecessary. The combination of endoscopic ultrasonography and somatostatin receptor scintigraphy allows visualization of gastrinomas in 90% of cases. Somatostatin receptor scintigraphy can also visualize metastatic lesions of gastrinomas and carcinoids in the whole body with high accuracy. In surgical management of a gastrinoma, duodenal transillumination and intraoperative ultrasound should be performed in all cases to exclude small duodenal or periduodenal, extrapancreatic tumors. US, CT, and MRI should be mainly used to exclude local and distant metastases. Angiography is helpful in detecting anatomical variations of abdominal vessels preoperatively. Due to the excellent results of endoscopic ultrasonography and somatostatin receptor scintigraphy in localizing insulinomas and gastrinomas, transhepatic portal venous sampling appears to be obsolete.

In a prospective study of 13 patients (three males and 10 females; mean age 53 [8-82] years) the value of somatostatin receptor scintigraphy (SRS) and endoscopic ultrasonography (EUS) was compared with transabdominal ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI) in the diagnosis of insulinoma (six patients) or gastrinoma (seven patients). There were ten separate primary lesions of each tumour type, proven histologically. For insulinoma the sensitivity of EUS was 90%, SRS 10% and CT, US and MRI together 20%. For gastrinoma the sensitivity of EUS was 90%, SRS 100%, and 30% for the other three methods together. Thus EUS had a high diagnostic localizing sensitivity for both tumours, while SRS was highly sensitive only in the diagnosis of gastrinoma, not insulinoma. The value of CT, MRI and conventional ultrasonography lies in their ability to visualize distant metastases.

Neurone und neuroendokrine Zellen haben eine Vielzahl von Merkmalen gemeinsam. Der Einsatz molekular- und zellbiologischer Verfahren hat das Verstandnis physiologischer und pathophysiologischer Vorgange wie Wachstum, Adhasion und Sekretion von neuroendokrinen Zellen verbessert. In dieser Arbeit werden aktuelle Befunde zum einem aus dem Bereich der Grundlagenforschung, aber auch der augenblicklichen, klinischen Forschung vorgestellt, die fur eine verbesserte Diagnostik und Therapie neuroendokriner Tumoren des gastroenteropankreatischen Systems relevant sind.

Eukaryonte Zellen verfugen uber ein intrazellulares Verteilersystem mit dem sie neu synthetisierte Proteine in verschiedene subzellulare Kompartimente und auch in den Extrazellularraum befordern konnen. Dabei werden Proteine, die fur den Export bestimmt sind, entweder kontinuierlich (konstitutiv) oder nach Stimulation der Zelle (reguliert) sezerniert. Wahrend der Translation der mRNA werden die neu entstehenden Proteinketten zu Translokationsstellen des endoplasmatischen Retikulums (ER) gebracht, wo sie entweder in der ER-Membran verbleiben oder uber spezifische Proteinkanale in das Lumen gelangen. Nach der Passage durch das ER werden die Proteine in Vesikel verpackt, wobei losliche Proteine luminal, und integrale Membranproteine membranstandig vorliegen. Mit einem Mantel (“coat”) aus zytoplasmatischen strukturgebenden Proteinen versehen, wandern die Vesikel zur nachsten Zielorganelle. Dort verschmelzen sie mit der Membran, wobei der Mantel unter Mitwirkung monomerer G-Proteine und Hydrolyse von GTP abgelegt wird. Die Membran der Vesikel wie auch die Membranen der jeweiligen Akzeptororganellen enthalten spezifische Proteine, die eine gerichtete Membranfusion und damit auch einen gerichteten Proteintransport sichern (SNARE-Hypothese). In dieser Arbeit werden die grundlegenden Mechanismen des Proteintransports erlautert und Zusammenhange mit bekannten, v. a. gastroenterologischen Krankheitsbildern hergestellt.

Clinical characteristics, echocardiographic and Doppler echocardiographic findings, as well as serum levels of chromogranin A were recorded on 62 patients (27 women, 35 men; mean age 55 [11-83] years) with histologically confirmed tumours of the gastroenteropancreatic (GEP) system. Changes in the right heart were found in 14 patients (22%), club-like thickening of tricuspid leaflets in 13, tricuspid regurgitation in 14, stenosis in 2 and right atrial or right ventricular dilatation in 11 and 5, respectively. There was no difference between the patients with or without right-heart changes in regard to age, presence of carcinoid syndrome, duration of symptoms, primary tumour site, pattern of metastases, treatment or chromogranin A level. Two patients with nonfunctioning tumours had right-heart changes. While clinical and biochemical parameters did not identify patients with right-heart changes, echocardiography demonstrated all haemodynamically significant endocardial changes. Even patients with nonfunctioning GEP tumours should be regularly monitored by echocardiography.

Localization of gastroenteropancreatic neuroendocrine tumors with conventional imaging techniques can be difficult. With the advent of the somatostatin-receptor scintigraphy the diagnosis of these could be improved. In this study 76 patients with immunohistologically proven neuroendocrine tumors were analysed by comparing somatostatin-receptor scintigraphy with conventional imaging techniques. Somatostatin-receptor-positive lesions were observed in 61 of all patients. Conventional imaging techniques (transabdominal ultrasonography, computerized tomographic scanning of the abdomen, magnetic resonance imaging of the abdomen) revealed neuroendocrine tumors in 42 patients (69%). A follow up of 19 patients with initially false positive somatostatin-receptor scintigrams showed that at least 6 patients had escaped the initial diagnosis by conventional techniques. All in all, our data support previous findings and demonstrate that somatostatin-receptor scintigraphy is a safe and sensitive procedure for in vivo imaging of gastroenteropancreatic neuroendocrine tumors. In addition, in certain cases, somatostatin-receptor scintigraphy is able to detect tumors that had escaped conventional imaging techniques.

Neuroendokrine gastroenteropankreatische (NE-GEP) Tumore exprimieren Somato-statin-Rezeptoren mit hoher Dichte und Affinitat [1]. Diese Rezeptoren konnen in vivo mittels eines Indium-111-markierten Somatostatinanalogons szintigraphisch dargestellt werden [2]. In dieser Untersuchung soll die diagnostische Relevanz der Somatostatin-Rezeptor-Szintigraphie (SRS) gegenuber etablierten bildgebenden Verfahren der Sonographie (US), der Endosonographie (EUS), der Kernspintomographie (NMR) und der Computertomographie (CT) evaluiert werden.

Background and aims: Tight junctions within the intestinal epithelium constitute pivotal elements of the intestinal barrier, perturbations of which have been associated with the pathogenesis and progression of inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. We previously demonstrated that glucocorticoid hormones stimulate intestinal tight junction sealing In Vitro by increasing the expression of claudin-4. The aim of this study was to identify the signaling pathways responsible. Methods: Caco-2 monolayers grown on semipermeable filter supports were treated with dexamethasone, the p38 inhibitor SB203580 or the MKP-1 inhibitor triptolide and tight junction function was assessed by measuring transepithelial electrical resistance (TEER). Activation of the claudin-4 promoter was analyzed in Caco-2 cells stably expressing the human claudin-4 promoter fused to a luciferase reporter. In addition, quantitative rt-PCR, Western Blotting and immunofluorescence were employed to analyze changes in the expression, activation and localization of claudin-4, MKP-1 and p38. Results: Treatment of Caco-2 cells with dexamethasone resulted in increased TEERs, increased expression of claudin-4 on the RNA and protein level and activation of the claudin-4 promoter. This was accompanied by decreased phosphorylation of the MAPK p38 as well as increased expression of the MAPK phosphatase MKP-1. Inhibition of p38 by SB203580 was sufficient to mimic the effect of dexamethasone both on TEER and claudin-4 expression as well as claudin-4 promoter activation. Conversely, inhibition of MKP-1 by triptolide prevented the dephosphorylation of p38 by dexamethasone and reversed its effect on TEER, claudin-4 expression and activation of the claudin-4 promoter. Localization of claudin-4 to the tight junction or cell viability was not affected by either of the compounds. Conclusions: Our data provide evidence that glucocorticoid induced up regulation of claudin-4 is mediated by dephosphorylation of p38 resulting from increased expression of theMAPK phosphatase MKP-1.We thereby identified a novel signaling pathway mediating an epithelial specific effect of glucocorticoids on intestinal tight junctions. Given the central role of barrier dysfunction in inflammatory bowel diseases, components of this pathway might represent promising new pharmacological targets for the treatment of these disorders.

Chromogranin A (Cg A) is a useful marker of neuroendocrine neoplasia in humans. Here we describe an immunoluminometric assay (ILMA) for measuring Cg A in serum, with use of a new tube as a solid phase. The new tube has a large surface area and is coated with a polyclonal antibody. Optimized coating conditions provide a high IgG adsorption to the polystyrene wall. Serum is added to the coated tube and incubated for 2 h or overnight. After washing the tubes, acridinium ester-labeled monoclonal antibody against Cg A is added and incubation is continued for 2 h. The tubes are washed again and the bound luminescence is measured. The assay is very sensitive (detection limit 1 microgram/L, if 25 microL of serum is used), is specific for human Cg A, and offers a wide dynamic range (1-1000 micrograms/L). The range in healthy humans is 10-53 micrograms/L (median 30 micrograms/L). The correlation of serum Cg A to creatinine and parathyroid hormone is described. We also report the influence of intravenous calcium injection on Cg A concentrations in serum.

The permanent cell culture line AR42J, derived from a rat pancreatic acinar carcinoma, is widely used for functional studies of exocrine pancreatic acinar cells. We now present evidence that these cells are amphicrine in that they contain zymogen granules as well as small (40-80 nm) neuroendocrine (NE) vesicles and typical neurotransmitters. Using the small NE vesicle-specific markers synaptophysin and "protein S.V.2", including synaptophysin cDNA probes, we have found that AR42J cells synthesize these proteins and contain vesicles harboring these proteins with biophysical properties similar to those of small NE vesicles. NE properties of these cells are further indicated by the presence of considerable amounts of stored amino acids (gamma-aminobutyric acid (GABA), glycine, glutamate) and by the presence of the GABA-synthesizing enzyme, glutamic acid decarboxylase. Finally, intermediate filament (IF) protein typing showed only cytokeratins 8 and 18, indicating that AR42J cells possess an IF protein complement indistinguishable from that of acinar and islet cells. Our results document the unusual case of a permanent cell line with combined exocrine and neuroendocrine properties that may be indicative of a derivation from a cell with multipotential character.

14684 Background: Vascular endothelial growth factors (VEGFs) and VEGF receptors (VEGFRs) are important mediators of tumor growth and metastasis. Since NEC of the gastroenteropancreatic system are ...

Chromogranin A (1, 2) ist ein hauptsachliches sekretorisches Protein des Nebennierenmarks. Nach der eingehenden Charakterisierung dieses Proteins zeigte sich, das dieses weit verbreitet in peptidsezernierenden neuroendokrinen Zellen vorkommt (s. hierzu Ubersichtsartikel WIEDENMANN amp; HUTTNER (3)). Da mittlerweile bekannt ist, das alle freigesetzten Hormone, Neuropeptide und Catecholamine mit Chromogranin zusammen freigesetzt werden, und diese „Peptidverpackungs“-Proteine nicht nur in normalen, sondern auch in fast alien neoplastischen neuroendokrinen Zellen des Gastrointestinaltraktes vorkommen, wurden Seren von Patienten mit unterschiedlichen gastroenteropankreatischen neuroendokrinen Tumoren mittels einer Radioimmunoassays fur Chromogranin, vergleichend mit konventionellen Untersuchungsmethoden, wie z.B. 5-Hydroxy-Indolessigsaure im Urin, Gastrin, VIP, Pankreatisches Polypeptid und anderen gastrointestinalen Hormonen untersucht.

4558 Background: Pasireotide is a novel multi-ligand somatostatin analogue with high affinity binding for four of the five somatostatin receptor subtypes (sst1,2,3 and sst5). This Phase II clinical trial showed that pasireotide is effective in controlling the symptoms of diarrhea and flushing in 27% of patients with metastatic carcinoid tumors refractory or resistant to octreotide LAR. The impact of pasireotide therapy on health-related quality of life (HRQL) was also evaluated. Methods: Patients in this open-label, multicenter study initially received pasireotide 300 μg sc bid which was escalated to a maximum dose of 1,200 μg sc bid until clinical response was achieved. Data are reported for the Functional Assessment of Chronic Illness Therapy-Diarrhea (FACIT-D) instrument. FACIT-D comprises the Functional Assessment of Cancer Therapy-General (FACT-G) score, which measures physical, social, emotional and functional well-being, plus a symptom-specific sub-scale which measures HRQL specific to diarrhea. FACIT-D baseline assessment was obtained on day 1 immediately before dosing, and then monthly thereafter. FACIT-D sub-scale and total scores at baseline and after 1, 2, and 3 months of pasireotide treatment are described by categories of clinical response. Results: 45 patients (mean age 61 years; range 40–83) received treatment, and 44 were eligible for the efficacy and HRQL analyses. Functional well-being scores, symptom-specific scores and total FACIT-D scores of non-responders tended to be lower at baseline and during treatment than those of responders. The three sub-scale and summary FACIT-D scores exhibited relatively stable mean HRQL scores and similar patterns of variability in clinical responders and non- responders through the third month of pasireotide treatment. Conclusions: HRQL was stable during treatment with pasireotide in patients with advanced metastatic disease refractory or resistant to octreotide LAR. Additional work in HRQL study design and evaluation of HRQL endpoints in patients with carcinoid disease is indicated, and will further improve our understanding of quality of life in patients with this disease. [Table: see text]

4082 Background: Pasireotide is a novel multiligand somatostatin analogue that exhibits high binding affinity to 4 of 5 somatostatin receptor subtypes: sst1,2,3 and sst5. Compared with octreotide, pasireotide has 30, 5 and 40 times greater affinity for sst1,3 and sst5 receptors respectively and a comparable affinity for sst2. Methods: This was a Phase II, open-label, multicenter study in patients with metastatic carcinoid tumors whose symptoms (diarrhea and flushing) were inadequately controlled by octreotide LAR. Patients had histopathologically confirmed disease, elevated 5-HIAA and/or CgA levels and at least one measurable lesion (excluding bone). Patients initially received pasireotide 300 μg sc bid and escalated to a maximum dose of 1200 μg sc bid every 3 days until clinical response was achieved. Partial response (PR) was defined as a mean of [Table: see text]

8024 Background: SOM230 is a novel multi-ligand somatostatin analog that exhibits high binding affinity to four somatostatin receptor subtypes: sst1,2,3 and sst5. Compared with octreotide, SOM230 has 30, 5 and 40 times greater affinity for sst1,3 and sst5 receptors, respectively, and a comparable affinity for sst2 receptors. Methods: This was a Phase II open-label, multicenter study in patients with metastatic carcinoid tumors whose symptoms (diarrhea and flushing) were refractory/resistant to octreotide LAR. Enrolled patients had histopathologically confirmed disease, elevated 5-HIAA and/or CgA levels and at least one measurable lesion (excluding bone). Symptom control with subcutaneous SOM230 was evaluated daily using patient diaries. Patients initially received SOM230 300 μg bid. Dose escalation of 150 μg bid, up to a maximum of 1200 μg bid, was allowed every 3 days until adequate symptom control was achieved. Adequate control or partial response (PR) was defined as a mean of

4086 Background Only few data exist on long-term outcome and independent prognostic factors of neuroendocrine tumors (NET) of the gastroenteropancreatic (GEP) system. Low incidence, heterogeneous c...

The need for standards in the management of patients with endocrine tumors of the digestive system prompted the European Neuroendocrine Tumor Society (ENETS) to organize a first Consensus Conference, which was held in Frascati (Rome) and was based on the recently published ENETS guidelines on the diagnosis and treatment of digestive neuroendocrine tumors (NET). Here, we report the tumor-node-metastasis proposal for foregut NETs of the stomach, duodenum, and pancreas that was designed, discussed, and consensually approved at this conference. In addition, we report the proposal for a working formulation for the grading of digestive NETs based on mitotic count and Ki-67 index. This proposal, which needs to be validated, is meant to help clinicians in the stratification, treatment, and follow-up of patients. © Springer-Verlag 2006.

We have defined the predominant site of p-nitrophenyl acetate reaction with hemoglobin. The site is involved with, at least, two modes of action: the imidazole catalysis of His beta 2 and the irreversible covalent acetylation of Lys beta 82. The effect of competitive inhibition of the reaction by 2,3-diphosphoglyceric acid, the dependence of the reaction rate on the protein conformation, hemoglobin mutants, and the diethylpyrocarbonate are consistent with the assignment of the active site. In addition, the results point to small conformational differences in the NH-terminal regions of the beta chains between Hb S and Hb A.

Synaptophysin, a mol. wt 38 000 glycopolypeptide of the synaptic vesicle membrane, was solubilized using Triton X-100 and purified by immunoaffinity or ion-exchange chromatography. From gel permeation and sucrose-density centrifugation in H2O/D2O, a Stokes radius of 7.3 nm, a partial specific volume of 0.830 and a total mol. wt of 119 000 were calculated for the native protein. Cross-linking of synaptic vesicles with glutaraldehyde, dimethylsuberimidate, or Cu2+ -o-phenantroline, resulted in the formation of a mol. wt 76 kd dimer of synaptophysin. Crosslinking of the purified protein in addition produced tri- and tetrameric adducts of the polypeptide. Native synaptophysin thus is a homooligomeric protein. Synaptophysin is N-glycosylated, since cultivation of the rat phaeochromocytoma cell line PC12 in the presence of tunicamycin reduced its mol. wt by about 6 kd. Upon transfer to nitrocellulose and incubation with 45Ca2+, synaptophysin behaved as one of the major calcium-binding proteins of the synaptic vesicle membrane. Pronase treatment of intact synaptic vesicles abolished this 45Ca2+ binding indicating that the Ca2+ binding site of synaptophysin must reside on a cytoplasmic domain of the transmembrane polypeptide. Based on these data, we propose that synaptophysin may play an important role in Ca2+-dependent neurotransmitter release.

Synaptophysin is an integral membrane glycoprotein originally isolated from presynaptic vesicles of bovine neurons. The authors have studied a wide spectrum of neuroendocrine (NE) neoplasms by immunofluorescence microscopy on cryostat sections of freshly frozen tissues using a monoclonal antibody to this protein (SY 38). Without exception, they found the identical--or a very similar--protein expressed in all neuroblastomas, ganglioneuroblastomas, ganglioneuromas, pheochromocytomas, and paragangliomas studied. In these "neural" type NE neoplasms, synaptophysin was coexpressed with neurofilament proteins. Synaptophysin was also demonstrated in NE neoplasms of "epithelial" type in which it was predominantly coexpressed with cytokeratins and desmoplakin. It was invariably found in all variants of islet cell neoplasms and in all medullary thyroid carcinomas. Synaptophysin was also demonstrated in several adenomas of the hypophysis and parathyroids, in the majority of carcinoids of the bronchopulmonary and gastrointestinal tracts, and in many, though not all, NE carcinomas of the same sites, and of the skin. Conversely, SY 38 did not immunostain any of a large number of benign and malignant non-NE epithelial neoplasms; nor was any immunostaining obtained in a group of mesenchymal tumors. It is remarkable that SY 38 did not immunostain a number of malignant melanomas, including several that were immunostained for neuron-specific enolase (NSE) and several neuropeptides. Parallel studies conducted on conventionally fixed, paraffin-embedded tissue sections immunostained by the use of the avidin-biotin complex technique yielded very similar results. The findings indicate that synaptophysin is expressed in the whole range of NE neoplasms without detectable relation to the expression of other NE markers such as NSE, serotonin, and neuropeptides. Nor could the expression of synaptophysin by these tumors be correlated with their epithelial and/or neural cytoskeletal characteristics, their clinical aggressiveness, or the presence or absence of endocrinologic abnormalities. While the consistent expression of synaptophysin by the "neural" type of NE neoplasms would seem predictable its presence in diverse benign and malignant NE tumors of "epithelial" type is remarkable. It is concluded that synaptophysin is a significant as well as novel NE marker, and the use of antibody SY 38 as a broad range marker for the study and diagnosis of NE neoplasms is proposed.

We have defined the predominant site of p-nitrophenyl acetate reaction with hemoglobin. The site is involved with, at least, two modes of action: the imidazole catalysis of His beta 2 and the irreversible covalent acetylation of Lys beta 82. The effect of competitive inhibition of the reaction by 2,3-diphosphoglyceric acid, the dependence of the reaction rate on the protein conformation, hemoglobin mutants, and the diethylpyrocarbonate are consistent with the assignment of the active site. In addition, the results point to small conformational differences in the NH-terminal regions of the beta chains between Hb S and Hb A.

Synaptophysin (SYP) is a glycoprotein recently isolated from presynaptic vesicles of bovine neurons. Initial studies have demonstrated its presence in neurons in the brain, spinal cord and retina, and in adrenal medullary cells. A subsequent study demonstrated it in pancreatic islet cells and certain neuroendocrine (NE) neoplasms, including several pancreatic islet cell tumors. Based on these preliminary observations, we examined, by immunohistochemistry, conventionally fixed, paraffin sections of 57 pancreatic endocrine tumors with a monoclonal antibody to SYP. Furthermore, we compared the SYP immunoreactivity of 30 of these same tumors with that of neuron-specific enolase (NSE) and of chromogranin (CG). SYP was demonstrated in all but one of the 57 tumors. In the comparative study, for which material was available in only 30 cases, SYP and NSE were present in 29 of the tumors, whereas CG was seen in only 15 cases. We conclude that SYP is a highly sensitive and useful marker for pancreatic NE neoplasms. Moreover, in view of the increasingly evident limited specificity of NSE, SYP should be considered the marker of choice for pancreatic NE neoplasms.

Tumor necrosis factor (TNF), a protein predominantly produced by activated macrophages/monocytes, is presently available in recombinant, purified form for clinical trials. Intensive studies in many laboratories have shown that besides the tumorcytotoxic effects, TNF acts on a large array of different cells and has potent immunomodulatory activities. In a clinical phase I study, some immunologic functional parameters of blood cells from patients who received 24-hour infusions of recombinant human TNF (rhTNF) were analyzed. Natural killer (NK) cell activity, TNF production, interleukin-1 (IL-1) production and mitogen-induced proliferation were measured either in whole blood samples or in cultures of peripheral mononuclear leukocytes of the patients directly before and after rhTNF infusion. NK cell activity, TNF and IL-1 production capacity and proliferative responses to concanavalin A (Con A) were significantly reduced after rhTNF application. We conclude from these observations that rhTNF in vivo acts directly or indirectly on NK cells and monocytes by either inactivating their functional capacity or by absorbing the relevant cells to the endothelial cell layer, thus removing them from circulation.