Mikael Lantz

Isolation and characterization of a tumor necrosis factor binding protein from urine

Tumor necrosis factor (TNF)/cachectin can produce both beneficial and harmful manifestations. Mechanisms may operate to counteract potentially harmful effects such as shock and cachexia. The TNF binding protein (TNF‐BP), which is found at increased levels in serum and urine of patients with chronic renal failure, may play such a role. TNF‐BP was purified 1000000–fold to homogeneity from urine of patients with chronic renal failure by use of ion exchange chromatography, affinity chromatography on TNF‐Sepharose and reverese phase chromatography. The purified protein contained only one chain with an apparent Mr on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of 30000. The aminoterminal amino acid sequence D‐S‐V‐X‐P‐Q‐G‐K‐Y‐I‐H‐P‐Q‐V‐N‐S‐I‐X‐K‐T revealed no significant homologies with previously described protein sequences. TNF‐BP may act as a regulator of the bioactivities of TNF/cachectin.

Infusion of tumor necrosis factor (TNF) causes an increase in circulating TNF-binding protein in humans

Serum samples from cancer patients receiving intravenous infusions of recombinant tumor necrosis factor (rTNF) and recombinant interferon-gamma (rIFN-gamma) were analyzed for TNF and the TNF-binding protein (TNF-BP). TNF-BP is a soluble fragment of the transmembrane TNF receptor with antagonistic effects to TNF and is released by proteolytic cleavage of the receptor. During a 60-min infusion of rTNF, peak serum levels of rTNF were observed after 30 to 60 min and a transient increase of circulating TNF-BP was observed with peak levels between 30 and 120 min. Injection of IFN-gamma alone did not affect the levels of TNF and TNF-BP. Thus administration of rTNF leads to release into the circulation of TNF-BP, which may modulate both systemic and local effects of TNF and influence its therapeutic efficacy.

Pregnant Women on Thyroxine Substitution Are Often Dysregulated in Early Pregnancy

BACKGROUND Thyroid hormones are important for normal fetal development. Maternal hypothyroidism during early pregnancy is associated with impaired neuropsychological development of children and other adverse outcomes. The primary aim of this prospective study was to determine whether thyroxine-treated pregnant women with hypothyroidism are adequately thyroxine substituted in early pregnancy. A secondary aim was to determine if fetal loss differed between females with thyrotropin (TSH) values within and outside the reference range at their first TSH test, scheduled for 1-2 weeks after verification of pregnancy. METHODS This was a prospective open-labeled study. During the years 1997-2002, 119 consecutive pregnancies in 101 females with thyroid diseases were followed at the Department of Endocrinology, Malmö University Hospital. At the first visit, 63 patients, median age 30 years (range 17-45 years), were on thyroxine substitution therapy for hypothyroidism. In these patients 83% were in their first trimester at the time of the initial test. RESULTS Of the 63 patients on thyroxine substitution for hypothyroidism 32 (51%; Group A) patients had serum TSH values within the reference range at their initial test and 31 (49%; Group B) had serum TSH values outside the reference range. Twelve (19%) had TSH values of <0.40 mIU=L and 19 (30%) had TSH values of >4.0 mIU=l. The fetal loss was 2 of 32 (6%) in Group A compared to 9 of 31 (29%) in Group B ( p < 0.05). CONCLUSIONS In 49% of pregnant women on thyroxine substitution, serum TSH values were outside the reference range when first tested, generally in the first trimester. Fetal loss was significantly greater in pregnant women with abnormal TSH values compared to those with normal TSH values. Thyroid function in pregnant women on thyroxine substitution should be monitored early in pregnancy and carefully followed during pregnancy. The thyroxine dose should be increased as needed early in pregnancy to avoid hypothyroidism.

Metalloproteases and serineproteases are involved in the cleavage of the two tumour necrosis factor (TNF) receptors to soluble forms in the myeloid cell lines U-937 and THP-1.

The proteolytic processing of the two TNF receptors (TNF‐R55 and TNF‐R75) into soluble forms was investigated in the myeloid cell lines U‐937 and THP‐1. Phorbol myristate acetate (PMA) rapidly stimulated release of soluble forms of both TNF‐receptors. Incubations were made with PMA and protease inhibitors directed against different target protease groups. The serineprotease inhibitors TPCK and dichloroisocoumarin and the metalloprotease inhibitor 1, 10‐phenanthroline reduced PMA‐induced release of both soluble receptor forms with about 60–70%. Furthermore, 1, 10‐phenanthroline also reduced PMA‐induced down‐regulation of TNF‐receptors in both cell lines as judged by TNF‐binding to cells. Reduced down‐regulation and TNF‐receptor shedding by 1, 10‐phenanthroline was reversed by Zn2+, indicating involvement of a Zn2+‐dependent metalloprotease. Thus, both serine proteases and metalloproteases are involved in the processing of TNF‐receptors.

The receptors for regulatory molecules of hematopoiesis

Abstract: Proliferation and differentiation of hematopoietic cells are controlled by pleiotropic regulatory molecules. While the sequences of these factors are not related, their membrane receptors are restricted to two gene families with homologous domains. The members of the hematopoietic (or cytokine) receptor family (for erythropoietin, interleukins‐2, ‐3, ‐4, ‐6 and ‐7, granulocyte‐macrophage and granulocyte colony‐stimulating factor) are composed of multiple subunits necessary for high‐affinity binding and cell signalling. Signal transducing mechanisms are largely unknown. The occurrence of variant signal transducers in different tissues could explain the pleiotropy of these regulatory molecules. Members of the receptor tyrosine kinase family bind dimeric forms of macrophage colony‐stimulating factor, stem cell factor and platelet‐derived growth factor leading to kinase activation and phosphorylation of many substrates involved in production of second messengers. Soluble forms (binding proteins) exist for members of both families. These may be proteolytic cleavage products of transmembrane receptors or naturally secreted products. Such binding proteins can potentially function as inhibitors in feedback regulation and in protection and transport of cytokines and would provide a rational therapy when cytokines are produced in excess. Knowledge of signal transduction mechanisms and of the three‐dimensional structure of ligands and receptors can lead to the design of drugs with cell‐specific effects.

Modulation of the constitutive gene expression of the 55 kD tumor necrosis factor receptor in hematopoietic cells

The expression of the 55 kD human tumor necrosis factor (TNF) receptor gene was investigated. By use of a 1.2 kb cDNA we demonstrated a constitutive expression of a single 2.3 kb transcript in cell lines and fresh blood cells. The TNF receptor gene expression was not affected by phorbol esters, dibutyryl cAMP (dbcAMP), interleukin-1 (IL-1), interferon-gamma (IFN-gamma) or by TNF, agents known to modulate functional TNF receptors. The phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) showed a dose dependent inhibition of the TNF receptor gene expression. This inhibition is not mediated by cAMP, since neither dbcAMP nor forskolin had any effects on the expression of the 55 kD receptor gene. The results suggest that the effects of phorbol diesters, IL-1, IFN-gamma, TNF and dbcAMP previously observed on binding of TNF to cells are limited to posttranscriptional regulation of the 55 kD TNF receptor.

Treatment with a thiazolidinedione increases eye protrusion in a subgroup of patients with type 2 diabetes.

Objective  Changes in eye protrusion in patients treated with pioglitazone.

Characterization of a relationship between the T‐lymphocyte derived differentiation inducing factor (DIF) and lymphotoxin: A common receptor system for DIF, lymphotoxin and tumor necrosis factor downregulated by phorbol diesters

Here we describe results which show that recombinant lymphotoxin (rLT), like the T‐lymphocyte derived differentiation inducing factor (DIF), inhibited the clonogenic growth of some myeloid leukemia cell lines by concentrations of 1 to 30 pmol/l. Wild type HL‐60 cells were resistant at these concentrations but responded with differentiation into monocyte‐like cells at higher concentrations. An antigenic relationship between DIF and LT was indicated because a neutralizing monoclonal anti‐LT anti‐body bound to and neutralized both differentiation and growth inhibitory effects of DIF. An activity, which cochromatographed with DIF during all purification steps, competed with binding of both rLT and recombinant tumor necrosis factor (rTNF) to HL‐60 cells. By use of radioiodinated ligand, 2100 binding sites for rLT were detected on HL‐60 cells with a Kd of 330 pmol/l. At 37°C bound ligand was transferred to lysosomes, followed by degradation. rTNF and rLT were shown to compete for binding sites on HL‐60 cells. Receptors for both rLT and rTNF were downregulated by activators of protein kinase C such as phorbol diester or diacylglycerol; the number of cell surface receptors decreased while the Kd remained unchanged. Our observations demonstrate a functional and antigenic relationship between DIF and LT and indicate that TNF, LT and DIF share binding sites on myeloid leukemia cells that are downregulated by activation of protein kinase‐C.

Proteomics of Orbital Tissue in Thyroid-Associated Orbitopathy.

CONTEXT A potentially altered protein expression profile in orbital tissue from patients with thyroid-associated orbitopathy (TAO) is suspected. OBJECTIVE To detect for the first time changes in proteomic patterns of orbital connective tissue in TAO and compare these with control tissue using mass spectrometry. DESIGN Proteomics cross-sectional, comparative study. SETTING Two academic endocrine institutions. SAMPLES A total of 64 orbital and peripheral adipose tissue samples were collected from 39 patients with TAO and 25 control subjects. METHODS Samples were analyzed and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technology. MAIN OUTCOME MEASURES Mean intensity values of all identified peptides per protein. RESULTS Thirty-one proteins were identified, of which 16 differentiated between controls and patients with TAO. Different protein patterns between orbital and peripheral adipose tissue were observed. Compared to controls, 10 proteins were markedly up-regulated (≥ 2-fold) in the orbital tissue of untreated patients: beta IV spectrin (6.2-fold), GTP binding G protein 2 (5.6-fold), POTE ankyrin domain family member F (5.4-fold), xylulokinase (4.1-fold), kinesin family member 1A and lipocalin 1 (both 3.6-fold), semicarbazide-sensitive metalloproteinase amine oxidase 3 and polymerase I transcript release factor (both 3.4-fold), cell-cycle protein elongin A binding protein 1 (3.3-fold), annexin A2 and cavin (both 3-fold), protein pointing to cell proliferation histone H4 (2.8-fold), and ADAM metallopeptidase with thrombospondin type 1 motif 14 (2.7-fold). The highest protein up-regulations were noted in the orbital tissue of medically untreated patients. Steroid therapy markedly reduced up-regulation of these proteins, foremost in nonsmokers. CONCLUSIONS Proteins involved in tissue inflammation, adipose tissue differentiation, lipid metabolism, and tissue remodeling were up-regulated in orbital tissue of untreated patients with TAO. Steroids decreased the expression of these proteins, whereas smoking attenuated such effect.

Effect of α-IFN on cytokine-induced antigen expression and secretion of TNF, LT and IgM in HCL

Abstract In order to investigate the possible mechanisms for the effect of alpha-interferon (α-IFN) in hairy cell leukaemia (HCL), blood cells from 4 cases were treated in vitro with α-IFN, tumour necrosis factor (TNF) and interleukin 2 (IL-2). Changes in the antigen expression, immunoglobulin (Ig) secretion and the production of TNF and lymphotoxin (LT) were investigated. TNF induced expression of CD4 and CD71, increased the intensity of HLA-DR, CD25, CD11c and CD13 expression and decreased both the intensity and frequency of sIg and cIg positivity. α-IFN decreased CD25 expression, the tartrate-resistant acid phosphatase activity (TRAP), reduced the TNF-induced CD4 and CD71 expression and antagonized the TNF effect on the Ig expression. Spontaneous TNF or LT production could not be detected in culture supernatants. However, TNF was found to induce LT production, an effect which α-IFN antagonized and IL-2 augmented. The reduction of CD25, TNF-induced CD71 and TRAP caused by α-IFN seems to represent a deactivation of the activated state of hairy cells (HCs). The failure of α-IFN to induce Ig secretion or CD38 expression in HCs speaks against a differentiation induction effect. The LT secretion induced by TNF suggests that other cytokines than TNF might be involved in the proliferation of HCs and that α-IFN by blocking the production of LT and perhaps other cytokines causes a growth arrest in HCs.