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Role of tumor necrosis factor system in pregnancy induced insulin

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Role of tumor necrosis factor system in pregnancy induced insulin Powered By Docstoc
					 Role of tumor necrosis factor system in pregnancy
induced insulin resistance and in the pathogenesis of
            cancer of the uterine cervix


                      Dr. Zsolt Melczer


    Semmelweis University Faculty of Medicine

   2nd Department of Obstetrics and Gynecology


Supervisors: Dr. Károly Cseh and Dr. András Falus

                            Budapest

                                2003.

      Ph.D. Course of Semmelweis University
     Molekuláris orvostudományok Tudományági Doktori Iskola /7/


          Szigorlati bizottság: Dr. Fekete Béla egyetemi tanár
                              Dr. Pajor Attila egyetemi tanár
                               Dr. László Ádám oszt. vez. főorvos


         Hivatalos bírálók: Dr. Csermely Péter egyetemi tanár
                            Dr. Pálfalvi László főorvos
       Biráló bizottság tagjai: Dr. Doszpod József egyetemi tanár
                              Dr. Rosivall László egyetemi tanár
                              Dr. Csapó Zsolt egyetemi docens
                              Dr. Lintner Ferenc oszt. vez. főorvos
                              Dr. Nékám Kristóf oszt. vez. főorvos
Abbreviations: ADAM: a disintegrin and metalloprotease, AP1: activator protein 1, ATCC:
American Type Culture Collection, BAFF: B cell activating factor belonging to TNF family, BCMA:
B cell maturation antigen, BMI: body mass index, CARD: caspase recruiting domain, CASPASE:
cistein aspartase, CD: cluster of differentiation, ConA: concanavalin A, CV: coefficient of variance,
DcR: decoy receptor, DD: death domain, DED: death effector domain, DM: diabetes mellitus, DNA:
desoxyribonucleic acid, DR: death receptor, EBP: enhancer binding protein, EDA: ectodysplasin A,
EDAR: ectodysplasin A receptor, EGF: epidermal growth factor receptor, FADD: Fas associated
death domain protein, FCS: fetal calf serum, FFA: free fatty acid, GITR: glucocorticoid induced
tumor necrosis factor receptor family related protein, GLUT 4: glucose transporter protein 4, GRB:
growth factor receptor binding protein, HRG: heregulin, HVEM: herpes virus entry mediator, ICAM
1: intercellular adhesion molecule, IFN: interferon, IL: interleukin, IRAK: interleunkin 1 receptor
associated kinase, IRS 1: insulin receptor substrate 1, JNK: c-jun-N-terminal kinase, kD: Kilodalton,
L: ligand, LIGHT: ligand of HVEM/TNF receptor 2, LPS: lipopolysaccharid, LT: lymphotoxin,
MAP: mitogen activated protein kinase, MAPK: MAPkinase, MHC: major histocompatibility
complex, mRNA: messenger ribonucleic acid, NFκB: nuclear factor κB, NGF: nerve growth factor,
OGTT: oral glucose tolerance test, PHA: phytohemagglutinin, PI3K: phosphatidil inositol 3 kinase,
PKC: protein kinase C, PLC: phospholipase C, PLE: placental enhancer, R. receptor, RAIDD:
receptor interacting protein- associated ICE homolog protein with death domain, RANK: receptor
activating nuclear factor κB, sf: surface, SOS: son of sevenless, TACE: TNF-alpha cleaving enzyme,
TACI: transmembrane activator and CAML (calcium modulator and cyclophyllin ligand) interactor,
TALL 1,2: TNF and apoptosis ligand-related leukocyte expressed ligand 1,2, TGF: transforming
growth factor, TLR: TOLL like receptor, TNF: tumor necrosis factor, TRADD: TNF receptor
associated death domain protein, TRAF: TNF receptor associated factor, TRAIL: TNF-related
apoptosis inducing ligand, TRAMP: TNF-receptor related apoptosis mediating protein, TWEAK:
TNF-like weak inducer of apoptosis, TRK: tyrosin kinase, VEGF: vascular endothelial growth factor,
VLDL: very low density lipoprotein, WHR: waist to hip ratio
Introduction

The biological role of TNF system

At the end of the 18th century, the New York surgeon Dr. William B. Coley observed the regression of
different tumors after erysipelas. His therapeutical experiences with heat destroyed Streptococcal
suspensions in the treatment of sarcomas was published in 1893 (1).
During the 40 years of his clinical praxis, about 200 patients have been treated with the „Coley-toxin”,
and at least one third of his patients- mostly with lympho- and osteosarcoma – recovered.
According to our present day knowledge, the bacterial extract induced TNF-α release from the
patients mononuclear cells.
During the past 100 years his initial observations have merged in the depth of oblivion.
In 1985 Aggarwal and co-workers published a cytokine, named tumor necrosis factor produced by
macrophages after the stimulation with bacterial LPS and caused the necrosis of different tumors.
In the same time, Beutler and co-workers isolated a macrophage cytokine, named cachectin from
animals suffering from chronic Trypanosomiasis and cachexia, and proved its identity with TNF-α (3).
These investigators observed for the first time the metabolic effect of the cytokine, and described the
elevation of the VLDL concentration in the sera of animals with cachexia. They have also published
the inhibition of the differentiation of adipocytes by TNF-α, the decreased mRNA content, the
diminished biosynthesis of lipoprotein lipase and FFA binding proteins of the fat cells. This group has
also revealed, that TNF-α is a key mediator of septic shock and multiorgan failure induced by
bacterial endotoxin (4).
In the past two decade thousands of articles have been published about the biological function of the
TNF system contributing to the understanding of the communication of the immune system, shedding
of cell surface structures and apoptosis.

Recently a great number of ligands and receptors belong to the TNF family (see below). The closest
relatives of TNF-α are LT-α (formerly TNF-β), LT-β and LIGHT.
The most prominent family member, TNF-α has a molecular mass of 17.5 kD containing 157 amino
acids. A signal peptide sequence of 76 amino acid is removed from the matured secreted protein. TNF-
α gene is located on the short arm of chromosome 6 between the class I and class II MHC region.
TNF related ligands could bind to four receptors belonging to the family of TNF receptors.
TNF-α associates with two receptors, a 55 kD (CD120a) TNFR-1 and a 75 kD( CD120b) TNFR-2,
similarly LT-α also binds to TNFR-1 and TNFR-2, LT-β can bind only to LT-βR, LIGHT to HVEM
and LT-βR. All of the members of TNF ligand and receptor families are active as trimers (except
NGFR and ligand). Except LT-β all of them form homotrimers, LT-β may contain one or two α or β
variants. TNF family members may exist in mono-, di-, tetra-, penta- and multimeric forms, however
these variants are biologically inactive.
TNF-α may occur in membrane bound and soluble forms, LT-α exists only in soluble state, however
exclusively membrane bound forms of LIGHT and LT-β are known.
TNFR-1 and TNFR-2 are also found in membrane bound and soluble state, HVEM and LIGHT can
only be detected in membrane bound forms. Both the soluble and membrane bound variants of TNF
ligands and receptors contribute to the diverse biological effects of the TNF system and may mediate
the auto-, juxta-, para- and endocrine effects (2, 5, 6).

According to a structural similarity, numerous ligands (CD27L/CD70, CD30L, CD40L/ CD154, FASL/CD95L,
CD134L/OX40L, CD137L, TALL-1/BAEF,TAA-2/APRIL, APO-2L/TRAIL, APO-3L/TWEAK, RANKL,
EDA, GITRL, NGF, VEGI) and its receptors
(CD27, CD30, CD40, FAS/CD95, CD134/OX40, CD137, TACI, BCMA, DR3/TRAMP, Fn14, DR 4/TRAIL-
R1, DR5/ TRAIL-R2, DR6, DcR1, DcR2, RANK, osteoprotegerin, EDAR, GITR, p75NGFR) belong now to the
TNF family.
A cistein reach aminoacid repeat (2x, 3x or 4x) is the characteristic motif of the TNF receptor family.
Recently a new nomenclature is used for the designation of the TNF family members, TNFSF for the
ligands and TNFRS for the receptors.
Many biological roles of the TNF family members have already been described, such as the regulation
of the adaptive immune system, bone, skin and neurological functions.
Several ligands and receptors may initiate the programmed cell death (TNF-α/TNFR-1, LT-α3/TNFR-
1, FasL/Fas, APO3L/DR3, TRAIL/DR4, TRAIL/DR5) (7, 8).
Shedding of the soluble form of the ligands and receptors from the cell membrane is catalysed by a
transmembrane metalloprotease, TACE (ADAM17) belonging to the ADAM family.

TNF-α is produced by different cell types and stimuli, mainly by the cells of the mononuclear
phagocyte system after the stimulation with bacterial lipopolysaccharid.
LPS binds to the TOLL-like cell surface receptors, and signals through the IL-1 signal transduction
system, eventually activating the NFκB transcription factor, leading to the activation of
proinflammatory cytokine, among them the TNF-α gene (9).

Binding sites for numerous transcription factors can be found in the TNF-α promoter region, such as
p300, CREB, CRE, Egr1, ETS, Elk1, Sp1, NFAT, ATF2 and LITAF.
Several single nucleotide polymorphisms were found in the TNF-α promoter in positions of –163, -
238, -244, -308, -574, -851, -856, -857, -862, -863, -1031.
Among them the –238 (G/A), -308 (G/A) and –863 (C/A) polymorphisms were found to be in an
association with immunological, infect and metabolic (obesity and insulin resistance) disorders by
influencing the production of TNF-α in mononuclear and fat cells (7).

Role of TNF-α in insulin resistance

Initially Beutler and Cerami observed during the course of cachexia that a cytokine secreted from
macrophages and named cachectin inhibited lipogenesis and stimulated lipolysis in adipocytes.
Cachectin proved to be identical to TNF-α (3, 4). These observations have raised the regulatory role of
the cytokine in metabolic processes. In the mid 90th, TNF-α turned to be expressed and secreted in fat
cells. Hotamisligil et al. described in animal models of obesity (ob/ob, db/db mice, fa/fa mice and rats)
and also in human obesity the overexpression of the protein in the fat tissue (10). These investigators
also observed that TNF-α decreased the signal transduction of insulin receptor in adipocytes resulting
in insulin resistance (11). In the past few years a great number of publications supported the initial
observation about the contribution of TNF-α in obesity-linked insulin resistance and in 2 DM. The
detailed molecular pathomechanism has not yet been fully revealed.
The main sites of insulin resistance in 2DM are the muscle, liver and fat tissues. Insulin resistance in
adipocytes is represented by the decreased insulin-mediated suppression of lipolysis and diminished
glucose uptake of the fat cells. In muscle tissue decreased insulin-stimulated glucose uptake and
glycogen synthesis is present in insulin resistant states. In the hepatic cells an increased glucose
production is observable without a suppressive effect of insulin in insulin resistant people. A
progressive decrease in insulin sensitivity may lead to the damage of the insulin production in the
pancreatic β-cells. TNF-α may contribute to each of these processes by auto- and paracrine
mechanisms. Moreover, by endocrine effect the cytokine secreted in to the circulation may inhibit the
secretion of insulin in the β-cells and may antagonise it’s signalization in the target tissues (12).
The activation and signal transduction of the insulin receptor is due to stepwise phosphorilation of
many interacting proteins. Eventually the GLUT-4 transporter protein translocates from its
intracellular compartment (tubulovesicular system) in to the cell membrane in the target tissues
(muscle and fat). After binding of the extracellular glucose to its transporter, GLUT-4 returns back to
its intracellular compartment.
Two signal transduction pathways are present in the cells after binding of insulin to its receptor. One
of them is working through the activation of PI3K and the other through the lipid „rafts” and several
adaptor proteins. Insulin binding to the α-chain of the receptor results in an autophosphorilation of
several tyrosine residues in the β-chain.
In the case of the first-one, IRS (1-4) adaptor proteins bind to the activated insulin receptor and after
phosphorilation PI3K subunits are docking and the enzyme is activated. Several other protein kinase
enzymes are phosphorilated and activated eventually through other unknown signal transducers and
the GLUT-4 transporter is translocated to the cell membrane.
The other pathway starts with the interaction of flotillin, a protein located in the lipid-rafts, and Cbl.
Through the activation of a kinase (CrkII) the consequent actin polymerisation leads to the
translocation of the GLUT-4 transporter to the cell membrane (13).
TNF-α may inhibit the autophosphorilation of the insulin receptor and IRS, subsequently the docking
of PI3K and the assembly of the Cbl/CrkII complex and eventually the translocation of the GLUT-4
transporter into the cell membrane. The effects of TNF-α on the phosphorilation pattern (serin vs.
tyrosine residues) are carried out by MAP-kinase, sphingomyelinase and several phosphatase
enzymes. The inhibitory effect of the cytokine is exerted by direct autocrat and paracrine manners.
Both receptor types type 1 and 2 are also expressed in adipocytes. Moreover elevated levels of the
soluble TNFR-s are also found in the circulation of obese subjects. A positive correlation was found
between serum TNF-α concentrations, BMI and the degree of insulin resistance in overweight/obese
subjects. Higher soluble TNFR-2 levels were suggested to be a predictor of insulin resistance and
onset of 2DM (14).
The effect of TNF-α on skeletal-muscle insulin resistance is far less clarified. TNF-α and its receptors
are expressed in muscle cells, too. However no differences were found in the rate of expression
between lean and obese subjects. TNF-α produced in the subcutaneous and visceral cell depots may
exert an endocrine effect on muscle cells. Theoretically a 3rd adipose tissue compartment, located
intramuscularly, may also contribute to TNF-α production and may exert a paracrine inhibitory effect
(11). Numerous questions are unanswered at present, e.g. the regulation of the expression of TNF-α
mRNA and protein in fat cells and its connection with the proliferation and differentiation of
adipocytes. TNF-system may inhibit the most important differentiation factors of the adipocytes, EBP
and PPARs leading to dedifferentiation and apoptosis of preadipocytes and matured fat cells.
Moreover TNF-α increases the expression of the leptin gene in the fat tissue, and leptin negatively
regulates TNF-α expression. Thiazolidine-diones, new insulin sensitising drugs, pharmacological
activators of PPAR-γ may inhibit the expression of TNF-α in the adipose tissue (15).
An indirect regulatory effect of TNF-α on insulin sensitivity has also been verified through other
adipocytokines (IL-6, resistin, adiponectin) and FFA. This later mechanism may rise the contribution
of the cytokine in hepatic insulin resistance, too (16).

The role of TNF-system and leptin in gynecological pathophysiology

TNF-a and both of its receptors TNFR-1 and –2 are expressed in placenta. Many cell types can
produce the proteins of the TNF-system, such as syntitio- and cytotrophoblasts, fetoplacentar
macrophages, cells of the spiral arteries and lipid-laden macrophages in the arterial wall. Probably the
most important function of TNF-system in the placenta is a non-specific antiviral immunological
defence together with IFN-α, β, and γ (17).
Moreover, the TNF-system may also be involved in the regulation of fetal development. This concept
is supported by the observations in the 60’s, concerning the developmental defects of the neonates of
women taking the sedative drug Thalidomide during the course of their pregnancy. The drug proved to
be a potent suppressor of the TNF-α production (18).
An increased TNF-α mRNA and protein production was measured in placenta and in the circulation of
pregnant women with eclampsia and preeclampsia. The elevated serum TNF-α concentrations were
suggested as a laboratory early diagnostic marker of preeclampsia. An elevated placental production of
cytokine was also detected in patients with premature delivery and abortion. Hypoxia and oxydative
stress was suggested to be the major inducers of the increased placental production of the cytokine
(19). Similar observations were found with leptin. This protein is also expressed in the fetoplacentar
unit. The concentration of leptin is also higher in women with preeclampsia. The main inducer of the
overproduction is also hypoxia. The most important function of leptin in pregnancy is its regulatory
effect on the maternal nutrition and the optimal consumption of energy depots to ensure the proper
neonatal development. This is supported by transgenic animal models, in leptin gene knock-out mice
the menstrual cycle is missing and the animals are infertile. In humans in puberty the leptin level
dramatically increase parallel with the elevation of the body fat content (20). In general, leptin can be
considered as the main integrator in the regulation of nutrition, appetite, energy depots, and metabolic
and reproductive processes.

The pathophysiological role of EGF family, erbB-2 and TNF-α in carcinogenesis

The members of the EGF family are EGF, amphiregulin, betacellulin, heparin-binding EGF and TGF-
α. Their close relatives are neuregulin family members, the heregulins (HRG-α and –β) and glial cell
growth factor. All of these ligands contain one or more homologous EGF-like aminoacid motives and
can bind to their cell surface receptors, the EGFR family members. During the secretion many splice
variants are produced resulting in numerous different ligands.
The EGF ligands can bind to the EGF-receptor (HER-1, human EGF receptor), the neuregulin ligands
to erbB-3 and erbB-4 (HER-3, HER-4) resulting in the dimerization and activation of the receptors.
The signal transduction of the EGF receptors is similar to the insulin receptor, using intracellular
tyrosine kinase activity.
The overexpression both EGF and EGFR family members is present in many cancers such as
epithelial, mammary, lung and bladder carcinomas in correlation with poor clinical outcome. Recently
monoclonal antibodies against EGFR family members are used in anticancer therapy (transtuzumab,
herceptin).
ErbB-2 (HER-2/neu) is a 185 kD transmembrane receptor with tyrosine kinase activity. At present no
specific ligand is known for erbB-2. The most important property of the EGFR family members is the
formation of heterodimers in different combinations with different ligand binding and signal
transduction properties. The most promiscuous family member in this respect is erbB-2.
Ligand independent activation of the receptors can be due to mutations resulting in spontaneous
heterodimerization. ErbB-2 overexpression in tumor cells may also lead to spontaneous
heterodimerization of HER family members and can cause continuous cell proliferation (21, 22).
ErbB-2 overexpression in tumors correlate with resistance to chemotherapy and is a poor prognostic
marker in gynecological malignancies such as mammary and ovarian cancer (23).
Activation of the erbB-2 may influence the transcriptional activity of NF-κB one of the main
transcriptional factors of TNF-α. Activation of the erbB-2 may inhibit apoptosis induced by the TNF-
system in mammary and ovarian cancer. This process can be reversed by inhibition of erbB-2 with a
monoclonal antibody (24).

Aims

During the course of pregnancy especially in the 3rd trimester, a marked decrease in insulin sensitivity
is present. The aim of these investigations is the evaluation of the pathophysiological role of the TNF-
system in the regulation of pregnancy-induced insulin resistance. In details,
1. to study the immunoreactive concentrations of TNF-α and sTNFR-s in the 1st, 2nd and 3rd
     trimesters of healthy pregnancy.
2. to investigate the relationship between the concentrations of the cytokine and it’s receptors and
     anthropometric parameters (BMI, DTC) and blood pressure of the pregnant women.
3. the comparison of the levels of TNF-a and it’s receptors and the indirect parameters of insulin
     resistance (fasting blood glucose and C-peptide concentrations and Cp/Bg ratio).
4. to evaluate the connection between the concentration of the components of the TNF-system and
     leptin during pregnancy.
5. to study the correlation among the indirect parameters of maternal insulin resistance (C-peptide
     concentration and Cp/Bg ratio) leptin, the components of TNF-system and anthropometric
     parameters of neonates, such as body weight, length and head circumference.

The TNF system plays an important role in the regulation of host defence against malignancies.
Therefore the aim of these investigations was to study the contribution of the TNF-system in the
pathomechanism of the cancer of the uterine cervix. Moreover, I studied the expression of the erbB-2
oncoprotein in the malignant tissue in comparison with the serum concentration of several components
of TNF-system. In details,
6. to measure the serum concentration of TNF-a and sTNFR-2 in patients with cancer of the uterine
    cervix.
7. to compare the production of TNF-α of peripheral blood mononuclear cells isolated from patients
    and healthy subjects stimulated with different mitogens such as LPS, ConA, PHA.
8. to detect the erbB-2 oncoprotein expression by immunohistochemical method in the cancer tissue
    of patients.
9. to study the connection between erbB-2 oncoprotein expression, serum concentration of TNF-α,
    sTNFR-2 and the TNF-α production of the isolated peripheral blood mononuclear cells.
10. to evaluate the prognostic value of erbB-2 oncoprotein expression in patients with the cancer of
    the uterine cervix.


Patients and methods

Forty-five healthy pregnant women 15 of them in the 1st, 15 in the 2nd and 15 in the 3rd trimester and 25 age-
matched healthy non-pregnant women as controls were included in the study after having their written
informed consent. The study was approved by the local ethical committee of the Semmelweis University. All
of the patients had normal glucose tolerance checked with 75 g oral glucose tolerance test at 20th week of
pregnancy. Pregnants with abnormal OGTT were excluded from the study. Fasting venous blood samples
were obtained from the patients and controls for the laboratory measurements. The data of twenty-three
newborns delivered from pregnancies of women in the 2nd and 3rd trimester were analysed. Determination of
cytokines at pregnants in the 2nd trimester were repeated in the 3rd trimester between 30th-35th weeks of
pregnancy and used for correlation analysis between maternal laboratory and anthropometrical parameters of
newborns. TNF-α (Sigma, St.Louis, USA, inter-assay CV: 7.0% intra-assay CV: 4.5% sensitivity: 0.5
pg/ml), TNFR-1 (inter-assay CV: 8.6%, intra-assay CV: 1.89%, sensitivity: 80 pg/ml) and -2
(BenderMedSystem, Vienna, Austria, inter-assay CV: 2.0%, intra-assay CV: 1.4%, sensitivity: 0.15 ng/ml)
and leptin (DRG, USA) concentrations were detected by ELISA, serum fasting C-peptide determinations
were carried out by RIA kit (Biodata, Rome, Italy, normal fasting range 0.66-2.50 ng/mL). Among
anthropometrical parameters BMI, and thigh circumference of the dominant lower limb (DTC) were analysed
in correlation with serum fasting glucose, C-peptide levels, their ratio, TNF-α, TNF-receptor (R)-1 and -2.
HbA1c was measured by HPLC (Bio-Rad, normal value in non-diabetics 4.3-5.8%); serum fructosamin
(normal value in non-diabetics 205-285 µmol/l) measurements were performed by Boehringer (Germany)
automatic analyser kits.
Ninety-one patients with carcinoma of the uterine cervix diagnosed and observed at the 2nd Department of
OB/GYN of Semmelweis University and Department of Gynaecological Oncology of St. Stephen Hospital
between 1995-2001 were included in this study after obtaining their informed consent. Tissue samples for
immunohistochemical detection of the erbB-2 protein expression was gained during the gynecological
examination (tissue samples for immunohistochemistry were obtained by Volkmann-device in patients with
UICC st. 3-4) and surgery (Wertheim-operation was carried out in patients with UICC st 1-2, and tissue samples
were taken from the surgically removed specimen). Tumour staging (UICC stage/st/ 1: 39, 2: 33, 3: 14 and 4: 5)
was performed by ultrasound and CT scans and histological evaluation of samples removed during the surgical
intervention. Serum samples for the determination of TNF-α and sTNFR-2 from the newly diagnosed subjects
were taken before surgical procedure from patient with UICC st. 3-4 of cervical cancer prior irradiation. Patients
with any clinical and laboratory sings of infection were excluded. Patients’ mortality was followed up between
1995-2001.
The expression of erbB-2 protein was detected by immunostaining on paraffin embedded sections using a
monoclonal mouse antibody to human erbB-2 (BioGenex, Mainz, Germany) in a working dilution of 1:180 as it
has already been described (5,12). The staining was evaluated semiquantitatively nostaining (score 0) less than
20% of tumour cells with positive staining (score 1) 20-50% of the cells with positive staining (score 2), more
then 50% of the cells with positive staining (score 3). Fasting serum TNF-α levels were determined by ELISA
kit (inter and intra assay variability: 7.0 and 4.5% respectively, sensitivity: 0.5 pg/ml, Sigma, St. Louis, USA).
Fasting sTNFR-2 concentrations were also measured by ELISA kit (BenderMedSystem, Austria, inter-assay CV:
2.0%, intra-assay CV: 1.4%, sensitivity: 0.15 ng/ml) Peripheral blood mononuclear cells (PBMNC) were
isolated from heparinized venous blood of 34 patients with cervical cancer (17 with and 17 without erbB-2
protein expression) and from 30 matched control subjects on Ficoll-Hypacue density gradient by centrifugation.
Cell viability was checked by trypan blue exclusion test. One million of peripheral blood mononuclear cells in
RPMI medium containing 10% FCS was stimulated with bacterial lipopolysaccharide (LPS, E.coli serotype
026:B6, Sigma, St. Louis, USA, 1 µg/ml final concentration), concanavaline A (ConA, Sigma, St. Louis, USA, 1
µg/ml final concentration) and phytohemaglutinine (PHA-P, Sigma, St. Louis, USA, 1 µg/ml final
concentration) for 24 hours on 24 well Greiner-plates in Forma Sci thermostat at 37 oC, 5% CO2. TNF-α
secreted to the medium was detected by L929 cell cytotoxicity bioassay. Human recombinant TNF-α (Sigma St
Louis USA) was used as a standard. Anti-human mouse monoclonal neutralising TNF-α antibodies (Boehringer,
Mannheim, Germany) was applied to verify the TNF-α cytotoxicity in the samples. Statistical analysis was
performed by the Mann-Whitney test, linear correlation analysis (Spearman) and Chi-square test with Yates’
correction. Prism3 program was used for the analysis and graphical illustration of the results.

Results

Significantly higher serum TNF-α levels were found in healthy pregnant women in the 3rd trimester
(X±SD: 5,33±0,43 pg/ml, p<0,01) as compared to patients in the 1st (4,05±0,26) trimester as well as
to the non-pregnant healthy controls (4,07±0,26). Serum TNF-α levels were slightly elevated in the
2nd trimester (4,35±0,28).
Both, serum soluble TNFR-1 and -2 were found to be elevated in the healthy pregnants in the 3rd
trimester of their pregnancy (TNFR-1: 2,86±0,78 ng/ml, TNFR-2: 5,75±2,10 ng/ml, p<0,01)
comparing to healthy female controls (TNFR-1: 2,07±0,38, -2: 3,27±0,76) and normal pregnants in
the 1st (TNFR-1: 1,97±0,38, -2: 3,78±1,11) and 2nd trimesters (TNFR-1: 2,10±0,20, -2: 3,89±0,62).
The ratio of sTNFR-2/-R-1 was significantly higher in the 3rd trimester (2.13±1.18) as compared to
healthy nonpregnant controls (1.60±0.41 p<0.01).
Significantly elevated serum leptin concentrations were measured in the 3rd trimester of pregnancy
(34.91±19.40 ng/ml, p<0,01) as compared to healthy female controls (11.91±7.74) and to normal
pregnants in the 1st (11.46±5.55), the 2nd (11.83±5.41) trimesters.
The BMI, dominant thigh circumference and blood pressure of patients were also elevated in the 3rd
trimester. The waist to hip ratio (WHR), the systolic (RRs) and diastolic (RRd) blood pressure in
the 3rd trimester were also found to be significantly higher. These parameters were even higher in
pregnant with BMI>27 kg/m2 . Fasting C-peptide levels were also found to be elevated in the 3rd
trimester (3.36±1.21 ng/ml p<0.01) as compared to the 1st (1.34±0.59) and 2nd (1.11±0.31) trimester
and to non-pregnant women (1.05±0.37). Values of the C-peptide/Blood glucose ratio showed the
same tendency (healthy pregnants, 1st trimester: 0.33±0.16, 2nd trimester: 0.28±0.10, 3rd trimester:
0.70±0.30, non-pregnants: 0.22±0.07, values of the 3rd trimester were significantly higher as
compared to controls 1st and 2nd trimester, p<0.01). We could calculate significant positive linear
correlation in our patients between gestational age and BMI (r=0.46, p<0.01), WHR (r=0.61,
p<0.01), dominant thigh circumference (r=0.59, p<0.001), systolic and diastolic blood pressure
(r=0.57, p<0.001). TNF-α was also found to be in a significant positive linear correlation with the
WHR (r=0.57, p<0.001), dominant thigh circumference (r=0.59, p<0.001). BMI was also in a
significant positive linear correlation with the WHR (r=0.64, p<0.001), dominant leg thigh
circumference (r=0.79, p<0.001) and fBGL (r=0.54, p<0.001).
After correcting for BMI, leptin, C-peptide, TNFR-2 concentrations and the C-peptide/Blood
glucose (Cp/Bg) ratio remained significantly elevated in the 3rd trimester. Serum TNF-α, TNFR-2
(TNFR-2 - Cp r=0.4950, p<0.0005, TNFR-2 - C-peptide/blood glucose ratio: r= 0.5141, p=
0.0007), C-peptide/blood glucose ratio, leptin concentrations and the BMI value were found to be
in a significant positive linear correlation with each other in pregnants.
In a multivariate analysis with forward stepwise linear regression the regression summary for C-
peptide as the dependent variable serum leptin (p=0.00004) and BMI (p=0.017) proved to be
significant.
The anthropometric parameters of 23 newborns of healthy pregnant women followed in the 2nd and
3rd trimesters of pregnancy were analysed in correlation with the maternal serum C-peptide, TNF-
α, TNFR-2 and leptin levels. The head circumference of the neonates was in a negative correlation
with the maternal leptin (r= -0.5888, p=0.0031) concentration and BMI value (r= -0.4711,
p=0.0233).
In patients with cancer of the uterine cervix significantly decreased serum TNF-α (X±SD: 2.70±0.69
pg/ml, p<0.0001), sTNFR-2 (3.85±1.05 ng/ml, p<0.0001) concentrations were found as compared to
the controls (TNF-α: 4.32±0.36, sTNFR-2: 4.85±0.82).
Significant positive linear correlation (Spearman) were found between serum TNF-α and sTNFR-2
levels both in cancer patients (r=0.3974, p<0.0001) and in the healthy controls (r=0.2878, p=0.0211).
The ratio of the sTNFR-2/TNF-α was significantly (p<0.0001) elevated in the cancer patients
(1.49±0.53) as compared to the control group (1.12±0.18). Serum TNF-α and sTNFR-2 concentrations
were significantly lower in patients with higher (3-4) UICC stages as compared to those with lower (1-
2) ones.
The mitogenic induced TNF-α production of the peripheral blood mononuclear cells (PBMNC)
isolated from cancer patients was also significantly decreased as compared to the healthy controls.
The expression of the erbB-2 protein was detected in the tumour samples of 16 patients. In 2 of them
(both UICC stage 4) the erbB-2 protein was also detectable not only in the malignant cells but also in
the histologically normal cervical cells. The erbB-2 protein expression was significantly more
prominent (score: 2+, 3+) and frequent (Chi-square test with Yates’ correction, p<0.0001) in patients
with UICC stage 3-4 (n= 14/19) comparing to those with stage 1-2 (2/72, score: 0, 1+). During a
follow-up period of 7 years 30 patients died. The erbB-2 protein positivity was significantly more
frequent (Chi-square test with Yates’ correction, p<0.0001) among them (14/30) comparing to the
surviving women (2/61, relative risk: 5.36, Odds ratio: 14.23).
The TNF-α (1.58±0.38) and sTNFR-2 (2.55±0.57) concentrations were significantly (p<0.0001) lower
in patients with erbB-2 positivity comparing to those with negativity (TNF-α: 2.82±0.62, TNFR-2:
4.06±0.87).
The mitogenic induced TNF-α production of the erbB-2 positive patients was significantly lower
comparing to the erbB-2 negative women.

Summary of the original observations

1. Significantly higher fasting TNF-α, sTNFR-1 and –2, leptin, C-peptide concentrations and Cp/Bg
    ratio were found in healthy pregnant women in the 3rd trimester as compared to those in the 1st and
    2nd trimester and non-pregnant subjects.
2. Significant positive linear correlation was calculated among indirect parameters of insulin
    resistance and maternal serum TNF-α, sTNFR-2 and leptin concentrations.
3. Significant negative linear correlation was detected between maternal BMI value, leptin
    concentration and the head circumference of neonates.
4. The role of adipocytokines, such as TNF-α, sTNFR-2 and leptin was emphasised in the
    pathomechanism of pregnancy-induced insulin resistance.
5. Among different adipocytokines, maternal leptin concentration may have the most important role
    in metabolic imprinting.
6. The expression of erbB-2 oncoprotein was detected in the malignant tissue with cancer of the
    uterine cervix especially in more advanced stages. ErbB-2 expression proved to be a poor
    prognostic marker in patients with the cancer of the uterine cervix.
7. Significantly lower serum TNF-a and sTNFR-2 concentrations were measured in patients as
    compared to healthy matched subjects. STNFR-2/TNF-a ratio was significantly greater in patients
    as compared to controls.
8. The mitogenic (LPS, ConA, PHA) induced TNF-a production of peripheral blood mononuclear
    cells isolated from patients was significantly lower especially in patients in more advanced stages
    as compared to healthy subjects.
9. Serum TNF-a and sTNFR-2 concentrations were significantly lower in patients with erbB-2
    protein expression as compared to those without erbB-2 positivity.
10. Mitogen stimulated TNF-a secretion of the peripheral blood mononuclear cells isolated from
    patients with erbB-2 positivity was significantly decreased as compared to those with erbB-2
    negativity.
11. The connection between the erbB oncoprotein family and the TNF-system was suggested in the
    pathogenesis of the cancer of the uterine cervix.
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List of publications


Winkler G., Lakatos P., Salamon F., Speer G., Baranyi É, Melczer Zs., Cseh K.: Contribution of
tumor necrosis factor (TNF)-α in insulis resistance in patients with android type obesity. (letter to
editor) Diabetes Care, 22: 870, 1999 /IF: 5.076/
Cseh K., Winkler G., Melczer Zs., Baranyi É.: The role of tumor necrosis factor (TNF)-α resistance
in obesity and insulin resistance. (research letter) Diabetologia, 43: 525, 2000 /IF: 4.986/
Melczer Zs., Bánhidy F., Csömör S., Siklós P., Baranyi É., Winkler G., Cseh K.: A tumor nekrózis
faktor α szerepe a terhesség során észlelhető inzulinrezisztencia pathomechanizmusában. MNL 64:
203-207, 2001
Winkler G., Cseh K., Baranyi É., Melczer Zs., Speer G., Hajós P., Salamon F., Túri Zs., Kovács
M., Vargha P., Karádi I.: Tumor necrosis factor system in insulin resistance in gestational diabetes.
Diab Res and Clin Pract 56: 93-99, 2002 /IF: 0.982/
Melczer Zs., Bánhidy F., Csömör S., Kovács M., Siklós P., Winkler G., Cseh K.: Role of Tumour
Necrosis Factor-α in insulin resistance during normal pregnancy. Eur J Obstet Gynecol Reprod Biol,
105: 7-10 2002 /IF: 0.884/
Cseh K., Baranyi É., Melczer Zs., Csákány M. Gy., Speer G., Kovács M., Gerö G, Karádi I.,
Winkler G.: The pathophysiological influence of leptin and the tumor necrosis factor system on
maternal insulin resistance: negative correlation with anthropometric parameters of neonates in
gestational diabetes. Gynecol Endocrinol 16: 453-460, 2002 /IF: 0.878/
Kalabay L., Cseh K., Pajor A., Baranyi É., Csákány M. Gy., Melczer Zs., Speer G., Kovács M.,
Siller Gy., Karádi I., Winkler G.: Correlation of maternal serum fetuin/α2-HS-glycoprotein
concentration with maternal insulin resistance and anthropometric parameters of neonates in normal
pregnancy and gestational diabetes. Eur J of Endocrinol 147: 243-248, 2002 /IF: 2.133/
Winkler G., Salamon F., Baranyi É., Speer G., Melczer Zs., Dworak O., Kovács M., Őry I.,
Csákány M. Gy., Lakatos P., Karádi I., Cseh K.: The role of the tumor necrosis factor system in
obesity-linked insulin resistance. Diab Hung 10 S2, 28-31, 2002
Melczer Zs., Bánhidy F., Csömör S., Siklós P., Winkler G., Dworak O., Cseh K.: ErbB-2/HER-
2 protein expression, serum Tumour Necrosis Factor-α and soluble Tumour Necrosis Factor
Receptor-2 concentrations in human carcinoma of the uterine cervix. Eur J of Gyn Oncol, XXIV
(2): 138-142, 2003 /IF: 0.562/
Melczer Zs., Bánhidy F., Csömör S., Tóth P., Kovács M., Cseh K., Winkler G.: Influence of
leptin and TNF system on insulin resistance in pregnancy and their effect on antropometric
parameters of newborns. Acta Obstet Gynecol Scand 82(5): 432-438, 2003 /IF: 1.284/
Melczer Zs., Bánhidy F., Csömör S., Siklós P., O. Dworak, Cseh K.: Szérum tumor nekrózis
faktor-α és szolubilis tumor nekrózis faktor-receptor-2 koncentrációk valamint ErbB2/HER-2 fehérje
expressziójának elemzése méhnyakrákos betegekben. Nőgyógyászati Onkológia 2003 (in press)


Abstracts

G.Speer, Zs.Nagy, Cs.Keszthelyi, D.Salamon, P.Szénási, S.Csömör, Zs.Melczer, K.Cseh
Decreased Tumor Necrosis Factor (TNF) production of peripherial blood mononuclear cells in
cervical cancer. European Federation of Immunological Societies 12th European Immunology
Meeting Barcelona, Spain 1994. p75
S.Csömör, Á.László, Zs.Melczer, Cs.Keszthelyi, K.Cseh Evaluation of cytotoxic activity of Tumor
Necrosis Factor (TNF) in cervical malignancies XIV.FIGO World Congress Montreal, Canada
PO.1367, 1994
Csömör S., Melczer Zs., Keszthelyi Cs., Cseh K. Tumor Necrosis Factor (TNF) termelődés
vizsgálata méhnyak neoplasiák esetében. Magyar Nőorvos Társaság 25.Nagygyűlése Debrecen p14,
1994
Zs.Melczer, F.Bánhidy, S.Csömör jr., G.Speer, D.Salamon, K.Cseh: Decreased cytotoxic cytokine
production in cervical intraepithelial neoplasia and cervical cancer 11th Cong.of EAGO, Budapest
P102, 1996
Zs.Melczer, S.Csömör, F.Bánhidy, G.Speer, D.Salamon, K.Cseh: Decreased mitogenic induced
tumor necrosis factor alpha production of peripheral blood mononuclear cells in cervical intraepithelial
neoplasm. Congress of ISOBM, San Diego, USA, Tumor Biology. S18: 1-136, 1997
Melczer Zs., Bánhidy F., Csömör S., Cseh K.: A Tumor Nekrózis Faktor a szerepe a terhesség
alatt észlelhető inzulin rezisztencia kialakulásában. Magyar Immunológiai Társaság Kongresszusa,
Bük, P 26, 1999
Winkler G., Baranyi É., Melczer Zs., Braun E., Szekeres O., Cseh K.: Leptin, TNF-receptor (R)-2
and TNF-α contribute to insulin resistance in normal pregnancy and gestational diabetes. Diab Res
and Clin Pract 50: S1, S426-427, 2000 /IF: 0.590/
Winkler G., Szekeres O., Braun E., Salamon F., Melczer Zs., Simon K., Cseh K.: Effect of
moderate weight reduction and pentoxyphyllin treatment on TNF-α and C-peptide levels in android
type obesity. Int J Obesity 24 (S1): S82, 2000 /IF: 3.003/
Winkler G., Baranyi É., Melczer Zs., Túri Zs., Speer G., Őri I., Braun E., Szekeres O., Szőcs A.,
Cseh K.: Role of the TNF-α system and leptin in insulin resistance in patients with gestational
diabetes Diabetologia 43, (S1): A172, 2000 /IF: 5.177/
Cseh K., Melczer Zs., Kovács M., Baranyi É., Simon K., Winkler G.: A tumor nekrózis faktor
(TNF)-α szerepe az élettani terhesség során kialakuló inzulinrezisztenciában. Diabetologia Hungarica
8, suppl. I. 13. 2000.
Baranyi É., Winkler G., Melczer Zs., Turi Zs., Őry I., Cseh K.: Emelkedett tumor nekrózis faktor-
α szint gestatiós diabetesben. Diab. Hung. 8, suppl. I. 7. 2000.
Winkler G., Baranyi É., Kovács M., Melczer Zs., Hajós P., Speer G., Cseh K.: A solubilis tumor
nekrózis faktor (TNF)-receptor 1-es és 2-es, valamint a szérum leptin szint alakulása gestatiós diabetes
(GDM)-ben. Diab. Hung. Suppl. I. 98-99. 2000.
Szekeres O., Melczer Zs., Turi Zs., Braun E., Speer G., Szőcs A., Őry I., Baranyi É., Kovács
M., Cseh K., Winkler G.: Emelkedett szérum tumor necrosis faktor-α szint gestatiós diabetesben és
szerepe az állapotot kísérő inzulinrezisztenciában. Diabetol Hung 9 (S1): 41, 2001
Melczer Zs., Bánhidy F., Csömör S., Cseh K., Winkler G.: Szérum Tumor Nekrózis Faktor
(TNF)-α, éhomi és posztprandiális C-peptid szintek alakulása sovány valamint gynoid és android
típusú elhízott nőkben. Magyar Szülészet-Nőgyógyászati Endokrinológiai Társaság II.
Kongresszusa, E17, Kecskemét, 2002


ΣIF: 25.555
Ebből közlemény és elismert levél (absztraktok nélkül): 16.785


Other publications


Csömör S.jr., Ujvári E., Melczer Zs.: A cervix-program hatásának vizsgálata klinikai beteganyagon.
MNL 57 (6).451-454. 1994
Bánhidy F.jr., Melczer Zs., Lukácsi L., Poller I., Bakács T.: A sejtes immunitás változása
méhnyakrákos betegek kuratív sugárkezelése során. Magyar Onkológia 42. 250-253. 1998
Bánhidy F.jr., Melczer Zs., Csömör S.jr., Poller I., Bakács T.: A sugárterápiás dózisfüggőség
hatása méhnyakrákos betegek sejtes immunitására. Magyar Onkológia 43. 69-72. 1999
Bánhidy F., Melczer Zs., Lukácsi L., Poller I., Bakács T.: A sejtes immunitás változása
méhnyakrákos betegek preoperatív sugárkezelése során. MNL 62. 31-34. 1999
Bánhidy F., Melczer Zs., Csömör S., Pálfalvy L.: Méhnyakrákos betegek sejtes immunitásának
vizsgálata a szövettani diagnózis alapján. MNL 62. 363-366. 1999
Bánhidy F., Melczer Zs., Lukácsi L., Pálfalvy L.: A jóindulatú petefészek daganatok hatása a
gazdaszervezet sejtes immunválaszára (K-, NK-sejt). MNL 62. 473-477. 1999
Bánhidy F.jr., Melczer Zs., Lukácsi L., Gimes G., Paulin F., Siklós P.: A sejtes immunitás (NK-,
K-sejtek) változása rosszindulatú petefészek-daganatos betegek műtéti kezelése során. Magyar
Onkológia 44. 149-152. 2000
Melczer Zs.: Bakteriális vaginosis terhesség alatt. (kommentár) Nőgyógyászati és Szülészeti
Továbbképző Szemle 3 (6) 387-397. 2001
Melczer Zs., Langmár Z., Paulin F.: Terhes és nem terhes nők bakteriális vaginosisának kezelése
során helyi ökoterápia alkalmazásával szerzett tapasztalataink. Magy Nőorv L 65: 319-323, 2002
Melczer Zs., Langmár Z., Paulin F.: Helyi ökoterápia alkalmazásával szerzett tapasztalataink terhes
és nem terhes nők bakteriális vaginosisának kezelése során. Magy Venerol Arch 234-238, 2002

				
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