Synergistic effect of interleukin-17 and tumour necrosis factor-α on inflammatory response in hepatocytes through interleukin-6-dependent and independent pathways.

The proinflammatory cytokines interleukin (IL)-17 and tumour necrosis factor (TNF)-α are targets for treatment in many chronic inflammatory diseases. Here, we examined their role in liver inflammatory response compared to that of IL-6. Human hepatoma cells (HepaRG, Huh7.5 and HepG2 cells) and primary human hepatocytes (PHH) were cultured with IL-6, IL-17 and/or TNF-α. To determine the contribution of the IL-6 pathway in the IL-17/TNF-α-mediated effect, an anti-IL-6 receptor antibody was used. IL-17 and TNF-α increased in synergy IL-6 secretion by HepaRG cells and PHH but not by Huh7.5 and HepG2 cells. This IL-17/TNF-α synergistic cooperation enhanced the levels of C-reactive protein (CRP) and aspartate aminotransferase (ASAT) in HepaRG cell and PHH cultures through the induction of IL-6. IL-17/TNF-α also up-regulated IL-8, monocyte chemoattractant protein (MCP)-1 and chemokine (C-C motif) ligand 20 (CCL20) chemokines in synergy through an IL-6-independent pathway. Interestingly, first exposure to IL-17, but not to TNF-α, was crucial for the initiation of the IL-17/TNF-α synergistic effect on IL-6 and IL-8 production. In HepaRG cells, IL-17 enhanced IL-6 mRNA stability resulting in increased IL-6 protein levels. The IL-17A/TNF-α synergistic effect on IL-6 and IL-8 induction was mediated through the activation of extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase, nuclear factor-κB and/or protein kinase B (Akt)-phosphatidylinositol 3-kinase signalling pathways. Therefore, the IL-17/TNF-α synergistic interaction mediates systemic inflammation and cell damage in hepatocytes mainly through IL-6 for CRP and ASAT induction. Independently of IL-6, the IL-17A/TNF-α combination may also induce immune cell recruitment by chemokine up-regulation. IL-17 and/or TNF-α neutralization can be a promising therapeutic strategy to control both systemic inflammation and liver cell attraction.

T- and B-cell responses to multivalent prime-boost DNA and viral vectored vaccine combinations against hepatitis C virus in non-human primates.

Immune responses against multiple epitopes are required for the prevention of hepatitis C virus (HCV) infection, and the progression to phase I trials of candidates may be guided by comparative immunogenicity studies in non-human primates. Four vectors, DNA, SFV, human serotype 5 adenovirus (HuAd5) and Modified Vaccinia Ankara (MVA) poxvirus, all expressing hepatitis C virus Core, E1, E2 and NS3, were combined in three prime-boost regimen, and their ability to elicit immune responses against HCV antigens in rhesus macaques was explored and compared. All combinations induced specific T-cell immune responses, including high IFN-γ production. The group immunized with the SFV+MVA regimen elicited higher E2-specific responses as compared with the two other modalities, while animals receiving HuAd5 injections elicited lower IL-4 responses as compared with those receiving MVA. The IFN-γ responses to NS3 were remarkably similar between groups. Only the adenovirus induced envelope-specific antibody responses, but these failed to show neutralizing activity. Therefore, the two novel regimens failed to induce superior responses as compared with already existing HCV vaccine candidates. Differences were found in response to envelope proteins, but the relevance of these remain uncertain given the surprisingly poor correlation with immunogenicity data in chimpanzees, underlining the difficulty to predict efficacy from immunology studies.

Metabolic reprogramming: a hallmark of viral oncogenesis.

More than 1 in 10 cases of cancer in the world are due to chronic viral infections. Viruses induce oncogenesis by targeting the same pathways known to be responsible for neoplasia in tumor cells, such as control of cell cycle progression, cell migration, proliferation and evasion from cell death and the host's immune defense. In addition, metabolic reprogramming has been identified over a century ago as a requirement for growth of transformed cells. Renewed interest in this topic has emerged recently with the discovery that basically all metabolic changes in tumor cells are finely orchestrated by oncogenes and tumor suppressors. Indeed, cancer cells activate biosynthetic pathways in order to provide them with sufficient levels of energy and building blocks to proliferate. Interestingly, viruses introduce into their host cells similar metabolic adaptations, and importantly, it seems that they depend on these changes for their persistence and amplification. The central carbon metabolism, for example, is not only frequently altered in tumor cells but also modulated by human papillomavirus, hepatitis B and C viruses, Epstein-Barr virus and Kaposi's Sarcoma-associated virus. Moreover, adenoviruses (Ad) and human cytomegalovirus, which are not directly oncogenic but present oncomodulatory properties, also divert cellular metabolism in a tumor cell-like mnner. Thus, metabolic reprogramming appears to be a hallmark of viral infection and provides an interesting therapeutic target, in particular, for oncogenic viruses. Therapeutic targeting of metabolic pathways may not only allow to eliminate or control the viral infection but also to prevent virus-induced carcinogenesis.

The long form of CDK2 arises via alternative splicing and forms an active protein kinase with cyclins A and E.

We have reinvestigated the long form of cyclin-dependent kinase (CDK)2 that is expressed in many rodent cells. We show that the mRNA encoding CDK2L arises by alternative splicing and that the encoded protein can bind to, and be activated by, cyclins A and E. The complex of CDK2L with cyclin A has about half the specific activity of the equivalent CDK2-cyclin A complex. Also, CDK2L--cyclin A is inhibited to the same extent and by the same concentrations of p21(CIP1) as CDK2--cyclin A. The nucleotide sequences of intron V in the human and murine CDK2 genes, where the sequences encoding the 48-residue insert in CDK2L are located, show very high conservation in the position of the alternatively spliced exon and its surroundings. Despite this, we were not able to detect significant expression of CDK2L in human cell lines, although a low level is expressed in COS-1 cells from monkeys.

p21WAF1 is dynamically associated with JNK in human T-lymphocytes during cell cycle progression.

We have examined the regulation of the c-Jun NH2-terminal kinase (JNK) subfamily of mitogen-activated protein kinases (MAPKs) in response to inhibition of DNA replication during the cell cycle of human T-lymphocytes. In this study, we demonstrate that JNK is rapidly activated following release of T-lymphocytes from G1/S-phase arrest and that this activation precedes resumption of DNA synthesis upon S-phase progression. We also show that activation of JNK correlates with dissociation of the cyclin-dependent protein kinase (CDK) inhibitor, p21WAF1, from JNK1. Since JNK1 isolated from T-lymphocytes by immunoprecipitation can be inhibited by recombinant p21WAF1 in vitro, these data suggest that JNK activation may be regulated in part by its dissociation from p21WAF1. The observation of a dynamic, physical association of native JNK1 and p21WAF1 in vivo has not previously been described and suggests a novel mechanism for JNK-mediated regulation of the cell cycle of human T-lymphocytes.

[The diagnostic potential of amniocentesis in the first 12 weeks of pregnancy]. / Das diagnostische Potential von Amniozentesen in den ersten 12 Schwangerschaftswochen.

Prenatal diagnosis of chromosomal and biochemical defects is accepted as a routine in high-risk patients. To eliminate the disadvantages of traditional amniocentesis (late diagnosis) and of chorion villus sampling (placentar mosaics, higher fetal loss rate) we evaluated the facts on amniocentesis during the first 12 weeks of pregnancy. 42 samples were analysed. 21 amniotic fluid samples were from pregnancies before the 13th week of gestation, 21 further punctures were performed between the 13th to 15th week and served as comparative figures. Of 21 samples, diagnosis was possible in 16 cases. Four cultures did not show any growth of cells; in one case, amniotic fluid could not be aspirated. In the comparison group chromosomal diagnosis could be done in every case. An average of 12.3 ml of amniotic fluid were taken. Chromosomal disorders found, included a translocation in chromosome 13/14 as well as a trisomy of chromosome 18. Biochemical defects such as Gaucher's disease and Niemann-Pick disease were excluded. 30 pregnancies without pathological symptoms were seen (mean birth weight 3200 g, mean duration of pregnancy 39 weeks). One case of abortion following amniocentesis was found in the 16th week of gestation. One case of premature delivery occurred in the 34th week and the pregnancy of a 40-year old women was complicated by EPH gestosis while 9 pregnancies were terminated by interruption following the patients' wish. Because chromosomal diagnosis could not be performed in five of 21 cases, amniocentesis during the 13th week of gestation cannot be recommended as a routine method. Early amniocentesis, however, is an alternative to the traditional amniocentesis at the 16th week of gestation.

Urinary 3-hydroxyproline excretion in Alport's syndrome: a non-invasive screening test?

Alport's syndrome is characterised by morphological and structural changes of the renal basement membranes. As the hydroxyproline content of isolated glomerular basement membranes is reduced in patients with Alport's syndrome, it is possible that the renal excretion of 3-hydroxproline (3-OHP), a key substrate of basement membrane collagen, may be altered in such patients. The urinary excretion of 3-OHP was determined by thin layer chromatography in 20 patients with Alport's syndrome, in healthy control subjects, and in patients with other renal diseases. These included patients with poststreptococcal glomerulonephritis, lower urinary tract infection, severe reflux nephropathy, lithium induced nephropathy, polycystic kidney disease, familiar benign haematuria, and renal graft rejection. Urinary excretion of 3-OHP was significantly higher in patients with Alport's syndrome compared with the patients with other renal diseases and the healthy control subjects. All other renal diseases investigated had 3-OHP values within the normal range. Urinary 3-OHP determination detected patients with Alport's syndrome with a high sensitivity (95.2%) and specificity (97.2%). We therefore suggest using urinary 3-OHP determinations as a simple non-invasive screening test for Alport's syndrome.

Influence ofL-arginine on glucose mediated collagen cross link precursors in patients with diabetes mellitus.

Long term complications of diabetes mellitus are largely due to chemical, structural and mechanical changes of connective tissue proteins involving glucose mediated collagen cross links (GMCC). To date there are only experimental therapeutic approaches for preventing long term complications of diabetes mellitus using the toxic substance aminoguanidine.L-arginine, a nontoxic substance, has been shown to reduce GMCC in animal models of diabetes mellitus. We have now performed a blind placebo controlled study with crossing over of two treatment periods of three months each in 29 patients with diabetes mellitus in order to examine the effect of treatment withL-arginine (2 × 1g daily) on glucose mediated collagen cross links (GMCC). GMCC was evaluated by determining glycosyl lysine (hexosyl lysine) levels in skin punch biopsies. Patients treated byL-arginine showed significantly lower GMCC precursors of skin collagen compared with the placebo treated group (difference of hexosyl lysine as counts/mL/ug hydroxyproline between the first and second skin biopsy 0.11 ± 4.44 vs. 4.03 ± 5.27,t = 2.17,p < 0.05). The only side effects ofL-arginine were gastric pain occurring only in patients who did not follow the instructions to takeL-arginine at meals. We conclude thatL-arginine could be useful for treating long term complications of diabetes mellitus.

The effect of substance L on glucose-mediated cross-links of collagen in the diabetic db/db mouse.

Genetically diabetic mice (db/db) were given 50 mg/kg body weight/day substance L, a nontoxic basic amino acid and compared to control diabetic mice without treatment. The oral administration of the compound was started at the age of 3 months and the animals were sacrificed at the age of 7 months. No adverse effects were observed in animals given the substance L. Total food consumption, drinking water intake and body weight were comparable between the groups. Nonenzymatic glycosylation of serum proteins and hemoglobin was not significantly different in the groups. Renal pathological lesions in the control diabetic mice showed glomerular mesangial expansion and on electron microscopy thickened glomerular basement membranes with a mean thickness of 3,204 +/- 186 A. Treated animals showed significantly less mesangial crescents and thinner glomerular basement membrane thickness of 2,520 +/- 252 A (p less than 0.01). The experimental animals showed in addition a lower mean kidney weight. Glomerular but not tubular proteinuria was reduced in the treated group. Basement membrane collagen type IV isolated from kidneys of experimental animals was more soluble in acidity and showed a lower degree of cross-linking as evaluated by SDS-polyacrylamide gel electrophoresis. We conclude that substance L is beneficial to diabetic renal changes. We suggest that this positive effect could be due to the inhibition of glucose-mediated abnormal cross-linking of collagenous structures by the interaction of substance L with reactive carbonyl residues of glycosylation adducts of collagen. Other possible mechanisms are discussed.

[Effect and adverse effects of epidural anesthesia during labor. With special reference to changes in the cardiotocogram]. / Effekt und Nebenwirkungen der Epiduralanänsthesie sub partu. Mit besonderer Berücksichtigung von Veränderungen der Kardiotokogramme.

In an Obstetric Department with approximately 2,000 deliveries yearly, offry third mother is delivered under epidural anesthesia. In a group of 600 maternity patients, the effectiveness, the side effects and the changes in the fetal cardiotocogram were examined during labour and delivery under epidural anesthesia. Evaluated were questionnaire to the mothers with mainly subjective impressions, the anesthetic protocol, and the protocol of labour and delivery including the internal cardiotocogram. 85% of the mothers would have a future delivery under an epidural anesthesia. The incidence of headaches and its independence of the type of obstetric analgesia and anesthesia is mentioned. The number of punctures, the technique of epidural anesthesia, the concentration of the local anesthetic used, failures and side effects are described. The incidence of cesarian sections, fetal morbidity and changes in the cardiotocogram are discussed in detail. The incidence of transilent silent decelerations and alarm dips in the cardiotocogram are mentioned. This study shows that epidural anesthesia is free of risks and a highly effective method for the conduct of obstetric analgesia.

[Illness due to enterolith in children (author's transl)]. / Krankheitsbilder im Kindesalter, die durch Kotsteine verursacht werden können.

The aim of this paper is to emphasize the extreme importance of the rectal examination and exact palpation of the abdomen in cases of obscure abdominal pain, especially as it is purely a question of a simple and inexpensive diagnostical procedure. The rectal examination should always be carried out prior to any large scale diagnostical procedures as it does, in the care of positive findings, spare the child the considerable burden of X-rays. If, after an appendectomy, the same pains that led to the operation continue, one must conclude that their cause has not been removed by the operation. An exact clinical classification should be undertaken to avoid the later occurrence of a critical illness (ileus). As with any diagnosis, it is necessary in the event of enterolith too, to regard it as a possibility. The anamnestic registration of a daily bowel movement should not be a reason for not carrying out the rectal examinations as large quantities of stool can collect.