Activation of the aryl hydrocarbon receptor by clozapine induces preadipocyte differentiation and contributes to endothelial dysfunction.

BACKGROUND: The superior therapeutic benefit of clozapine is often associated with metabolic disruptions as obesity, insulin resistance, tachycardia, higher blood pressure, and even hypertension. AIMS: These adverse vascular/ metabolic events under clozapine are similar to those caused by polycyclic aromatic hydrocarbons (PAHs), and clozapine shows structural similarity to well-known ligands of the aryl hydrocarbon receptor (AhR). Therefore, we speculated that the side effects caused by clozapine might rely on AhR signaling. METHODS: We examined clozapine-induced AhR activation by luciferase reporter assays in hepatoma HepG2 cells and we proved upregulation of the prototypical AhR target gene Cyp1A1 by realtime-PCR (RT-PCR) analysis and enzyme activity. Next we studied the physiological role of AhR in clozapine's effects on human preadipocyte differentiation and on vasodilatation by myography in wild-type and AhR-/- mice. RESULTS: In contrast to other antipsychotic drugs (APDs), clozapine triggered AhR activation and Cyp1A1 expression in HepG2 cells and adipocytes. Clozapine induced adipogenesis via AhR signaling. After PGF2α-induced constriction of mouse aortic rings, clozapine strongly reduced the maximal vasorelaxation under acetylcholine in rings from wild-type mice, but only slightly in rings from AhR-/- mice. The reduction was also prevented by pretreatment with the AhR antagonist CH-223191. CONCLUSION: Identification of clozapine as a ligand for the AhR opens new perspectives to explain common clozapine therapy-associated adverse effects at the molecular level.

[Microbiome, diabetes and heart: a novel link?] / Mikrobiom, Diabetes und Herz: neue Zusammenhänge?

Patients with type 2 diabetes suffer from a high cardiovascular risk. The underlying pathomechanisms are not fully understood and treatment options are correspondingly limited. The gut microbiome could be a new important player in cardiometabolic diseases. Dysbiosis of the intestinal flora has been associated with insulin resistance, diabetes mellitus and cardiovascular diseases, such as atherosclerosis and heart failure. The negative cardiovascular effects of type 2 diabetes mellitus could therefore partly be mediated by gut microbiota. This review article discusses specific gut microbiome-associated mechanisms, which are modulated in both type 2 diabetes and cardiovascular diseases. It is presented how intestinal bacteria may contribute to systemic low-grade inflammation. Furthermore, it is shown how the intestinal microbiome as a complex metabolic organ is able to influence the cardiometabolic phenotype via production of bioactive metabolites. Further studies will have to demonstrate whether these mechanisms contribute to the high cardiovascular risk in type 2 diabetes.

Oral hypoglycemic agents and the heart failure conundrum: Lessons from and for outcome trials.

AIM: Type 2 diabetes is not only an independent risk factor for cardiovascular (CV) disease but is also associated with a greater incidence of heart failure (HF). The aim of this review is to examine the effects of oral antidiabetic drugs on CV disease and HF. DATA SYNTHESIS: Trials of anti-diabetic agents are now designed to assess CV safety, but frequently HF is not included as a primary endpoint. However, HF in patients with diabetes is more frequent than other CV events and seems to be underestimated. A burning question is therefore if the most used trial design to monitor CV safety, i.e. non-inferiority, allows clinical translation of trial findings. Available data further suggest that the CV effects of anti-diabetic drugs may be rather class-specific and are only partly due to their glucose-lowering actions. Metformin, recommended as first line in most guidelines, shows positive CV effects while other classes like thiazolidinediones may precipitate HF. Experimental results on the relatively novel dipeptidyl peptidase IV (DPP IV) inhibitors imply CV protective effects, but the non-inferiority trials published to date show an overall neutral CV outcome and a potential increase in HF by saxagliptin. However, results on sitagliptin of the recently released TECOS indicate that HF is not a class-dependent effect of DPP IV inhibitors. CONCLUSION: Further basic research and long-term outcome studies to clarify the effects of antidiabetic agents on CV and HF are required so that we can select the optimal antidiabetic therapy for our patients.

ITCH modulates SIRT6 and SREBP2 to influence lipid metabolism and atherosclerosis in ApoE null mice.

Atherosclerosis is a chronic inflammatory disease characterized by the infiltration of pro-inflammatory macrophages into a lipid-laden plaque. ITCH is an E3 ubiquitin ligase that has been shown to polarize macrophages to an anti-inflammatory phenotype. We therefore investigated the effect of ITCH deficiency on the development of atherosclerosis. ApoE-/-ITCH-/- mice fed a western diet for 12 weeks showed increased circulating M2 macrophages together with a reduction in plaque formation. Bone marrow transplantation recreated the haemopoietic phenotype of increased circulating M2 macrophages but failed to affect plaque development. Intriguingly, the loss of ITCH lead to a reduction in circulating cholesterol levels through interference with nuclear SREBP2 clearance. This resulted in increased LDL reuptake through upregulation of LDL receptor expression. Furthermore, ApoE-/-ITCH-/- mice exhibit reduced hepatic steatosis, increased mitochondrial oxidative capacity and an increased reliance on fatty acids as energy source. We found that ITCH ubiquitinates SIRT6, leading to its breakdown, and thus promoting hepatic lipid infiltration through reduced fatty acid oxidation. The E3 Ubiquitin Ligase ITCH modulates lipid metabolism impacting on atherosclerosis progression independently from effects on myeloid cells polarization through control of SIRT6 and SREBP2 ubiquitination. Thus, modulation of ITCH may provide a target for the treatment of hypercholesterolemia and hyperlipidemia.

Improved human T-cell responses against synthetic HLA-0201 analog peptides derived from the WT1 oncoprotein.

Wilms tumor protein 1 (WT1) is a transcription factor overexpressed in several types of leukemia and solid tumors. For this reason, WT1 is an attractive target for immunotherapy. Four peptide nonamers from WT1 have been identified by others to generate a WT1-specific cytotoxic response in the context of human leukocyte antigen (HLA)-A0201 and A2402. However, as WT1 is a self-antigen, breaking tolerance is a potential obstacle to vaccination. Here, we use a strategy to circumvent tolerance by designing synthetic immunogenic analog peptides that could crossreact to the native peptides (a heteroclitic response). A number of synthetic peptides derived from nonamer sequences of the WT1 protein were designed in which single amino-acid substitutions were introduced at HLA-A0201 major histocompatibility complex (MHC)-binding positions. Several of new peptides could stabilize MHC class I A0201 molecules better than native sequences. Some analogs were also able to elicit WT1-specific T-cell recognition and cytotoxic T-cell lymphocytes more effectively than native sequences. Importantly, T cells stimulated with the new analogs crossreacted with the native WT1 peptide sequence and were able to kill HLA-matched chronic myeloid leukemia cell lines. In conclusion, analog heteroclitic WT1 peptides with increased immunogenicity can be synthesized and are potential cancer vaccine candidates.

Administration of interleukin-7 after allogeneic bone marrow transplantation improves immune reconstitution without aggravating graft-versus-host disease.

Prolonged immunodeficiency after allogeneic bone marrow transplantation (BMT) causes significant morbidity and mortality from infection. This study examined in murine models the effects of interleukin-7 (IL-7) given to young and middle-aged (9-month-old) recipients of major histocompatibility complex (MHC)-matched or -mismatched allogeneic BMT. Although administration of IL-7 from day 0 to 14 after syngeneic BMT promoted lymphoid reconstitution, this regimen was ineffective after allogeneic BMT. However, IL-7 administration from day 14 (or 21) to 27 after allogeneic BMT accelerated restoration of the major lymphoid cell populations even in middle-aged recipients. This regimen significantly expanded donor-derived thymocytes and peripheral T cells, B-lineage cells in bone marrow and spleen, splenic natural killer (NK) cells, NK T cells, and monocytes and macrophages. Interestingly, although recipients treated with IL-7 had significant increases in CD4(+) and CD8(+) memory T-cell populations, increases in naive T cells were less profound. Most notable, however, were the observations that IL-7 treatment did not exacerbate graft-versus-host disease (GVHD) in recipients of an MHC-matched BMT, and would ameliorate GVHD in recipients of a MHC-mismatched BMT. Nonetheless, graft-versus-leukemia (GVL) activity (measured against 32Dp210 leukemia) remained intact. Although activated and memory CD4(+) and CD8(+) T cells normally express high levels of IL-7 receptor (IL-7R, CD127), activated and memory alloreactive donor-derived T cells from recipients of allogeneic BMT expressed little IL-7R. This might explain the failure of IL-7 administration to exacerbate GVHD. In conclusion, posttransplant IL-7 administration to recipients of an allogeneic BMT enhances lymphoid reconstitution without aggravating GVHD while preserving GVL.

Differential use of Fas ligand and perforin cytotoxic pathways by donor T cells in graft-versus-host disease and graft-versus-leukemia effect.

In allogeneic bone marrow transplantation (BMT) donor T cells are primarily responsible for antihost activity, resulting in graft-versus-host disease (GVHD), and for antileukemia activity, resulting in the graft-versus-leukemia (GVL) effect. The relative contributions of the Fas ligand (FasL) and perforin cytotoxic pathways in GVHD and GVL activity were studied by using FasL-defective or perforin-deficient donor T cells in murine parent --> F1 models for allogeneic bone marrow transplantation. It was found that FasL-defective B6.gld donor T cells display diminished GVHD activity but have intact GVL activity. In contrast, perforin-deficient B6.pfp(-/-) donor T cells have intact GVHD activity but display diminished GVL activity. Splenic T cells from recipients of B6.gld or B6.pfp(-/-) T cells had identical proliferative and cytokine responses to host antigens; however, splenic T cells from recipients of B6.pfp(-/-) T cells had no cytolytic activity against leukemia cells in a cytotoxicity assay. In experiments with selected CD4(+) or CD8(+) donor T cells, the FasL pathway was important for GVHD activity by both CD4(+) and CD8(+) T cells, whereas the perforin pathway was required for CD8-mediated GVL activity. These data demonstrate in a murine model for allogeneic bone marrow transplantation that donor T cells mediate GVHD activity primarily through the FasL effector pathway and GVL activity through the perforin pathway. This suggests that donor T cells make differential use of cytolytic pathways and that the specific blockade of one cytotoxic pathway may be used to prevent GVHD without interfering with GVL activity.

Molecular characterization of a cDNA encoding functional human deoxyhypusine synthase and chromosomal mapping of the corresponding gene locus.

Deoxyhypusine synthase is essentially required for the post-translational formation of hypusine, a modification of a specific lysine residue in eukaryotic initiation factor 5A, which appears to be pivotal for cell proliferation. From a human peripheral blood mononuclear cells cDNA library we isolated two independent sequences encoding biologically active deoxyhypusine synthase. DNA sequence analysis revealed a 369 amino acid protein with a molecular mass of 41.055 kDa. This recombinant deoxyhypusine synthase showed significant catalytic activity in synthesis of deoxyhypusine after in vitro transcription and translation as well as upon expression in Escherichia coli. Using a panel of somatic rodent-human cell hybrids we localized the deoxyhypusine synthase gene to human chromosome 19.

Identification of a new member of the human eIF-5A gene family.

Using an oligodeoxynucleotide generated by rapid PCR amplification of 5'-cDNA ends (5'-RACE) as a detection probe, we have isolated a new genomic clone encoding the human eukaryotic initiation factor 5A (eIF-5A). Sequence analysis revealed that the eIF-5A coding region is identical to the corresponding cDNA but interrupted by three introns. In a plasmid shuffle experiment we show functional replacement of the essential homologous gene in Saccharomyces cerevisiae by this human eIF-5A.

The genomic structure encoding human initiation factor eIF-5A.

A genomic clone encoding the human eukaryotic initiation factor 5A (eIF-5A) was isolated and its entire nucleotide sequence was determined. The whole eIF-5A coding region is not interrupted by introns. The functional eIF-5A gene is highly homologous to the corresponding complementary DNA. One single 1.4-kb transcript thereof is expressed in human cell lines. Furthermore, we also isolated and sequenced two additional eIF-5A-related sequences which are, by expression and sequence analyses, identified as pseudogenes of the functional eIF-5A. The sequence homology between these pseudogenes and the functional eIF-5A is 71 and 87%.

A genotoxic study of metal workers exposed to trichloroethylene. Sperm parameters and chromosome aberrations in lymphocytes.

Metal workers exposed to trichloroethylene for the degreasing of metals were studied to evaluate the genotoxicity of this exposure. For 15 workers presently exposed to high doses of trichloroethylene there was no difference from unexposed persons with respect to sperm count and morphology, and a small increase of two fluorescent bodies (YFF%) in spermatozoa. In contrast, there was a highly significant increase in frequency of structural aberrations (breaks, gaps, translocation, deletions, inversions) and hyperdiploid cells in cultured lymphocytes from trichloroethylene degreasers. As control groups, physicians from chemically non-exposed surroundings and a concurrently sampled reference from cytogenetic investigations were used. This study indicates positive correlations between exposure to trichloroethylene and somatic chromosome aberrations, whereas no effect on male germ cells could be demonstrated.

Spontaneous abortion following mid-trimester amniocentesis. Clinical significance of placental perforation and blood-stained amniotic fluid.

The clinical significance of placental perforation and blood-stained amniotic fluid was studied in a group of 7238 Danish women undergoing mid-trimester amniocentesis for prenatal diagnosis under ultrasound guidance. The risk of spontaneous abortion was significantly increased both in pregnancies where the placenta was perforated and in those with blood-stained amniotic fluid. The risk estimate nearly doubled after placental perforation and more than doubled with a bloody tap. It is concluded that for women at relatively low risk of a fetal genetic abnormality, the indication of the amniocentesis should be reconsidered if a placental perforation is unavoidable.

Short stature in Scandinavian women. An obstetrical risk factor.

We have carried out a case-controlled study on relations between short stature (i.e. less than 156 cm tall) and problems with childbirth in Danish women. Data obtained from 182 pregnant, short women (short mothers) were compared with those obtained from a control group of 2116 pregnant women who were between 166 and 175 cm tall (control mothers). The prevalence rate for acute cesarean section was three-fold greater in short mothers than in controls, and the prevalence rate for elective cesarean section was twice as high in short mothers as in controls. Moreover, the prevalence rates of intra-uterine asphyxia, intra-uterine growth retardation and low Apgar scores were higher in babies of short mothers than in those of control mothers, despite the increased level of obstetric intervention in the former group. Since the findings show that short stature in pregnant women is an obstetrical risk factor, we recommend that it should be given attention in order to detect early signs of intra-uterine asphyxia and to apply the best form of active management of labor if necessary.

Human placental lactogen and dU-estrogen levels in normal twin pregnancies.

Reference intervals for human placental lactogen (hPL) and dU-estrogens (dU-E) in uncomplicated twin pregnancies are found to be higher than singleton pregnancies and considerably wider. Significantly more monozygotic than dizygotic pregnancies, and significantly more monoplacental than diplacental pregnancies showed hPL values below the median on the normal range curve. The same was not found for dU-E. Significantly more growth-retarded fetuses were found in monoplacental than diplacental pregnancies. The benefit of measuring the two parameters in order to identify the intrauterine growth retarded fetuses were evaluated in terms of sensitivity and specificity. Both parameters were found less suitable for the purpose.

Bed rest in twin pregnancy.

A total of 146 twin pregnancies were studied to evaluate the importance of bed rest. A significantly reduced frequency of preterm delivery was found after bed rest in hospital as compared to bed rest at home or no bed rest at all. An increase in gestational age and in birth weight was seen after bed rest. No effect was found on the intrauterine increase of fetal weight per time. Early routine ultrasound is recommended, so that effective bed rest may be instituted in due time.