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A Temme

Publications and source records attributed to A Temme.

12 recordsLinked to original sources
PubMed2002

Efficient transduction and long-term retroviral expression of the melanoma-associated tumor antigen tyrosinase in CD34(+) cord blood-derived dendritic cells.

Differentiation of genetically modified CD34(+) hematopoietic stem cells into dendritic cells (DCs) will contribute to the development of immunotherapeutic anticancer protocols. Retroviral vectors that have been used for the transduction of CD34(+) cells face the problem of gene silencing when integrated into the genome of repopulating stem cells. We reasoned that a high copy number of retroviral DNA sequences might overcome silencing of transgene expression during expansion and differentiation of progenitor cells into functional DCs. To prove this, we utilized a retroviral vector with bicistronic expression of the melanoma-associated antigen tyrosinase and the enhanced green fluorescent protein (EGFP). Human cord blood CD34(+) cells were transduced with vesicular stomatitis virus G-protein (VSV-G) pseudotyped Moloney murine leukemia virus (MoMuLV) particles using 100-150 multiplicity of infection. During expansion of transduced cells with immature phenotype, transgene expression was strongly silenced, but upon differentiation into mature DCs, residual transgene expression was retained. Intracellular processing of the provirally expressed tyrosinase was tested in a chromium release assay utilizing a cytotoxic T cell clone specific for a HLA-A*0201-restricted tyrosinase peptide. We suggest that retroviral transduction of tumor-associated antigens in hematopoietic progenitor cells and subsequent differentiation into DCs is a suitable basis for the development of potent anti-tumor vaccines.

Antigens, CD34↗
PubMed2001

Dilated bile canaliculi and attenuated decrease of nerve-dependent bile secretion in connexin32-deficient mouse liver.

Gap junction channels in the rodent liver are composed of connexin26 (Cx26) and connexin32 (Cx32) proteins. Gap junctional intercellular communication in the mouse liver enhances the effects of hormonal or sympathetic stimulation of glucose release from glycogen stores. To determine whether contraction of bile canaliculi and bile secretion are dependent on the function of gap junction channels, we compared wild-type and connexin32-deficient mice. Confocal laser scanning microscopy of the wild-type mouse liver confirmed the close association of connexin26 and -32 proteins with the zona occludens-1 protein and actin filaments of the bile canaliculi. The decrease of bile flow after electrical stimulation of sympathetic nerves in the perfused liver was attenuated in the Cx32-deficient liver compared with wild-type controls. The amount of secreted bile, however, was similar in wild-type and Cx32-deficient livers. Furthermore, Cx32-deficient mice exhibited dilated bile canaliculi, suggesting that the contraction of bile canaliculi could be impaired in these animals.

Alanine Transaminase↗
PubMed2000

Acute-phase response and circadian expression of connexin26 are not altered in connexin32-deficient mouse liver.

In mouse hepatocytes, the gap junctional proteins connexin32 (Cx32) and connexin26 (Cx26) are expressed in the same gap junctional plaque. Expression of the major Cx32 protein is downregulated during liver regeneration and cholestasis. Here we have analyzed the acute-phase response (after experimental inflammation) and circadian connexin expression in Cx32-deficient and wild-type mouse liver. Acute-phase response was triggered by intraperitoneal injection of lipopolysaccharide (LPS). Injection of recombinant mouse interleukin-1beta (mIL-1beta), mIL-6 or tumor necrosis factor alpha (mTNF-alpha) had no inflammatory effect. Northern blot analysis of positive and negative acute-phase transcripts following stimulation with cytokine or LPS revealed no difference between Cx32-deficient livers and wild-type controls, suggesting that loss of the Cx32 gene had no effect on experimental liver inflammation. Actin, beta-fibrinogen and Cx26 transcripts were increased after endotoxin stimulation. Under conditions of hepatic acute-phase response, Cx32 transcripts were not detected in LPS-treated livers of wild-type mice. Immunoblot analysis of proteins from inflamed wild-type livers indicated a strongly diminished amount of Cx32 protein, whereas the level of Cx26 protein was increased. Although intraperitoneal injection of mIL-1, mIL-6 as well as mTNF-alpha did not induce an acute-phase response, Cx32 protein expression was diminished, suggesting that post-transcriptional downregulation of Cx32 preceded the acute-phase response. Northern blot hybridization of RNA from wild-type and Cx32-deficient mouse liver revealed a similar circadian regulation of Cx26 and GAPDH transcripts with maximal expression around 2 p.m. and a minimum after midnight.

Actins↗
PubMed2000

The extent of synchronous initiation and termination of DNA synthesis in regenerating mouse liver is dependent on connexin32 expressing gap junctions.

BACKGROUND/AIMS: It has previously been shown in rat liver that the gap junctional proteins connexin32 and connexin26 are downregulated when murine hepatocytes are in the S-phase of the cell cycle. Therefore, it has been hypothesized that loss of functional gap junctions could affect proliferation of hepatocytes. This study aimed to check this hypothesis. METHODS: We searched for differences in liver regeneration after two-thirds partial hepatectomy between connexin32-deficient and wild-type mice. RESULTS: The ratio of liver to body weight in regenerating liver was not affected by loss of the connexin32 gene. The peak of DNA synthesis occurred at the same time, i.e. 36 to 96 h after partial hepatectomy, in connexin32-deficient and wild-type liver. During this time, however, only about half as many nuclei of hepatocytes in connexin32-deficient liver incorporated bromodeoxyuridine, compared to wild-type liver. Furthermore, 1-2 weeks after full recovery of liver mass, we detected a higher level of bromodeoxyuridine incorporation into hepatocytes of connexin32-deficient than in wild-type liver. CONCLUSIONS: Loss of connexin32 protein and/or diminished expression of connexin26 did not promote G0/1-S transition of hepatocytes in two-thirds hepatectomized mouse livers. Instead, the extent of synchronous initiation and termination of DNA synthesis in regenerating liver was altered in connexin32-deficient mice.

Animals↗
PubMed1999

Biological functions of connexin genes revealed by human genetic defects, dominant negative approaches and targeted deletions in the mouse.

Gap junction channels in mammalian organs can be built up of at least 13 different connexin proteins, most of which are expressed in only few cell types, although many cells express more than one connexin protein. Recently, the consequences of missing or defective connexin proteins were studied in human patients with defects in connexin32 (Cx32; beta 1; X-linked Charcot-Marie-Tooth disease) or in Cx26 (beta 2; non-syndromic sensorineural deafness), and in mice with targeted deletions in the Cx26, Cx32, Cx37 (alpha 4), Cx43 (alpha 1), Cx46 (alpha 3) or Cx50 (alpha 8) genes. Some effects of dominant negative mutations in connexin genes have been characterized in Xenopus oocytes and transfected mammalian cells in culture. Here we review results of these different experimental approaches and report new findings regarding the characterization of Cx40 (alpha 5)- and Cx31 (beta 3)-deficient mice. The phenotypic alterations, caused by different defective connexin genes in mice or humans, are divergent, although in most known cases the viability is not affected. When more than one connexin gene, coexpressed in the same cell, is inactivated, development or maturation can be more severely affected at an earlier stage. Some connexin proteins, if present in the same cell, can partially replace each other in certain functions. Thus, the diversity of connexin proteins in mammalian cells may provide functional overlap and complementation.

Animals↗
PubMed1998

Transplacental uptake of glucose is decreased in embryonic lethal connexin26-deficient mice.

Mice that harbor a targeted homozygous defect in the gene coding for the gap junctional protein connexin26 died in utero during the transient phase from early to midgestation. From day 10 post coitum onwards, development of homozygous embryos was retarded, which led to death around day 11 post coitum. Except for growth retardation, no gross morphological alterations were detected between homozygous connexin26-defective embryos and wild-type littermates. At day 9 postcoitum, when chorioallantoic placenta started to function, connexin26 was weakly expressed in the yolk sac epithelium, between syncytiotrophoblasts I and II in the labyrinth region of the placenta, and in the skin of the embryo. At day 10 post coitum, expression of connexin26 in the placenta was much stronger than at the other locations. To analyze involvement of connexin26 in the placental transfer of nutrients, we have measured embryonic uptake of the nonmetabolizable glucose analogue 3-O-[14C]methylglucose, injected into the maternal tail vein. At day 10 post coitum, viable, homozygous connexin26-defective embryos accumulated only approximately 40% of the radioactivity measured in wild-type and heterozygous littermates of the same size. We conclude that the uptake of glucose, and presumably other nutrients as well, from maternal blood into connexin26-deficient mouse embryos was severely impaired and apparently not sufficient to support the rapid organogenesis during midgestation. Our results suggest that connexin26 gap junction channels likely fulfill an essential role in the transfer of maternal nutrients and embryonic waste products between syncytiotrophoblast I and II in the labyrinth layer of the mouse placenta.

3-O-Methylglucose↗
PubMed1998

Downregulation of connexin32 protein and gap-junctional intercellular communication by cytokine-mediated acute-phase response in immortalized mouse hepatocytes.

In the present study, we have analyzed the direct effects of cytokines, which mediate the acute-phase response in liver, on connexin expression and gap-junctional intercellular communication in immortalized MHSV12 mouse hepatocytes. When these cells were stimulated for 24 h with interleukin 1 and interleukin 6, the amount of connexin26 (Cx26) mRNA increased together with beta-fibrinogen mRNA, as expected for this positive acute-phase gene. In contrast, connexin32 (Cx32) mRNA expression was not affected under these conditions. Indirect immunfluorescence revealed a drastic decrease in Cx32 signals, whereas slightly more Cx26 signals were found. Stronger stimulation with interleukin 1 and tumor necrosis factor alpha gave a dose-dependent increase in steady state levels of Cx26 and beta-fibrinogen mRNA, but no further change in Cx32 mRNA level was seen. However, when Cx32 protein was analyzed on immunoblots, we found a 5-fold decrease in expression even at low cytokine doses that did not affect Cx32 mRNA expression. Under these conditions, cell to cell transfer of Lucifer yellow, microinjected into immortalized hepatocytes, was decreased by 70%, suggesting that intercellular communication through Cx32 channels was partially inhibited earlier than other genetic alterations characteristic of the acute-phase response. Thus, the major hepatic gap junction protein was largely downregulated at the beginning of the experimental inflammatory reaction, but about 30% of gap-junctional intercellular communication was maintained. This suggests that, during the acute-phase response, the second hepatic Cx26 protein may compensate in part for the downregulation of the Cx32 protein.

Acute-Phase Reaction↗
PubMed1997

High incidence of spontaneous and chemically induced liver tumors in mice deficient for connexin32.

Connexins are subunits of gap junction channels, which mediate the direct transfer of ions, second messenger molecules and other metabolites between contacting cells. Gap junctions are thought to be involved in tissue homeostasis, embryonic development and the control of cell proliferation [1,2]. It has also been suggested that the loss of intercellular communication via gap junctions may contribute to multistage carcinogenesis [3-5]. We have previously shown that transgenic mice that lack connexin32 (Cx32), the major gap junction protein expressed in hepatocytes, express lower levels of a second hepatic gap junction protein, Cx26, suggesting that Cx32 has a stabilizing effect on Cx26 [6]. Here, we report that male and female one-year-old mice deficient for Cx32 had 25-fold more and 8-fold more spontaneous liver tumors than wild-type mice, respectively. Incorporation of bromodeoxyuridine (BrdU) into the liver was higher for Cx32-deficient mice than for wild-type mice, suggesting that their hepatocyte proliferation rate was higher. Furthermore, intraperitoneal injection, two weeks after birth, of the carcinogen diethylnitrosamine (DEN) led, after one year, both to more liver tumors in Cx32-deficient mice than in controls, and to accelerated tumor growth. Loss of Cx32 protein from hepatic gap junctions is therefore likely to cause enhanced clonal survival and expansion of mutated ('initiated') cells, which results in a higher susceptibility to hepatic tumors. Our results demonstrate that functional gap junctions inhibit the development of spontaneous and chemically induced tumors in mouse liver.

Animals↗
PubMed1996

Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice.

The gap junctional protein connexin32 is expressed in hepatocytes, exocrine pancreatic cells, Schwann cells, and other cell types. We have inactivated the connexin32 gene by homologous recombination in the mouse genome and have generated homozygous connexin32-deficient mice that were viable and fertile but weighed on the average approximately 17% less than wild-type controls. Electrical stimulation of sympathetic nerves in connexin32-deficient liver triggered a 78% lower amount of glucose mobilization from glycogen stores, when compared with wild-type liver. Thus, connexin32-containing gap junctions are essential in mouse liver for maximal intercellular propagation of the noradrenaline signal from the periportal (upstream) area, where it is received from sympathetic nerve endings, to perivenous (downstream) hepatocytes. In connexin32-defective liver, the amount of connexin26 protein expressed was found to be lower than in wild-type liver, and the total area of gap junction plaques was approximately 1000-fold smaller than in wild-type liver. In contrast to patients with connexin32 defects suffering from X chromosome-linked Charcot-Marie-Tooth disease (CMTX) due to demyelination in Schwann cells of peripheral nerves, connexin32-deficient mice did not show neurological abnormalities when analyzed at 3 months of age. It is possible, however, that they may develop neurodegenerative symptoms at older age.

Animals↗
PubMed1984

Dosimetrical verification of calculated total and gamma absorbed dose distributions DT, respectively DG, for fast neutron therapy.

The physical part of treatment planning for neutron therapy is highly important but quite more complicated than for photons and electrons due to the necessity of separation of total and gamma absorbed dose distributions (DT and DG). Therefore, dosimetrical verifications of dose distributions of complex treatment plans are very rare, and the experimental difficulties are enormous. A method using TLD-300 (CaF2: Tm) detectors is described with the ability to provide from each detector's readings both DT and DG using the different LET dependence of the main glow-curve peaks. The principle of an on-line computer program is given for an automatic mathematical glow-curve analysis which is necessary to achieve the accuracy of about +/- 5% (DT) and +/- 10% (DG) in single fixed fields. Dosimetrically relevant features of the TLD-300 method are discussed. Examples of dosimetrical verifications of calculated dose distributions (DT and DG) for a treatment with one single fixed field, with two wedge filter fields and with a rotational field with d(14)+Be neutrons are shown. Deviations exceeding significantly the uncertainties above are found only in the case of the wedge filter treatment for the absolute (quite less for the relative) values of the DG dose distribution. Probable reasons are mostly the reduced contribution of scattered radiation for phantom sizes, which laterally scarcely exceed the useful beam, and slight neutron energy spectrum changes by the wedge filter.

Dose-Response Relationship, Radiation↗
PubMedDate not supplied

Tissue equivalence in clinical neutron dosimetry: comparison of dose distributions in nine tissue substitutes for a d(14)Be neutron beam.

Depth dose distributions for a d(14)Be neutron beam, measured separately for total and gamma absorbed dose, are presented for nine tissue substitutes and for two field sizes. These data are used to examine methods to transform depth dose curves from one material to another. Best results are found when the local depths are transformed by constant empirical factors which are independent of depth and field size. As a physical interpretation of the empirical factors, kerma-weighted mean-free-path lengths are calculated for the interaction of the Essen neutron beam with the materials. The ratios of these free path lengths agree with the empirical factors within +/- 10%. However, for clinical dosimetry, a direct comparability of spatial absorbed dose distributions measured in two different phantom materials is only given if their corresponding transformation factor is near unity.

Animals↗