Neurochemical profile of the mouse hypothalamus using in vivo 1H MRS at 14.1T.

The hypothalamus plays an essential role in the central nervous system of mammals by among others regulating glucose homeostasis, food intake, temperature, and to some extent blood pressure. Assessments of hypothalamic metabolism using, e.g. (1)H MRS in mouse models can provide important insights into its function. To date, direct in vivo (1)H MRS measurements of hypothalamus have not been reported. Here, we report that in vivo single voxel measurements of mouse hypothalamus are feasible using (1)H MRS at 14.1T. Localized (1)H MR spectra from hypothalamus were obtained unilaterally (2-2.2 microL, VOI) and bilaterally (4-4.4 microL) with a quality comparable to that of hippocampus (3-3.5 microL). Using LCModel, a neurochemical profile consisting of 21 metabolites was quantified for both hypothalamus and hippocampus with most of the Cramér-Rao lower bounds within 20%. Relative to the hippocampus, the hypothalamus was characterized by high gamma-aminobutryric acid and myo-inositol, and low taurine concentrations. When studying transgenic mice with no glucose transporter isoform 8 expressed, small metabolic changes were observed, yet glucose homeostasis was well maintained. We conclude that a specific neurochemical profile of mouse hypothalamus can be measured by (1)H MRS which will allow identifying and following metabolic alterations longitudinally in the hypothalamus of genetic modified models.

Lack of antitumour activity of human recombinant tumour necrosis factor-alpha, alone or in combination with melphalan in a nude mouse human melanoma xenograft system.

The most promising developments in the field of isolated limb perfusion have centred around the use of the recombinant cytokine tumour necrosis factor-alpha (rTNF-alpha) in combination with melphalan. While the results of clinical trials are impressive, the exact antitumour mechanisms of rTNF-alpha and its role in combination with melphalan remain unclear. Our aim was to study the antitumour activity of human rTNF-alpha with or without the combination of melphalan in a nude mouse human melanoma xenograft system. In a first attempt to define the maximal tolerated single dose of rTNF-alpha in this setting, 15 animals were exposed to increasing doses of rTNF-alpha (60-2500 microg/kg intraperitoneally). All but one animal survived and tumour growth was not influenced by these single dose applications of rTNF-alpha even at the very high doses. Anti-tumour activity of repeated application of melphalan (three times 9 mg/kg in group 2 and three times 6 mg/kg in group 3), of rTNF-alpha alone (nine doses of 50 microg/kg in group 4), and of rTNF-alpha in combination with melphalan (nine doses of 50 microg/kg rTNF-alpha and three times 6 mg/kg melphalan in group 5) was further compared with non-treated animals (group 1). Tumour growth was significantly inhibited in all animals treated with melphalan (group 2, 3 and 5), but was not decreased in animals treated with rTNF-alpha alone (group 4). Mean final tumour volumes and mean tumour weight were not different in group 2 (789 +/- 836 mm3, 0.38 +/- 0.20 g), group 3 (1173 +/- 591 mm3, 0.55 +/- 0.29 g) and group 5 (230 +/- 632 mm3, 0.37 +/- 0.29 g), but significant lower than group 1 (3156 +/- 1512 mm3, 2.35 +/- 0.90 g) and group 4 (3228 +/- 1990 mm3, 2.00 +/- 1.16 g). There were no significant differences between high and low dose melphalan treatment and between melphalan treatment in combination with rTNF-alpha. Histological examination did not show differences between treated and non-treated animals besides slightly inhibited mitotic activities of tumour cells in melphalan-treated animals. While tumour growth of human xenotransplanted melanoma in nude mice could be inhibited by melphalan, we failed to demonstrate any antitumour effect of rTNF-alpha. The combination of melphalan and rTNF-alpha did not enhance the antiproliferative effect of melphalan alone. Human xenotransplanted tumours on nude mice might not be the ideal experimental setting for studies of potential direct antineoplastic activity of rTNF-alpha, and these results support the concept that TNF-alpha exerts its antitumour activity indirectly, possibly by impairing the tumour vasculature and by activating the immune system.

Altered expression of the transcription factor Mef2c during retinal degeneration in Rpe65-/- mice.

Purpose. To investigate the role of the myocyte enhancer factor 2 (Mef2) transcription factor family in retinal diseases, Mef2c expression was assessed during retinal degeneration in the Rpe65(-/-) mouse model of Leber's congenital amaurosis (LCA). Mef2c-dependent expression of photoreceptor-specific genes was further addressed. Methods. Expression of Mef2 members was analyzed by oligonucleotide microarray, quantitative PCR (qPCR) and in situ hybridization. Mef2c-dependent transcriptional activity was assayed by luciferase assay in HEK293T cells. Results. Mef2c was the only Mef2 member markedly downregulated during retinal degeneration in Rpe65(-/-) mice. Mef2c mRNA level was decreased by more than 2 fold at 2 and 4 months and by 3.5 fold at 6 months in retinas of Rpe65(-/-) mice. Downregulation of Mef2c at the protein level was confirmed in Rpe65(-/-) retinas. The decrease in Mef2c mRNA levels in the developing Rpe65(-/-) retinas, from post-natal day (P)13 onward, was concomitant with the decreased expression of the rod-specific transcription factors Nrl and Nr2e3. Nrl was further shown to drive Mef2c transcriptional activity, supporting a physiological role for Mef2c in the retina. In addition, Mef2c appeared to act as a transcriptional repressor of its own expression, as well as those of the retina-specific retinal G-protein coupled receptor (Rgr), rhodopsin and M-opsin genes. Conclusions. These findings highlight the early altered regulation of the rod-specific transcriptional network in Rpe65-related disease. They further indicate that Mef2c may act as a novel transcription factor involved in the development and the maintenance of photoreceptor cells.

Interferon-gamma impacts at multiple points during the progression of autoimmune diabetes.

The role of interferon-gamma in autoimmune diabetes was assessed by breeding a null mutation of the interferon-gamma receptor alpha chain into the nonobese diabetic mouse strain, as well as into a simplified T cell receptor transgenic model of diabetes. In contrast to a previous report on abrogation of the interferon-gamma gene, mutation of the gene encoding its receptor led to drastic effects on disease in both mouse lines. Nonobese diabetic mice showed a marked inhibition of insulitis-both the kinetics and penetrance-and no signs of diabetes; the transgenic model exhibited near-normal insulitis, but this never evolved into diabetes, either spontaneously or after experimental provocation. This failure could not be explained by perturbations in the ratio of T helper cell phenotypes; rather, it reflected a defect in antigen-presenting cells or in the islet beta cell targets.

Monoclonal antibodies against carcinoembryonic antigen (CEA) used in a solid-phase enzyme immunoassay. First clinical results.

A solid-phase enzyme immunoassay using both mouse monoclonal and goat polyclonal antibodies against carcinoembryonic antigen (CEA) was developed. The assay detects 0.6 to 1.2 ng of CEA per ml of serum and has 3 incubation steps which can be performed in 1 day. Polystyrene balls coated with polyclonal goat anti-CEA antibodies are first incubated with heat-extracted serum samples. Bound CEA is then detected by addition of mouse monoclonal antibodies, followed by goat IgG anti-mouse IgG1 coupled to alkaline phosphatase. Results with this enzyme immunoassay using monoclonal antibodies (M-EIA) have been compared with those obtained by the conventional inhibition radioimmunoassay (RIA) using goat antiserum. Three hundred and eighty serum samples from 167 patients with malignant or non-malignant diseases and from 134 normal individuals with or without heavy smoking habits were analyzed by the 2 assays. Excellent correlation between the results of the 2 assays was obtained, but the M-EIA, using monoclonal antibodies from a single hybridoma, did not discriminate better than the conventional RIA between CEA produced by different types of carcinoma and between CEA associated with malignant or non-malignant diseases. Follow-up studies of several patients by sequential CEA determinations with the 2 assays showed that the M-EIA was as accurate as the RIA for the detection of tumor recurrences.

Multilayered genetic and omics dissection of mitochondrial activity in a mouse reference population

The manner by which genotype and environment affect complex phenotypes is one of the fundamental questions in biology. In this study, we quantified the transcriptome--a subset of the metabolome--and, using targeted proteomics, quantified a subset of the liver proteome from 40 strains of the BXD mouse genetic reference population on two diverse diets. We discovered dozens of transcript, protein, and metabolite QTLs, several of which linked to metabolic phenotypes. Most prominently, Dhtkd1 was identified as a primary regulator of 2-aminoadipate, explaining variance in fasted glucose and diabetes status in both mice and humans. These integrated molecular profiles also allowed further characterization of complex pathways, particularly the mitochondrial unfolded protein response (UPR(mt)). UPR(mt) shows strikingly variant responses at the transcript and protein level that are remarkably conserved among C. elegans, mice, and humans. Overall, these examples demonstrate the value of an integrated multilayered omics approach to characterize complex metabolic phenotypes.

Regional variations in ABC transporter expression along the mouse intestinal tract.

The ATP-binding cassette (ABC) family of proteins comprise a group of membrane transporters involved in the transport of a wide variety of compounds, such as xenobiotics, vitamins, lipids, amino acids, and carbohydrates. Determining their regional expression patterns along the intestinal tract will further characterize their transport functions in the gut. The mRNA expression levels of murine ABC transporters in the duodenum, jejunum, ileum, and colon were examined using the Affymetrix MuU74v2 GeneChip set. Eight ABC transporters (Abcb2, Abcb3, Abcb9, Abcc3, Abcc6, Abcd1, Abcg5, and Abcg8) displayed significant differential gene expression along the intestinal tract, as determined by two statistical models (a global error assessment model and a classic ANOVA, both with a P < 0.01). Concordance with semiquantitative real-time PCR was high. Analyzing the promoters of the differentially expressed ABC transporters did not identify common transcriptional motifs between family members or with other genes; however, the expression profile for Abcb9 was highly correlated with fibulin-1, and both genes share a common complex promoter model involving the NFkappaB, zinc binding protein factor (ZBPF), GC-box factors SP1/GC (SP1F), and early growth response factor (EGRF) transcription binding motifs. The cellular location of another of the differentially expressed ABC transporters, Abcc3, was examined by immunohistochemistry. Staining revealed that the protein is consistently expressed in the basolateral compartment of enterocytes along the anterior-posterior axis of the intestine. Furthermore, the intensity of the staining pattern is concordant with the expression profile. This agrees with previous findings in which the mRNA, protein, and transport function of Abcc3 were increased in the rat distal intestine. These data reveal regional differences in gene expression profiles along the intestinal tract and demonstrate that a complete understanding of intestinal ABC transporter function can only be achieved by examining the physiologically distinct regions of the gut.

Continuous arterial spin labeling of mouse cerebral blood flow using an actively-detuned two-coil system at 9.4T.

Among numerous magnetic resonance imaging (MRI) techniques, perfusion MRI provides insight into the passage of blood through the brain's vascular network non-invasively. Studying disease models and transgenic mice would intrinsically help understanding the underlying brain functions, cerebrovascular disease and brain disorders. This study evaluates the feasibility of performing continuous arterial spin labeling (CASL) on all cranial arteries for mapping murine cerebral blood flow at 9.4 T. We showed that with an active-detuned two-coil system, a labeling efficiency of 0.82 ± 0.03 was achieved with minimal magnetization transfer residuals in brain. The resulting cerebral blood flow of healthy mouse was 99 ± 26 mL/100g/min, in excellent agreement with other techniques. In conclusion, high magnetic fields deliver high sensitivity and allowing not only CASL but also other MR techniques, i.e. (1)H MRS and diffusion MRI etc, in studying murine brains.

Superantigen-reactive CD4+ T cells are required to stimulate B cells after infection with mouse mammary tumor virus.

Superantigens are defined by their ability to stimulate a large fraction of T cells via interaction with the T cell receptor (TCR) V beta domain. Endogenous superantigens, classically termed minor lymphocyte-stimulating (Mls) antigens, were recently identified as products of open reading frames (ORF) in integrated proviral copies of mouse mammary tumor virus (MMTV). We have described an infectious MMTV homologue of the classical endogenous superantigen Mls-1a (Mtv-7). The ORF molecules of both the endogenous Mtv-7 and the infectious MMTV(SW) interact with T cells expressing the TCR V beta 6, 7, 8.1, and 9 domains. Furthermore, the COOH termini of their ORF molecules, thought to confer TCR specificity, are very similar. Since successful transport of MMTV from the site of infection in the gut to the mammary gland depends on a functional immune system, we were interested in determining the early events after and requirements for MMTV infection. We show that MMTV(SW) infection induces a massive response of V beta 6+ CDC4+ T cells, which interact with the viral ORF. Concomitantly, we observed a B cell response and differentiation that depends on both the presence and stimulation of the superantigen-reactive T cells. Furthermore, we show that B cells are the main target of the initial MMTV infection as judged by the presence of the reverse-transcribed viral genome and ORF transcripts. Thus, we suggest that MMTV infection of B cells leads to ORF-mediated B-T cell interaction, which maintains and possibly amplifies viral infection.

Physiologically based pharmacokinetic modeling of arsenic in the mouse

A remarkable feature of the carcinogenicity of inorganic arsenic is that while human exposures to high concentrations of inorganic arsenic in drinking water are associated with increases in skin, lung, and bladder cancer, inorganic arsenic has not typically caused tumors in standard laboratory animal test protocols. Inorganic arsenic administered for periods of up to 2 yr to various strains of laboratory mice, including the Swiss CD-1, Swiss CR:NIH(S), C57Bl/6p53(+/-), and C57Bl/6p53(+/+), has not resulted in significant increases in tumor incidence. However, Ng et al. (1999) have reported a 40% tumor incidence in C57Bl/6J mice exposed to arsenic in their drinking water throughout their lifetime, with no tumors reported in controls. In order to investigate the potential role of tissue dosimetry in differential susceptibility to arsenic carcinogenicity, a physiologically based pharmacokinetic (PBPK) model for inorganic arsenic in the rat, hamster, monkey, and human (Mann et al., 1996a, 1996b) was extended to describe the kinetics in the mouse. The PBPK model was parameterized in the mouse using published data from acute exposures of B6C3F1 mice to arsenate, arsenite, monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) and validated using data from acute exposures of C57Black mice. Predictions of the acute model were then compared with data from chronic exposures. There was no evidence of changes in the apparent volume of distribution or in the tissue-plasma concentration ratios between acute and chronic exposure that might support the possibility of inducible arsenite efflux. The PBPK model was also used to project tissue dosimetry in the C57Bl/6J study, in comparison with tissue levels in studies having shorter duration but higher arsenic treatment concentrations. The model evaluation indicates that pharmacokinetic factors do not provide an explanation for the difference in outcomes across the various mouse bioassays. Other possible explanations may relate to strain-specific differences, or to the different durations of dosing in each of the mouse studies, given the evidence that inorganic arsenic is likely to be active in the later stages of the carcinogenic process. [Authors]

Expression and presentation of endogenous mouse mammary tumor virus superantigens by thymic and splenic dendritic cells and B cells.

Tolerance against superantigens (SAgs) encoded by endogenous mouse mammary tumor virus (Mtv) loci involves the intrathymic deletion of SAg-reactive T cells expressing a particular TCR V beta-chain, presumably upon presentation of the SAg by specialized APC. However, although the role of dendritic cells (DC) in the induction of tolerance against conventional Ags has been demonstrated, little is known about the role played by DC in tolerance induction against Mtv SAgs. Moreover, there is conflicting evidence concerning the capacity of DC to express and present Mtv SAgs. In this report we have analyzed the expression of Mtv SAgs in highly purified thymic and splenic DC and B cells by reverse transcriptase-PCR, using primers amplifying Mtv SAg-specific spliced mRNAs. DC express Mtv SAgs at levels comparable to B cells, but display a differential expression pattern of the various Mtv loci compared with B cells. Furthermore, our results show that DC are able to induce the deletion of SAg-reactive thymocytes in an in vitro assay, indicating that Mtv SAgs are functionally expressed on the DC surface. Collectively, our data are consistent with the hypothesis that DC play a role in the induction of intrathymic tolerance to Mtv SAgs.

Recirculating CD4 memory T cells mount rapid secondary responses without major contributions from follicular CD4 effectors and B cells.

For weeks after primary immunization with thymus-dependent antigens the responding lymph nodes contain effector CD4 T cells in T zones and germinal centers as well as recirculating memory T cells. Conversely, remote nodes, not exposed to antigen, only receive recirculating memory cells. We assessed whether lymph nodes with follicular effector CD4 T cells in addition to recirculating memory CD4 T cells mount a more rapid secondary response than nodes that only contain recirculating memory cells. Also, the extent to which T cell frequency governs accelerated CD4 T cell recall responses was tested. For this, secondary antibody responses to a superantigen, where the frequency of responding T cells is not increased at the time of challenge, were compared with those to conventional protein antigens. With both types of antigens similar accelerated responses were elicited in the node draining the site of primary immunization and in the contralateral node, not previously exposed to antigen. Thus recirculating memory cells are fully capable of mounting accelerated secondary responses, without the assistance of CD4 effector T cells, and accelerated memory responses are not solely dependent on higher T cell frequencies. Accelerated memory CD4 T cell responses were also seen in B cell-deficient mice.

Genes encoding tumor necrosis factor alpha and granzyme A are expressed during development of autoimmune diabetes.

Progressive destruction of the insulin-producing beta cells in nonobese diabetic mice is observed after infiltration of the pancreas with lymphocytes [Makino, S., Kunimoto, K., Muraoka, Y., Mizushima, Y., Katagiri, K. &amp; Tochino, Y. (1980) Exp. Anim. (Tokyo) 29, 1-13]. We show that the genes for tumor necrosis factor alpha and granzyme A, a serine protease associated with cytoplasmic granules of cytotoxic cells, are expressed during the development of spontaneous diabetes mellitus in the nonobese diabetic mouse. Granzyme A-positive cells are found both in and surrounding the islets, implying induction prior to islet infiltration. Tumor necrosis factor alpha expression is exclusively observed in the intra-islet infiltrate, predominantly in lymphocytes adjacent to insulin-producing beta cells, the targets of the autoimmune destruction, implying that tumor necrosis factor alpha expression is induced locally--i.e., in the islet. A considerable portion of cells expressing tumor necrosis factor alpha appear to be CD4+ T cells. This T-cell subset was previously shown to be necessary for development of the disease. Thus, these findings may be important for understanding the pathogenesis of autoimmune diabetes mellitus and potentially also for that of other T-cell-mediated autoimmune diseases.

Differential reactivity of TCR Vbeta10 alleles to a mouse mammary tumor virus superantigen.

Mouse mammary tumor virus (MMTV) expresses a superantigen (SAg) which plays a critical role in the viral life cycle. We have recently described the new infectious MMTV (SIM) encoding a Vbeta4-specific SAg in mice with a TCR-Vbeta(b) haplotype. We have now compared the SAg activity of this virus in BALB/c mice harboring the TCR-Vbeta(a), TCR-Vbeta(b) or TCR-Vbeta(c) haplotypes which differ by a central deletion in the TCR-Vbeta(a) and TCR-Vbeta(c) locus and by mutations in some of the remaining Vbeta elements. Injection of MMTV (SIM) led to a strong stimulation of Vbeta4+ CD4+ T cells in TCR-Vbeta(b) mice, but only to a weak stimulation of these cells in TCR-Vbeta(a) or TCR-Vbeta(c) mice. A large increase in the percentage of Vbeta10+ cells was observed among CD4+ T cells in mice with the Vbeta(a) or Vbeta(c), but not the Vbeta(b) TCR-Vbeta haplotype. Vbeta10+ cells dominated the response when Vbeta10(a/c) and Vbeta4 subsets were present together. This is the first report of a viral SAg interacting with murine Vbeta10+ cells. Six amino acid differences between Vbeta10(a/c) and Vbeta10(b) could account for the gain of reactivity of Vbeta10(a/c) to the MMTV(SIM) SAg. No mutations were found in the hypervariable region 4 (HV4) of the TCR. Mutations at positions 22 and 28 introduce into Vbeta10(a/c) the same amino acids which are found at these positions in the MMTV(SIM)-reactive Vbeta4. Tridimensional models indicated that these amino acids lie close to HV4 and are likely to be important for the interaction of the SAg with the TCR.

Preferential binding of mouse mammary tumor virus to B lymphocytes.

Mouse mammary tumor virus (MMTV) has been shown to preferentially infect B lymphocytes in vivo. We have used recombinant envelope-coated fluospheres and highly purified MMTV particles to study the distribution of the viral receptors on fresh mouse lymphocytes. A preferential dose-dependent binding to B lymphocytes was observed which could be competed with neutralizing antibodies. In contrast, T-lymphocyte binding remained at background levels. These results strongly suggest a higher density of viral receptor molecules on B lymphocytes than on T lymphocytes and correlate with the preferential initial infection of B lymphocytes observed in vivo.