Increase of fat oxidation and weight loss in obese mice caused by chronic treatment with human growth hormone or a modified C-terminal fragment

OBJECTIVE: To observe the chronic effects of human growth hormone (hGH) and AOD9604 (a C-terminal fragment of hGH) on body weight, energy balance, and substrate oxidation rates in obese (ob/ob) and lean C57BL/6Jmice. In vitro assays were used to confirm whether the effects of AOD9604 are mediated through the hGH receptor, and if this peptide is capable of cell proliferation via the hGH receptor. METHOD: Obese and lean mice were treated with hGH, AOD or saline for 14 days using mini-osmotic pumps. Body weight, caloric intake, resting energy expenditure, fat oxidation, glucose oxidation, and plasma glucose, insulin and glycerol were measured before and after treatment. BaF-BO3 cells transfected with the hGH receptor were used to measure in Vitro I-125-hGH receptor binding and cell proliferation. RESULTS: Both hGH and AOD significantly reduced body weight gain in obese mice. This was associated with increased in vivo fat oxidation and increased plasma glycerol levels (an index of lipolysis). Unlike hGH, however, AOD9604 did not induce hyperglycaemia or reduce insulin secretion. AOD9604 does not compete for the hGH receptor and nor does it induce cell proliferation, unlike hGH. CONCLUSIONS: Both hGH and its C-terminal fragment reduce body weight gain, increase fat oxidation, and stimulate lipolysis in obese mice, yet AOD9604 does not interact with the hGH receptor. Thus, the concept of hGH behaving as a pro-hormone is further confirmed. This data shows that fragments of hGH can act in a manner novel to traditional hGH-stimulated pathways.

Persistence of recipient lymphocytes in NOD mice after irradiation and bone marrow transplantation

The non-obese diabetic (NOD) mouse is a unique and invaluable model of autoimmune disease, in particular type I diabetes. Bone marrow transplantation as a therapy for type I diabetes has been explored in NOD mice. NOD mice require higher doses of conditioning irradiation for successful allogeneic bone marrow transplantation, suggesting that NOD hematopoietic cells are radioresistant compared to those of other mouse strains. However, studies of hematopoietic reconstitution in NOD mice are hampered by the lack of mice bearing a suitable cell-surface marker that would allow transferred cells or their progeny to be distinguished. In order to monitor hematopoietic reconstitution in NOD mice we generated congenic NOD mice that carry the alternative allelic form of the pan-leukocyte alloantigen CD45. Following irradiation and congenic bone marrow transplantation, we found that the myeloid lineage was rapidly reconstituted by cells of donor origin but substantial numbers of recipient T lymphocytes persisted even after supra-lethal irradiation. This indicates that radiation resistance in the NOD hematopoietic compartment is a property primarily of mature T lymphocytes. (C) 2004 Elsevier Ltd. All rights reserved.

Fms-like tyrosine kinase 3 ligand administration overcomes a genetically determined dendritic cell deficiency in NOD mice and protects against diabetes development

A dendritic cell (DC) imbalance with a marked deficiency in CD4(-)8(+) DC occurs in non-obese diabetic (NOD) mice, a model of human autoimmune diabetes mellitus. Using a NOD congenic mouse strain, we find that this CD4(-)8(+) DC deficiency is associated with a gene segment on chromosome 4, which also encompasses non-MHC diabetes susceptibility loci. Treatment of NOD mice with fms-like tyrosine kinase 3 ligand (FL) enhances the level of CD4(-)8(+) DC, temporarily reversing the DC subtype imbalance. At the same time, fms-like tryosine kinase 3 ligand treatment blocks early stages of the diabetogenic process and with appropriately timed administration can completely prevent diabetes development. This points to a possible clinical use of FL to prevent autoimmune disease.

A peptide-binding motif for I-A(g7), the class II major histocompatibility complex (MHC) molecule of NOD and Biozzi AB/H mice

The class II major histocompatibility complex molecule I-A(g7) is strongly linked to the development of spontaneous insulin-dependent diabetes mellitus (IDDM) in non obese diabetic mice and to the induction of experimental allergic encephalomyelitis in Biozzi AB/H mice. Structurally, it resembles the HLA-DQ molecules associated with human IDDM, in having a non-Asp residue at position 57 in its beta chain. To identify the requirements for peptide binding to I-A(g7) and thereby potentially pathogenic T cell epitopes, we analyzed a known I-A(g7)-restricted T cell epitope, hen egg white lysozyme (HEL) amino acids 9-27. NH2- and COOH-terminal truncations demonstrated that the minimal epitope for activation of the T cell hybridoma 2D12.1 was M12-R21 and the minimum sequence for direct binding to purified I-A(g7) M12-Y20/K13-R21. Alanine (A) scanning revealed two primary anchors for binding at relative positions (p) 6 (L) and 9 (Y) in the HEL epitope. The critical role of both anchors was demonstrated by incorporating L and Y in poly(A) backbones at the same relative positions as in the HEL epitope. Well-tolerated, weakly tolerated, and nontolerated residues were identified by analyzing the binding of peptides containing multiple substitutions at individual positions. Optimally, p6 was a large, hydrophobic residue (L, I, V, M), whereas p9 was aromatic and hydrophobic (Y or F) or positively charged (K, R). Specific residues were not tolerated at these and some other positions. A motif for binding to I-A(g7) deduced from analysis of the model HEL epitope was present in 27/30 (90%) of peptides reported to be I-A(g7)-restricted T cell epitopes or eluted from I-A(g7). Scanning a set of overlapping peptides encompassing human proinsulin revealed the motif in 6/6 good binders (sensitivity = 100%) and 4/13 weak or non-binders (specificity = 70%). This motif should facilitate identification of autoantigenic epitopes relevant to the pathogenesis and immunotherapy of IDDM.

The origin of a Robertsonian chromosomal translocation in house mice inferred from linked microsatellite markers

The Western European house mouse, Mus domesticus, includes many distinct Robertsonian (Rb) chromosomal races. Two competing hypotheses may explain the distribution of Rb translocations found in different populations: they may have arisen independently multiple times or they may have arisen once and been spread through long distance dispersal. We investigated the origin of the Rb 5.15 translocation using 6 microsatellite loci linked to the centromeres of chromosomes 5 and 15 in 84 individuals from 3 Rb populations and 4 neighboring standard-karyotype populations. Microsatellite variation on the 5.15 metacentric chromosomes was significantly reduced relative to the amount of variation found on acrocentric chromosomes 5 and 15, suggesting that linked microsatellite loci can track specific mutational events. Phylogenetic analyses resulted in trees which are consistent with multiple origins of the 5.15 metacentric found in the three Rb populations. These results suggest that cytologically indistinguishable mutations have arisen independently in natural populations of house mice.

(-)-CGP 12177 causes cardiostimulation and binds to cardiac putative beta(4)-adrenoceptors in both wild-type and beta(3)-adrenoceptor knockout mice

Some blockers of beta(1)- and beta(2)-adrenoceptors cause cardiostimulant effects through an atypical beta-adrenoceptor (putative beta(4)-adrenoceptor) that resembles the beta(3)-adrenoceptor. It is likely but not proven that the putative beta(4)-adrenoceptor is genetically distinct from the beta(3)-adrenoceptor. We therefore investigated whether or not the cardiac atypical beta-adrenoceptor could mediate agonist effects in mice lacking a functional beta(3)-adrenoceptor gene (beta(3)KO). (-)-CGP 12177, a beta(1)- and beta(2)-adrenoceptor blocker that causes agonist effects through both beta(3)-adrenoceptors and cardiac putative beta(4)-adrenoceptors, caused cardiostimulant effects that were not different in atria from wild-type (WT) mice and beta(3)KO mice. The effects of (-)-CGP 12177 were resistant to blockade by (-)-propranolol (200 nM) but were blocked by (-)-bupranolol (1 mu M) with an equilibrium dissociation constant of 15 nM in WT and 17 nM in beta(3)KO. (-)-[H-3]CGP 12177 labeled a similar density of the putative beta(4)-adrenoceptor in ventricular membranes from the hearts of both WT (B-max = 52 fmol/mg protein) and beta(3)KO (B-max = 53 fmol/mg protein) mice. The affinity of (-)-[H-3]CGP 12177 for the cardiac putative beta(4)-adrenoceptor was not different between WT (K-d = 46 nM) and beta(3)KO (K-d = 40 nM). These results provide definitive evidence that the cardiac putative beta(4)-adrenoceptor is distinct from the beta(3)-adrenoceptor.

Mice expressing the E7 oncogene of HPV16 in epithelium show central tolerance, and evidence of peripheral anergising tolerance, to E7-encoded cytotoxic T-lymphocyte epitopes

In order to derive mice which expressed both the E7 open reading frame transgene of human papillomavirus type 16 in skin and MHC class 1 restriction elements for several E7-encoded cytotoxic T-lymphocyte (CTL) epitopes, K14.HPV16E7 mice which express E7 in basal keratinocytes were crossed to the F1 generation with A2.1 K-b transgenic mice which express the MHC binding cleft domains of human HLA A*0201, and murine H-2(b). F1 mice (denoted K14E7xA2.1) expressed E7 in the thymus at least as early as 2-5 days before birth. Immunisation of FVBxA2.1 control mice (transgenic for HLA A*0201 and H-2(b) but not for E7), with two HLA A*0201-restricted epitopes of E7 and one H-2(b)-restricted CTL epitope of E7, gave strong primary CTL responses recognising epitope-pulsed or constitutively E7-expressing syngeneic target cells. In contrast, in immunised K14E7xA2.1 mice, the CTL responses to the H-2(b) epitope and one of the HLA A*0201 CTL epitopes were strongly down-regulated, and to the other HLA A*0201 epitope, completely abolished, as demonstrated by percentage specific killing by bulk splenocyte cultures in cyrotoxicity assays, and by CTL precursor frequency analysis, In thymus-transplanted bone marrow radiation chimeras in which the immune system of K14E7xA2.1 mice was replaced by a FVBxA2.1 immune system, specific immunisation did not result in reemergence of strong E7-directed CTL responses. In agreement with these in vitro findings, specific immunisation failed to significantly alter the course of E7-associated tumour development in K14E7xA2.1 mice. These data are consistent with a model of central deletional CTL tolerance to E7-encoded epitopes recognised in the context of two distinct MHC class 1 restriction elements, and with the possibility of peripheral T-cell anergy maintained by expression of E7 in the skin. (C) 1998 Academic Press.

Dysregulated hematopoiesis and a progressive neurological disorder induced by expression of an activated form of the human common beta chain in transgenic mice

Previously we described activating mutations of h beta(c), the common signaling subunit of the receptors for the hematopoietic and inflammatory cytokines, GM-CSF, IL-3, and IL-5. The activated mutant, h beta(c)FI Delta, is able to confer growth factor-independent proliferation on the murine myeloid cell line FDC-P1, and on primary committed myeloid progenitors. We have used this activating mutation to study the effects of chronic cytokine receptor stimulation. Transgenic mice were produced carrying the h beta(c)FI Delta cDNA linked to the constitutive promoter derived from the phosphoglycerate kinase gene, PGK-1. Transgene expression was demonstrated in several tissues and functional activity of the mutant receptor was confirmed in hematopoietic tissues by the presence of granulocyte macrophage and macrophage colony-forming cells (CFU-GM and CFU-M) in the absence of added cytokines. All transgenic mice display a myeloproliferative disorder characterized by splenomegaly, erythrocytosis, and granulocytic and megakaryocytic hyperplasia. This disorder resembles the human disease polycythemia vera, suggesting that activating mutations in h beta(c) may play a role in the pathogenesis of this myeloproliferative disorder. In addition, these transgenic mice develop a sporadic, progressive neurological disease and display bilateral, symmetrical foci of necrosis in the white matter of brain stem associated with an accumulation of macrophages. Thus, chronic h beta(c) activation has the potential to contribute to pathological events in the central nervous system.

Mucosal immunisation with papillomavirus virus-like particles elicits systemic and mucosal immunity in mice

It has been shown previously that recombinant virus-like particles (VLPs) of papillomavirus can induce VLP-specific humoral and cellular immune responses following parenteral administration. To test whether mucosal administration of bovine papillomavirus type 1 (BPV1) VLPs could produce mucosal as well as systemic immune responses to VLPs, 50 mu g chimeric BPV1 VLPs containing an HPV16 E7 CTL epitope (BPVL1/E7 VLP) was administered intranasally to mice. After two immunisations, L1-specific serum IgG and IgA were observed. L1-specific IgG and IgA were also found in respiratory and vaginal secretions. Both serum and mucosal antibody inhibited papillomavirus VLP-induced agglutination of RBC, indicating that the antibody induced by mucosal immunisation may recognize conformational determinants associated with virus neutralisation. For comparison, VLPs were given intramuscularly, and systemic and mucosal immune responses were generally comparable following systemic or mucosal delivery. However, intranasal administration of VLP induced significantly higher local IgA response in lung, suggesting that mucosally delivered HPV VLP may be more effective for mediating local mucosal immune responses. Intranasal immunisation with HPV6b L1 VLP produced VLP-specific T proliferative responses in splenocytes, and immunisation with BPVL1 VLP containing an HPV16 E7 CTL epitope induced E7-specific CTL responses. We conclude that immunisation with papillomavirus VLPs via mucosal and intramuscular routes, without adjuvant, can elicit specific antibody at mucosal surfaces and also systemic VLP epitope specific T cell responses. These findings suggest that mucosally delivered VLPs may offer an alternative HPV VLP vaccine strategy for inducing protective humoral immunity to anogenital HPV infection, together with cell-mediated immune responses to eliminate any cells which become infected. (C) 1998 Academic Press.

Complement-mediated regulation of Trichomonas vaginalis infection in mice

Trichomonas vaginalis is a flagellated protozoan which causes trichomoniasis, a sexually transmitted disease of the human genitourinary tract, The importance of the alternative complement pathway in host defence against T. vaginalis was investigated in vitro. Kinetic studies utilising immunofixation following electrophoresis showed that both a strongly and weakly virulent strain of T, vaginalis activated murine serum C3. In vivo studies with congenic-resistant, C5-deficient, B10.D2/OSn- and C5-sufficient, B10.D2/nSn mice showed that the presence of C5 is a significant factor in the innate host resistance to primary infection with a strongly virulent, but not a weakly virulent trichomonad strain.

T cell-mediated and non-specific inflammatory mechanisms contribute to the skin pathology of HPV 16 E6E7 transgenic mice

One of three lines of mice transgenic for the E6 and E7 genes of human papillomavirus type 16 (HPV16) expressed from an alpha A-crystallin promoter also expresses the transgene ectopically in the skin. This line, designated alpha ACE6E7#19, develops skin disease from 3 months of age, characterised by epidermal hyperplasia and eventual skin loss. Administration of complete Freund's adjuvant (CFA) to alpha ACE6E7#19 mice, but not to nontransgenic littermate controls, induced local epidermal hyperplasia which was histologically similar to the spontaneously arising skin pathology. Local application of 2,4-dinitrochlorobenzene (DNCB) to DNCB-sensitised aACE6E7#19 mice, but not DNCB-sensitised controls, also induced hyperplasia. Treatment with cyclosporin A (CsA) or systemic depletion of CD4+ cells significantly reduced the incidence of skin disease. These data suggest that local inflammation, and cytokines produced by T helper cells, contribute to the induction of hyperplastic skin disease in alpha ACE6E7#19 mice. Spontaneous skin disease with similar histological appearance, frequency, age of onset and severity in alpha ACE6E7#19 mice was observed in scid-/- aACE6E7#19 mice, despite immune paresis. Antigen-specific immune responses and T-cell cytokines a re therefore not necessary for the induction of skin disease. We propose that epidermal hyperplasia associated with HPV16 E6 and E7 expression in skin is accelerated by local secretion of pro-inflammatory cytokines, whose production can be enhanced by activated CD4+ T cells.

Development of P2 olfactory glomeruli in P2-internal ribosome entry site-tau-lacZ transgenic mice

Primary olfactory neurons project their axons to the olfactory bulb, where they terminate in discrete loci called glomeruli. All neurons expressing the same odorant receptor appear to terminate in a few glomeruli in each olfactory bulb. In the P2-IRES-tau-LacZ line of transgenic mice, LacZ is expressed in the perikarya and axons of primary olfactory neurons that express the P2 odorant receptor. In the present study, we examined the developmental appearance of P2 neurons, the topographical targeting of P2 axons, as well as the formation of P2 glomeruli in the olfactory bulb. P2 axons were first detected in the olfactory nerve fiber layer at embryonic day 14.5 (E14.5), and by E15.5 these axons terminated in a broad locus in the presumptive glomerular layer. During the next 5 embryonic days, the elongated cluster of axons developed into discrete glomerulus-like structures. In many cases, glomeruli appeared as pairs, which were initially connected by a fascicle of P2 axons. This connection was lost by postnatal day 7.5, and double glomeruli at the same locus were observed in 85% of adult animals. During the early postnatal period, there was considerable mistargeting of P2 axons. In some cases P2 axons entered inappropriate glomeruli or continued to grow past the glomerular layer into the deeper layers of the olfactory bulb. These aberrant axons were not observed in adult animals. These results indicate that olfactory axons exhibit errors while converging onto a specific glomerulus and suggest that guidance cues may be diffusely distributed at target sites in the olfactory bulb.