Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice.

Adipocyte complement-related protein (30 kDa) (Acrp30), a secreted protein of unknown function, is exclusively expressed in differentiated adipocytes; its mRNA is decreased in obese humans and mice. Here we describe novel pharmacological properties of the protease-generated globular head domain of Acrp30 (gAcrp30). Acute treatment of mice with gAcrp30 significantly decreased the elevated levels of plasma free fatty acids caused either by administration of a high fat test meal or by i.v. injection of Intralipid. This effect of gAcrp30 was caused, at least in part, by an acute increase in fatty acid oxidation by muscle. As a result, daily administration of a very low dose of gAcrp30 to mice consuming a high-fat/sucrose diet caused profound and sustainable weight reduction without affecting food intake. Thus, gAcrp30 is a novel pharmacological compound that controls energy homeostasis and exerts its effect primarily at the peripheral level.

Optimizing preparation of normal dendritic cells and bcr-abl+ mature dendritic cells derived from immunomagnetically purified CD14+ cells.

The goal of this work was to optimize dendritic cell (DC) preparations obtained from patients suffering from chronic myeloid leukemia (CML) and compare them with DC prepared from normal CD14+ mononuclear cells (MNC). We studied normal DC and bcr-abl+ leukemic DC (CML-DC) yields, expression of membrane molecules, differentiation status, and ability to stimulate T cells. We isolated DC precursors from PBMC by CD14-specific immunoadsorption and cultured them for 7 days in GM-CSF and IL-4, followed by a 3-day incubation to fully differentiate the cells. We evaluated cultures of CML-DC using RPMI 1640 medium supplemented with FBS and X-VIVO 15 medium containing human AB serum. In contrast to cells matured in RPMI 1640, virtually all cells incubated in X-VIVO 15 expressed CD83, a marker of mature DC. CML-DC and normal DC were indistinguishable in expression of CD83, resulting in the highest percentage reported so far. The yields of normal DC and CML-DC from CD14+ cells were indistinguishable. The percentage of bcr-abl+ cells in PBMC varied among patients between 65% and 97% and the final CML-DC preparations were >98% bcr-abl+ the highest purity of bcr-abl+ cells to date. Normal DC and CML-DC were equally effective in stimulating proliferation of allogeneic and autologous T cells. These techniques provide highly enriched, mature, functional CML-DC.

Identification of a Drosophila homologue to vertebrate Crk by interaction with MBC.

The vertebrate adapter protein termed Crk was initially identified from the chicken CT10 retrovirus on the basis of its transforming activity (Mayer et al., 1988. Nature 332, 272-275). We have identified a Drosophila protein with homology to vertebrate Crk, termed dCRK, by interaction with the protein encoded by the Drosophila myoblast city (mbc) gene. The dCRK protein has extensive homology to the both the Crk-II form of vertebrate Crk and the Crk-related protein CRKL, and includes one SH2 domain followed by two SH3 domains. A single protein of approx. 37kDa is detected in extracts from embryos, and Northern analysis revealed a single transcript of 1.3kb. The dCrk mRNA is abundant throughout embryogenesis, declines during the larval stages, and reappears during pupation. In addition, maternally-provided transcripts have been detected. During embryogenesis, the spatial distribution of this transcript is relatively broad and appears to include all germ layers. Finally, dCrk is located on the fourth chromosome, approximately at cytological position 101F-102A.

Drosophila myoblast city encodes a conserved protein that is essential for myoblast fusion, dorsal closure, and cytoskeletal organization.

The Drosophila myoblast city (mbc) locus was previously identified on the basis of a defect in myoblast fusion (Rushton et al., 1995. Development [Camb.]. 121:1979-1988). We describe herein the isolation and characterization of the mbc gene. The mbc transcript and its encoded protein are expressed in a broad range of tissues, including somatic myoblasts, cardial cells, and visceral mesoderm. It is also expressed in the pole cells and in ectodermally derived tissues, including the epidermis. Consistent with this latter expression, mbc mutant embryos exhibit defects in dorsal closure and cytoskeletal organization in the migrating epidermis. Both the mesodermal and ectodermal defects are reminiscent of those induced by altered forms of Drac1 and suggest that mbc may function in the same pathway. MBC bears striking homology to human DOCK180, which interacts with the SH2-SH3 adapter protein Crk and may play a role in signal transduction from focal adhesions. Taken together, these results suggest the possibility that MBC is an intermediate in a signal transduction pathway from the rho/rac family of GTPases to events in the cytoskeleton and that this pathway may be used during myoblast fusion and dorsal closure.

Immunological interactions between Trichinella spiralis and Heligmosomoides polygyrus: cross reactivity between muscle larvae and antibodies raised to unrelated antigens.

It is documented that concurrent infections in mice with the 2 unrelated nematode parasites, Heligmosomoides polygyrus and Trichinella spiralis, can result in delayed rejection of the latter species. The basis of this immunological interference is not completely understood, but a possibility exists that antibodies induced by 1 species may interact with antigens produced by the other parasite. Therefore, it was investigated whether H. polygyrus infections may induce the production of antibodies which could cross react with T. spiralis muscle larval (m.l.) antigens. The results shown here indicate that this assumption is correct, and there is cross reactivity between antibodies produced against H. polygyrus, and T. spiralis m.l. antigens. Furthermore, antibodies which were not specific for either species were also able to bind to T. spiralis m.l. This is in agreement with recent evidence which has shown that antibodies that are not specific for H. polygyrus may still be absorbed by an H. polygyrus homogenate. It is considered that the binding of these antibodies may be involved in manipulation of the host immune response by T. spiralis.

Non-specific binding of IgG1 to Heligmosomoides polygyrus: adult worm homogenate superantigen is a target for immunoglobulin-induced inhibition.

Previous evidence had indicated that selected antigens contained in H. polygyrus adult worm homogenate (AWH) could bind non-specifically to mouse IgG1. To determine whether H. polygyrus superantigen was one of these binding molecules, an inhibition assay was carried out using monoclonal antibodies (MoAb) to block the in vitro superantigen response. The results indicated that non-specific IgG1 binding could inhibit the cellular response to the superantigen. This assumption was tested using affinity chromatography to extract those antigens which bound non-specifically to mouse IgG1. Both the protein fraction which bound to the column and the unfractionated AWH demonstrated superantigen activity, as described previously. In contrast, the unbound fraction contained no superantigen activity. None of the tested fractions exhibited non-specific mitogen activity. These results indicate that the superantigen produced by H. polygyrus binds to host IgG1 of any specificity and this binding can inhibit further host recognition of this molecule. Additionally, it was demonstrated that an apparently similar superantigen is also contained in L4 homogenate, and is strongly represented in the excretory/secretory (E/S) proteins produced by both adult and L4 parasitic stages. Therefore, it is probable that H. polygyrus superantigen influences the host during both the L4 and adult stages of the life-cycle.