Crystallization and preliminary X-ray crystallographic studies on a Fab fragment of the mouse anti-human Fas monoclonal antibody HFE7A.

The Fas-Fas ligand system is involved in apoptosis. The mouse anti-human Fas monoclonal antibody HFE7A (m-HFE7A) has a potential use in human therapy against autoimmune diseases such as rheumatoid arthritis. Information on the three-dimensional structure is essential for antibody humanization. Crystals of an antigen-binding fragment (Fab) of m-HFE7A were obtained by the hanging-drop vapour-diffusion method using sodium citrate as a precipitant and 2-methyl-2,4-pentanediol as an additive. Fast optimization to produce single crystals suitable for X-ray analysis was achieved by the streak-seeding technique. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 43.4, b = 74.0, c = 133.8 A. The crystals diffract at least to 2.5 A resolution.

Increased expression of the sterol regulatory element-binding protein-1 gene in insulin receptor substrate-2(-/-) mouse liver.

Insulin receptor substrate (IRS)-2(-/-) mice develop diabetes because of insulin resistance in the liver and failure to undergo beta-cell hyperplasia. Here we show by DNA chip microarray analysis that expression of the sterol regulatory element-binding protein (SREBP)-1 gene, a downstream target of insulin, was paradoxically increased in 16-week-old IRS-2(-/-) mouse liver, where insulin-mediated intracellular signaling events were substantially attenuated. The expression of SREBP-1 downstream genes, such as the spot 14, ATP citrate-lyase, and fatty acid synthase genes, was also increased. Increased liver triglyceride content in IRS-2(-/-) mice assures the physiological importance of SREBP-1 gene induction. IRS-2(-/-) mice showed leptin resistance; low dose leptin administration, enough to reduce food intake and body weight in wild-type mice, failed to do so in IRS-2(-/-) mice. Interestingly, high dose leptin administration reduced SREBP-1 expression in IRS-2(-/-) mouse liver. Thus, IRS-2 gene disruption results in leptin resistance, causing an SREBP-1 gene induction, obesity, fatty liver, and diabetes.

Cloning and expression of a novel murine anti-human Fas antibody.

Agonistic anti-human Fas antibodies that can induce apoptosis are thought to have therapeutic effects for various diseases resulting from an abnormality of the Fas/FasL system. However, some anti-Fas antibodies show toxicity, and it is difficult to investigate their therapeutic and toxicological effect using animals because of their species specificity. We previously obtained a murine anti-human Fas mAb, HFE7A. HFE7A reacted with both human and murine Fas, and mitigated lymphadenopathy without any sign of hepatotoxicity in MRLgld/gld mice. It is suggested that humanized HFE7A would be a therapeutic treatment for various diseases resulting from an abnormality of the Fas/FasL system. Here we isolated the cDNAs that code for the heavy and light chains of HFE7A and identified the corresponding nucleotide sequences. The recombinant HFE7A was indistinguishable in binding and apoptosis-inducing activity to that from a hybridoma cell line. These data provide essential information for the humanization and clinical application of the humanized HFE7A.

Molecular design, synthesis, and hypoglycemic activity of a series of thiazolidine-2,4-diones.

A series of imidazopyridine thiazolidine-2,4-diones were designed and synthesized from their corresponding pyridines. These compounds represent conformationally restricted analogues of the novel hypoglycemic compound rosiglitazone (5). The series was evaluated for its effect on insulin-induced 3T3-L1 adipocyte differentiation in vitro and its hypoglycemic activity in the genetically diabetic KK mouse in vivo. The structure-activity relationships are discussed. On the basis of the in vivo potency, 5-[4-(5-methoxy-3-methyl-3H-imidazo[4, 5-b]pyridin-2-ylmethoxy)benzyl]thiazolidine-2,4-dione (19a) was selected as the candidate for further studies in a clinical setting.

A novel murine anti-human Fas mAb which mitigates lymphadenopathy without hepatotoxicity.

Defects in Fas-mediated apoptosis are implicated in autoimmune diseases including rheumatoid arthritis (RA). Although induction of Fas-mediated apoptosis could have therapeutic effects on these diseases, it might cause deleterious effects in liver as Fas ligand or an agonistic anti-murine Fas antibody Jo2 causes severe hepatic injury in mice. We report here on the interesting characteristics of the newly obtained anti-Fas mAb, HFE7A, which cross-reacts with the Fas molecules of various species ranging from human to mouse and mitigates autoimmune symptoms without hepatotoxicity in mice. The administration of HFE7A to mice induced apoptosis in the thymocytes, although administration of HFE7A to mice or to marmosets did not induce any sign of hepatitis. The effect of HFE7A on liver is different from that of anti-murine Fas antibody Jo2, which causes acute and lethal hepatic injury to mice. Administration of HFE7A reduced lymphadenopathy and abnormal T cells in MRL-gld/gld mice. HFE7A induced apoptosis in synovial cells prepared from RA patients. Surprisingly, HFE7A protected mice from fulminant hepatitis induced by Jo2. Therefore, HFE7A is a potential therapeutic antibody not only for autoimmune diseases including RA but also for fulminant hepatitis.

Importance of the carboxy-terminus of human interleukin-11 in conserving its biological activity.

Biological activities of the carboxy-terminal (C-terminal) deletion mutants of human interleukin-11 (IL-11) were analyzed. Removal of only 1 amino-acid residue (leucine) from the C-terminus caused nearly an 80% loss of its biological activity. This shows the importance of C-terminus of human IL-11 in terms of conserving the biological activity.

Troglitazone prevents the inhibitory effects of inflammatory cytokines on insulin-induced adipocyte differentiation in 3T3-L1 cells.

Tumor necrosis factor (TNF) is implicated in wasting syndromes and insulin resistance in chronic infection and obese-linked diabetes. TNF (10 ng/ml) inhibited adipocyte differentiation of 3T3-L1 cells, and in these TNF treated cells little insulin-stimulated glucose uptake was observed. Treatment of 3T3-L1 cells with troglitazone (1-10 microM) partially prevented this inhibitory effect of TNF on adipogenesis, and enhanced expression of C/EBP alpha and GLUT4, even in the presence of TNF. Troglitazone also prevented the inhibitory effects of interleukin-1, interleukin-6, and leukemia inhibitory factor, but not of transforming growth factor beta on adipocyte differentiation of 3T3-L1 cells. These effects might contribute to the antidiabetic effect of troglitazone in obese diabetic animals.

Regulation of lipoprotein lipase synthesis in 3T3-L1 adipocytes by interleukin-11/adipogenesis inhibitory factor.

Interleukin-11/adipogenesis inhibitory factor (IL-11/AGIF) inhibits adipogenesis and suppresses lipoprotein lipase (EC3.1.1.34, LPL) activity in adipocytes (1,2). We investigated the mechanism of suppression of LPL activity in 3T3-L1 adipocytes by IL-11/AGIF. Incubation of adipocytes with 50 ng/ml of IL-11/AGIF led to a 75% decrease in LPL activity within 8 hours, whereas LPL mRNA level decreased by less than 30%. The LPL synthesis, as judged by the incorporation of 35S-label into immunoprecipitable LPL, decreased at almost the same rate over the same time period as enzyme activity. The degradation rate was not significantly affected by IL-11/AGIF. These data suggest that regulation of the synthesis of the enzyme protein is at least one of the main steps in the suppression of LPL by IL-11/AGIF in 3T3-L1 adipocytes.

[Function, molecular structure and gene expression of interleukin-11 (IL-11/AGIF)].

Interleukin-11 (IL-11) is a novel cytokine that was identified in a medium conditioned by the primate bone marrow-derived stromal cell line PU-34. It was originally identified as a growth factor for the IL-6-dependent plasmacytoma cell line T1165. Adipogenesis inhibitory factor (AGIF) was cloned from the human bone marrow-derived cell line KM-102. The AGIF cDNA sequence was revealed to be identical to that of the IL-11 cDNA. AGIF inhibits the process of adipogenesis of the bone marrow-derived preadipocyte cell line H-1/A. Other biological activities of IL-11/AGIF, megakaryocytopoiesis, stem-cell proliferation, hepatic acute phase responses and antigen-specific antibody responses are also summarized.

Adipogenesis inhibitory factor. A novel inhibitory regulator of adipose conversion in bone marrow.

Recombinant adipogenesis inhibitory factor (AGIF) was purified to homogeneity from the conditioned medium of COS-1 cells transfected with human AGIF cDNA. The amino-terminal sequence analysis of the mature AGIF revealed that AGIF was produced as a precursor consisting of 199 amino acids and processed into a mature form of 178 amino acids by a cleavage between Ala(-1) and Pro(+1). The purified AGIF inhibited the process of adipogenesis in mouse 3T3-L1 preadipocytes, indicating that AGIF directly acts on the cells. AGIF acted as an adipogenic antagonist not only on the extramedullary cell line 3T3-L1 but also on the mouse bone marrow stroma-derived cell line H-1/A, suggesting that this cytokine may regulate adipogenesis in bone marrow.

Molecular cloning of cDNA encoding adipogenesis inhibitory factor and identity with interleukin-11.

A cDNA encoding a novel adipogenesis inhibitory factor (AGIF) that inhibits the process of adipogenesis in mouse 3T3-L1 preadipocytes was cloned from a cDNA library of the human bone marrow-derived stromal cell line KM-102. The cloned cDNA contains an open reading frame coding for an AGIF precursor of 199 amino acids. Analysis of the sequence of this cDNA revealed identity of this factor with a recently reported novel cytokine, designated interleukin-11 (IL-11). AGIF/IL-11 may play an important role in stromal cell-associated hematopoeisis through its regulatory action on adipocyte differentiation in the bone marrow microenvironment.

Identification of a novel class of elastase isozyme, human pancreatic elastase III, by cDNA and genomic gene cloning.

By using porcine elastase I cDNA as a probe, we have isolated two different but closely related cDNAs encoding elastase-like proteases from a human pancreatic cDNA library. The amino acid sequences deduced from the cloned cDNA sequences showed 56-61% identity with those of both pancreatic elastases I and II, similar to the homology between elastases I and II. The active form of the elastase-like proteases appeared to be composed of 242 amino acids and preceded by a signal peptide and propeptide of 28 amino acids. Dot blot analysis of various tissue mRNAs demonstrated that the genes for the cloned cDNAs are expressed at a high level only in the pancreas. In addition, sequence analysis of the cloned genomic genes corresponding to one of the cDNAs showed that they are members of the elastase gene family. These results indicate that the two enzymes encoded by the cDNAs should be classified into a third class of elastase isozyme. Therefore, we designated them as human pancreatic elastases IIIA and IIIB. They strongly resembled cholesterol-binding pancreatic protease, suggesting that they may possess not only a digestive function but also function(s) related to cholesterol metabolism or transport in the intestine.