Neonatal death in mice lacking cardiotrophin-like cytokine is associated with multifocal neuronal hypoplasia.

Mice with null mutations of ciliary neurotrophic factor (Cntf) receptor alpha (Cntf-Ralpha), or cytokine-like factor 1 (Clf), one component of Cntf-II (a heterodimeric Cntf-Ralpha ligand), die as neonates from motor neuron loss affecting the facial nucleus and ventral horn of the lumbar spinal cord. Exposure to cardiotrophin-like cytokine (Clc), the other putative Cntf-II element, supports motor neuron survival in vitro and in ovo. Confirmation that Clc ablation induces an equivalent phenotype to Clf deletion would support a role for Clc in the functional Cntf-II complex. In this study, Clc knockout mice had decreased facial motility, did not suckle, died within 24 hours, and had 32% and 29% fewer motor neurons in the facial nucleus and lumbar ventral horn, respectively; thus, Clc is essential for motor neuron survival during development. The concordance of the Clc knockout phenotype with those of mice lacking Cntf-Ralpha or Clf bolsters the hypothesis that Clc participates in Cntf-II.

Anti-CD45RB monoclonal antibody prolongs renal allograft survival in cynomolgus monkeys.

Previously, an anti-CD45RB monoclonal antibody (mAb) has been shown to induce murine allograft tolerance. The present study was performed to assess the ability of an anti-human CD45RB mAb to prevent rejection in a monkey MHC-mismatched kidney transplant model. The recipients were allocated into the following treatment groups: (1) isotype control IgG; (2) mouse anti-human CD45RB IgG1 (6G3); (3) human-mouse chimeric anti-CD45RB-IgG1 (C6G3-IgG1); (4) human-mouse chimeric anti-CD45RB-IgG2 (C6G3-IgG2); (5) tacrolimus at a subtherapeutic dose and (6) tacrolimus and C6G3-IgG1 in combination. Monotherapy with anti-CD45RB mAb significantly prolonged renal allograft survival to a median survival of 21 days. Adding a subtherapeutic dose of tacrolimus improved the efficacy of the anti-CD45RB mAb, achieving a median survival of 85 days, whereas a subtherapeutic dose of tacrolimus alone only moderately prolonged survival to 27 days. Treatment with anti-CD45RB mAb resulted in an alteration of the CD45RB(hi) : CD45RB(lo) cell ratio in the peripheral blood. We have, for the first time, demonstrated that an anti-human CD45RB mAb (6G3) can prolong graft survival. Induction with an anti-CD45RB mAb improves the efficacy of tacrolimus in the prevention of rejection. These encouraging results indicate that an anti-CD45RB mAb may be valuable in future clinical transplantation.

CD4+CD45RBHi T cell transfer induced colitis in mice is accompanied by osteopenia which is treatable with recombinant human osteoprotegerin.

BACKGROUND AND AIMS: Transfer of CD4+CD45RBHi T cells into semi syngeneic immunodeficient mice represents a model of inflammatory bowel disease (IBD). As patients with IBD often suffer from osteopenia, we studied if this T cell transfer in mice results in osteopenia in addition to colitis, and if treatment with osteoprotegerin (OPG) has effects on the bone mineral density of T cell transferred mice. We also investigated whether osteopenia was due to malabsorption as a result of a dysregulated digestive tract or as a consequence of the inflammatory process. METHODS: CD4+CD45RBHi or CD4+CD45RBLo T cells (4 x 10(5)) were sorted from CB6F1 and transferred into C.B.17 scid/scid mice. Recipient mice were treated with human IgG1 Fc (control) or Fc-OPG three times per week in a prophylactic regimen as well as a therapeutic regimen (after 10% body weight loss) and were evaluated for osteopenia and colitis. RESULTS: Mice that received CD4+CD45RBHi T cells developed osteopenia (as indicated by decreased bone density accompanied by decreased osteoblasts and increased osteoclasts) and colitis (as indicated by histological changes in the large intestine). Mice that received CD4+CD45RBLo T cells developed neither osteopenia nor colitis. All animals consumed, on average, the same amount of food and water over the course of the study. Prophylactic treatment with Fc-OPG increased bone density in mice that received either CD4+CD45RBHi or CD4+CD45RBLo T cells but had no effects on the gastrointestinal tract. Fc-OPG treatment of osteopenic mice with established IBD caused the normalisation of bone density. Osteopenia in CD4+CD45RBHi T cell recipients was accompanied by hypoparathyroidism that was partially normalised by treatment with Fc-OPG. CD4+CD45RBHi T cell recipients also had a bone marrow inflammatory cell infiltrate expressing tumour necrosis factor alpha which was unaffected by treatment with Fc-OPG. CONCLUSIONS: CD4+CD45RBHi T cell transfer results in osteopenia in addition to colitis. Evidence suggests that this osteopenia was induced by inflammatory cell infiltration and not by malabsorption of calcium. Recombinant human osteoprotegerin effectively treated the osteopenia. OPG may be a useful therapeutic option for treating osteopenia in patients with IBD.

Role of IL-18 in acute lung inflammation.

We have examined the role of IL-18 after acute lung inflammation in rats caused by intrapulmonary deposition of IgG immune complexes. Constitutive IL-18 mRNA and protein expression (precursor form, 26 kDa) were found in normal rat lung, whereas in inflamed lungs, IL-18 mRNA was up-regulated; in bronchoalveolar (BAL) fluids, the 26-kDa protein form of IL-18 was increased at 2-4 h in inflamed lungs and remained elevated at 24 h, and the "mature" protein form of IL-18 (18 kDa) appeared in BAL fluids 1-8 h after onset of inflammation. ELISA studies confirmed induction of IL-18 in inflamed lungs (in lung homogenates and in BAL fluids). Prominent immunostaining for IL-18 was found in alveolar macrophages from inflamed lungs. When rat lung macrophages, fibroblasts, type II cells, and endothelial cells were cultured in vitro with LPS, only the first two produced IL-18. Intratracheal administration of rat recombinant IL-18 in the lung model caused significant increases in lung vascular permeability and in BAL content of neutrophils and in BAL content of TNF-alpha, IL-1beta, and cytokine-induced neutrophil chemoattractant, whereas intratracheal instillation of anti-IL-18 greatly reduced these changes and prevented increases in BAL content of IFN-gamma. Intratracheal administration of the natural antagonist of IL-18, IL-18 binding protein, resulted in suppressed lung vascular permeability and decreased BAL content of neutrophils, cytokines, and chemokines. These findings suggest that endogenous IL-18 functions as a proinflammatory cytokine in this model of acute lung inflammation, serving as an autocrine activator to bring about expression of other inflammatory mediators.

A novel Notch ligand, Dll4, induces T-cell leukemia/lymphoma when overexpressed in mice by retroviral-mediated gene transfer.

Notch receptors mediate cell-fate decisions through interaction with specific ligands during development. The biological role of a novel Notch ligand, Dll4, in mice was explored by reconstituting lethally irradiated mice with bone marrow (BM) cells transduced with Dll4 retroviral vector. White blood cell and lymphocyte counts in Dll4-overexpressing mice were reduced at the early stage of reconstitution but increased significantly at approximately 10 weeks after BM transplantation. BM, spleen, lymph nodes, and peripheral blood of Dll4-overexpressing mice contained predominantly CD4(+)CD8(+) T cells and virtually lacked B cells. The Dll4-overexpressing mice eventually developed a lethal phenotype that was characterized by the progression of a T-cell lymphoproliferative disease (restricted to BM and lymphoid tissues) to transplantable monoclonal T-cell leukemia/lymphoma scattered to multiple organs. Results suggest that the interaction of Dll4 with Notch1 may provide key signals for T-cell development.

IL-18-binding protein protects against lipopolysaccharide- induced lethality and prevents the development of Fas/Fas ligand-mediated models of liver disease in mice.

IL-18-binding protein (IL-18BP) is a natural IL-18 inhibitor. Human IL-18BP isoform a was produced as fusion construct with human IgG1 Fc and assessed for binding and neutralizing IL-18. IL-18BP-Fc binds human, mouse, and rat IL-18 with high affinity (K(D) 0.3-5 nM) in a BIAcore-based assay. In vitro, IL-18BP-Fc blocks IL-18 (100 ng/ml)-induced IFN-gamma production by KG1 cells (EC(50) = 0.3 microg/ml). In mice challenged with an LD(90) of LPS (15 mg/kg), IL-18BP-Fc (5 mg/kg) administered 10 min before LPS blocks IFN-gamma production and protects against lethality. IL-18BP-Fc administered 10 min before LPS blocks IFN-gamma production induced by LPS (5 mg/kg) with ED(50) of 0.005 mg/kg. Furthermore, IL-18BP-Fc (5 mg/kg) abrogates LPS (5 mg/kg)-induced IFN-gamma production even when administered 6 days before LPS but shows no effect when administered 9 or 12 days before LPS. Given 10 min before LPS challenge to mice primed 12 days in advance with heat-killed Propionibacterium acnes, IL-18BP-Fc prevents LPS-induced liver damage and IFN-gamma and Fas ligand expression. Given at the moment of priming with P. acnes, IL-18BP-Fc decreases P. acnes-induced granuloma formation, macrophage-inflammatory protein-1alpha and macrophage-inflammatory protein-2 production and prevents sensitization to LPS. IL-18BP-Fc also prevents Con A-induced liver damage and IFN-gamma and Fas ligand expression as well as liver damage induced by Pseudomonas aeruginosa exotoxin A or by anti-Fas agonistic Ab. In conclusion, IL-18BP can be engineered and produced in recombinant form to generate an IL-18 inhibitor, IL-18BP-Fc, endowed with remarkable in vitro and in vivo properties of binding and neutralizing IL-18.

WSX-1 is required for the initiation of Th1 responses and resistance to L. major infection.

WSX-1 is a class I cytokine receptor with homology to the IL-12 receptors. The physiological role of WSX-1, which is expressed mainly in T cells, was investigated in gene-targeted WSX-1-deficient mice. IFN-gamma production was reduced in isolated WSX-1(-/-) T cells subjected to primary stimulation in vitro to induce Th1 differentiation but was normal in fully differentiated and activated WSX-1(-/-) Th1 cells that had received secondary stimulation. WSX-1(-/-) mice were remarkably susceptible to Leishmania major infection, showing impaired IFN-gamma production early in the infection. However, IFN-gamma production during the later phases of the infection was not impaired in the knockout. WSX-1(-/-) mice also showed poorly differentiated granulomas with dispersed accumulations of mononuclear cells when infected with bacillus Calmette-Guerin (BCG). Thus, WSX-1 is essential for the initial mounting of Th1 responses but dispensable for their maintenance.

Stimulatory effects of B7-related protein-1 on cellular and humoral immune responses in mice.

Inducible costimulator (ICOS) and B7-related protein-1 (B7RP-1) constitute a receptor-ligand pair involved in T cell costimulation. In this study, the stimulatory effects of B7RP-1 on cellular and humoral immune responses were investigated giving mice a construct with the extracellular domain of murine B7RP-1 fused with human IgG1 Fc (B7RP-1-Fc). B7RP-1-Fc stimulated contact hypersensitivity (CH) given near either the time of sensitization or challenge with oxazolone. When given near challenge time, B7RP-1-Fc stimulated CH more than a construct containing the extracellular domain of murine B7.2 and Fc (B7.2-Fc). B7RP-1-Fc increased the number of cells in lymph nodes draining the skin sensitized with oxazolone, especially activated T cells. B7RP-1-Fc also increased the ability of the cells in these lymph nodes to induce CH when transfused into naive mice. B7RP-1-Fc stimulated the production of anti-keyhole limpet hemocyanin (KLH) Ab, increasing anti-KLH IgG, IgG2a, and IgE, whereas B7.2-Fc did not affect this production. B7RP-1-Fc also increased the number of cells in lymph nodes draining the skin immunized with KLH and their production of IFN-gamma, IL-4, and IL-10 in response to KLH. Finally, B7RP-1-Fc increased the presence of eosinophils in the bronchoalveolar lavage and lungs of mice sensitized and challenged with OVA so to mount an asthmatic reaction. B7RP-1-Fc stimulates both cellular and humoral immune responses in vivo by increasing number and function of T and B cells reacting to Ag exposure.

Role of interferon regulatory factor-1 in the regulation of IL-18 production and activity.

Interferon regulatory factor-1 (IRF-1) is a transcription factor regulating the expression of several cytokines. Using IRF-1 knockout (KO) mice, the role of IRF-1 in the production and activity of IL-18 was evaluated. Administration of IL-12 or concanavalin A significantly increased levels of circulating IL-18 in wild-type (WT), but not in IRF-1 KO mice. However, despite these differences in circulating IL-18 levels, no or only minor differences in constitutive or inducible IL-18 mRNA and tissue-associated protein levels were observed between WT and IRF-1 KO mice. On the other hand, we observed that constitutive and inducible levels of the IL-18-processing enzyme caspase-1 were markedly reduced in the spleen and the liver of IRF-1 KO compared to WT mice. In addition, both constitutive and inducible liver mRNA levels for the IL-18 binding protein (IL-18BP), a specific IL-18 antagonist, were significantly lower in IRF-1 KO than in WT mice. Compared to IL-12, IL-18 only weakly induced IRF-1 mRNA in cultured splenocytes. However, IL-18-induced IFN-gamma production was strongly reduced in splenocytes from IRF-1 KO compared to WT cells. In conclusion, IRF-1 regulates IL-18 production and activity mostly by modulating expression of caspase-1 and IL-18BP. In addition, IRF-1 participates in the induction of IFN-gamma by IL-18.

TACI is a TRAF-interacting receptor for TALL-1, a tumor necrosis factor family member involved in B cell regulation.

We and others recently reported tumor necrosis factor (TNF) and apoptosis ligand-related leukocyte-expressed ligand 1 (TALL-1) as a novel member of the TNF ligand family that is functionally involved in B cell proliferation. Transgenic mice overexpressing TALL-1 have severe B cell hyperplasia and lupus-like autoimmune disease. Here, we describe expression cloning of a cell surface receptor for TALL-1 from a human Burkitt's lymphoma RAJI cell library. The cloned receptor is identical to the previously reported TNF receptor (TNFR) homologue transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI). Murine TACI was subsequently isolated from the mouse B lymphoma A20 cells. Human and murine TACI share 54% identity overall. Human TACI exhibits high binding affinities to both human and murine TALL-1. Soluble TACI extracellular domain protein specifically blocks TALL-1-mediated B cell proliferation without affecting CD40- or lipopolysaccharide-mediated B cell proliferation in vitro. In addition, when injected into mice, soluble TACI inhibits antibody production to both T cell-dependent and -independent antigens. By yeast two-hybrid screening of a B cell library with TACI intracellular domain, we identified that, like many other TNFR family members, TACI intracellular domain interacts with TNFR-associated factor (TRAF)2, 5, and 6. Correspondingly, TACI activation in a B cell line results in nuclear factor kappaB and c-Jun NH(2)-terminal kinase activation. The identification and characterization of the receptor for TALL-1 provides useful information for the development of a treatment for B cell-mediated autoimmune diseases such as systemic lupus erythematosus.

Delayed mortality and attenuated thrombocytopenia associated with severe malaria in urokinase- and urokinase receptor-deficient mice.

We explored the role of urokinase and tissue-type plasminogen activators (uPA and tPA), as well as the uPA receptor (uPAR; CD87) in mouse severe malaria (SM), using genetically deficient (-/-) mice. The mortality resulting from Plasmodium berghei ANKA infection was delayed in uPA(-/-) and uPAR(-/-) mice but was similar to that of the wild type (+/+) in tPA(-/-) mice. Parasitemia levels were similar in uPA(-/-), uPAR(-/-), and +/+ mice. Production of tumor necrosis factor, as judged from the plasma level and the mRNA levels in brain and lung, was markedly increased by infection in both +/+ and uPAR(-/-) mice. Breakdown of the blood-brain barrier, as evidenced by the leakage of Evans Blue, was similar in +/+ and uPAR(-/-) mice. SM was associated with a profound thrombocytopenia, which was attenuated in uPA(-/-) and uPAR(-/-) mice. Administration of aprotinin, a plasmin antagonist, also delayed mortality and attenuated thrombocytopenia. Platelet trapping in cerebral venules or alveolar capillaries was evident in +/+ mice but absent in uPAR(-/-) mice. In contrast, macrophage sequestration in cerebral venules or alveolar capillaries was evident in both +/+ and uPAR(-/-) mice. Polymorphonuclear leukocyte sequestration in alveolar capillaries was similar in +/+ and uPAR(-/-) mice. These results demonstrate that the uPAR deficiency attenuates the severity of SM, probably by its important role in platelet kinetics and trapping. These results therefore suggest that platelet sequestration contributes to the pathogenesis of SM.

APRIL and TALL-I and receptors BCMA and TACI: system for regulating humoral immunity.

We report that the tumor neurosis factor homolog APRIL (a proliferation-inducing ligand) stimulates in vitro proliferation of primary B and T cells and increases spleen weight due to accumulation of B cells in vivo. APRIL functions via binding to BCMA (B cell maturation antigen) and TACI (transmembrane activator and CAML-interactor) and competes with TALL-I (also called BLyS or BAFF) for receptor binding. Soluble BCMA and TACI specifically prevent binding of APRIL and block APRIL-stimulated proliferation of primary B cells. BCMA-Fc also inhibits production of antibodies against keyhole limpet hemocyanin and Pneumovax in mice, indicating that APRIL and/or TALL-I signaling via BCMA and/or TACI are required for generation of humoral immunity. Thus, APRIL-TALL-I and BCMA-TACI form a two ligands-two receptors pathway involved in stimulation of B and T cell function.

Protection against the mortality associated with disease models mediated by TNF and IFN-gamma in mice lacking IFN regulatory factor-1.

Mortality and cytokine production associated with disease models mediated by TNF- and IFN-gamma were studied in mice lacking IFN regulatory factor-1 (IRF-1). IRF-1 knockout (KO) mice showed no mortality after the injection of a dose of LPS lethal in intact control mice (LD95). KO mice showed lower circulating levels of TNF and IFN-gamma than controls. KO mice also showed lower TNF and IFN-gamma mRNA in the spleen or liver than controls. KO mice had smaller spleens than controls, which contained similar percentage but lower absolute count of macrophages and lower percentage and absolute count of NK cells. IRF-1 KO mice survived longer than controls after the coinjection of LPS and galactosamine. IRF-1 KO mice also showed less mortality than controls after the injection of Con A and in a model of cerebral malaria. After the injection of a lethal dose of TNF (LD88), mortality was similar between KO and intact mice. Mortality was also similar after the coinjection of two nonlethal doses of TNF and IFN-gamma, a lethal combination (LD100). This study shows that the lack of IRF-1 protects against the mortality associated with disease models mediated by TNF and IFN-gamma but has no effect on the mortality directly induced by TNF and IFN-gamma. The lack of IRF-1 appears to result in impaired production of TNF and IFN-gamma, reflecting a down-regulation of gene expression in the liver and spleen as well as a reduction in the number of splenic cells.

Novel neurotrophin-1/B cell-stimulating factor-3: a cytokine of the IL-6 family.

We have identified a cytokine of the IL-6 family and named it novel neurotrophin-1/B cell-stimulating factor-3 (NNT-1/BSF-3). NNT-1/BSF-3 cDNA was cloned from activated Jurkat human T cell lymphoma cells. Its sequence predicts a 225-aa protein with a 27-aa signal peptide, a molecular mass of 22 kDa in mature form, and the highest homology to cardiotrophin-1 and ciliary neurotrophic factor. The gene for NNT-1/BSF-3 is on chromosome 11q13. A murine equivalent to NNT-1/BSF-3 also was identified, which shows 96% homology to human NNT-1/BSF-3. NNT-1/BSF-3 mRNA is found mainly in lymph nodes and spleen. NNT-1/BSF-3 induces tyrosine phosphorylation of glycoprotein 130 (gp130), leukemia inhibitory factor receptor beta, and signal transducer and activator of transcription 3 in the SK-N-MC human neuroblastoma cells. NNT-1/BSF-3 shows activities typical of IL-6 family members. In vitro, it supports the survival of chicken embryo motor and sympathetic neurons. In mice, it induces serum amyloid A, potentiates the induction by IL-1 of corticosterone and IL-6, and causes body weight loss and B cell hyperplasia with serum IgG and IgM increase. NNT-1/BSF-3 is a gp130 activator with B-cell stimulating capability.

Corynebacterium parvum- and Mycobacterium bovis Bacillus Calmette and Guerin-induced granuloma formation in mice lacking CD4 and CD8.

Granuloma formation is a T-cell-dependent inflammatory response that is important in the host defense against intracellular bacteria. The role of CD4 and CD8 molecules in the development of Corynebacterium parvum- and Mycobacterium bovis Bacillus Calmette and Guerin (BCG)-induced granulomas was examined in CD4/CD8 knockout (KO) mice. CD4/CD8 KO mice developed a greater granulomatous response to heat-killed C. parvum and heat-killed BCG than did control mice. Thus, granuloma formation is not dependent upon the presence of CD4 and CD8. On the other hand, CD4/CD8 KO mice challenged with live BCG showed initially fewer and smaller granulomas but later more and larger granulomas than control mice. CD4/CD8 KO mice had a greater BCG load than control mice. The absence of CD4 and CD8 therefore impaired the host defense against infection with BCG. alphabeta T-cells were present in the granulomas of both CD4/CD8 KO and control mice in similar numbers. Also the production of IFN-gamma mRNA was similar in the two groups. In conclusion, CD4 and CD8 are not essential to the granulomatous response against C. parvum and BCG, but contribute to the host defense against live BCG infection.

Genetic evidence for functional redundancy of Platelet/Endothelial cell adhesion molecule-1 (PECAM-1): CD31-deficient mice reveal PECAM-1-dependent and PECAM-1-independent functions.

Platelet/endothelial cell adhesion molecule-1 (PECAM-1; CD31), a member of the Ig superfamily, is expressed strongly at endothelial cell-cell junctions, on platelets, and on most leukocytes. CD31 has been postulated to play a role in vasculogenesis and angiogenesis, and has been implicated as a key mediator of the transendothelial migration of leukocytes. To further define the physiologic role of CD31, we used targeted gene disruption of the CD31 gene in embryonic stem cells to generate CD31-deficient mice. CD31-deficient mice (CD31KO) are viable and born at the expected Mendelian frequency, remain healthy, and exhibit no obvious vascular developmental defects. In response to inflammatory challenge, polymorphonuclear leukocytes of CD31KO mice are arrested between the vascular endothelium and the basement membrane of inflammatory site mesenteric microvessels, confirming a role for CD31 in the migration of neutrophils through the subendothelial extracellular matrix. Normal numbers of leukocytes are recovered from inflammatory sites in CD31KO mice, however, suggesting that the defect in leukocyte migration across basal lamina observed in the absence of CD31 may be compensated for by the use of other adhesion molecules, or possibly an increased rate of migration. Homing of T lymphocytes in vivo is normal, and CD31KO mice are able to mount a cutaneous hypersensitivity response normally. In addition, CD31-mediated homophilic adhesion does not appear to play a role in platelet aggregation in vitro. This study provides genetic evidence that CD31 is involved in transbasement membrane migration, but does not play an obligatory role in either vascular development or leukocyte migration.

The prolonged hematologic effects of a single injection of PEG-rHuMGDF in normal and thrombocytopenic mice.

A single injection of > or =10 microg/kg PEG-rHuMGDF in mice causes a dose-dependent increase in circulating platelets beginning on day 3 and peaking on days 5-6. The mean platelet volume and platelet distribution width at doses > or =100 microg/kg initially increase in a dose-dependent fashion and later decrease. However, the mean platelet volume does not change when platelets are incubated with PEG-rHuMGDF in vitro. The number of marrow megakaryocytes increases in a dose-dependent fashion as early as day 1 and peaks on day 3. Marrow megakaryocyte colony-forming units (CFU-Meg) do not increase on days 1-3 at a dose of 100 microg/kg (a dose that increases platelet numbers two- to threefold and may be clinically relevant), but the relative frequency of high ploidy megakaryocytes and the proportion of large marrow megakaryocytes (29-50 microm in diameter) increases. After a dose of 1,000 microg/kg the percentage of megakaryocytes in mitosis peaks at 24-48 hours and the percentage of megakaryocytes incorporating BrdU is maximal at 48 hours, the relatively delayed peak of BrdU incorporation most likely representing endomitosis. The relative frequency of type II and III megakaryocytes peaks on days 3 and 4, respectively. Pharmacokinetic analysis of PEG-rHuMGDF shows peak serum concentrations at 2-4 hours and a terminal half-life of 11.4+/-2.5 hours. A single injection of PEG-rHuMGDF ameliorates carboplatin-induced megakaryocytopenia and thrombocytopenia in a dose-response dependent fashion. In conclusion, a single injection of PEG-rHuMGDF increases megakaryocyte and platelet production in normal and myelo-suppressed mice.

T-cell co-stimulation through B7RP-1 and ICOS.

T-cell activation requires co-stimulation through receptors such as CD28 and antigen-specific signalling through the T-cell antigen receptor. Here we describe a new murine costimulatory receptor-ligand pair. The receptor, which is related to CD28 and is the homologue of the human protein ICOS, is expressed on activated T cells and resting memory T cells. The ligand, which has homology to B7 molecules and is called B7-related protein-1 (B7RP-1), is expressed on B cells and macrophages. ICOS and B7RP-I do not interact with proteins in the CD28-B7 pathway, and B7RP-1 co-stimulates T cells in vitro independently of CD28. Transgenic mice expressing a B7RP-1-Fc fusion protein show lymphoid hyperplasia in the spleen, lymph nodes and Peyer's patches. Presensitized mice treated with B7RP-1-Fc during antigen challenge show enhanced hypersensitivity. Therefore, B7RP-1 exhibits co-stimulatory activities in vitro and in vivo. ICOS and B7RP-1 define a new and distinct receptor-ligand pair that is structurally related to CD28-B7 and is involved in the adaptive immune response.

PEG-rHuMGDF promotes multilineage hematopoietic recovery in myelosuppressed mice.

PEG-rHuMGDF administered to normal mice is a lineage-specific growth factor for megakaryocytes and platelets as judged by morphologic examination of hematologic cells in marrow and peripheral blood smears. The purpose of this study was to document that PEG-rHuMGDF in myelosuppressed mice promotes multilineage hematopoietic recovery. High-dose 5-fluorouracil (5-FU) in mice results in profound myelosuppression and 0-30% survival. Mice receiving a single dose of PEG-rHuMGDF (1000 microg/kg) 1 day after 5-FU (225 mg/kg) demonstrate an increased survival (76% vs 27% in control mice at 14 days). Compared to surviving controls, PEG-rHuMGDF-treated mice not only show the expected higher platelet counts, but also increased marrow colony-forming unit granulocyte-macrophage, increased multilineage marrow cellularity, and increased neutrophil, monocyte, and lymphocyte counts in peripheral blood. PEG-rHuMGDF- and vehicle-treated mice both develop hepatic abscesses after 5-FU treatment, but the abscesses in the PEG-rHuMGDF-treated mice contain more neutrophils, suggesting that myeloid reconstitution contributes to their survival. Furthermore, survival in 5-FU-treated mice is significantly improved by granulocyte colony-stimulating factor and antibiotics, suggesting that infection rather than thrombocytopenia is the predominant cause of death. PEG-rHuMGDF after 5-FU promotes survival accompanied by accelerated lymphohematopoietic repopulation, suggesting that PEG-rHuMGDF, a lineage-specific thrombopoietic factor in normal mice, promotes multilineage hematopoietic recovery in myelosuppressed mice.

Leptin causes body weight loss in the absence of in vivo activities typical of cytokines of the IL-6 family.

To investigate if leptin shares in vivo activities with interleukin (IL)-6 family cytokines, it was tested in normal mice for the ability, after a single injection, to induce the acute-phase protein serum amyloid A, to potentiate the induction by IL-1 of serum corticosterone and IL-6, and to inhibit the induction by lipopolysaccharide of serum tumor necrosis factor and, after seven daily injections, to cause body weight loss and to change peripheral blood cell counts. At a 0.5 mg/kg dose, leptin caused body weight loss but did not show any of the other activities above. At a dose of 5 mg/kg, which also caused body weight loss, leptin potentiated the induction by IL-1 of serum corticosterone and IL-6 but did not show any other activity. In addition to causing body weight loss, leptin shows only some of the in vivo activities typical of IL-6 family cytokines and only if used at a dose that exceeds the one sufficient to affect body weight. In vivo, leptin seems to chiefly control body weight and not inflammatory or hematopoietic processes.