IL-12 contributes to allergen-induced airway inflammation in experimental asthma

Lack of sufficient IL-12 production has been suggested to be one of the basic underlying mechanisms in atopy, but a potential role of IL-12 in established allergic airway disease remains unclear. We took advantage of a mouse model of experimental asthma to study the role of IL-12 during the development of bronchial inflammation. Administration of anti-IL-12p35 or anti-IL-12p40 mAb to previously OVA-sensitized BALB/c mice concomitantly with exposure to nebulized OVA, abolished both the development of bronchial hyperresponsiveness to metacholine as well as the eosinophilia in bronchoalveolar lavage fluid and peripheral blood. Anti-IL-12 treatment reduced CD4(+) T cell numbers and IL-4, IL-5, and IL-13 levels in the bronchoalveolar lavage fluid and the mRNA expression of IL-10, eotaxin, RANTES, MCP-1, and VCAM-1 in the lung. Anti-IL-12p35 treatment failed to show these effects in IFN-gamma knockout mice pointing to the essential role of IFN-gamma in IL-12-induced effects. Neutralization of IL-12 during the sensitization process aggravated the subsequent development of allergic airway inflammation. These data together with recent information on the role of dendritic cells in both the sensitization and effector phase of allergic respiratory diseases demonstrate a dual role of IL-12. Whereas IL-12 counteracts Th2 sensitization, it contributes to full-blown allergic airway disease upon airway allergen exposure in the postsensitization phase, with enhanced recruitment of CD4(+) T cells and eosinophils and with up-regulation of Th2 cytokines, chemokines, and VCAM-1. IFN-gamma-producing cells or cells dependent on IFN-gamma activity, play a major role in this unexpected proinflammatory effect of IL-12 in allergic airway disease.     

Andes Virus Recognition of Human and Syrian Hamster β3 Integrins Is Determined by an L33P Substitution in the PSI Domain

Andes virus (ANDV) causes a fatal hantavirus pulmonary syndrome (HPS) in humans and Syrian hamsters. Human α_vβ₃ integrins are receptors for several pathogenic hantaviruses, and the function of α_vβ₃ integrins on endothelial cells suggests a role for α_vβ₃ in hantavirus directed vascular permeability. We determined here that ANDV infection of human endothelial cells or Syrian hamster-derived BHK-21 cells was selectively inhibited by the high-affinity α_vβ₃ integrin ligand vitronectin and by antibodies to α_vβ₃ integrins. Further, antibodies to the β₃ integrin PSI domain, as well as PSI domain polypeptides derived from human and Syrian hamster β₃ subunits, but not murine or bovine β₃, inhibited ANDV infection of both BHK-21 and human endothelial cells. These findings suggest that ANDV interacts with β₃ subunits through PSI domain residues conserved in both Syrian hamster and human β₃ integrins. Sequencing the Syrian hamster β₃ integrin PSI domain revealed eight differences between Syrian hamster and human β₃ integrins. Analysis of residues within the PSI domains of human, Syrian hamster, murine, and bovine β₃ integrins identified unique proline substitutions at residues 32 and 33 of murine and bovine PSI domains that could determine ANDV recognition. Mutagenizing the human β₃ PSI domain to contain the L33P substitution present in bovine β₃ integrin abolished the ability of the PSI domain to inhibit ANDV infectivity. Conversely, mutagenizing either the bovine PSI domain, P33L, or the murine PSI domain, S32P, to the residue present human β₃ permitted PSI mutants to inhibit ANDV infection. Similarly, CHO cells transfected with the full-length bovine β₃ integrin containing the P33L mutation permitted infection by ANDV. These findings indicate that human and Syrian hamster α_vβ₃ integrins are key receptors for ANDV and that specific residues within the β₃ integrin PSI domain are required for ANDV infection. Since L33P is a naturally occurring human β₃ polymorphism, these findings further suggest the importance of specific β₃ integrin residues in hantavirus infection. These findings rationalize determining the role of β₃ integrins in hantavirus pathogenesis in the Syrian hamster model.Hantaviruses persistently infect specific small mammal hosts and are spread to humans by the inhalation of aerosolized excreted virus (41, 42). Hantaviruses predominantly infect endothelial cells and cause one of two vascular leak-based diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) (41). Hantavirus diseases are characterized by increased vascular permeability and acute thrombocytopenia in the absence of endothelial cell lysis (36, 41, 42, 54). In general, hantaviruses are not spread from person to person; however, the Andes hantavirus (ANDV) is an exception, since there are several reports of person-to-person transmission of ANDV infection (11, 37, 47, 52). ANDV is also unique in its ability to cause an HPS-like disease in Syrian hamsters and serves as the best-characterized hantavirus disease model with a long onset, symptoms, and pathogenesis nearly identical to that of HPS patients (20, 21, 50).Hantavirus infection of the endothelium alters endothelial cell barrier functions through direct and immunological responses (8, 14). Although the means by which hantaviruses cause pulmonary edema or hemorrhagic disease has been widely conjectured, the mechanisms by which hantaviruses elicit pathogenic human responses have yet to be defined. Hantaviruses coat the surface of infected VeroE6 cells days after infection (17), and this further suggests that dynamic hantavirus interactions with immune and endothelial cells are likely to contribute to viral pathogenesis. Hantavirus pathogenesis has been suggested to involve CD8⁺ T cells, tumor necrosis factor alpha or other cytokines, viremia, and the dysregulation of β₃ integrins (7, 8, 13-16, 25-28, 32, 34, 38, 44-46). However, these responses have not been demonstrated to contribute to hantavirus pathogenesis, and in some cases there are conflicting data on their involvement (18, 25-28, 34, 35, 44, 45, 48). Immune complex deposition clearly contributes to HFRS patient disease and renal sequelae (4, 7), but it is unclear what triggers vascular permeability in HPS and HFRS diseases or why hemorrhage occurs in HFRS patients but not in HPS patients (8, 36, 54). Acute thrombocytopenia is common to both diseases, and platelet dysfunction resulting from defective platelet aggregation is reported in HFRS patients (7, 8).Pathogenic hantaviruses have in common their ability to interact with α_IIbβ₃ and α_vβ₃ integrins present on platelets and endothelial cells (13, 16), and β₃ integrins have primary roles in regulating vascular integrity (1, 2, 6, 19, 22, 39, 40). Consistent with the presence of cell surface displayed virus (17), pathogenic hantaviruses uniquely block α_vβ₃ directed endothelial cell migration and enhance endothelial cell permeability for 3 to 5 days postinfection (14, 15). Pathogenic hantaviruses dysregulate β₃ integrin functions by binding domains present at the apex of inactive β₃ integrin conformers (38). α_vβ₃ forms a complex with vascular endothelial cell growth factor receptor 2 (VEGFR2) and normally regulates VEGF-directed endothelial cell permeability (2, 3, 10, 39, 40). However, both β₃ integrin knockouts and hantavirus-infected endothelial cells result in increased VEGF-induced permeability, presumably by disrupting VEGFR2-β₃ integrin complex formation (2, 14, 19, 39, 40). This suggests that at least one means for hantaviruses to increase vascular permeability occurs through interactions with β₃ integrins that are required for normal platelet and endothelial cell functions.α_vβ₃ and α_IIbβ₃ integrins exist in two conformations: an active extended conformation where the ligand binding head domain is present at the apex of the heterodimer and a basal, inactive bent conformation where the globular head of the integrin is folded toward the cell membrane (30, 53, 55). Pathogenic HTN and NY-1 hantaviruses bind to the N-terminal plexin-semaphorin-integrin (PSI) domain of β₃ integrin subunits and are selective for bent, inactive α_vβ₃ integrin conformers (38). Pathogenic hantavirus binding to inactive α_vβ₃ integrins is consistent with the selective inhibitory effect of hantaviruses on α_vβ₃ function and endothelial cell permeability (14, 15, 38). Although the mechanism of hantavirus induced vascular permeability has yet to be defined, there is a clear role for β₃ integrin dysfunction in vascular permeability deficits (5, 6, 22, 29, 39, 40, 51) which make an understanding of hantavirus interactions with β₃ subunits important for both entry and disease processes.The similarity between HPS disease in humans and Syrian hamsters (20, 21) suggests that pathogenic mechanisms of ANDV disease are likely to be coincident. Curiously, other hantaviruses (Sin Nombre virus [SNV] and Hantaan virus [HTNV]) are restricted in Syrian hamsters and fail to cause disease in this animal, even though they are prominent causes of human disease (50). Although the host range restriction for SNV and HTNV in Syrian hamsters has not been defined (33), the pathogenesis of ANDV in Syrian hamsters suggests that both human and Syrian hamster β₃ integrins may similarly be used by ANDV and contribute to pathogenesis.We demonstrate here that ANDV infection of the Syrian hamster BHK-21 cell line and human endothelial cells is dependent on α_vβ₃ and inhibited by α_vβ₃ specific ligands and antibodies. Further, polypeptides expressing the N-terminal 53 residues of human and Syrian hamster β₃ subunits block ANDV infection. This further indicates that ANDV interaction with the N-terminal 53 residues of both human and Syrian hamster β₃ integrins is required for viral entry. We also demonstrate that ANDV recognition of human and Syrian hamster β₃ integrins is determined by proline substitutions at residues 32/33 within the β₃ integrin PSI domain. These results define unique ANDV interactions with human and Syrian hamster β₃ integrins.     

Development of oligodendrocytes. Studies of rat glial cells cultured in chemically-defined medium

Oligodendrocytes are macroglial cells that synthesize and maintain myelin in the central nervous system. Oligodendrocytes in rodent brain are formed postnatally from glial progenitor cells. These progenitors cells are bipotential and differentiate in a later stage of development into type-2 astrocytes. Recent studies with cultured cells indicate that growth factors such as platelet-derived growth factor and ciliary neurotrophic factor are instrumental in the control of these events. This paper discusses various methods for the isolation of oligodendrocytes and for their maintenance in culture. We use cerebra or spinal cords from one-week old rat pups to prepare glial cultures that are enriched in oligodendrocytes (60-80% or greater than or equal to 90%, respectively). After one day in serum-containing medium the cells are kept in chemically-defined medium, supplemented with the hormones insulin, T3 and hydrocortisone. The activities of astrocyte-and oligodendrocyte-specific marker enzymes were measured to evaluate the influence of these hormones on the differentiation of the oligodendrocytes. Finally, glial energy metabolism and the utilization of ketone bodies and of fatty acids are discussed briefly.     

RhoBTB3 interacts with the 5-HT7a receptor and inhibits its proteasomal degradation

The 5-hydroxytryptamine (5-HT)7 receptor is the most recently identified serotonin receptor and is involved in a wide variety of central nervous system (CNS) functions, namely circadian rhythm, REM sleep, depression, thermoregulation, obsessive-compulsive disorder (OCD), anxiety, schizophrenia, epilepsy, nociception, migraine, sensation-seeking behavior, impulsivity, learning and memory. These numerous (patho)physiological processes of the CNS, in which the 5-HT7 receptor is involved, most likely reflect a diverse set of signaling pathways arising from this receptor. In order to reveal new interaction partners and possibly new signaling and/or trafficking pathways, we performed a yeast two-hybrid screening, using the C-terminal tail of the 5-HT7a receptor as bait and an adult-human brain cDNA library as prey. In this way we identified RhoBTB3 as a new interaction partner of the 5-HT7a receptor. By means of co-immunoprecipitation we were able to confirm the interaction between full length 5-HT7a receptor and RhoBTB3 in HEK293T cells. Subsequent domain mapping of this interaction revealed that not only the C-terminal tail, but also the third intracellular loop of the 5-HT7a receptor is involved. In addition, immunofluorescence microscopy showed clear co-localization between the 5-HT7a receptor and RhoBTB3 at the plasma membrane and in the endoplasmic reticulum. Despite the fact that RhoBTB3 has been shown to interact with Cul3, which in turn interacts with the E3 ubiquitin ligase, Roc1, we show here that RhoBTB3 neither recruits Cul3/Roc1 to the 5-HT7a receptor nor does it mediate ubiquitination of this receptor. Instead, we demonstrate that RhoBTB3 strongly inhibits proteasomal degradation of the 5-HT7a receptor.     

In vivo gait analysis in a mouse femur fracture model

Although the mouse has become a preferred species for molecular studies on fracture healing, gait analysis after fracture fixation and during bone healing has not yet been performed in mice. Herein, we introduce a novel technique for gait analysis in mice and report the change of motion pattern after fracture and fixation. A standardized femur fracture was stabilized by a common pin. The non-fractured tibia was additionally marked with a pin, allowing continuous analysis of the tibio-femoral angle by digital video-radiography. Dynamic gait analysis was performed at day fourteen after surgery in a radio-opaque running wheel. Fracture fixation resulted in a significantly reduced range and maximum of the tibio-femoral angle compared to non-fractured controls. This was associated with a significantly reduced stride length. Because stride frequency was slightly increased and, thus, stride time diminished, stride velocity was not significantly reduced compared to controls. Thus, our study demonstrates distinct alterations of the gait of mice at 2 weeks after femur fracture and stabilization. Our results support the need of gait analysis in fracture healing studies to assess the animals’ well-being.     

Role of endogenous interleukin-12 (IL-12) in induced and spontaneous relapses of experimental autoimmune encephalomyelitis in mice.

Actively induced, chronic relapsing experimental autoimmune encephalomyelitis (CREAE) was studied in SJL/J and in Biozzi ABH mice. In Biozzi ABH mice, relapses occurred spontaneously with high frequency. In SJL/J mice, spontaneous relapses occurred infrequently; however they could be induced reproducibly by reimmunization. In both models, moderately increased levels of serum IL-12(p40) were consistently found shortly before primary attacks, but irregularly at later times. Injections of anti-IL-12 antibody inhibited disease development in both SJL/J and in Biozzi ABH mice. The time window during which treatment needed to be initiated in order to be effective, ranged from before induction until shortly before the symptoms of primary attacks emerged. Such treatment inhibited not only the first attack but also the spontaneous or induced relapses. Most significantly, anti-IL-12 antibody given during remission of primary disease inhibited actively re-induced relapses in SJL/J, but not spontaneous relapses in Biozzi ABH mice. These results indicate that endogenous IL-12 favours EAE development by crucially affecting the active induction process, but that a second burst of IL-12 production may not be necessary for triggering spontaneous relapses.     

Revascularization of ischemic tissues by PlGF treatment,and inhibition of tumor angiogenesis,arthritis and atherosclerosis by anti-Flt1

The therapeutic potential of placental growth factor (PlGF) and its receptor Flt1 in angiogenesis is poorly understood. Here, we report that PlGF stimulated angiogenesis and collateral growth in ischemic heart and limb with at least a comparable efficiency to vascular endothelial growth factor (VEGF). An antibody against Flt1 suppressed neovascularization in tumors and ischemic retina, and angiogenesis and inflammatory joint destruction in autoimmune arthritis. Anti-Flt1 also reduced atherosclerotic plaque growth and vulnerability, but the atheroprotective effect was not attributable to reduced plaque neovascularization. Inhibition of VEGF receptor Flk1 did not affect arthritis or atherosclerosis, indicating that inhibition of Flk1-driven angiogenesis alone was not sufficient to halt disease progression. The anti-inflammatory effects of anti-Flt1 were attributable to reduced mobilization of bone marrow-derived myeloid progenitors into the peripheral blood; impaired infiltration of Flt1-expressing leukocytes in inflamed tissues; and defective activation of myeloid cells. Thus, PlGF and Flt1 constitute potential candidates for therapeutic modulation of angiogenesis and inflammation.     

RELATIONSHIP BETWEEN THE 4-AMINOBUTYRATE BYPATH AND THE OXIDATION OF 2-OXOGLUTARATE IN RAT BRAIN MITOCHONDRIA

Abstract— A method is described to evaluate simultaneously the contributions of 2-oxoglutarate oxidation and the GABA bypath to succinate production in isolated rat brain mitochondria.
2-Oxoglutarate oxidation is under respiratory control whereas the activity of the GABA shunt is but slightly affected by the mitochondrial energy state.
The oxidation of GABA is half-maximal with 5m m -GABA. GABA does not affect 2-oxoglutarate oxidation. 1 m m -2-oxoglutarate is optimal for GABA oxidation, whereas higher concentrations inhibit the shunt activity.
The rate of GABA oxidation observed in vitro (5 nmol/min.mg mitochondrial protein) is comparable to the activity of the shunt under in viuo conditions.
The control and the compartmentation of GABA oxidation are discussed.