Factor Knockout Mice Research Articles

Non-clinical pharmacokinetics and pharmacodynamics of rVIII-SingleChain, a novel recombinant single-chain factor VIII

IntroductionrVIII-SingleChain (CSL627), a novel recombinant coagulation factor VIII (FVIII), is under investigation in a phase I/III clinical programme (AFFINITY) for the treatment of haemophilia A. Non-clinical studies were conducted to investigate the pharmacokinetic/pharmacodynamic profile of rVIII-SingleChain in comparison with full-length recombinant FVIII. Materials and MethodsBinding affinity of rVIII-SingleChain for von Willebrand factor was investigated by surface plasmon resonance analysis. The pharmacokinetic profile of rVIII-SingleChain was compared with a marketed full-length recombinant FVIII concentrate (Advate®) in haemophilia A mice, von Willebrand factor knock-out mice, Crl:CD (SD) rats, rabbits and cynomolgus monkeys. Systemic FVIII activity or antigen levels were recorded. Procoagulant activity was measured in an FeCl3-induced arterial occlusion model and by recording thrombin generation activity (ex vivo) after administration of 200–250IU/kg rVIII-SingleChain or full-length FVIII to haemophilia A mice. ResultsrVIII-SingleChain displayed a high affinity for von Willebrand factor (KD=44pM vs. 139 pM for full-length recombinant FVIII). In all animal species tested, rVIII-SingleChain had more favourable pharmacokinetic properties than full-length recombinant FVIII: clearance was decreased and area under the curve and terminal half-life were enhanced vs. full-length recombinant FVIII, while in vivo recovery and volume of distribution were equivalent. rVIII-SingleChain showed a prolonged thrombin generation potential and prolonged procoagulant activity vs. full-length recombinant FVIII in an FeCl3-induced arterial occlusion model. ConclusionsrVIII-SingleChain had a higher affinity for von Willebrand factor than full-length recombinant FVIII and displayed favourable pharmacokinetic/pharmacodynamic properties in non-clinical models.

GM-CSF knockout mice for preclinical testing of agents with antimicrobial activity against Mycobacterium abscessus

Of the non-tuberculous mycobacteria, Mycobacterium abscessus is particularly refractory to antimicrobial therapy and new agents with activity against these pathogens are urgently needed. The screening of candidate antimicrobial agents against M. abscessus requires a relevant and reproducible animal model of chronic infection. Granulocyte-macrophage colony-stimulating factor knockout (GM-CSF KO) mice were used to develop a new animal model of chronic pulmonary M. abscessus infection that can be used for preclinical efficacy testing of antimicrobial drugs. GM-CSF KO mice were infected with a clinical isolate of M. abscessus via intrapulmonary aerosol delivery using a microsprayer device. The clinical condition, histology and cfu of M. abscessus-infected GM-CSF KO mice were evaluated over a period of 4 months. Mice were treated with azithromycin (100 mg/kg) by oral gavage and the clinical condition, histology and bacterial burden was determined after 2 weeks of treatment. We show that pulmonary infection of GM-CSF KO mice with M. abscessus results in a chronic pulmonary infection that lends itself to preclinical testing of new antimicrobial drugs against this bacterium. Azithromycin treatment of M. abscessus-infected GM-CSF KO mice resulted in a lower bacterial burden in the lungs and spleen, weight gain and significant improvement in lung pathology. Intrapulmonary aerosol infection of GM-CSF KO mice with M. abscessus is a useful animal model for studying pathogenesis as well as pre-clinical testing of new compounds against M. abscessus in acute or chronic phases of infection.

Altered Responses to Cold Environment in Urocortin 1 and Corticotropin-Releasing Factor Deficient Mice

We examined core body temperature (CBT) of urocortin 1 (UCN1) and corticotropin releasing factor (CRF) knockout (KO) mice exposed to 4°C for 2 h. UCN1KO mice showed higher average CBT during cold exposure as compared to WT. The CBT of male and female WT mice dropped significantly to34.1 ± 2.4and34.9 ± 3.1 C at 4°C, respectively. In contrast, the CBT of male and female UCN1KO mice dropped only slightly after 2 h at 4°C to36.8 ± 0.7and38.1 ± 0.5 C, respectively. WT female and male UCN1KO mice showed significant acclimatization to cold; however, female UCN1KO mice did not show such a significant acclimatization. CRFKO mice showed a dramatic decline in CBT from38.2 ± 0.4at 22°C to26.1 ± 9.8at 4°C for 2 h. The CRF/UCN1 double KO (dKO) mice dropped their CBT to32.5 ± 4.0after 2 h exposure to 4°C. Dexamethasone treatment prevented the decline in CBT of the CRFKO and the dKO mice. Taken together, the data suggest a novel role for UCN1 in thermoregulation. The role of CRF is likely secondary to adrenal glucocorticoids, which have an important regulatory role on carbohydrate, fat, and protein metabolism.

Circulating Antibodies and Macrophages as Modulators of Adenovirus Pharmacology

Adenovirus serotype 5 (Ad5) naturally infects the liver after intravenous injection, making it a candidate for hepatocyte-directed gene transfer. While Ad5 can be efficient, most of the dose is destroyed by liver Kupffer cells before it can reach hepatocytes. In contrast, Ad5 bearing the hexon from Ad6 (Ad5/6) evades Kupffer cells. While Ad5/6 dramatically increases hepatocyte transduction in BALB/c mice, it has surprisingly little effect on C57BL/6 mice. To determine the source of this strain-specific difference, the roles of Kupffer cells, liver sinusoidal endothelial cells (LSECs), hepatocytes, scavenger receptors, clotting factors, and immunoglobulins were analyzed. The numbers of Kupffer cells and LSECs, the level of clotting factor X, and hepatocyte infectibility did not differ between different strains of mice. In contrast, high levels of immunoglobulins correlated negatively with Ad5 liver transduction in different mouse strains. Removal of immunoglobulins by use of Rag-deficient mice restored Ad5 transduction to maximal levels. Removal of Kupffer cells by predosing or by testing in colony-stimulating factor knockout mice restored Ad5 transduction in the presence of immunoglobulins. Partial reconstitution of IgM in Rag mice resulted in significant reductions in liver transduction by Ad5 but not by Ad5/6. These data suggest a role for IgM-mediated clearance of Ad5 via Kupffer cells and may explain the mechanism by which Ad5/6 evades these cells. These mechanisms may play a vital role in Ad pharmacology in animals and in humans.

Mechanism of hyperphagia contributing to obesity in brain-derived neurotrophic factor knockout mice

Global-heterozygous and brain-specific homozygous knockouts (KOs) of brain-derived neurotrophic factor (BDNF) cause late- and early-onset obesity, respectively, both involving hyperphagia. Little is known about the mechanism underlying this hyperphagia or whether BDNF loss from peripheral tissues could contribute to overeating. Since global-homozygous BDNF-KO is perinatal lethal, a BDNF-KO that spared sufficient brainstem BDNF to support normal health was utilized to begin to address these issues. Meal pattern and microstructure analyses suggested overeating of BDNF-KO mice was mediated by deficits in both satiation and satiety that resulted in increased meal size and frequency and implicated a reduction of vagal signaling from the gut to the brain. Meal-induced c-Fos activation in the nucleus of the solitary tract, a more direct measure of vagal afferent signaling, however, was not decreased in BDNF-KO mice, and thus was not consistent with a vagal afferent role. Interestingly though, meal-induced c-Fos activation was increased in the dorsal motor nucleus of the vagus nerve (DMV) of BDNF-KO mice. This could imply that augmentation of vago-vagal digestive reflexes occurred (e.g., accommodation), which would support increased meal size and possibly increased meal number by reducing the increase in intragastric pressure produced by a given amount of ingesta. Additionally, vagal sensory neuron number in BDNF-KO mice was altered in a manner consistent with the increased meal-induced activation of the DMV. These results suggest reduced BDNF causes satiety and satiation deficits that support hyperphagia, possibly involving augmentation of vago-vagal reflexes mediated by central pathways or vagal afferents regulated by BDNF levels.

Native-Like Aggregates of Factor VIII Are Immunogenic in von Willebrand Factor Deficient and Hemophilia a Mice

The administration of recombinant factor VIII (FVIII) is the first-line therapy for hemophilia A (HA), but 25%-35% of patients develop an inhibitory antibody response. In general, the presence of aggregates contributes to unwanted immunogenic responses against therapeutic proteins. FVIII has been shown to form both native-like and nonnative aggregates. Previously, we showed that nonnative aggregates of FVIII are less immunogenic than the native protein. Here, we investigated the effect of native-like aggregates of FVIII on immunogenicity in HA and von Willebrand factor knockout (vWF(-/-)) mice. Mice immunized with native-like aggregates showed significantly higher inhibitory antibody titers than animals that received native FVIII. Following restimulation in vitro with native FVIII, the activation of CD4+ T-cells isolated from mice immunized with native-like aggregates is approximately fourfold higher than mice immunized with the native protein. Furthermore, this is associated with increases in the secretion of proinflammatory cytokines IL-6 and IL-17 in the native-like aggregate treatment group. The results indicate that the native-like aggregates of FVIII are more immunogenic than native FVIII for both the B-cell and the T-cell responses.

Placental Growth Factor Regulates Cardiac Inflammation Through the Tissue Inhibitor of Metalloproteinases-3/Tumor Necrosis Factor-α–Converting Enzyme Axis

Background— Heart failure is one of the leading causes of mortality and is primarily the final stage of several overload cardiomyopathies, preceded by an early adaptive hypertrophic response and characterized by coordinated cardiomyocyte growth, angiogenesis, and inflammation. Therefore, growth factors and cytokines have to be critically regulated during cardiac response to transverse aortic constriction. Interestingly, the dual properties of placental growth factor as an angiogenic factor and cytokine make it a candidate to participate in cardiac remodeling in response to hemodynamic overload. Methods and Results— After transverse aortic constriction, placental growth factor knockout mice displayed a dysregulation of cardiac remodeling, negatively affecting muscle growth. Molecular insights underscored that this effect was ascribable mainly to a failure in the establishment of adequate inflammatory response owing to an impaired activity of tumor necrosis factor-α–converting enzyme. Interestingly, after transverse aortic constriction, placental growth factor knockout mice had strongly increased levels of tissue inhibitor of metalloproteinases-3, the main natural TACE inhibitor, thus indicating an unbalance of the tissue inhibitor of metalloproteinases-3/tumor necrosis factor-α–converting enzyme axis. Strikingly, when we used an in vivo RNA interference approach to reduce tissue inhibitor of metalloproteinases-3 levels in placental growth factor knockout mice during transverse aortic constriction, we obtained a complete phenotype rescue of early dilated cardiomyopathy. Conclusions— Our results demonstrate that placental growth factor finely tunes a balanced regulation of the tissue inhibitor of metalloproteinases-3/tumor necrosis factor-α–converting enzyme axis and the consequent TNF-α activation in response to transverse aortic constriction, thus allowing the establishment of an inflammatory response necessary for adaptive cardiac remodeling.

Forebrain specific heparin-binding epidermal growth factor-like growth factor knockout mice show exacerbated ischemia and reperfusion injury

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a hypoxia-inducible neuroprotective protein that also stimulates proliferation of neuronal precursor cells. In this study, we investigated the possible role of HB-EGF in ischemia and reperfusion injury by measuring the changes in its mRNA expression following focal cerebral ischemia. We also examined neural damage after a middle cerebral artery occlusion (MCAO) and reperfusion in ventral forebrain specific HB-EGF knockout (KO) mice. The levels of HB-EGF mRNA in the cerebral cortex of wild-type (WT) mice were significantly increased 3-24 h after MCAO and reperfusion. Cerebral infraction in HB-EGF KO mice was aggravated at 1 day and 6 days after MCAO and reperfusion compared with WT mice. The number of terminal deoxynucleotidyl transferase (TdT)-mediated dNTP nick end labeling (TUNEL) and an oxidative stress marker, 8-hydroxy-2'-deoxyguanosine (8-OHdG) positive cells, were higher in HB-EGF KO mice than in WT mice. On the other hand, fewer bromodeoxyuridine (BrdU) positive cells were found in the subventricular zone in HB-EGF KO mice compared with WT mice. These results indicate that HB-EGF may play a pivotal role in ischemia and reperfusion injury and that endogenously synthesized HB-EGF is necessary for both the neuroprotective effect and for regulation of cell proliferation in the subventricular zone.

Complement expression in mice throughout neural tube closure

A role for the complement cascade in mammalian neural development is becoming apparent. There is evidence that complement factors 3a (C3a) and 5a (C5a), are regulators of neural differentiation and proliferation. Our lab has also demonstrated a very high incidence of neural tube defects in mice lacking C5a receptor (C5aR) signaling under maternal folate-deficient conditions. We are currently investigating specific complement factor and receptor knockout mice in our folate-deficiency model to elucidate their particular functional roles. These results support the hypothesis of a novel role for C3a and C5a in foetal development, with selective localisation to neural proliferative zones, and, in particular, a crucial developmental role following dietary folate deficiency.

Targeted PPARγ deficiency in alveolar macrophages disrupts surfactant catabolism

Surfactant accumulates in alveolar macrophages of granulocyte-macrophage colony-stimulating factor (GM-CSF) knockout (KO) mice and pulmonary alveolar proteinosis (PAP) patients with a functional loss of GM-CSF resulting from neutralizing anti-GM-CSF antibody. Alveolar macrophages from PAP patients and GM-CSF KO mice are de-ficient in peroxisome proliferator-activated receptor-gamma (PPARgamma) and ATP-binding cassette (ABC) lipid transporter ABCG1. Previous studies have demonstrated that GM-CSF induces PPARgamma. We therefore hypothesized that PPARgamma promotes surfactant catabolism through regulation of ABCG1. To address this hypothesis, macrophage-specific PPARgamma (MacPPARgamma) knockout mice were utilized. MacPPARgamma KO mice develop foamy, lipid-engorged Oil Red O positive alveolar macrophages. Lipid analyses revealed significant increases in the cholesterol and phospholipid contents of MacPPARgamma KO alveolar macrophages and extracellular bronchoalveolar lavage (BAL)-derived fluids. MacPPARgamma KO alveolar macrophages showed decreased expression of ABCG1 and a deficiency in ABCG1-mediated cholesterol efflux to HDL. Lipid metabolism may also be regulated by liver X receptor (LXR)-ABCA1 pathways. Interestingly, ABCA1 and LXRbeta expression were elevated, indicating that this pathway is not sufficient to prevent surfactant accumulation in alveolar macrophages. These results suggest that PPARgamma mediates a critical role in surfactant homeostasis through the regulation of ABCG1.

Changes in 5-HT2A-mediated behavior and 5-HT2A- and 5-HT1A receptor binding and expression in conditional brain-derived neurotrophic factor knock-out mice

Changes in brain-derived neurotrophic factor (BDNF) expression have been implicated in the etiology of psychiatric disorders. To investigate pathological mechanisms elicited by perturbed BDNF signaling, we examined mutant mice with central depletion of BDNF (BDNF2L/2LCk-cre). A severe impairment specific for the serotonin 2A receptor (5-HT2AR) in prefrontal cortex was described previously in these mice. This is of much interest, as 5-HT2ARs have been linked to neuropsychiatric disorders and anxiety-related behavior. Here we further characterized the serotonin receptor alterations triggered by BDNF depletion. 5-HT2A ([3H]-MDL100907) and 5-HT1A ([3H]-WAY100635) receptor autoradiography revealed site-specific alterations in BDNF mutant mice. They exhibited lower 5-HT2A receptor binding in frontal cortex but increased binding in hippocampus. Additionally, 5-HT1A receptor binding was decreased in hippocampus of BDNF mutants, but unchanged in frontal cortex. Molecular analysis indicated corresponding changes in 5-HT2A and 5-HT1A mRNA expression but normal 5-HT2C content in these brain regions in BDNF2L/2LCk-cre mice. We investigated whether the reduction in frontal 5-HT2AR binding was reflected in reduced functional output in two 5-HT2A-receptor mediated behavioral tests, the head-twitch response (HTR) and the ear-scratch response (ESR). BDNF2L/2LCk-cre mutants treated with the 5-HT2A receptor agonist (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) showed a clearly diminished ESR but no differences in HTR compared to wildtypes. These findings illustrate the context-dependent effects of deficient BDNF signaling on the 5-HT receptor system and 5-HT2A-receptor functional output.

Prevention of elastase-induced emphysema in placenta growth factor knock-out mice

BackgroundAlthough both animal and human studies suggested the association between placenta growth factor (PlGF) and chronic obstructive pulmonary disease (COPD), especially lung emphysema, the role of PlGF in the pathogenesis of emphysema remains to be clarified. This study hypothesizes that blocking PlGF prevents the development of emphysema.MethodsPulmonary emphysema was induced in PlGF knock-out (KO) and wild type (WT) mice by intra-tracheal instillation of porcine pancreatic elastase (PPE). A group of KO mice was then treated with exogenous PlGF and WT mice with neutralizing anti-VEGFR1 antibody. Tumor necrosis factor alpha (TNF-α), matrix metalloproteinase-9 (MMP-9), and VEGF were quantified. Apoptosis measurement and immuno-histochemical staining for VEGF R1 and R2 were performed in emphysematous lung tissues.ResultsAfter 4 weeks of PPE instillation, lung airspaces enlarged more significantly in WT than in KO mice. The levels of TNF-α and MMP-9, but not VEGF, increased in the lungs of WT compared with those of KO mice. There was also increased in apoptosis of alveolar septal cells in WT mice. Instillation of exogenous PlGF in KO mice restored the emphysematous changes. The expression of both VEGF R1 and R2 decreased in the emphysematous lungs.ConclusionIn this animal model, pulmonary emphysema is prevented by depleting PlGF. When exogenous PlGF is administered to PlGF KO mice, emphysema re-develops, implying that PlGF contributes to the pathogenesis of emphysema.

Generation and Characterization of Conditional Heparin-Binding EGF-Like Growth Factor Knockout Mice

Recently, neurotrophic factors and cytokines have been shown to be associated in psychiatric disorders, such as schizophrenia, bipolar disorder, and depression. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family, serves as a neurotrophic molecular and plays a significant role in the brain. We generated mice in which HB-EGF activity is disrupted specifically in the ventral forebrain. These knockout mice showed (a) behavioral abnormalities similar to those described in psychiatric disorders, which were ameliorated by typical or atypical antipsychotics, (b) altered dopamine and serotonin levels in the brain, (c) decreases in spine density in neurons of the prefrontal cortex, (d) reductions in the protein levels of the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor and post-synaptic protein-95 (PSD-95), (e) decreases in the EGF receptor, and in the calcium/calmodulin-dependent protein kinase II (CaMK II) signal cascade. These results suggest the alterations affecting HB-EGF signaling could comprise a contributing factor in psychiatric disorder.

Physiology, Injury, and Recovery of Interstitial Cells of Cajal: Basic and Clinical Science

In the last 15 years, our understanding of the cellular basis of gastrointestinal function has been altered irreversibly by the discovery that normal gastrointestinal (GI) motility requires interstitial cells of Cajal (ICC). Research in this relatively short time period has modified our original concept that the core unit that controls motility is made up of nerves and smooth muscle, to one that now includes ICC. This concept has now expanded to beyond the GI tract, suggesting that it may be a fundamental property of the regulation of smooth muscle function that requires rhythmic contraction. ICC are distributed throughout the GI tract, have important functions in the control of GI motility, and are often abnormal in diseased states. Recently, significant steps forward have been made in our understanding of the physiology of ICC as well as mechanisms of injury and recovery. These advances are the focus of this review. The Physiology of ICC Unique motor patterns are intrinsic to every organ of the GI tract, which suit their functions related to mixing, absorption, and anally directed movement. The ICC are an integral part of the control of these motor activities. The distribution of ICC throughout the musculature is associated with nerve structures. Myenteric pacemaker ICC surround the myenteric or Auerbach’s plexus and intramuscular ICC are associated with nerve varicosities throughout the muscle layers (Figures 1 and 2). Other subpopulations of ICC are associated with nonganglionated plexuses of nerve varicosities at the inner borders of the circular muscle layers in the intestine and colon (Figures 1 and 2). The best understood function is that of pacemaker activity in the stomach and small intestine where the ICC generate a periodic depolarization at a characteristic frequency in each of these organs that is called the slow wave or pacemaker activity.

The Membrane Form of Tumor Necrosis Factor Is Sufficient to Mediate Partial Innate Immunity toFrancisella tularensisLive Vaccine Strain

Here we characterize Francisella tularensis live vaccine strain (LVS) infection in total tumor necrosis factor (TNF) knockout (KO) mice and in transgenic mice expressing only the membrane form of TNF (memTNF). MemTNF mice, but not TNF KO mice, survived low-dose, sublethal LVS infections. Splenic nitric oxide production was impaired in infected memTNF mice and was absent in infected TNF KO mice. Spleen cell production of interferon-gamma, RANTES, and monocyte chemotactic protein-1 was elevated in TNF KO mice, compared with that in WT mice, by days 4-5 after infection, along with transiently increased numbers of CCR2(+) cells, whereas memTNF mice had an intermediate phenotype. By day 6 after infection, TNF KO mice, but not memTNF mice, exhibited massive apoptosis in spleens and livers, which shortly preceded their death. Thus, memTNF partially functions to regulate chemokine expression, cell recruitment, and nitric oxide production during primary LVS infection and protects against the induction of apoptosis observed in TNF KO mice.

Targets of anticytokine therapy and the risk of infections in humans and mice

To examine the type and risk of infections in humans and mice deficient in proinflammatory cytokines. Naturally occurring or manipulated genetic defects of tumor necrosis factor, interleukins-1, -6, -12, and -15, and interferon-gamma are examined for their increased susceptibility to, or protection from, infection. Interleukin-12p40 and interferon-gamma-blockers may lead to increased incidence of infections with intracellular bacteria, parasites, and fungi. In addition, we may see viral infections with interferon-gamma-blockers. Increased risk of infections is unlikely with either interleukin-1- or interleukin-15-blockers. Interleukin-6-blockers may lead to increased risk of infection with extracellular bacteria, viruses, parasites and fungi. In tumor necrosis factor knockout mice, increased susceptibility to pathogens are reported that are normally controlled by granuloma formation. In patients treated with tumor necrosis factor-blockers, a two-fold increase of granulomatous infections, predominantly reactivation of latent tuberculosis, is found. The infections detected in tumor necrosis factor knockout mice were accurate for predicting the infections observed when using tumor necrosis factor-blockers. If a similar correlation exists for other cytokines, the use of interferon-gamma and interleukin-12p40 blockers, and possibly interleukin-6 blockers, will lead to an increased risk for severe infections. Care should be taken when new cytokine blockers/antagonists are introduced.

Pulmonary Alveolar Proteinosis

Abstract: Pulmonary alveolar proteinosis (PAP) is a rare disease characterized by the accumulation of surfactant phospholipids and proteins in the alveoli (Trapnell et al, N Engl J Med. 2003;349:2527–2539). Less than 500 separate cases of PAP have been reported in the literature from the time of first description in 1958. These reports suggest an annual incidence of 0.36 and prevalence of 3.7 cases per million population (Seymour et al, Am J Respir Crit Care Med. 2002;166:215–235). The clinical course of PAP ranges from spontaneous resolution to death because of superimposed infection or respiratory failure. A number of important milestones mark the key advances in the study of this disease, which have led to an improved classification based on pathogenesis. In the last decade, the serendipitous finding of a lung disease reminiscent of PAP in transgenic granulocyte-macrophage colony-stimulating factor knockout mice (GM-CSF−/−) led to the link between GM-CSF and PAP (Dranoff et al. Science. 1994;264:713–716; Stanley et al. Proc Natl Acad Sci USA. 1994;91:5592–5596). The finding of neutralizing antibodies to GM-CSF suggest an autoimmune basis to idiopathic PAP (Kitamura et al. J Exp Med. 1999;190:875–880). These findings have resulted in innovative treatment approaches to enhance the current standard of care. This review summarizes some of these landmark studies and provides a rationale and plan for investigations that lead to the diagnosis, classification, and management of PAP.

Re-Expression of a Developmentally Restricted Potassium Channel in Autoimmune Demyelination: Kv1.4 Is Implicated in Oligodendroglial Proliferation

Mechanisms of lesion repair in multiple sclerosis are incompletely understood. To some degree, remyelination can occur, associated with an increase of proliferating oligodendroglial cells. Recently, the expression of potassium channels has been implicated in the control of oligodendrocyte precursor cell proliferation in vitro. We investigated the expression of Kv1.4 potassium channels in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, a model of multiple sclerosis. Confocal microscopy revealed expression of Kv1.4 in AN2-positive oligodendrocyte precursor cells and premyelinating oligodendrocytes in vitro but neither in mature oligodendrocytes nor in the spinal cords of healthy adult mice. After induction of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, Kv1.4 immunoreactivity was detected in or around lesions already during disease onset with a peak early and a subsequent decrease in the late phase of the disease. Kv1.4 expression was confined to 2',3'-cyclic nucleotide 3'-phosphodiesterase-positive oligodendroglial cells, which were actively proliferating and ensheathed naked axons. After a demyelinating episode, the number of Kv1.4 and 2',3'-cyclic nucleotide 3'-phosphodiesterase double-positive cells was greatly reduced in ciliary neurotrophic factor knockout mice, a model with impaired lesion repair. In summary, the re-expression of an oligodendroglial potassium channel may have a functional implication on oligodendroglial cell cycle progression, thus influencing tissue repair in experimental autoimmune encephalomyelitis and multiple sclerosis.

Impaired cutaneous wound healing in granulocyte/ macrophage colony-stimulating factor knockout mice

Wound healing involves various cells and cytokines, resulting in the regular progression of remodelling events. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a multifunctional pleiotropic cytokine and is known to facilitate wound healing, although the precise molecular and cellular mechanisms remain to be explored. To use GM-CSF gene knockout (GM-CSF KO) mice to investigate the role of GM-CSF in cutaneous wound healing following full-thickness skin injury. Full-thickness skin wounds were made in GM-CSF KO and wild-type mice. The wound closure, leucocyte infiltration, vascularization and extent of cytokine production were determined. Wound healing was significantly delayed in GM-CSF KO mice, accompanied by reduced cytokine production (interleukin-6, monocyte chemoattractant protein-1 and macrophage inflammatory protein-2), and platelet-endothelial cell adhesion molecule-1 expression. Consequently there was reduced recruitment of neutrophils and macrophages and reduced vascularization in the wounds of GM-CSF KO mice. Although collagen deposition was delayed, it was significantly increased in the wounds of the GM-CSF KO mice in the later stages of wound healing. We conclude that GM-CSF plays an important role in the complex network of effector molecules that regulate keratinocyte proliferation and the inflammatory response. These data have important implications for further development of the therapeutic manipulation of wound healing using GM-CSF.

Cytokines, Signaling Pathways, and Effector Molecules Required for the Control ofLeishmania(Viannia)braziliensisin Mice

Cutaneous leishmaniasis is caused by protozoan parasites of the genus Leishmania. The mechanisms of pathogen control have been established primarily in the mouse model of Leishmania major infection, but they might not hold true for other Leishmania species associated with cutaneous disease. Here, we analyzed the role of cytokines, signaling components, and effector molecules in the control of New World cutaneous leishmaniasis due to L. braziliensis. Unlike L. major, L. braziliensis caused small, nonulcerative, and self-healing skin swelling in C57BL/6 mice, as well as BALB/c mice. In contrast to the results obtained for L. mexicana, mice deficient for interleukin-12 or its key signaling molecule, signal transducer and activator of transcription 4, rapidly succumbed to severe visceral leishmaniasis. Infection of tumor necrosis factor knockout mice with L. braziliensis led to progressive, nonhealing skin lesions with erosions and hemorrhagic ulcerations, but in contrast to the results with L. major, only 20 to 30% of the mice developed fatal visceral disease. As seen with L. major, mice with a deleted inducible nitric oxide synthase gene (iNOS(-/-)) were unable to contain L. braziliensis in the skin, whereas the control of the parasite in the spleen remained unimpaired. Unlike what happens in L. major infections, NADPH oxidase had no impact on the course of disease in L. braziliensis-infected mice. These results not only define essential components of a protective immune response to L. braziliensis but also illustrate that the requirements for the control of cutaneous leishmaniasis vary between different parasite species.