Antibody-Directed Myostatin Inhibition Improves Diaphragm Pathology in Young but not Adult Dystrophic mdx Mice

Duchenne muscular dystrophy (DMD) is characterized by progressive skeletal muscle wasting and weakness, leading to premature death from respiratory and/or cardiac failure. A clinically relevant question is whether myostatin inhibition can improve function of the diaphragm, which exhibits a severe and progressive pathology comparable with that in DMD. We hypothesized that antibody-directed myostatin inhibition would improve the pathophysiology of diaphragm muscle strips from young mdx mice (when the pathology is mild) and adult mdx mice (when the pathology is quite marked). Five weeks treatment with a mouse chimera of anti-human myostatin antibody (PF-354, 10 mg/kg/week) increased muscle mass (P < 0.05) and increased diaphragm median fiber cross-sectional area (CSA, P < 0.05) in young C57BL/10 and mdx mice, compared with saline-treated controls. PF-354 had no effect on specific force (sP_o, maximum force normalized to muscle CSA) of diaphragm muscle strips from young C57BL/10 mice, but increased sP_o by 84% (P < 0.05) in young mdx mice. In contrast, 8 weeks of PF-354 treatment did not improve muscle mass, median fiber CSA, collagen infiltration, or sP_o of diaphragm muscle strips from adult mdx mice. PF-354 antibody-directed myostatin inhibition completely restored the functional capacity of diaphragm strips to control levels when treatment was initiated early, but not in the later stages of disease progression, suggesting that such therapies may only have a limited window of efficacy for DMD and related conditions.Duchenne muscular dystrophy (DMD) is the most severe of the muscular dystrophies and affects approximately 1 in 3500 live male births. It is characterized by progressive skeletal muscle weakness and wasting that leads to premature death caused by respiratory or cardiac failure.¹ DMD is caused by the absence of dystrophin, a membrane stabilizing cytoskeletal protein that confers protection from contraction-mediated trauma.² The fragility of dystrophic muscle fibers renders them susceptible to injury and ongoing cycles of damage, degeneration, and incomplete regeneration. Currently, there is no cure for DMD, and despite their potential, widely lauded gene therapies have yet to be perfected nor will they be optimized in time to treat current patients.³ Therefore, it is crucial to develop therapeutic strategies that can increase muscle strength, enhance muscle fiber regeneration and/or reduce degeneration, and protect muscles from contraction-mediated injury.^4,5Myostatin, originally termed growth and differentiation factor-8, is a member of the transforming growth factor-β superfamily. Myostatin negatively regulates skeletal muscle growth,⁶ an effect attributed to inhibition of both myoblast proliferation and differentiation.⁷ Livestock and humans with a loss-of-function mutation in the myostatin gene exhibit hypermuscularity.^6,7,8,9 Numerous studies have demonstrated that myostatin inhibition, via genetic deletion or pharmacological inactivation, can increase skeletal muscle size and strength.^{10,11,12,13,14} Not surprisingly, there is considerable interest in developing strategies to modulate myostatin activity in clinical situations where enhancing muscle growth and strength may have beneficial effects for age-related muscle wasting, cancer cachexia, denervation, sepsis, and the muscular dystrophies.^{15,16,17,18,19,20}Several strategies have been used to inhibit myostatin in dystrophic mdx mice. Transgenic deletion of myostatin¹⁸ or overexpression of follistatin, an endogenous antagonist of myostatin,²¹ in 5-week-old to 9-month-old mdx mice increased muscle mass and fiber cross-sectional area (CSA), improved diaphragm pathology, and reduced infiltration of connective tissue in the diaphragm. Similar improvements in limb muscle mass and fiber CSA as well as in diaphragm pathology were also found after 3 months administration of a myostatin inhibitory antibody (JA16)¹⁹ or myostatin propeptide²⁰ to 4-week-old mdx mice. However, the limb muscles of mdx mice undergo the first profound bout of muscle degeneration at 19 to 21 days after birth, and this is when the pathology in the limb muscles of mdx mice most closely resembles that in DMD.²² Early treatment in mdx mice is sometimes difficult to translate to humans, because DMD is usually detected only when the condition has progressed to a stage when functional impairments are evident. Therefore, to comprehensively assess the therapeutic potential of such interventions, it is recommended that studies in mdx mice should examine effects in young (2- to 3-week-old) mdx mice before or during the initial bout of severe muscle fiber degeneration and in older mice after several cycles of degeneration and less than successful regeneration, at the time when clinical treatments for DMD are usually first implemented.²³Although the mdx mouse is a commonly used model of DMD, the limb muscles have only a relatively mild myopathy.²⁴ In contrast, the diaphragm exhibits a more severe and progressive dystrophic pathology.²⁴ To assess the therapeutic potential of myostatin inhibition for improving the dystrophic pathology in the mdx mouse, the effects on diaphragm muscle function are important clinically because respiratory insufficiency is a predictor of mortality in DMD.The aim of this study was to investigate the therapeutic potential of myostatin inhibition, administered via a novel myostatin blocking antibody (PF-354), on the pathology and function of the diaphragm muscle of young (16- to 17-day-old) and adult (12-week-old) mdx mice. The efficacy of PF-354 for inhibiting myostatin activity has been shown previously in muscles from aged mice with 4 weeks treatment with PF-354 reducing Smad3 phosphorylation (a downstream event involved in myostatin signaling) and increasing skeletal muscle mass.¹⁵ We tested the hypothesis that PF-354 antibody mediated myostatin inhibition would improve the pathology and function of the diaphragm muscle of young and adult mdx mice.     

Delayed Cardiomyopathy in Dystrophin Deficient mdx Mice Relies on Intrinsic Glutathione Resource

Oxidative stress contributes to the pathogenesis of Duchenne muscular dystrophy (DMD). Although they have been a model for DMD, mdx mice exhibit slowly developing cardiomyopathy. We hypothesized that disease process was delayed owing to the development of an adaptive mechanism against oxidative stress, involving glutathione synthesis. At 15 to 20 weeks of age, mdx mice displayed a 33% increase in blood glutathione levels compared with age-matched C57BL/6 mice. In contrast, cardiac glutathione content was similar in mdx and C57BL/6 mice as a result of the balanced increased expression of glutamate cysteine ligase catalytic and regulatory subunits ensuring glutathione synthesis in the mdx mouse heart, as well as increased glutathione peroxidase-1 using glutathione. Oral administration from 10 weeks of age of the glutamate cysteine ligase inhibitor, l-buthionine(S,R)-sulfoximine (BSO, 5 mmol/L), led to a 33% and 50% drop in blood and cardiac glutathione, respectively, in 15- to 20-week-old mdx mice. Moreover, 20-week-old BSO-treated mdx mice displayed left ventricular hypertrophy associated with diastolic dysfunction, discontinuities in β-dystroglycan expression, micronecrosis and microangiopathic injuries. Examination of the glutathione status in four DMD patients showed that three displayed systemic glutathione deficiency as well. In conclusion, low glutathione resource hastens the onset of cardiomyopathy linked to a defect in dystrophin in mdx mice. This is relevant to the glutathione deficiency that DMD patients may suffer.Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by a defect in dystrophin, a subsarcolemmal structural protein of the cardiac and skeletal muscles that contributes to the integrity of the cellular membrane.¹ Dystrophin-deficient mdx mice are the most commonly used experimental model of DMD.^2,3,4,5,6,7 In DMD patients, as well as in mdx mice, dystrophin defect alters the sarcolemmal integrity of cardiac and skeletal myocytes,^8,9,10 with increased susceptibility to oxidative stress^11,12,13 leading to necrotic cell death and inflammation.^14,15 Further evidence in support of oxidative stress as a cause of the dystrophic pathophysiology comes from in vivo studies demonstrating reduced signs of muscle damages in mdx mice given antioxidants.^16,17The tripeptide glutathione (l-gamma glutamyl-cysteinyl-glycine) is a major antioxidant, which plays a central role in cell redox regulation and controls multiple cellular processes. In particular, glutathione, through the inhibition of the neutral sphingomyelinase-dependent apoptotic pathway,^18,19 determines cardiac myocyte function and survival.^20,21,22 Alterations in glutathione homeostasis and metabolism are associated with inflammation²³ and characterize several human inflammatory chronic diseases.^24,25 Thus, fibroblasts from DMD patients show a low capacity for glutathione synthesis.²⁶ In contrast, a noticeable feature of dystrophic muscles in mdx mice is the up-regulation of glutathione peroxidase and glutathione reductase, which respectively use and recycle reduced glutathione.²⁷ However, this protective mechanism does not entirely compensate the chronic oxidative challenge since glutathione levels in 6- to 8-week-old mdx mice tibialis anterior muscles are decreased by 20% compared with control mice.²⁷ Accordingly, green tea polyphenols, which beyond their inherent antioxidant properties, promote glutathione synthesis, and reduce muscle damage and necrosis in mdx mice.^16,17 This does not rule out that the intrinsic increased turnover of glutathione in mdx mice may delay the progression of the dystrophic disease, although it cannot fully protect against it.Cardiac manifestations of DMD are recognized later than those of skeletal muscles, but are present in 90% of DMD patients by the age of 18 years, progressing to fatal heart failure in 20% of patients.²⁸ Similar to DMD patients, mdx mice experience a progressive development of cardiac defects. However, the disease develops with less severity than in patients and in old mice only; young adult mice show few echocardiographic signs of cardiomyopathy before 10 months of age.^7,29 Glutathione plays a central role in protecting cardiac vasculature,³⁰ and cardiac glutathione deficiency is a common feature of cardiomyopathies in animal models,^20,21 as in patients.^21,22,31We hypothesized that the progression of cardiomyopathy in mdx mice was slow owing to a boost in glutathione synthesis and resources, set as an adaptative mechanism against oxidative stress. Mice administered oral l-buthionine sulfoximine (BSO), a specific inhibitor of glutamate cysteine ligase (GLCL), the rate-limiting enzyme of glutathione synthesis, are a suitable, well-characterized model for glutathione deficiency.³² From 10 weeks of age, mdx mice experience a continuing cycle of skeletal muscle fiber degeneration and regeneration.^33,34 We chose this age to start a 10-week treatment with BSO added to the drinking water at a low dose (5 mmol/L) devoid of toxicity.³² At variance from 20-week-old untreated mdx mice receiving water, 20-week-old BSO-treated mdx mice displayed cardiac left ventricular (LV) structural abnormalities and hypertrophy together with altered diastolic function, reminiscent of the early stage of the cardiomyopathy in DMD patients before the manifestations of clinical symptoms.^35,36 To get a first insight into the glutathione status of DMD patients, we measured blood glutathione in four DMD patients with mean age of 28 ± 2 years and on wheelchair dependency. We found that three of them demonstrated systemic glutathione deficiency.     

Inhibition of Prostaglandin D Synthase Suppresses Muscular Necrosis

Duchenne muscular dystrophy is a fatal muscle wasting disease that is characterized by a deficiency in the protein dystrophin. Previously, we reported that the expression of hematopoietic prostaglandin D synthase (HPGDS) appeared in necrotic muscle fibers from patients with either Duchenne muscular dystrophy or polymyositis. HPGDS is responsible for the production of the inflammatory mediator, prostaglandin D₂. In this paper, we validated the hypothesis that HPGDS has a role in the etiology of muscular necrosis. We investigated the expression of HPGDS/ prostaglandin D₂ signaling using two different mouse models of muscle necrosis, that is, bupivacaine-induced muscle necrosis and the mdx mouse, which has a genetic muscular dystrophy. We treated each mouse model with the HPGDS-specific inhibitor, HQL-79, and measured both necrotic muscle volume and selected cytokine mRNA levels. We confirmed that HPGDS expression was induced in necrotic muscle fibers in both bupivacaine-injected muscle and mdx mice. After administration of HQL-79, necrotic muscle volume was significantly decreased in both mouse models. Additionally, mRNA levels of both CD11b and transforming growth factor β1 were significantly lower in HQL-79-treated mdx mice than in vehicle-treated animals. We also demonstrated that HQL-79 suppressed prostaglandin D₂ production and improved muscle strength in the mdx mouse. Our results show that HPGDS augments inflammation, which is followed by muscle injury. Furthermore, the inhibition of HPGDS ameliorates muscle necrosis even in cases of genetic muscular dystrophy.Duchenne muscular dystrophy (DMD) is one of the most common types of muscular dystrophy, affecting approximately 1 out of 3500 boys.¹ Progressive muscular dystrophy in DMD is caused by membrane vulnerability,² which results from a defect in the muscle protein dystrophin,^3,4 but the precise pathophysiology of the disease progression is not known. There is still no complete cure for this disastrous disease, albeit gene transfer has been extensively tried in mammalian models. Glucocorticoids^5,6 and their analogs⁷ are effective in suppressing the disease only to some degree. In DMD, these steroids reduce the infiltration of inflammatory cells into the muscle⁸ and down-regulate the expression of genes involved in the immune response.⁹ These data suggest inflammation may play a role in the progression of the disease.Earlier we reported the expression of hematopoietic prostaglandin (PG) D synthase (HPGDS), the enzyme responsible for the production of PGD₂,¹⁰ in necrotic muscle fibers, mainly in the focus of grouped necrosis, in patients with DMD or polymyositis.¹¹ We recently reported that overproduction of PGD₂ produced by HPGDS aggravates inflammation and causes profound tissue damage in twitcher, a genetic demyelinating mouse model.¹² The biosynthesis of PGs was also suppressed by glucocorticoids, via suppression of enzymes in the overall synthesis of PGs including phospholipase A₂ and cyclooxygenase. PGD₂ mediates inflammatory responses through two specific receptors, DP1¹³ and DP2,¹⁴ causing peripheral vasodilatation, augmentation of vascular permeability, and chemotaxis.¹⁵ Based on these findings, we hypothesized that HPGDS augments the inflammation that is followed by the muscle injury, especially in the foci of grouped necrosis. Here, using bupivacaine hydrochloride (BPVC)-induced muscle necrosis, where sequences of muscle necrosis are similar to that of progressive muscular dystrophy,¹⁶ and the mdx mouse as a DMD model, we clarified the role of PGD₂ in the pathogenesis and investigated the therapeutic potentials of blockade of HPGDS/PGD₂/DP signaling on the muscular necrosis.     

Myotendinous Junction Defects and Reduced Force Transmission in Mice that Lack α7 Integrin and Utrophin

The α7β1 integrin, dystrophin, and utrophin glycoprotein complexes are the major laminin receptors in skeletal muscle. Loss of dystrophin causes Duchenne muscular dystrophy, a lethal muscle wasting disease. Duchenne muscular dystrophy-affected muscle exhibits increased expression of α7β1 integrin and utrophin, which suggests that these laminin binding complexes may act as surrogates in the absence of dystrophin. Indeed, mice that lack dystrophin and α7 integrin (mdx/α7^−/−), or dystrophin and utrophin (mdx/utr^−/−), exhibit severe muscle pathology and die prematurely. To explore the contribution of the α7β1 integrin and utrophin to muscle integrity and function, we generated mice lacking both α7 integrin and utrophin. Surprisingly, mice that lack both α7 integrin and utrophin (α7/utr^−/−) were viable and fertile. However, these mice had partial embryonic lethality and mild muscle pathology, similar to α7 integrin-deficient mice. Dystrophin levels were increased 1.4-fold in α7/utr^−/− skeletal muscle and were enriched at neuromuscular junctions. Ultrastructural analysis revealed abnormal myotendinous junctions, and functional tests showed a ninefold reduction in endurance and 1.6-fold decrease in muscle strength in these mice. The α7/utr^−/− mouse, therefore, demonstrates the critical roles of α7 integrin and utrophin in maintaining myotendinous junction structure and enabling force transmission during muscle contraction. Together, these results indicate that the α7β1 integrin, dystrophin, and utrophin complexes act in a concerted manner to maintain the structural and functional integrity of skeletal muscle.Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease that affects 1 in every 3500 live male births. Patients with DMD have impaired mobility, are restricted to a wheelchair by their teens, and die from cardiopulmonary failure in their early twenties.^1,2 Currently, there is no cure or effective treatment for this devastating disease. Mutations in the dystrophin gene resulting in loss of the dystrophin protein are the cause of disease in DMD patients and the mdx mouse model.^3,4,5,6,7The dystrophin glycoprotein complex links laminin in the extracellular matrix to the actin cytoskeleton. The N-terminal region of dystrophin interacts with cytoskeletal F-actin⁸ and the C-terminal region associates with the dystrophin-associated protein complex, which include α- and β-dystroglycan, α- and β-syntrophin, the sarcoglycans, and sarcospan.⁹ In DMD, the absence of dystrophin leads to disruption of the dystrophin glycoprotein complex, resulting in increased muscle fragility and altered cell signaling.⁹ Loss of this critical transmembrane linkage complex in DMD patients and mdx mice results in progressive muscle damage and weakness, inflammation, necrosis, and fibrosis. Lack of dystrophin also leads to abnormalities at myotendinous and neuromuscular junctions (MTJ and NMJ), which further contribute to skeletal muscle damage.^{10,11,12,13,14,15,16,17} In addition, defective muscle repair in DMD patients eventually results in muscle degeneration exceeding the rate of regeneration.¹⁸ Overall, dystrophin is critical for muscle function, structure, and stability, and its absence results in progressive muscle wasting and severe muscular dystrophy. In the absence of dystrophin two additional laminin-binding receptors, the α7β1 integrin and utrophin, are up-regulated in the skeletal muscle of DMD patients and mdx mice, which may compensate for the loss of the dystrophin glycoprotein complex.^19,20,21The α7β1 integrin is a heterodimeric laminin receptor involved in bidirectional cell signaling and is localized at junctional and extrajunctional sites in skeletal muscle.^22,23 At least six α7 integrin isoforms produced by developmentally regulated RNA splicing are expressed in skeletal muscle.²⁴ Mutations in the α7 integrin gene (ITGA7) cause myopathy in humans.²⁵ Mice lacking the α7 integrin develop myopathy, exhibit vascular smooth muscle defects and have altered extracellular matrix deposition.^{26,27,28,29,30} The observation that the α7β1 integrin is elevated in the muscle of DMD patients and mdx mice led to the hypothesis that the α7β1 integrin may compensate for the loss of dystrophin.¹⁹ Enhanced expression of the α7 integrin in the skeletal muscle of severely dystrophic mice reduced muscle pathology and increased lifespan by threefold.^10,11 In contrast, loss of both dystrophin and α7 integrin in mice results in severe muscular dystrophy and premature death by 4 weeks of age.^28,31 The α7β1 integrin is therefore a major modifier of disease progression in DMD.The utrophin glycoprotein complex is a third major laminin receptor in skeletal muscle. Utrophin has significant sequence homology to dystrophin.^32,33 In normal adult muscle utrophin is restricted to neuromuscular and myotendinous junctions.³⁴ During development or in damaged or diseased muscle, utrophin expression is increased and becomes localized at extrajunctional sites.^35,36 Utrophin interacts with the same proteins as dystrophin, but binds to actin filaments at different sites.³⁷ In mice, loss of utrophin results in a mild form of myasthenia with reduced sarcolemmal folding at the postsynaptic membrane of the neuromuscular junction.^12,15 Transgenic overexpression of utrophin has been shown to rescue mdx mice.³⁸ Mice that lack both dystrophin and utrophin exhibit severe muscular dystrophy and die by 14 weeks of age.^13,14 Thus, utrophin is also a major laminin receptor that modifies disease progression in DMD.To understand the functional overlap between the α7β1 integrin and utrophin in skeletal muscle, we produced mice that lack both α7 integrin and utrophin (α7/utr^−/−). Since both complexes are highly enriched at the MTJ and NMJ, we hypothesized that α7/utr^−/− mice may have severe abnormalities at these critical junctional sites. Our study demonstrates α7/utr^−/− mice exhibit partial embryonic lethality comparable with that observed in α7^−/− mice. Dystrophin is increased in these animals and enriched at the NMJ but not the MTJ. α7/utr^−/− mice display ultrastructural defects in their MTJ and compromised force transmission. Together, these results indicate that the α7β1 integrin, dystrophin and utrophin laminin binding complexes provide continuity between laminin in the extracellular matrix and the cell cytoskeleton, which are necessary for the normal structural and functional properties of skeletal muscle.     

Mast Cell-Derived TNF Can Exacerbate Mortality during Severe Bacterial Infections in C57BL/6-KitW-sh/W-sh Mice

We used mast cell-engrafted genetically mast cell-deficient C57BL/6-Kit^W-sh/W-sh mice to investigate the roles of mast cells and mast cell-derived tumor necrosis factor in two models of severe bacterial infection. In these mice, we confirmed findings derived from studies of mast cell-deficient WBB6F₁-Kit^W/W-v mice indicating that mast cells can promote survival in cecal ligation and puncture (CLP) of moderate severity. However, we found that the beneficial role of mast cells in this setting can occur independently of mast cell-derived tumor necrosis factor. By contrast, using mast cell-engrafted C57BL/6-Kit^W-sh/W-sh mice, we found that mast cell-derived tumor necrosis factor can increase mortality during severe CLP and can also enhance bacterial growth and hasten death after intraperitoneal inoculation of Salmonella typhimurium. In WBB6F₁-Kit^W-sh/W-sh mice, mast cells enhanced survival during moderately severe CLP but did not significantly change the survival observed in severe CLP. Our findings in three types of genetically mast cell-deficient mice thus support the hypothesis that, depending on the circumstances (including mouse strain background, the nature of the mutation resulting in a mast cell deficiency, and type and severity of infection), mast cells can have either no detectable effect or opposite effects on survival during bacterial infections, eg, promoting survival during moderately severe CLP associated with low mortality but, in C57BL/6-Kit^W-sh/W-sh mice, increasing mortality during severe CLP or infection with S. typhimurium.The factors determining whether particular infections will be successfully controlled or progress to death are incompletely understood. Studies conducted using genetically mast cell-deficient (WB/ReJ × C57BL/6)F₁-Kit^W/W-v mice (WBB6F₁-Kit^W/W-v mice), the corresponding normal (WBB6F₁-Kit^+/+) mice, and mast cell-engrafted WBB6F₁-Kit^W/W-v mice have indicated that mast cells can increase survival during various models of bacterial infection of moderate severity,^{1,2,3,4,5,6,7,8,9,10,11,12,13} defined herein as infections that result in relatively low mortality in normal mice. Although mast cells can contribute to host defense against bacteria by multiple direct and indirect mechanisms,^{1,2,3,4,5,7,8,9,10,11,12,13} several groups have focused on the potential role of mast cell-derived tumor necrosis factor (TNF) in such settings.^1,2,3,4,5,7 Results obtained in work using TNF-deficient mice⁴ or mice in which the actions of TNF are blocked by neutralizing antibodies^1,2 have clearly demonstrated that TNF can have protective functions during some bacterial infections, and that such TNF-dependent effects may include the enhancement of neutrophil recruitment and/or function and the promotion of bacterial clearance.However, such genetic or neutralizing antibody-based approaches eliminate or reduce the function of TNF derived from all cellular sources, not just mast cell-derived TNF. Thus, there has been no direct evidence yet published that shows that mast cells represent a critical source of TNF in such settings. Moreover, WBB6F₁-Kit^W/W-v mice have a modest deficiency in neutrophils,^14,15,16 and this abnormality, as well as their virtual lack of mast cells, might contribute to the increased pathology observed in these mice during certain models of bacterial infection. Therefore, in the present study, we used genetically mast cell-deficient C57BL/6-Kit^W-sh/W-sh mice (produced as in ref.¹⁷) engrafted with either wild-type mast cells or mast cells unable to produce TNF to re-examine the roles of mast cells, and mast cell-derived TNF, in two different models of bacterial infection.Adult C57BL/6-Kit^W-sh/W-sh mice are profoundly mast cell-deficient,^17,18,19 as are WBB6F₁-Kit^W/W-v mice,^20,21 but, unlike WBB6F₁-Kit^W/W-v mice, C57BL/6-Kit^W-sh/W-sh mice have been reported to have increased levels of blood and bone marrow neutrophils.^15,16 We used both WBB6F₁-Kit^W/W-v mice and C57BL/6-Kit^W-sh/W-sh mice to investigate the role of mast cells in moderately severe versus severe cecal ligation and puncture (CLP), with moderately severe versus severe CLP defined herein as models of CLP in which no more than 50% (in the moderately severe CLP model) vs. more than 50% (in the severe CLP model) of normal mice succumb during the first 4 days after surgery to induce acute bacterial peritonitis. Our observations in C57BL/6-Kit^W-sh/W-sh mice confirm work in WBB6F₁-Kit^W/W-v mice^1,3,4,5,6,8 in providing evidence that mast cells can enhance host resistance and survival during moderately severe CLP, a well known model of mouse bacterial peritonitis, but provide evidence that mast cells can enhance survival in this model independently of their ability to produce TNF. By contrast, mast cell-derived TNF increased mortality during severe CLP in C57BL/6-Kit^W-sh/W-sh mice. We found that mast cell-derived TNF also can hasten mortality in C57BL/6-Kit^W-sh/W-sh mice subjected to a different model of severe bacterial infection: that induced by the intraperitoneal inoculation of Salmonella typhimurium.     

PTEN Contributes to Profound PI3K/Akt Signaling Pathway Deregulation in Dystrophin-Deficient Dog Muscle

Duchenne muscular dystrophy is the most common and severe form of muscular dystrophy, and although the genetic basis of this disease is well defined, the overall mechanisms that define its pathogenesis remain obscure. Alterations in individual signaling pathways have been described, but little information is available regarding their putative implications in Duchenne muscular dystrophy pathogenesis. Here, we studied the status of various major signaling pathways in the Golden Retriever muscular dystrophy dog that specifically reproduces the full spectrum of human pathology. Using antibody arrays, we found that Akt1, glycogen synthase kinase-3β (GSK3β), 70-kDa ribosomal protein S6 kinase (p70S6K), extracellular signal-regulated kinases 1/2, and p38δ and p38γ kinases all exhibited decreased phosphorylation in muscle from a 4-month-old animal with Golden Retriever muscular dystrophy, revealing a deep alteration of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase pathways. Immunohistochemistry analysis revealed the presence of muscle fibers exhibiting a cytosolic accumulation of Akt1, GSK3β, and phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (PTEN), an enzyme counteracting PI3K-mediated Akt activation. Enzymatic assays established that these alterations in phosphorylation and expression levels were associated with decreased Akt and increased GSK3β and PTEN activities. PTEN/GSK3β-positive fibers were also observed in muscle sections from 3- and 36-month-old animals, indicating long-term PI3K/Akt pathway alteration. Collectively, our data suggest that increased PTEN expression and activity play a central role in PI3K/Akt/GSK3β and p70S6K pathway modulation, which could exacerbate the consequences of dystrophin deficiency.Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disorder that affects 1 newborn boy in 3500. This recessive disease is caused by mutations in the dystrophin gene, resulting in total lack of the protein,^1,2,3 and is characterized by severe degeneration of muscle fibers, progressive paralysis, and death. Dystrophin is located under the sarcolemma of muscle fibers, and is associated with a complex comprising several integral, peripheral membrane and cytoplasmic proteins: the dystrophin-glycoprotein complex (DGC).^4,5,6,7 By providing a strong physical link between the cytoskeleton network and the extracellular matrix, the DGC ensures the integrity of skeletal muscle fibers. In the absence of dystrophin, the complex is destabilized and this integrity is lost.^5,8 However, the impaired structural role of the DGC alone may not be sufficient to account for the massive degenerative process observed in DMD muscles. Numerous observations suggest that signaling pathway alterations may also participate in DMD pathogenesis.Dystrophin and various DGC proteins have been demonstrated to interact with a number of signaling proteins, including growth factor receptor-bound protein 2,⁹ neuronal nitric oxide synthase,¹⁰ calmodulin,¹¹ focal adhesion kinase,¹² and caveolin-3.^13,14,15 Moreover, studies of the X chromosome-linked muscular dystrophy (mdx) mouse¹⁶ revealed modulations in mitogen-activated protein kinase (MAPK) signaling cascades, as dystrophic animals exhibited increased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2)^17,18 and c-jun N-terminal kinases 1 and 2 (JNK1/2),^19,20,21 and decreased phosphorylation of p38.¹⁸ Also, the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway has been shown to be affected in the mdx mouse, with increased synthesis and phosphorylation of Akt.^22,23In addition to the limited information related to the origin of signal perturbations in dystrophic muscle, almost no information is available regarding signaling pathways in clinically relevant animal models or human tissue samples.²³ It is noteworthy that the mdx mouse model of DMD is characterized by successive degeneration/regeneration processes, but does not exhibit the progressive muscle wasting and accumulation of connective tissue observed during the development of the human disease.^24,25,26 The Golden Retriever muscular dystrophy (GRMD) dog, characterized by rapidly progressive clinical dysfunction, severe muscle weakness, and abundant fiber necrosis, displays a disease progression that is far more similar to human DMD.^27,28In this study, we used antibody arrays to assess the global phosphorylation status of key proteins of the PI3K/Akt and MAPK signaling pathways in skeletal muscles of 4-month-old healthy and GRMD dogs. Our data indicated that Akt1, glycogen synthase kinase-3β (GSK3β) and p70S6K, as well as ERK1/2 and p38δ and γ kinases all displayed a decreased phosphorylation level in GRMD muscle. Western immunoblot, immunohistochemistry analysis, and enzymatic assays allowed us to confirm these results and demonstrated that they were associated with a reduction in Akt activity and with enhanced GSK3β expression and activity. Analysis of key enzymes involved in Akt regulation revealed that phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (PTEN) was present at a much higher level and was more active in GRMD muscle. Moreover, immunohistochemistry analysis showed that all of the GSK3β-positive fibers observed in GRMD muscle sections exhibited a strong cytosolic labeling of PTEN, suggesting that the accumulation of the phosphatase could play a central role in PI3K/Akt signaling pathway deregulation. The observation of PTEN/GSK3β-positive fibers in muscle sections from 3- and 36-month-old GRMD dogs further demonstrated that both the early and late stages of the disease share deregulation of the pathway. Collectively, our findings highly suggest that alterations in PTEN exist in GRMD muscle, which leads to long-term and deep modulation of the PI3K/Akt signaling pathway.     

Therapeutic Potential of Proteasome Inhibition in Duchenne and Becker Muscular Dystrophies

Duchenne muscular dystrophy (DMD) and its milder allelic variant, Becker muscular dystrophy (BMD), result from mutations of the dystrophin gene and lead to progressive muscle deterioration. Enhanced activation of proteasomal degradation underlies critical steps in the pathogenesis of the DMD/BMD dystrophic process. Previously, we demonstrated that treatment with the proteasome inhibitor MG-132 rescues the cell membrane localization of dystrophin and the dystrophin glycoprotein complex in mdx mice, a natural genetic mouse model of DMD. The current work aims to thoroughly define the therapeutic potential in dystrophinopathies of Velcade, a drug that selectively blocks the ubiquitin-proteasome pathway. Velcade is particularly intriguing since it has been approved for the treatment of multiple myeloma. Therefore, its side effects in humans have been explored. Velcade effects were analyzed through two independent methodological approaches. First, we administered the drug systemically in mdx mice over a 2-week period. In this system, Velcade restores the membrane expression of dystrophin and dystrophin glycoprotein complex members and improves the dystrophic phenotype. In a second approach, we treated with the compound explants from muscle biopsies of DMD or BMD patients. We show that the inhibition of the proteasome pathway up-regulates dystrophin, α-sarcoglycan, and β-dystroglycan protein levels in explants from BMD patients, whereas it increases the proteins of the dystrophin glycoprotein complex in DMD cases.Duchenne muscular dystrophy (DMD) is an incurable inherited disease, characterized by progressive muscle degeneration and weakness. The pathology results from the mutation of the DMD gene, which leads to deficiency of dystrophin, a 427-kDa protein found throughout the cytoplasmic face of the plasma membrane in both skeletal and cardiac muscle. Becker muscular dystrophy (BMD), a much milder allelic form of the disease, is caused by a reduction in the amount, or an alteration in the size, of dystrophin protein. In muscle cells, dystrophin binds through its amino-terminus to cytoskeletal F-actin and through the carboxyl-terminus to β-dystroglycan, a transmembrane component of a multimolecular complex [the dystrophin-glycoprotein complex (DGC)], which includes α, β, γ, δ-sarcoglycan, α, β-dystroglycan, the syntrophins, and dystrobrevin. In the DGC, dystroglycan appears to play a critical role, since it connects dystrophin with the DGC and it also binds laminin in the extracellular space, thus creating a mechanical linkage between cytoskeleton and extracellular matrix.¹Dystrophin deficiency is associated with a severe decrease or absence in the expression and membrane localization of the members of the DGC.² It is feasible that dystrophin primary genetic loss leads to plasma membrane instability, thus causing a disarray of the multimeric complex and addressing its components to intracellular proteolysis. Indeed, different reports indicated that activation of protein degradation through the ubiquitin-proteasome system occurs in the pathogenesis of the DMD/BMD degenerative process.³Consistently, our initial studies showed that local and systemic treatment with the proteasome inhibitor MG-132 rescues the expression of the DGC in mdx mice, a natural genetic mouse model of DMD.⁴ The effects of MG-132 were confirmed in freshly isolated skeletal muscle biopsies from patients affected by DMD or BMD, thus suggesting a possible therapeutic implication for these agents.⁵In the present study we investigated the effects of the drug Velcade (bortezomib, PS-341), a proteasome inhibitor that belongs to the class of peptide boronates. Velcade is particularly appealing to us for different reasons.First, while MG-132 exerts an inhibitory action on both the proteasome and the calpain system, Velcade displays a selective and high affinity to the proteasome. Therefore, this drug is an optimal probe to test the therapeutic potential of the inhibition of the proteasome pathway in muscular dystrophies. Secondarily, Velcade has been already approved by the Food and Drug Administration and the European Medicines Agency for the treatment of multiple myeloma. Thus, its side effects have been already explored and managed.^6,7 Finally, Velcade is able to reduce the activity of nuclear factor-kappa B (NF-κB).⁸ Because NF-κB pathway has been shown to be involved in inflammation responses in myopathies and DMD, Velcade effects on this signaling molecule may have important clinical implications.⁹Our previous results had indicated that Velcade, once injected locally into the gastrocnemius muscles of mdx mice, could up-regulate the expression and membrane localization of dystrophin and members of the DGC.¹⁰ However, the long-term actions of this proteasome inhibitor and its possible side effects in dystrophic animals are not known yet. To thoroughly evaluate Velcade’s therapeutic potential in dystrophinopathies, we adopted an integrated approach. First, we analyzed its efficacy using a systemic treatment and assessed the rescue of the dystrophic phenotype of mdx mice.The mdx mouse model is characterized by a primary genetic defect of dystrophin due to a missense mutation in exon 23, which leads to a premature stop codon. Skeletal muscle tissue from mdx mutants is histologically normal early in postnatal development, but starting around 3 to 4 weeks of age, muscle necrosis develops, with some visible muscle weakness. Although skeletal limb muscles are characterized by a persistent and progressive degeneration and necrosis, at approximately 6 to 8 weeks of age, the muscle damage appears to plateau and it is possible to observe the beginning of a regenerative response activated by satellite cells, which continues until 12 weeks of age.¹¹Concurrently, we investigated the effects of the drug in human muscle explants of fresh biopsies from patients with DMD and BMD.     

Dual Roles of CD40 on Microbial Containment and the Development of Immunopathology in Response to Persistent Fungal Infection in the Lung

Persistent pulmonary infection with Cryptococcus neoformans in C57BL/6 mice results in chronic inflammation that is characterized by an injurious Th2 immune response. In this study, we performed a comparative analysis of cryptococcal infection in wild-type versus CD40-deficient mice (in a C57BL/6 genetic background) to define two important roles of CD40 in the modulation of fungal clearance as well as Th2-mediated immunopathology. First, CD40 promoted microanatomic containment of the organism within the lung tissue. This protective effect was associated with: i) a late reduction in fungal burden within the lung; ii) a late accumulation of lung leukocytes, including macrophages, CD4⁺ T cells, and CD8+ T cells; iii) both early and late production of tumor necrosis factor-α and interferon-γ by lung leukocytes; and iv) early IFN-γ production at the site of T cell priming in the regional lymph nodes. In the absence of CD40, systemic cryptococcal dissemination was increased, and mice died of central nervous system infection. Second, CD40 promoted pathological changes in the airways, including intraluminal mucus production and subepithelial collagen deposition, but did not alter eosinophil recruitment or the alternative activation of lung macrophages. Collectively, these results demonstrate that CD40 helps limit progressive cryptococcal growth in the lung and protects against lethal central nervous system dissemination. CD40 also promotes some, but not all, elements of Th2-mediated immunopathology in response to persistent fungal infection in the lung.CD40, a 48-kDa type I transmembrane protein and member of the tumor necrosis factor receptor family, is a well-described costimulatory molecule expressed on B cells, dendritic cells (DC), macrophages, basophils, and platelets as well as nonhematopoietic cells including fibroblasts, epithelial, and endothelial cells. The ligand for CD40, known as CD154 or CD40L, is a type II transmembrane protein member of the tumor necrosis factor (TNF) superfamily expressed primarily by activated T cells, B cells, and platelets.^1,2,3 CD40 can be induced on DC, monocytes, and macrophages under inflammatory conditions.^4,5 Signaling via the CD40/CD40L pathway exerts numerous biological effects including: i) increased cytokine expression (especially TNF-α and Th1 cytokines interleukin (IL)-12 and interferon (IFN)-α) and nitric oxide production; ii) upregulation of additional costimulatory molecules (CD80 and CD86) on antigen-presenting cells (APC); iii) enhanced cell survival (particularly of B and T cells, DC, and endothelial cells); iv) Ig isotype switching; and v) somatic hypermutation of Ig.^1,4,5The CD40/CD40L signaling pathway contributes to adaptive Th1 immune responses required to clear Leishmanisa spp.,^6,7,8 Trypanosoma spp.,^6,7,8,9 Shistosoma mansoini,¹⁰ and the fungi Candida albicans¹¹ and Pneumocystis spp.¹² The enhanced production of IFN-γ, TNF-α, and nitric oxide associated with CD40/CD40L signaling is thought to be responsible for this protective effect. However, other studies have suggest that CD40/CD40L signaling is not required for successful host defense against Listeria monocytogenes,^13,14 Toxoplasma gondi,¹⁵ lymphocytic choriomeningitis virus,^16,17 or the fungus Hisoplasma capsulatum.^18,19 In models of Mycobacterium spp. infection, CD40 appears dispensable for clearance of an i.v. infection,^20,21 but essential for clearing the organism in response to aerosolized infection in the lungs.^22,23 Thus, the role of CD40 in antimicrobial host defense varies and depends not only on the specific pathogen but also on the primary site of infection.Cryptococcus neoformans, an opportunistic fungal pathogen acquired through inhalation, causes significant morbidity and mortality primarily in patients with AIDS, lymphoid or hematological malignancies, or patients receiving immunosuppressive therapy secondary to autoimmune disease or organ transplantation.^24,25 Infection in non-immunocompromised patients has been reported.^26,27,28 Murine models of cryptococcal infection in CBA/J or BALB/c mice demonstrate that development of a Th1 antigen-specific immune response characterized by IFN-γ production and classical activation of macrophages is required to eradicate the organism.^{29,30,31,32,33,34,35,36,37,38,39,40} In contrast, a model of persistent cryptococcal infection has been developed using C57BL/6 mice;^{41,42,43,44,45,46,47} this model reflects many features observed in humans diagnosed with allergic bronchopulmonary mycosis.⁴⁸ Specifically, these mice fail to clear the organism from the lung and develop characteristic Th2-mediated immunopathology including: i) tissue eosinophilia; ii) airway hyperreactivity, mucus production, and fibrosis; and iii) alternative macrophage activation associated with YM1 crystal deposition.The molecular mechanisms responsible for the immunopathologic response associated with persistent cryptococcal infection are not clearly defined. These features are abrogated in the absence of IL-4,⁴⁵ whereas more severe Th2-mediated lung injury occurs in the absence of IFN-γ.^29,41 TNF-α exerts a protective effect by enhancing IFN- γ production and the subsequent classical activation of lung macrophages.^31,35,49,50 Lymphocytes are critical mediators of this Th2 response as the pathological features of chronic cryptococcal infection are substantially diminished in CD4 T cell-depleted mice despite no change in fungal clearance.⁴²Although interactions between CD4 T cells and APC are critical determinants of T cell polarization in response to cryptococcal lung infection,^{49,51,52,53,54,55} the contribution of specific costimulatory molecules including the CD40/CD40L signaling pathway has not been fully elucidated. In vitro studies suggest that activation of the CD40/CD40L pathway in response to Cryptococcus promotes IFN-γ production by T cells and TNF-α, and nitric oxide (NO) production by monocytes.⁵⁶ In the absence of CD40L, primary pulmonary infection with a weakly virulent strain of C. neoformans was associated with impaired fungal clearance; however, measurements of immune function at the site of infection in the lung or evidence of systemic fungal dissemination were not evaluated.⁵⁷ The potential to target CD40 therapeutically is highlighted by studies showing that treatment of mice with disseminated or intracerebral cryptococcal infection with an agonist antibody to CD40 in combination with IL-2 improves survival.^58,59 In this study, we used gene-targeted CD40-deficient mice (on a C57BL/6 genetic background), a clinically relevant model, and assessments of immune function and histopathology in the lung to identify two unique roles for the CD40-signaling pathway in response to persistent cryptococcal lung infection.     

mdx(⁵cv) mice manifest more severe muscle dysfunction and diaphragm force deficits than do mdx Mice

Duchenne muscular dystrophy (DMD) is characterized by progressive skeletal muscle dysfunction leading to premature death by the third decade of life. The mdx mouse, the most widely used animal model of DMD, has been extremely useful to study disease mechanisms and to screen new therapeutics. However, unlike patients with DMD, mdx mice have a very mild motor function deficit, posing significant limitations for its use as a platform to assess the impact of treatments on motor function. It has been suggested that an mdx variant, the mdx^5cv mouse, might be more severely affected. Here, we compared the motor activity, histopathology, and individual muscle force measurements of mdx and mdx^5cv mice. Our study revealed that mdx^5cv mice showed more severe exercise-induced fatigue, Rotarod performance deficits, and gait anomalies than mdx mice and that these deficits began at a younger age. Muscle force studies showed more severe strength deficits in the diaphragm of mdx^5cv mice compared to mdx mice, but similar force generation in the extensor digitorum longus. Muscle histology was similar between the two strains. Differences in genetic background (genetic modifiers) probably account for these functional differences between mdx strains. Overall, our findings indicate that the mdx and mdx^5cv mouse models of DMD are not interchangeable and identify the mdx^5cv mouse as a valuable platform for preclinical studies that require assessment of muscle function in live animals.Duchenne muscular dystrophy (DMD), the most prevalent muscular dystrophy, is an X-linked recessive disorder affecting 1 in 3500 male births. DMD is typically diagnosed around 3 years of age with a rapid progression of muscle weakness that results in wheelchair dependence by 12 years of age and death by the third decade of life.¹ Molecularly, DMD stems from mutations in the dystrophin gene that typically result in a lack of dystrophin protein expression in skeletal and cardiac muscles.^2,3 Loss of dystrophin compromises the muscle fiber membrane, leading to cycles of muscle fiber degeneration and regeneration, chronic inflammation, and accumulation of fibrotic tissue.^4–6 Currently, there is no definitive treatment for DMD, but several gene-, cell-, and drug-based therapeutic approaches are being evaluated.^7–12 Assessing and comparing the efficacy of these treatments require an easily available, well-characterized animal model with measurable and reproducible motor deficits, histologic pathology, and physiological alterations of muscle function.The mdx mouse is the most widely studied animal model of DMD and has been extensively used in preclinical studies over the past 20 years. It arose from a spontaneous nonsense mutation in exon 23 of the dystrophin gene in an inbred C57BL/10 background.¹³ The mdx muscles lack dystrophin expression and show histopathological features similar to DMD.^13–16 However, contrary to DMD patients, mdx mice have a milder phenotype, with minimal fibrosis in all muscles with the exception of the diaphragm,¹⁷ a near normal life span,¹⁸ and only minimal motor deficits.^13,19–23 In particular, treatment effect on overall motor function has been difficult to assess, posing a challenge for preclinical studies.^24,25As a result, mdx variants were generated with the hope of obtaining a more severe phenotype.^26–28 However, these alternative DMD mouse models had histopathological features and mild functional impairment similar to the mdx mouse. Among these, mdx^5cv mice harbor a mutation affecting exon 10 that selectively disrupts expression of full-length dystrophin such as in mdx mice.²⁹ However, mdx^5cv mice have recently been preferred to mdx mice because of their very low level of dystrophin-expressing revertant fibers,³⁰ allowing a more accurate quantification of the efficiency of gene and cell therapy in restoring dystrophin expression. In addition, mdx^5cv mice are on the C57BL/6 background that allows rapid transfer of the dystrophin mutation onto transgenic and knock-out mice to dissect molecular aspects of disease.Although mdx and mdx^5cv mice have been generally regarded as similar, a recent study found significant differences in gene expression profiles in all muscle groups examined, and a qualitative histologic examination suggested a more severe pathology in mdx^5cv mice.³¹ We therefore sought to determine whether mdx^5cv mice have more severe motor function deficits that render them distinct from mdx mice and better suited to study the effect of treatments on motor function and endurance.     

Increased Resistance to Taenia crassiceps Murine Cysticercosis in Qa-2 Transgenic Mice

We previously reported important differences in resistance to Taenia crassiceps murine cysticercosis between BALB/c substrains. It was suggested that resistance might correlate with expression of the nonclassic class I major histocompatibility complex (MHC) Qa-2 antigen; BALB/cAnN is Qa-2 negative and highly susceptible to T. crassiceps, whereas BALB/cJ expresses Qa-2 and is highly resistant. In this study, we investigated the role of Qa-2 in mediating resistance to cysticercosis by linkage analysis and infection of Qa-2 transgenic mice. In BALB/cAnN × (C57BL/6J × BALB/cAnN)F₁ and BALB/cAnN × (BALB/cJ × BALB/cAnN)F₁ backcrosses, the expression of Qa-2 antigen correlated with resistance to cysticercosis. Significantly fewer parasites were recovered from infected Qa-2 transgenic male and female mice than from nontransgenic mice of similar genetic background. These results clearly demonstrate that the Qa-2 MHC antigen is involved in resistance to T. crassiceps cysticercosis.

Taenia solium cysticercosis is a parasitic disease that seriously affects human health (24) and causes important economic losses in pig farming of developing countries (1) where conditions that favor parasite transmission persist. The essential role of pigs as an obligatory intermediate host in the parasite life cycle offers the opportunity to interfere with transmission by inducing acquired immunity through vaccination (10, 13, 18), by decreasing susceptibility through genetic manipulation (14), or both. Systematic exploration of the role of genetic factors in cysticercosis and the identification of protective immunogens are hampered by the high costs and slow data retrieval involved in studies with pigs. However, another cestode, Taenia crassiceps, that naturally infects rodents (3) is highly suitable for experimentation. It shows extensive antigenic cross-reactivity and cross-protective immunity with T. solium (7, 21); the antigenic similarity is such that T. crassiceps antigens can be used for immunodiagnosis of human cysticercosis (9). Furthermore, T. crassiceps and T. solium both have a typical two-host taeniid life cycle and morphologically and structurally related larval stages. Since T. crassiceps can reproduce asexually, experimental infection is readily attained by injecting the cysticerci in the peritoneal cavity of the mouse (3). Thus, T. crassiceps murine cysticercosis has been shown to be a useful experimental model of metacestode infection in the study of genetic factors involved in host resistance (2, 20) and underlying immunological mechanisms (19, 23, 25).Initial findings showed that genes linked to H-2 affect T. crassiceps growth in mice (20). Thus, significant differences in the extent of the parasitosis were found between mice carrying the H-2^d (BALB/cAnN and DBA/2) haplotype, which were the most susceptible, and mice with H-2^b (BALB/B, C57BL/6J, and C57BL/10J) or H-2^k (BALB/K, C3H/HeJ, and C3H/FeJ) haplotype, which were comparatively resistant. Further studies (2) showed low susceptibility of congenic and recombinant B10 mice, regardless of H-2 haplotype, indicating that genes in C57BL background confer resistance to the parasitosis such that they override the effect of H-2. The effect of genes outside H-2 on the control of parasite growth was also revealed by the differential susceptibility of three H-2^d BALB/c substrains, of which BALB/cAnN was highly susceptible, whereas BALB/cJ was highly resistant and BALB/cByJ displayed and intermediate degree of susceptibility (2). BALB/cAnN and BALB/cJ, which are genetically quite similar strains, differ in several phenotypes, including the expression of the Qa-2 antigen (11, 16). This antigen is a nonclassical class I major histocompatibility complex (MHC) molecule encoded by four genes (Q6 to Q9) located telomeric to the H-2D loci (11, 22). BALB/cJ (Qa-2^low), a Qa-2 expressor substrain, has only active Q6 and Q7 genes because Q8 and Q9 have fused, resulting in an inactive Q8/Q9^d gene (11, 15). In BALB/cAnN (Qa-2^null), an additional deletion of genomic DNA has occurred between the Q6 and Q7 genes, leading to their inactivation and accounting for the Qa-2 null expression (11). We proposed previously that differences in susceptibility to T. crassiceps observed between BALB/cJ and BALB/cAnN might be related to Qa-2 antigen expression (2). Here we describe that results of genetic linkage studies are entirely consistent with this hypothesis. Furthermore, a role of Qa-2 in mediating resistance to T. crassiceps was directly established by the diminution of parasite loads in infected Qa-2 transgenic mice.     

Chemokine CXCL16 Regulates Neutrophil and Macrophage Infiltration into Injured Muscle,Promoting Muscle Regeneration

Only a few specific chemokines that mediate interactions between inflammatory and satellite cells in muscle regeneration have been identified. The chemokine CXCL16 differs from other chemokines because it has both a transmembrane region and active, soluble chemokine forms. Indeed, we found increased expression of CXCL16 and its receptor, CXCR6, in regenerating myofibers. Muscle regeneration in CXCL16-deficient (CXCL16KO) mice was severely impaired compared with regeneration in wild-type mice. In addition, there was decreased MyoD and myogenin expression in regenerating muscle in CXCL16KO mice, indicating impaired satellite cell proliferation and differentiation. After 1 month, new myofibers in CXCL16KO mice remained significantly smaller than those in muscle of wild-type mice. To understand how CXCL16 regulates muscle regeneration, we examined cells infiltrating injured muscle. There were more infiltrating neutrophils and fewer macrophages in injured muscle of CXCL16KO mice compared with events in wild-type mice. Moreover, absence of CXCL16 led to different expression of cytokines/chemokines in injured muscles: mRNAs of macrophage-inflammatory protein (MIP)-1α, MIP-1β, and MIP-2 were increased, whereas regulated on activation normal T cell expressed and secreted, T-cell activation-3, and monocyte chemoattractant protein-1 mRNAs were lower compared with results in muscles of wild-type mice. Impaired muscle regeneration in CXCL16KO mice also resulted in fibrosis, which was linked to transforming growth factor-β1 expression. Thus, CXCL16 expression is a critical mediator of muscle regeneration, and it suppresses the development of fibrosis.Skeletal muscle regeneration following injury involves proliferation and differentiation of satellite cells leading to the formation of new myofibers.¹ The regeneration process initially involves infiltration of inflammatory cells into injured muscle, including neutrophils, monocytes and macrophages; these accumulate in response to cytokines and chemokines.² This is important because the types of infiltrating cells influence the severity of the injury and the regeneration processes. For example, when neutrophils were depleted by administering an antibody, muscle regeneration following lipopolysaccharide-induced muscle fiber damage was accelerated.³ Neutrophil infiltration was emphasized because these cells cause tissue damage by processes that are related to the production of reactive oxygen species.^4,5,6 The respiratory bursts from infiltrating leukocytes produce oxidizing reactions that damage cells during the early inflammatory period. Indeed, neutrophils obtained from humans or rodents were shown to damage cell membranes of C2C12 myotubes.⁷In contrast to the adverse influence of infiltrating neutrophils on injured muscle, infiltration of monocytes/macrophages can be beneficial.^8,9,10,11,12 For example, when macrophage infiltration into injured muscle was suppressed, muscle regeneration was sharply impaired and this was associated with the development of muscle fibrosis.^13,14 Macrophages not only remove necrotic myofibers by phagocytosis, they also release cytokines as well as growth factors including hepatocyte growth factor, insulin-like growth factor-1, fibroblast growth factor, and tumor necrosis factor-α.^8,9,10,12,15 Release of these cytokines and growth factors stimulate satellite cells, which are closely linked to the processes of muscle regeneration.The recruitment of neutrophils and macrophages into injured muscles is at least partially mediated by chemokines, and consequently, their influence has been examined extensively. For example, the reports of Warren et al¹⁵ and Shireman et al¹⁶ provided the critical evidence that the CC chemokine, monocyte chemoattractant protein-1 (MCP-1), and its receptor, CCR2, were critical for the regeneration processes occurring in injured muscle. Specifically, knocking out of the CCR2 receptor or blocking the action of MCP-1 significantly delayed the muscle regeneration occurring in injured tissue. There is evidence, however, that changes in the expression of cytokines besides MCP-1 contribute to muscle regeneration.¹⁷Structurally and functionally, CXCL16 differs from MCP-1 and other chemokines.¹⁸ MCP-1 and the majority of other chemokines are small molecules secreted by inflammatory cells, whereas CXCL16 is synthesized as a transmembrane multidomain molecule consisting of a chemokine domain plus a glycosylated mucin-like stalk linked to a single transmembrane helix. There are two forms of CXCL16 resulting from cleavage at the cell surface. The soluble form of CXCL16 is composed of the extracellular stalk and the chemokine domain. It functions as chemoattractant to promote cell migration and changes in the functions of recruited cells.¹⁹ The remaining transmembrane structure of CXCL16 interacts with its receptor, CXCR6, to establish cell to cell adhesion. Indeed, CXCR6 is expressed on several types of inflammatory cells including macrophages.^{18,20,21,22,23,24,25,26} Previously, we found that inhibition of CXCL16 significantly reduces the infiltration of macrophages into the kidney of rats with anti-glomerular basement membrane antibody-associated glomerulonephritis.²⁷ Given the unique features of CXCL16 and the importance of macrophages in the processes of muscle regeneration, we studied the role of CXCL16 in regulating muscle regeneration. We studied CXCL16 knockout (CXCL16KO) mice using a standard model of muscle injury and regeneration, cardiotoxin injection into tibialis anterior (TA) muscles. Our results reveal that CXCL16 is critical for recruitment of macrophages, which are essential for satellite cell proliferation and differentiation in vivo.     

Carbon Monoxide Rescues Heme Oxygenase-1-Deficient Mice from Arterial Thrombosis in Allogeneic Aortic Transplantation

Heme oxygenase-1 (HO-1) catalyzes the conversion of heme into carbon monoxide (CO), iron, and biliverdin. In preliminary studies, we observed that the absence of HO-1 in aortic allograft recipients resulted in 100% mortality within 4 days due to arterial thrombosis. In contrast, recipients normally expressing HO-1 showed 100% graft patency and survival for more than 56 days. Abdominal aortic transplants were performed using Balb/cJ mice as donors and either HO-1^+/+ or HO-1^−/− (C57BL/6×FVB) mice as recipients. Light and electron microscopy revealed extensive platelet-rich thrombi along the entire length of the graft in HO-1^−/− recipients at 24 hours. Treatment of recipients with CORM-2, a CO-releasing molecule (10 mg/kg of body weight intravenously), 1 hour prior and 1, 3, and 6 days after transplantation, significantly improved survival (62% at >56 days, P < 0.001) compared with HO-1^−/− recipients treated with inactive CORM-2 (median survival 1 day). Histological analyses revealed that CO treatment markedly reduced platelet aggregation within the graft. Adoptive transfer of wild-type platelets to HO-1^−/− recipients also conferred protection and increased survival. Aortic transplants from either HO-1^−/− or HO-1^+/+ C57BL/6 donors into HO-1^+/+ (Balb/cJ) mice did not develop arterial thrombosis, surviving more than 56 days. These studies demonstrate an important role for systemic HO-1/CO for protection against vascular arterial thrombosis in murine aortic allotransplantation.Heme oxygenase-1 (HO-1) is an inducible enzyme that catalyzes the rate-limiting step in heme degradation, leading to the generation of equimolar amounts of iron, biliverdin, and carbon monoxide (CO). Biliverdin is then converted to bilirubin by biliverdin reductase.^1,2 HO-1 is highly up-regulated in mammalian tissues in response to a wide variety of conditions including vascular injury, ischemia, inflammation, immune injury, oxidative stress, cell cycle dysregulation, and sublethal and lethal cell damage.^3,4,5 The wide range of inducers of HO-1 provides support for a vital role in maintenance of cellular homeostasis under different pathophysiological conditions including inflammatory diseases such as septic shock and asthma,^6,7 cardiovascular diseases such as myocardial infarction and atherosclerosis,^8,9 ischemia-reperfusion injury in multiple organ systems,^8,10 and transplant rejection.^11,12One of the products of HO-1-mediated heme degradation, CO, is known to be toxic at high concentrations due to its high affinity for hemoglobin. However, there is substantial evidence that lower concentrations of CO endogenously generated from the breakdown of heme by HO serves essential regulatory roles in a variety of physiological and pathophysiological processes.¹³ Exogenous or endogenous CO can confer some of the cytoprotective effects attributed to HO-1.^14,15Transitional metal carbonyls, CO-releasing molecules (CORMs), have been used to deliver CO in a controlled manner without altering carboxyhemoglobin levels.^16,17,18 A wide range of CORMs containing manganese (CORM-1), ruthenium (CORM-2 and −3), boron (CORM-A1), and iron (CORM-F3) are currently being investigated to facilitate the pharmaceutical use of CO for the prevention of vascular dysfunction, inflammation, ischemia-reperfusion injury, and transplant rejection.^{19,20,21,22,23}Thrombosis is a major complication during multiple vascular pathological conditions during which HO-1 and its byproduct CO could provide significant protection through attenuation of inflammation, endothelial cell damage, and apoptosis, as well as modulation of vascular tone.^6,8,9 However, very little is known regarding the potential roles of HO-1 and CO in modulating platelet-dependent effects after vascular injury in the setting of transplantation. In these studies, we show that expression of HO-1 plays a critical role in the development of post-transplant arterial thrombosis immediately following abdominal aortic transplantation. We tested the hypothesis that CO, a product of the HO-1 reaction, mediates anti-thrombotic effects in vivo by inhibition of platelet mediated thrombus formation within the graft. We found that HO-1-deficient mice develop vascular thrombosis following aortic transplantation and that the development of thrombosis can be prevented by systemic administration of CORM-2.     

Neuroprotective Effect of Oligodendrocyte Precursor Cell Transplantation in a Long-Term Model of Periventricular Leukomalacia

Perinatal white matter injury, or periventricular leukomalacia (PVL), is the most common cause of brain injury in premature infants and is the leading cause of cerebral palsy. Despite increasing numbers of surviving extreme premature infants and associated long-term neurological morbidity, our understanding and treatment of PVL remains incomplete. Inflammation- or ischemia/hypoxia-based rodent models, although immensely valuable, are largely restricted to reproducing short-term features of up to 3 weeks after injury. Given the long-term sequelae of PVL, there is a need for subchronic models that will enable testing of putative neuroprotective therapies. Here, we report long term characterization of a neonatal inflammation-induced rat model of PVL. We show bilateral ventriculomegaly, inflammation, reactive astrogliosis, injury to pre-oligodendrocytes, and neuronal loss 8 weeks after injury. We demonstrate neuroprotective effects of oligodendrocyte precursor cell transplantation. Our findings present a subchronic model of PVL and highlight the tissue protective effects of oligodendrocyte precursor cell transplants that demonstrate the potential of cell-based therapy for PVL.Premature, low-birth weight infants are commonly diagnosed with perinatal white matter damage, which leads to long-term neurological deficits including cerebral palsy.^1,2,3,4 Congenital encephalomyelitis was described almost 150 years ago, a disease in newborn children characterized by pale softened zones of degeneration within the deep white matter surrounding lateral ventricles and is now often referred to as periventricular leukomalacia (PVL).⁵ The white matter damage that occurs in preterm infants has more recently been shown to be also accompanied by significant cerebral-cortex and deep-gray matter abnormalities leading to neurodegeneration and altered neurobehavioral performance.⁶PVL is characterized by selective oligodendrocyte precursor cell (OPC) loss resulting in delayed or disrupted myelination, white matter atrophy and ventriculomegaly, neuronal loss, and cyst and scar formation. Extreme premature birth (approximately 23 to 32 weeks) accompanied by inflammation or infection corresponds to the period when immature dividing and differentiating oligodendrocytes predominate in the cerebral white matter.^7,8,9,10 PVL has a complex etiology. The two most important determinants are cerebral hypoperfusion and maternal intrauterine infection. Periventricular white matter is susceptible to hypoperfusion due to the comparative immaturity of the periventricular vasculature of the preterm infant.^11,12 Early oligodendrocyte lineage cells are vulnerable to the consequences of hypoperfusion and subsequent microglial and astrocytic activation on account of amplified glutamate receptor-mediated responses and lack of efficient antioxidant protection.^{13,14,15,16,17,18} Maternal infection, recently implicated as a causative factor in the pathogenesis of PVL,^{19,20,21,22,23} is believed to initiate an inflammatory/cytokine cascade that results in the release of early-response pro-inflammatory cytokines such as tumor necrosis factor–α (TNF-α), interleukin−1 β (IL-1β) and interleukin−6 (IL-6), and causes damage to immature oligodendrocytes.²⁴ TNF-α appears to have a particularly important role in PVL pathogenesis with in vitro evidence suggesting direct damage to OPCs,^25,26 while IL-1β and IL-6 modulate injury indirectly.^27,28Although cells of the oligodendrocyte lineage are regarded as the primary target in the pathogenesis of PVL, there is increasing evidence that neonatal white matter damage is accompanied by gray matter abnormalities, including neuronal loss, impaired axonal guidance, and altered synaptogenesis.^6,29 Premature newborns affected by PVL often have smaller cerebral cortex and deep gray matter volumes, reduced cortical neurons, and alterations in the orientation of central white matter fiber tracts.^6,30,31,32 Together, these data suggest that developing neurons, like immature oligodendrocyte lineage cells, are also vulnerable to injury caused by inflammatory response or hypoxia.Aspects of PVL have been modeled by ischemia/hypoxia and inflammation-mediated rodent models. Animal models of hypoxia-ischemia have clearly shown that following brain injury there is reduced myelination, enlarged ventricles, loss of neurons and damage to axons and dendrites and altered neurobehavioral performances.^{33,34,35,36,37,38,39} Experimentally induced inflammation has been used in a number of studies to model PVL pathology and test potential treatments.^23,40,41,42 Administration of the endotoxin lipopolysaccharide (LPS), a potent inducer of innate immune response and inflammation,^43,44 either intracerebrally during early neonatal period, intrauterine or peritoneally to a pregnant mother results in inflammation and hypomyelination.^20,22,23,38 Moreover, in animal models of LPS-induced PVL neuronal loss and a reduction in neurite length in the parietal cortex has been observed.^42,45 In vitro evidence suggests LPS is not directly toxic to OPCs, but causes injury through the activation of Toll-like receptor 4-positive microglia, which is a source of pro-inflammatory cytokines, nitric oxide, and free radicals.^44,45,46 Furthermore, it has been shown that LPS-induced inflammation increases the susceptibility of white matter to injury in response to otherwise “harmless” subthreshold hypoxic-ischemic insult.⁴⁷ Current models of PVL are typically short term, with studies using either hypoxia-ischemia or inflammation rarely extending analysis beyond 14 days. There is therefore a need to evaluate the longer-term, subchronic consequences of LPS injury and specifically address whether hypomyelination and neuronal injury are self-limiting or indeed spontaneously repair.Cellular therapeutic strategies are predicated on cell replacement and/or tissue protection independent of specific cellular differentiation. Progenitor cells including OPCs have previously been used for cellular replacement of damaged or lost cells in a variety of CNS injury models where damage to myelin and neuronal loss occur.^{48,49,50,51,52,53} Furthermore, there is accumulating evidence that implicates progenitor cell mediated neuroprotection through a variety of mechanisms including graft derived neurotrophic support independent of directed differentiation.^54,55 Moreover, there is evidence to suggest that OPCs secrete factors that are capable of supporting neuronal survival,^{56,57,58,59,60,61} thus suggesting they may be a potential therapeutic source for replacing lost or damaged cells and protecting healthy tissue following neonatal brain injury. In this study we have examined the subchronic effects of LPS induced injury and then examined the putative neuroprotective effects of OPC transplantation.     

A Protein Kinase Cδ-Dependent Protein Kinase D Pathway Modulates ERK1/2 and JNK1/2 Phosphorylation and Bim-Associated Apoptosis by Asbestos

Inhalation of asbestos and oxidant-generating pollutants causes injury and compensatory proliferation of lung epithelium, but the signaling mechanisms that lead to these responses are unclear. We hypothesized that a protein kinase (PK)Cδ-dependent PKD pathway was able to regulate downstream mitogen-activated protein kinases, affecting pro- and anti-apoptotic responses to asbestos. Elevated levels of phosphorylated PKD (p-PKD) were observed in distal bronchiolar epithelial cells of mice inhaling asbestos. In contrast, PKCδ−/− mice showed significantly lower levels of p-PKD in lung homogenates and in situ after asbestos inhalation. In a murine lung epithelial cell line, asbestos caused significant increases in the phosphorylation of PKCδ-dependent PKD, ERK1/2, and JNK1/2/c-Jun that occurred with decreases in the BH3-only pro-apoptotic protein, Bim. Silencing of PKCδ, PKD, and use of small molecule inhibitors linked the ERK1/2 pathway to the prevention of Bim-associated apoptosis as well as the JNK1/2/c-Jun pathway to the induction of apoptosis. Our studies are the first to show that asbestos induces PKD phosphorylation in lung epithelial cells both in vivo and in vitro. PKCδ-dependent PKD phosphorylation by asbestos is causally linked to a cellular pathway that involves the phosphorylation of both ERK1/2 and JNK1/2, which play opposing roles in the apoptotic response induced by asbestos.Asbestos is a group of naturally occurring mineral fibers that are linked to the development of lung cancer, mesothelioma, and pleural and pulmonary fibrosis, ie, asbestosis.^1,2 The mechanisms leading to asbestos-related diseases are still unclear, but oxidative stress due to phagocytosis of longer fibers, iron-driven generation of oxidants from fiber surfaces, and depletion of cellular antioxidants are linked to cell injury and inflammation.^3,4,5,6Bronchiolar and alveolar type II epithelial cells, which first encounter asbestos fibers after inhalation, are key cell types in asbestos-associated inflammation and fibroproliferation.² Initial cell reactions to asbestos include epithelial cell injury, ie, apoptosis and necrosis,^5,6 which may lead to compensatory cell proliferation^7,8 and the production of inflammatory and fibrogenic cytokines.^8,9,10 Asbestos-induced signaling mechanisms governing these cell responses appear to involve a broad variety of cascades including the mitogen-activated protein kinases (MAPK),^3,7,11,12 nuclear factor-κB (NF-κB),^9,13,14 and the protein kinase (PK)C^10,12,15,16 and A families.¹⁷A critical signaling protein involved in asbestos signaling is PKCδ, which is known to be activated in bronchiolar and alveolar epithelial cells in vivo and in vitro^10,12,16 via increased formation of diacylglycerol.¹⁸ We have shown that PKCδ governs apoptosis via an oxidant-dependent mitochondrial pathway after exposure of lung epithelial cells to asbestos fibers.¹⁶ Recent studies comparing PKCδ +/+ and PKCδ −/− mice also reveal an important role of PKCδ in metalloproteinase expression as well as cytokine production in vitro and in vivo.^10,15 A variety of other studies also link PKCδ to either pro-apoptotic or anti-apoptotic events depending on the stimulus and cell type.^19,20In this study, we focused on PKD as a potential link between PKCδ, activation of MAPKs and downstream repercussions such as expression of fos/jun proto-oncogenes and apoptosis in asbestos-exposed lung epithelium. PKD is a serine/threonine protein kinase classified as a subfamily of the Ca²⁺/calmodulin-dependent kinase superfamily.²¹ PKD1, which includes mouse PKD and its human homolog PKCμ, is the most extensively studied PKD.²² The other two members of this family include PKD2²³ and PKD3, (originally PKCν).²⁴ Conserved regions of PKDs include a phosphorylation-dependent catalytic domain, a pleckstrin-homology domain that inhibits the catalytic activity, and cysteine-rich motifs that recruit PKD to the plasma membrane. PKCδ is proposed to interact with the pleckstrin-homology domain of PKD, transphosphorylating its activation loop at Ser744 and Ser748, and leading to PKD activation.²⁵ In addition, PKD can be activated through the Src-Abl pathway by tyrosine phosphorylation of Tyr463 (T463) in the pleckstrin-homology domain after oxidative stress,²⁶ as well as by caspase-mediated proteolytic cleavage 27 and by bone morphogenetic protein 2.²⁸ Downstream targets of PKD signaling include several important signaling molecules such as ERK1/2, JNK1/2, and NF-κB,^21,26,29,30 but how these affect functional ramifications of carcinogens, such as asbestos, are unclear.The BH3-only protein, Bim, is a pro-apoptotic member of the Bcl-2 family that links stress-induced signals to the core apoptotic machinery.^31,32 There are three different splice variants of the Bim gene encoding short, long, and extra-long Bim proteins (BimS, BimL, and BimEL).³³ BimS-induced apoptosis requires mitochondrial localization but not interaction with anti-apoptosis proteins,³⁴ whereas BimL is bound to microtubules and is less cytotoxic.³⁵ Disruption of BimL binding to microtubules via JNK-dependent phosphorylation can cause its redistribution to the mitochondria and induction of pro-apoptotic machinery.³⁶ BimEL is post-translationally regulated by ERK1/2, which promotes its phosphorylation and rapid dissociation from Mcl-1 and Bcl-x(L)³⁷ and proteasomal degradation.³⁸We reveal here that PKD is involved in multiple signaling events after asbestos inhalation and in vitro. Specifically, PKD is a downstream effector of PKCδ and modulates phosphorylation of both ERK1/2 and JNK1/2 in lung epithelial cells after asbestos exposure. Our data also suggest that PKD inhibits apoptosis through an ERK1/2-mediated destabilization of the pro-apoptotic BH3-only protein, BimEL. The fact that PKD is an important signaling molecule in MAPK signaling and survival after cell injury by asbestos may have important therapeutic implications in asbestos-related diseases.     

Gastrin Is an Essential Cofactor for Helicobacter-Associated Gastric Corpus Carcinogenesis in C57BL/6 Mice

We have previously described a synergistic interaction between hypergastrinemia and Helicobacter felis infection on gastric corpus carcinogenesis in FVB/N mice housed under specific-pathogen-free conditions. However, gastrin-deficient (GAS-KO) mice on a mixed C57BL/6/129Sv genetic background maintained in conventional housing were reported to develop spontaneous gastric antral tumors. Therefore, we investigated the role of gastrin in Helicobacter-associated gastric carcinogenesis in H. felis-infected mice on a uniform C57BL/6 background housed in specific-pathogen-free conditions. Hypergastrinemic transgenic (INS-GAS) mice, GAS-KO mice, and C57BL/6 wild-type mice were infected with H. felis for either 12 or 18 months. At 12 months postinfection, INS-GAS mice had mild corpus dysplasia, while B6 wild-type mice had either severe gastritis or metaplasia, and GAS-KO mice had only mild to moderate gastritis. At 18 months postinfection, both INS-GAS and B6 wild-type mice had both severe atrophic gastritis and corpus dysplasia, while GAS-KO mice had severe gastritis with mild gastric atrophy, but no corpus dysplasia. In contrast, both GAS-KO and B6 wild-type mice had mild to moderate antral dysplasia, while INS-GAS mice did not. H. felis antral colonization remained stable over time among the three groups of mice. These results point to a distinct effect of gastrin on carcinogenesis of both the gastric corpus and antrum, suggesting that gastrin is an essential cofactor for gastric corpus carcinogenesis in C57BL/6 mice.Gastric cancer remains the second leading cause of cancer-related mortality in the world, although its incidence and mortality rates have been decreasing in the United States over the past 70 years.^1,2,3 The risk of developing gastric adenocarcinoma is strongly associated with Helicobacter pylori infection, which is gradually disappearing from western societies. Despite the overall decline in gastric cancer prevalence, the treatment of stomach cancer remains a challenging clinical problem, since most patients who undergo surgical resection develop regional or distant recurrences and the overall 5-year survival rate for gastric cancer patients remains around 20% in western countries.³H. pylori, first identified in the gastric antrum of patients with active chronic gastritis and peptic ulcers,⁴ is now recognized as the major cause of gastric cancer, and has been classified as a group I carcinogen by World Health Organization.^5,6 H. pylori infection causes persistent chronic gastritis, which in susceptible individuals may progress to atrophy, intestinal metaplasia, dysplasia, and finally, intestinal-type gastric cancer. This sequence, commonly referred to as Correa’s cascade, is considered the primary histological pathway for the development of intestinal type of gastric cancer,⁷ and is both initiated and promoted by H. pylori infection.It has generally been recognized that H. pylori infection results in a mild (1.5- to 2-fold elevation) hypergastrinemia that occurs early on in the course of the infection in many individuals. Given the known properties of gastrin as a mucosal growth factor, hypergastrinemia was postulated to be a factor promoting the development of gastric cancer. Indeed, previous studies have suggested a possible association between hypergastrinemia, Helicobacter infection, and gastric cancer.^8,9,10,11,12 Therefore, to study the role of gastrin and the potential mechanisms involved in gastric carcinogenesis, we developed a mouse model of gastric cancer through the generation of insulin-gastrin (INS-GAS) transgenic mice that overexpressed human amidated gastrin. In the absence of Helicobacter infection, INS-GAS mice on an FVB/N genetic background exhibited mild hypergastrinemia in association with elevated gastric acid secretion and an increased parietal cell number at 1 to 3 months of age. With increasing age, the INS-GAS mice showed progressive loss of parietal cells and significant changes in the corpus, including hypochlorhydria, gastric atrophy, metaplasia, and dysplasia. At 20 months of age, INS-GAS mice developed invasive gastric cancer.⁹ The gastric cancer phenotype was accelerated by gastric Helicobacter spp. infection, and lesion severity was more profound in male INS-GAS mice.¹⁰ The cause of this gender-specific incidence was due in part to ovarian-dependent estrogen production, since H. pylori infected ovariectomized female INS-GAS mice also developed severe gastric neoplasia, and 17beta-estradiol treatment significantly suppressed this phenotype.¹²However, determining the role of gastrin in predisposing individuals to gastric cancer has not been straightforward. Some H. pylori-infected patients have lower levels of gastrin and acid secretion relative to non-infected healthy persons, and hypochlorhydria probably plays an important role in the carcinogenic process through altered bacterial colonization along with changes in nitrite levels.¹³ Gastrin-deficient mice on a mixed C57BL6/129Sv background developed spontaneous gastric antral tumors when maintained under conventional housing conditions at 12 months of age, while C57BL/6 wild-type and somatostatin-deficient mice did not develop tumors.¹⁴ The authors concluded that neoplastic transformation of the antrum does not require gastrin, and that gastrin may actually suppress the development of gastric antral tumors. In addition, there have been other genetic models reported, such as the gp130^757F/F mouse, which do not appear to be dependent on gastrin for tumor development.¹⁵ It has been difficult to reconcile these observations regarding the influence of gastrin on gastric cancer, given the different genetic backgrounds, housing conditions, and Helicobacter infection status. Thus, the purpose of this study is to examine the effect of gastrin in Helicobacter-associated gastric carcinogenesis using hypergastrinemic (INS-GAS) mice and gastrin deficient (GAS-KO) mice on a uniform C57BL/6 background and housed under SPF conditions.