Inflammasome Up-Regulation and Activation in Dysferlin-Deficient Skeletal Muscle

A deficiency of the dysferlin protein results in limb girdle muscular dystrophy type 2B and Miyoshi myopathy, with resulting plasma membrane abnormalities in myofibers. Many patients show muscle inflammation, but the molecular mechanisms that initiate and perpetuate this inflammation are not well understood. We previously showed abnormal activation of macrophages and hypothesized that activation of the inflammasome pathway may play a role in disease progression. To test this, we studied the inflammasome molecular platform in dysferlin-deficient human and mouse muscle. Consistent with our model, components of the NACHT, LRR and PYD-containing proteins (NALP)-3 inflammasome pathway were specifically up-regulated and activated in dysferlin-deficient but not in dystrophin-deficient and normal muscle. We demonstrate for the first time that normal primary skeletal muscle cells are capable of secreting IL-1β in response to combined treatment with lipopolysaccharide and the P2X7 receptor agonist, benzylated ATP, suggesting that not only immune cells but also muscle cells can actively participate in inflammasome formation. In addition, we show that dysferlin-deficient primary muscle cells express toll-like receptors (TLRs; TLR-2 and TLR-4) and can efficiently produce IL-1β in response to lipopolysaccharide and benzylated ATP. These data indicate that skeletal muscle is an active contributor of IL-1β and strategies that interfere with this pathway may be therapeutically useful for patients with limb girdle muscular dystrophy type 2B.Genetic defects in the dysferlin gene result in limb girdle muscular dystrophy (LGMD2B) and distal muscular dystrophy of the Miyoshi type in human patients.^1,2 The clinical presentation and progression of patients with LGMD2B/Miyoshi show enigmatic histological and clinical features that are not entirely explained by the myofiber defect.^3,4 Patients are quite healthy until their late teens. Although there are presymptomatic elevations of serum creatine kinase, there is little evidence of weakness before disease onset, which can appear more acute than that of other dystrophies. The exact nature of the trigger and the molecular pathways that initiate and perpetuate muscle fiber damage and dysfunction in LGMD2B are still unclear.We and others have previously shown that muscle inflammation is often present in LGMD2B patient biopsies,^5,6,7 and dysferlin-deficient monocytes show increased phagocytic activity when compared with control cells.⁷ In addition, we found that smallinterfering RNA-mediated inhibition of dysferlin expression in the J774 macrophage cell line resulted in significantly enhanced phagocytosis. Importantly, this experiment demonstrated that the phagocytotic defect seen in both human and murine monocytes is likely a direct consequence of dysferlin deficiency, rather than a downstream effect on monocyte activation in vivo in the dystrophic organism. Dysferlin-deficient mice also showed strong up-regulation of the endocytic proteins cation-independent mannose 6-phosphate receptor (CIMPR), clathrin, and adaptin-α, and LGMD2B muscle exhibited a decreased expression of decay accelerating factor that was not dysferlin-specific. We further showed that the expression levels of the small Rho family GTPases, RhoA, Rac1, and Cdc 42 were increased in the immune cells of dysferlin-deficient mice when compared with control cells, indicating that plasma membrane reorganization and remodeling are active in dysferlin deficiency.⁷ Dysferlin plays a role in vesicle traffic and membrane repair,^8,9 and recent data from our group have also indicated that dysferlin-deficient muscle, but not Fukutin-related protein- or dystrophin-deficient muscle, shows increased levels of vesicle trafficking pathway proteins (eg, synaptotagmin-like protein, Slp2a and the small GTPase, Rab27A), suggesting that dysferlin-deficient cells may release excess amounts of vesicle contents and contribute to the inflammation and muscle fiber damage associated with this genetic defect.¹⁰ These data strongly suggest that mild myofiber damage in dysferlin-deficient muscle stimulates an inflammatory cascade (eg, inflammasome) that may initiate, exacerbate, and possibly perpetuate the underlying myofiber-specific dystrophic process.⁷ However, the events that initiate this inflammatory cascade are not yet well characterized.The molecular platform that triggers the activation of inflammatory caspases and processing of pro-interleukin (IL)-1β to mature (active) IL-1β is termed the inflammasome.¹¹ The inflammasome is a multimeric protein complex composed of the NACHT, LRR and PYD-containing proteins (NALP)-3 protein, the apotosis-associated speck-like protein containing a caspase recruitment domain (ASC-1), caspase-1, and pro-IL-1β. The inflammasome pathway has been well characterized in the cells that participate in innate immunity;¹¹ however, there is very little information regarding its expression and activation in nonhematopoietic cells such as skeletal muscle.Pro-IL-1β and pro-caspase-1 are stored in secretory lysosomes, where they await an exocytosis-inducing stimulus; in the absence of such a stimulus, these molecules may undergo lysosomal degradation.¹² Lysosome exocytosis and IL-1β secretion are facilitated by extracellular ATP. There is evidence that ATP triggers via P2X7 receptors an efflux of K⁺ from the cell, followed by a Ca²⁺ influx and the activation of phosphatidylcholine-specific phospholipase C and Ca²⁺ -independent and -dependent phospholipase A₂, leading to the secretion of IL-1β.¹² Recent data further support the concept that inflammasome activation (caspase-1 and IL-1β activation) and the unconventional protein secretion of leaderless proteins such as IL-1β and fibroblast growth factor 2 are interdependent.¹³The present study is based on the hypothesis that an increase in vesicular trafficking and plasma membrane repair defects result in the release of ATP and other endogenous danger/alarm signals (eg, the high mobility group box 1 [HMGB1] and S100 proteins) in dysferlin-deficient muscle. These, in turn, bind to their cellular receptors and activate the inflammasome pathway. We now present evidence that inflammasome components are indeed present in muscle and are activated in dysferlin-deficient patients and mice. Hence, the endogenous danger signals generated as a result of the primary genetic defect in LGMD2B may contribute to the activation of the muscle inflammasome, which in turn generates a proinflammatory environment in muscle that leads to increased muscle inflammation and damage.     

Expression and localization of the multidrug resistance protein 5 (MRP5/ABCC5), a cellular export pump for cyclic nucleotides, in human heart

The multidrug resistance protein 5 (MRP5/ABCC5) has been recently identified as cellular export pump for cyclic nucleotides with 3′,5′-cyclic GMP (cGMP) as a high-affinity substrate. In view of the important role of cGMP for cardiovascular function, expression of this transport protein in human heart is of relevance. We analyzed the expression and localization of MRP5 in human heart [21 auricular (AS) and 15 left ventricular samples (LV) including 5 samples of dilated and ischemic cardiomyopathy]. Quantitative real-time polymerase chain reaction normalized to β-actin revealed expression of the MRP5 gene in all samples (LV, 38.5 ± 12.9; AS, 12.7 ± 5.6; P < 0.001). An MRP5-specific polyclonal antibody detected a glycoprotein of ~190 kd in crude cell membrane fractions from these samples. Immunohistochemistry with the affinity-purified antibody revealed localization of MRP5 in cardiomyocytes as well as in cardiovascular endothelial and smooth muscle cells. Furthermore, we could detect MRP5 and ATP-dependent transport of [³H]cGMP in sarcolemma vesicles of human heart. Quantitative analysis of the immunoblots indicated an interindividual variability with a higher expression of MRP5 in the ischemic (104 ± 38% of recombinant MRP5 standard) compared to normal ventricular samples (53 ± 36%, P < 0.05). In addition, we screened genomic DNA from our samples for 20 single-nucleotide polymorphisms in the MRP5 gene. These results indicate that MRP5 is localized in cardiac and cardiovascular myocytes as well as endothelial cells with increased expression in ischemic cardiomyopathy. Therefore, MRP5-mediated cellular export may represent a novel, disease-dependent pathway for cGMP removal from cardiac cells.     

Dysferlin Deficiency and the Development of Cardiomyopathy in a Mouse Model of Limb-Girdle Muscular Dystrophy 2B

Limb-girdle muscular dystrophy 2B, Miyoshi myopathy, and distal myopathy of anterior tibialis are severely debilitating muscular dystrophies caused by genetically determined dysferlin deficiency. In these muscular dystrophies, it is the repair, not the structure, of the plasma membrane that is impaired. Though much is known about the effects of dysferlin deficiency in skeletal muscle, little is known about the role of dysferlin in maintenance of cardiomyocytes. Recent evidence suggests that dysferlin deficiency affects cardiac muscle, leading to cardiomyopathy when stressed. However, neither the morphological location of dysferlin in the cardiomyocyte nor the progression of the disease with age are known. In this study, we examined a mouse model of dysferlinopathy using light and electron microscopy as well as echocardiography and conscious electrocardiography. We determined that dysferlin is normally localized to the intercalated disk and sarcoplasm of the cardiomyocytes. In the absence of dysferlin, cardiomyocyte membrane damage occurs and is localized to the intercalated disk and sarcoplasm. This damage results in transient functional deficits at 10 months of age, but, unlike in skeletal muscle, the cell injury is sublethal and causes only mild cardiomyopathy even at advanced ages.Plasma membrane damage in mechanically active cells such as the myocyte is inevitable even under normal physiological conditions.^1,2 Since membranes are not self-sealing, effective and efficient repair mechanisms are necessary to maintain cell viability. Dysferlin plays a central role in this active repair mechanism in skeletal muscle. In the absence of dysferlin disruptions of the skeletal muscle plasma membrane are not repaired leading to cell death.³ Skeletal muscle can regenerate new cells from satellite cells but eventually even this response is exhausted, and lost myocytes are replaced by fat and fibrosis resulting in debilitating muscular dystrophy.Limb-girdle muscular dystrophy type 2 B (LGMD2B), Miyoshi myopathy, and distal myopathy of anterior tibialis are three clinically distinct forms of muscular dystrophy that are caused by mutations within the dysferlin (DYSF) gene resulting in severe to complete deficiency of dysferlin expression.^4,5 Clinically, these dysferlinopathies start in young adulthood with progressive muscle weakness and atrophy that advances to severe disability in older adulthood.Dysferlin is a 273 kDa membrane-spanning protein with multiple C2 domains that bind calcium, phospholipids, and proteins to then trigger signaling events, vesicle trafficking, and membrane fusion.^6,7 The name “dysferlin” reflects the homology with FER-1, the Caenorhabditis elegans spermatogenesis factor involved in the fusion of vesicles with the plasma membrane, as well as the dystrophic phenotype associated with its deficiency.⁵ Dysferlin is crucial to calcium dependent membrane repair in muscle cells.^3,8 In normal skeletal muscle, sarcolemma injuries lead to the accumulation of dysferlin-enriched membrane patches and resealing of the membrane in the presence of Ca²⁺.^3,9While the profound effect of dysferlin deficiency in skeletal muscle has been the subject of much investigation, the effect of dysferlin deficiency in cardiac muscle has largely been ignored. However, in 2004, Kuru et al¹⁰ reported on a 57-year-old Japanese woman with LGMD2B and dilated cardiomyopathy; more recently, Wenzel et al¹¹ described dilated cardiomyopathy in two out of seven patients with LGMD2B and other cardiac abnormalities in three of the others. These observations suggest that dysferlin deficiency can lead to cardiomyopathy as well as to muscular dystrophy. However, neither the morphological location of dysferlin in the cardiomyocyte nor the progression of the disease with age are known.Spontaneous mutations in the mouse are valuable resources in understanding human disease processes. Genetically defined mice develop dysferlinopathies closely resembling LGMD2B, Miyoshi myopathy, and distal myopathy of anterior tibialis.¹² In 2004, Ho et al¹² identified A/J mice as dysferlin deficient. A retrotransposon insertion in the dysferlin gene was found to result in a null allele, resulting in skeletal muscle dystrophy that shows histopathological and ultrastructural features that closely resemble the human dysferlinopathies of LGMD2B, Miyoshi myopathy, and distal myopathy of anterior tibialis.¹² The onset of dystrophic features in A/J mice begins in proximal limb muscles at 4 to 5 months of age and progresses to severe debilitating muscular dystrophy over several months. Ho et al¹² also found that human and murine dysferlin share very similar (approximately 90% identity) amino acid sequences. Cardiac muscle was not included in their study.Recently, Han et al,⁸ using sucrose gradient membrane fractionation on homogenates of wild-type C57BL/6J mouse heart muscle, showed that dysferlin is present in the cardiomyocyte plasma membrane and intracellular vesicle fractions. It was proposed that dysferlin is localized to the cardiomyocyte sarcolemma and some unidentified type of vesicles.⁸ Han et al⁸ in one study and Wenzel et al¹¹ in another study showed that the induction of significant cardiac stress lead to cardiac dysfunction in dysferlin-deficient mice, but to what extent dysferlin deficiency causes cardiomyopathy by aging alone in patients clinically affected with the debilitating effects of LGMD2B, Miyoshi myopathy, or distal myopathy of anterior tibialis is unknown.In this study, we used the A/J mouse model to study the effects of aging in mice affected by genetically determined dysferlin deficiency by using echocardiography and conscious electrocardiography to determine functional changes in vivo, followed postmortem by light and electron microscopy to determine associated morphological changes. We have determined that the normal primary location for dysferlin in the cardiomyocyte of control A/HeJ mice is the intercalated disk (ID), and to a lesser extent, to a distinctive transverse banding pattern within the sarcoplasm of the cardiomyocyte. We have also determined that in the dysferlin-deficient cardiomyocyte there is evidence of membrane damage at these locations. We also present data that show functional cardiac deficits were present in vivo at around 10 months of age then recovered by 12 months. Histopathology showed that under normal laboratory conditions dysferlin deficiency causes only a mild cardiomyopathy even at advanced ages, suggesting the possibility of dysferlin-independent membrane repair mechanisms in cardiac muscle that do not exist in skeletal muscle.     

Amyloidose bei Muskeldystrophie

Mutations in the gene encoding dysferlin (DYSF) cause limb-girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy (MM). We were able to examine eight patients suspected of LGMD2B clinically, histochemically. The genotype was determined in every case. We found sarcolemmal and interstitial amyloid deposits in four muscle sections. All of the mutations associated with amyloid were located in the N-terminal region of dysferlin, and dysferlin clearly proved to be a component of the amyloid deposits. Dysferlin-deficient muscular dystrophy is the first muscular dystrophy in which amyloidosis is involved. This fact must be considered in the process of developing therapeutic strategies. The influence of the amyloid deposits on the pathogenesis of the disease and the possible involvement of other organs in the progressive course are as yet unclear.     

Genetic Manipulation of Dysferlin Expression in Skeletal Muscle : Novel Insights into Muscular Dystrophy

Mutations in the gene DYSF, which codes for the protein dysferlin, underlie Miyoshi myopathy and limb-girdle muscular dystrophy 2B in humans and produce a slowly progressing skeletal muscle degenerative disease in mice. Dysferlin is a Ca²⁺-sensing, regulatory protein that is involved in membrane repair after injury. To assess the function of dysferlin in healthy and dystrophic skeletal muscle, we generated skeletal muscle–specific transgenic mice with threefold overexpression of this protein. These mice were phenotypically indistinguishable from wild-type, and more importantly, the transgene completely rescued the muscular dystrophy (MD) disease in Dysf-null A/J mice. The dysferlin transgene rescued all histopathology and macrophage infiltration in skeletal muscle of Dysf^−/− A/J mice, as well as promoted the rapid recovery of muscle function after forced lengthening contractions. These results indicate that MD in A/J mice is autonomous to skeletal muscle and not initiated by any other cell type. However, overexpression of dysferlin did not improve dystrophic symptoms or membrane instability in the dystrophin-glycoprotein complex–lacking Scgd (δ-sarcoglycan) null mouse, indicating that dysferlin functionality is not a limiting factor underlying membrane repair in other models of MD. In summary, the restoration of dysferlin in skeletal muscle fibers is sufficient to rescue the MD in Dysf-deficient mice, although its mild overexpression does not appear to functionally enhance membrane repair in other models of MD.The muscular dystrophies (MD) are a diverse group of genetic muscle wasting disorders that typically result in premature death due to cardiac or respiratory failure.¹ Most characterized mutations in humans that cause MD result from alterations in structural proteins that connect the underlying contractile proteins to the basal lamina, providing rigidity to the skeletal muscle cell membrane, or in proteins that directly stabilize or repair the cell membrane.^1,2 For example, loss of dystrophin in Duchenne’s MD or mutations in other components of the dystrophin-glycoprotein complex (DGC) leads to a fundamental alteration in the physical properties of the sarcolemma, permitting contraction-induced microtears and the unregulated exchange of ions such as Ca²⁺, leading to necrosis and degeneration of myofibers.^1,2 The DGC is a multisubunit complex organized at the sarcolemma that links the underlying contractile proteins to the extracellular matrix, providing the sarcolemma with cytoskeletal support and protecting it from damage incurred during the contractile cycle. The DGC contains structural proteins such as dystrophin, dystroglycans, sarcoglycans, dystrobrevin, sarcospan, and syntrophins, as well as a number of signaling proteins.^1,2 That membrane instability and aberrant repair capacity underlie myofiber degeneration in MD was further suggested by the observation that mutations in the putative membrane repair protein dysferlin cause limb girdle muscular dystrophy 2B and Miyoshi myopathy.³ Limb girdle muscular dystrophy 2B/Miyoshi myopathy typically presents in early adulthood or the late teen years, and muscle biopsies from these patients show a striking inflammatory response.³Dysferlin is a 230 kDa, Ca²⁺ sensitive protein that participates in membrane resealing events following injury, but does not directly interact with components of the DGC. Mice lacking dysferlin (Dysf) exhibit progressive disease in skeletal muscle and cardiac tissue despite having a functional DGC, which is characterized by myofiber necrosis, cycles of degeneration and regeneration, inflammation, and adipocyte replacement.^4,5 Lack of dysferlin also results in the accumulation of vesicles and structural membrane defects as analyzed by electron microscopy, suggesting a role in normal membrane turnover and recycling.^4,6Disease in dystrophic skeletal muscle is dramatically influenced by the inflammatory response, achieved mainly by infiltration of cytotoxic T-lymphocytes and macrophages.^7,8 It has been suggested that Dysf-null inflammatory cells may initiate the disease process in the muscles of Dysf-null mice and humans, as these immune cells appear to be hyperactive and abnormal.^9,10,11 For example, macrophages and dendritic-T cell activation markers are elevated in the SJL mouse model for Dysf deficiency and in human limb girdle muscular dystrophy 2B.⁹ Thus, it is unclear whether disease due to dysferlin deficiency is primarily due to an autonomous effect in immune cells or skeletal muscle fibers. To address this issue, we generated transgenic mice that express dysferlin specifically in skeletal muscle and used them to evaluate the necessity of dysferlin within myofibers to initiate muscle disease in Dysf null A/J mice. Moreover, we assessed the ability of increased dysferlin expression to alleviate pathology in a MD model that lacks a component of the DGC, Scgd (δ-sarcoglycan).     

Failure of Pelvic Organ Support in Mice Deficient In Fibulin-3

Fibulin-5 is crucial for normal elastic fiber synthesis in the vaginal wall; more than 90% of fibulin-5-knockout mice develop pelvic organ prolapse by 20 weeks of age. In contrast, fibulin-1 and -2 deficiencies do not result in similar pathologies, and fibulin-4-knockout mice die shortly after birth. EFEMP1 encodes fibulin-3, an extracellular matrix protein important in the maintenance of abdominal fascia. Herein, we evaluated the role of fibulin-3 in pelvic organ support. Pelvic organ support was impaired significantly in female Efemp1 knockout mice (Fbln3^{−[supi]/−}), and overt vaginal, perineal, and rectal prolapse occurred in 26.9% of animals. Prolapse severity increased with age but not parity. Fibulin-5 was up-regulated in vaginal tissues from Fbln3^{−[supi]/−} mice regardless of prolapse. Despite increased expression of fibulin-5 in the vaginal wall, pelvic organ support failure occurred in Fbln3^{−[supi]/−} animals, suggesting that factors related to aging led to prolapse. Elastic fiber abnormalities in vaginal tissues from young Fbln3^{−[supi]/−} mice progressed to severe elastic fiber disruption with age, and vaginal matrix metalloprotease activity was increased significantly in Fbln3^{−[supi]/−} animals with prolapse compared with Fbln3^{−[supi]/−} mice without prolapse. Overall, these results indicate that both fibulin-3 and -5 are important in maintaining pelvic organ support in mice. We suggest that increased vaginal protease activity and abnormal elastic fibers in the vaginal wall are important components in the pathogenesis of pelvic organ prolapse.     

Dysferlin deficiency enhances monocyte phagocytosis: a model for the inflammatory onset of limb-girdle muscular dystrophy 2B

Dysferlin deficiency causes limb-girdle muscular dystrophy type 2B (LGMD2B; proximal weakness) and Miyoshi myopathy (distal weakness). Muscle inflammation is often present in dysferlin deficiency, and patients are frequently misdiagnosed as having polymyositis. Because monocytes normally express dysferlin, we hypothesized that monocyte/macrophage dysfunction in dysferlin-deficient patients might contribute to disease onset and progression. We therefore examined phagocytic activity, in the presence and absence of cytokines, in freshly isolated peripheral blood monocytes from LGMD2B patients and in the SJL dysferlin-deficient mouse model. Dysferlin-deficient monocytes showed increased phagocytic activity compared with control cells. siRNA-mediated inhibition of dysferlin expression in the J774 macrophage cell line resulted in significantly enhanced phagocytosis, both at baseline and in response to tumor necrosis factor-alpha. Immunohistochemical analysis revealed positive staining for several mononuclear cell activation markers in LGMD2B human muscle and SJL mouse muscle. SJL muscle showed strong up-regulation of endocytic proteins CIMPR, clathrin, and adaptin-alpha, and LGMD2B muscle exhibited decreased expression of decay accelerating factor, which was not dysferlin-specific. We further showed that expression levels of small Rho family GTPases RhoA, Rac1, and Cdc 42 were increased in dysferlin-deficient murine immune cells compared with control cells. Therefore, we hypothesize that mild myofiber damage in dysferlin-deficient muscle stimulates an inflammatory cascade that may initiate, exacerbate, and possibly perpetuate the underlying myofiber-specific dystrophic process.     

Dysferlin deficiency blunts β‐adrenergic‐dependent lusitropic function of mouse heart

AbstractDysferlin is a cell membrane bound protein with a role in the repair of skeletal and cardiac muscle cells. Deficiency of dysferlin leads to limb‐girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy. In cardiac muscle, dysferlin is located at the intercalated disc and transverse tubule membranes. Loss of dysferlin causes death of cardiomyocytes, notably in ageing hearts, leading to dilated cardiomyopathy and heart failure in LGM2B patients. To understand the primary pathogenesis and pathophysiology of dysferlin cardiomyopathy, we studied cardiac phenotypes of young adult dysferlin knockout mice and found early myocardial hypertrophy with largely compensated baseline cardiac function. Cardiomyocytes isolated from dysferlin‐deficient mice showed normal shortening and re‐lengthening velocities in the absence of external load with normal peak systolic Ca²⁺ but slower Ca²⁺ re‐sequestration than wild‐type controls. The effects of isoproterenol on relaxation velocity, left ventricular systolic pressure and stroke volume were blunted in dysferlin‐deficient mouse hearts compared with that in wild‐type hearts. Young dysferlin‐deficient mouse hearts expressed normal isoforms of myofilament proteins whereas the phosphorylation of ventricular myosin light chain 2 was significantly increased, implying a molecular response to the impaired lusitropic function. These early phenotypes of diastolic cardiac dysfunction and blunted lusitropic response of cardiac muscle to β‐adrenergic stimulation indicate a novel pathogenic mechanism of dysferlin cardiomyopathy.

Abbreviations

CaMKII

calmodulin kinase II

cMyBP‐C

cardiac myosin binding protein‐C

LGMD2B

limb‐girdle muscular dystrophy 2B

DTT

dithiothreitol

LVP_max

left ventricular systolic peak pressure

LVP_min

left ventricular end diastolic pressure

mAb

monoclonal antibody

MHC

myosin heavy chain

MLC2v

ventricular myosin light chain 2

MM

Myoshimyopathy

TnI

troponin I

TnT

troponin T

T_P

time for reaching peak cytosolic calcium

TR₂₅

time for 25% calcium re‐sequestration

TR₇₅

time for 75% calcium re‐sequestration

     

Foxp3+ T-regulatory cells in Sjogren's syndrome: correlation with the grade of the autoimmune lesion and certain adverse prognostic factors

Sjögren’s syndrome (SS) is a chronic autoimmune exocrinopathy associated with variable lymphocytic infiltration of the affected organs (primarily salivary and lacrimal glands) and broad clinical manifestations, including lymphoma development. To investigate the potential implication of Foxp3⁺ T-regulatory cells in the regulation of SS inflammatory responses, we studied their incidence in the minor salivary glands (MSGs) and their relationship with histopathological and clinical disease parameters. Similar percentages of infiltrating Foxp3⁺ cells were observed in the MSG lesions of all SS patients (n = 30) and non-SS sialadenitis controls (n = 7). Foxp3⁺ cells were not detected in sicca-complaining controls with negative biopsy (n = 6). In SS patients, Foxp3⁺ cell frequency varied according to lesion severity, with the highest and lowest frequencies obtained in intermediate and mild MSG lesions, respectively. In the peripheral blood of these patients, reverse distribution of Foxp3⁺ cells was observed. Furthermore, the frequency of Foxp3⁺ cells in the MSG lesions and peripheral blood was negatively associated (r = −0.6679, P = 0.0065). MSG-infiltrating Foxp3⁺ cells were found to positively correlate with biopsy focus score (P = 0.05), infiltrating mononuclear cells, dendritic cells, and macrophages (P ≤ 0.024 each), and serum C4 levels (P = 0.0328), whereas lower Foxp3⁺ cell incidence correlated with adverse predictors for lymphoma development, such as the presence of C4 hypocomplementemia (P = 0.012) and SG enlargement (tendency, P = 0.067). Our findings suggest that the Foxp3⁺ T-regulatory cell frequency in the MSG lesions of SS patients correlates with inflammation grade and certain risk factors for lymphoma development.     

Clinical and pathological characteristics of four Korean patients with limb-girdle muscular dystrophy type 2B

Limb-girdle muscular dystrophy type 2B (LGMD2B), a subtype of autosomal recessive limb-girdle muscular dystrophy (ARLGMD), is characterized by a relatively late onset and slow progressive course. LGMD2B is known to be caused by the loss of the dysferlin protein at sarcolemma in muscle fibers. In this study, the clinical and pathological characteristics of Korean LGMD2B patients were investigated. Seventeen patients with ARLGMD underwent muscle biopsy and the histochemical examination was performed. For the immunocytochemistry, a set of antibodies against dystrophin, alpha, beta, gamma, delta-sarcoglycans, dysferlin, caveolin-3, and beta-dystroglycan was used. Four patients (24%) showed selective loss of immunoreactivity against dysferlin at the sarcolemma on the muscle specimens. Therefore, they were classified into the LGMD2B category. The age at the onset of disease ranged from 9 yr to 33 yr, and none of the patients was wheelchair bound at the neurological examination. The serum creatine kinase (CK) was high in all the patients (4010-5310 IU/L). The pathologic examination showed mild to moderate dystrophic features. These are the first Korean LGMD2B cases with a dysferlin deficiency confirmed by immunocytochemistry. The clinical, pathological, and immunocytochemical findings of the patients with LGMD2B in this study were in accordance with those of other previous reports.     

Role for the alternative complement pathway in ischemia/reperfusion injury

The terminal complement components play an important role in mediating tissue injury after ischemia and reperfusion (I/R) injury in rats and mice. However, the specific complement pathways involved in I/R injury are unknown. The role of the alternative pathway in I/R injury may be particularly important, as it amplifies complement activation and deposition. In this study, the role of the alternative pathway in I/R injury was evaluated using factor D-deficient (−/−) and heterozygote (+/−) mice. Gastrointestinal ischemia (GI) was induced by clamping the mesenteric artery for 20 minutes and then reperfused for 3 hours. Sham-operated control mice (+/− versus −/−) had similar baseline intestinal lactate dehydrogenase activity (P = ns). Intestinal lactate dehydrogenase activity was greater in −/− mice compared to +/− mice after GI/R (P = 0.02) thus demonstrating protection in the −/− mice. Intestinal myeloperoxidase activity in +/− mice was significantly greater than −/− mice after GI/R (P < 0.001). Pulmonary myeloperoxidase activity after GI/R was significantly higher in +/− than −/− mice (P = 0.03). Addition of human factor D to −/− animals restored GI/R injury and was prevented by a functionally inhibitory antibody against human factor D. These data suggest that the alternative complement pathway plays an important role in local and remote tissue injury after GI/R. Inhibition of factor D may represent an effective therapeutic approach for GI/R injury.     

Quantitation of Bacteria in Blood of Typhoid Fever Patients and Relationship between Counts and Clinical Features, Transmissibility, and Antibiotic Resistance

Salmonella typhi was isolated from 369 and Salmonella paratyphi A was isolated from 6 of 515 Vietnamese patients with suspected enteric fever. Compared with conventional broth culture of blood, direct plating of the buffy coat had a diagnostic sensitivity of 99.5% (95% confidence interval [CI], 97.1 to 100%). Blood bacterial counts were estimated by the pour plate method. The median S. typhi count in blood was 1 CFU/ml (range, <0.3 to 387 CFU/ml), of which a mean of 63% (95% CI, 58 to 67%) were intracellular. The mean number of bacteria per infected leukocyte was 1.3 (interquartile range [IQR], 0.7 to 2.4) CFU/cell (n = 81). Children (<15 years old; n = 115) had higher median blood bacterial counts than adults (n = 262): 1.5 (range, <0.3 to 387) versus 0.6 (range, <0.3 to 17.7) CFU/ml (P = 0.008), and patients who excreted S. typhi in feces had higher bacteremias than those who did not: a median of 3 (range, <0.3 to 32) versus 1 (range, <0.3 to 68) CFU/ml (P = 0.02). Blood bacterial counts declined with increasing duration of illness (P = 0.002) and were higher in infections caused by multidrug-resistant S. typhi (1.3 [range, <0.3 to 387] CFU/ml; n = 313) than in infections caused by antibiotic-sensitive S. typhi (0.5 [range, <0.3 to 32] CFU/ml; n = 62) (P = 0.006). In a multivariate analysis this proved to be an independent association, suggesting a relationship between antibiotic resistance and virulence in S. typhi.     

Regulatory T-Cell Depletion in Angioimmunoblastic T-Cell Lymphoma

Angioimmunoblastic T-cell lymphoma (AITL) is the most frequent nodal T-cell lymphoma and is characterized by a polymorphic lymph node infiltrate, various dysimmune disorders, and a poor prognosis. Regulatory T-cells (Treg) play an emerging role in the prognosis of non-Hodgkin B-cell lymphoma and mediate significant autoreactive T-cell suppression. In this report, we demonstrate that numbers of Treg are significantly decreased in AITL lymph nodes [n = 30, 91 (40–195) per high power fields] compared with follicular lymphoma [n = 19, 179 (86–355)] and reactive lymph nodes [n = 8, 186 (140–265)]. Moreover, the few Treg in lymph nodes of AITL are resting Treg (rTreg) and have a naive CD45RA+, PD1−, and ICOS− phenotype [n = 5, 57% of Treg are CD45RA+ (16–96)], in contrast to the Treg in follicular lymphomas [n = 5, 7.4% (1–13)] or reactive lymph nodes [n = 7, 18.6% (6–48)]. Interestingly, Treg depletion was not observed in AITL peripheral blood at diagnosis. Altogether, these data suggest that Treg depletion could contribute to the nodal neoplastic T_FH expansion and dysimmune symptoms in AITL.Angioimmunoblastic T-cell lymphoma (AITL), one of the most frequent entities among peripheral T-cell lymphoma,¹ is characterized by lymphadenopathy, B-symptoms, and an aggressive behavior.^2,3 Its natural history has been the subject of controversy, having been considered for many years to be a nonmalignant disorder or a dysimmune disease, until the clonal nature of AITL was proven by molecular studies.⁴The neoplastic cells in AITL show an immune profile closely related to that of follicular T-helper cells (T_FH), characterized by the expression of markers such as CD4, CD10, CXCL13, ICOS, and PD1.^5,6,7 The putative derivation of AITL from T_FH cells has also been demonstrated recently by gene profiling studies.⁸ Because AITL is characterized by a polymorphic proliferation and a dysimmune disorder, a pathogenic role for the peritumoral immune cells is possible. Regulatory T-cells (Treg) CD4⁺ CD25^hi FOXP3+ CD127^low in the tumoral microenvironment play an emerging role in lymphoma growth regulation.⁹ They are also responsible for autoreactive T-cell suppression in T-cell lymphopoiesis, and they can control inappropriate autoimmune responses.¹⁰ Furthermore, they comprise different subsets referred to as CD45RA− activated Treg (aTreg) and CD45RA+ resting Treg (rTreg).¹¹ Several recent studies underline the prognostic impact of Treg lymph node involvement in B-cell non-Hodgkin lymphoma and Hodgkin lymphoma.^12,13,14 However, Treg activity in T-cell non-Hodgkin lymphoma has not been evaluated. We report here quantitative and qualitative Treg evaluation in involved lymph nodes and blood samples of AITL patients, which suggest that Treg alteration could contribute to tumor cell development and autoimmune associated symptoms.     

Identical mutation in patients with limb girdle muscular dystrophy type 2B or Miyoshi myopathy suggests a role for modifier gene(s)

Limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM), a distal muscular dystrophy, are both caused by mutations in the recently cloned gene dysferlin, gene symbol DYSF. Two large pedigrees have been described which have both types of patient in the same families. Moreover, in both pedigrees LGMD2B and MM patients are homozygous for haplotypes of the critical region. This suggested that the same mutation in the same gene would lead to both LGMD2B or MM in these families and that additional factors were needed to explain the development of the different clinical phenotypes. In the present paper we show that in one of these families Pro791 of dysferlin is changed to an Arg residue. Both the LGMD2B and MM patients in this kindred are homozygous for this mutation, as are four additional patients from two previously unpublished families. Haplotype analyses suggest a common origin of the mutation in all the patients. On western blots of muscle, LGMD2B and MM patients show a similar abundance in dysferlin staining of 15 and 11%, respectively. Normal tissue sections show that dysferlin localizes to the sarcolemma while tissue sections from MM and LGMD patients show minimal staining which is indistinguishable between the two types. These findings emphasize the role for the dysferlin gene as being responsible for both LGMD2B and MM, but that the distinction between these two clinical phenotypes requires the identification of additional factor(s), such as modifier gene(s).     

CD4+ cells,macrophages, MHC-I and C5b-9 involve the pathogenesis of dysferlinopathy

Objective: Dysferlin is a sarcolemmal protein that plays an important role in membrane repair by regulating vesicle fusion with the sarcolemma. Mutations in the dysferlin gene (DYSF) lead to multiple clinical phenotypes, including Miyoshi myopathy (MM), limb girdle muscular dystrophy type 2B (LGMD 2B), and distal myopathy with anterior tibial onset (DMAT). Patients with dysferlinopathy also show muscle inflammation, which often leads to a misdiagnosis as inflammatory myopathy. In this study, we examined and analyzed the dyferlinopathy-associated immunological features. Methods: Comparative immunohistochemical analysis of inflammatory cell infiltration, and muscle expression of MHC-I and C5b-9 was performed using muscle biopsy samples from 14 patients with dysferlinopathy, 7 patients with polymyositis, and 8 patients with either Duchenne muscular dystrophy or Becker muscular dystrophy (DMD/BMD). Results: Immunohistochemical analysis revealed positive staining for immune response-related CD4⁺ cells, macrophages, MHC-I and C5b-9 in dysferlinopathy, which is in a different mode of polymyositis and DMD/BMD. Conclusion: These results demonstrated the involvement of immune factors in the pathogenesis of dysferlinopathy.     

Virulence Properties of Shiga Toxin-Producing Escherichia coli (STEC) Strains of Serogroup O118, a Major Group of STEC Pathogens in Calves

Shiga toxin-producing Escherichia coli (STEC) strains of serogroup O118 are the most prevalent group among STEC strains in diarrheic calves in Germany (L. H. Wieler, Ph.D. thesis, University of Giessen, 1997). To define their virulence properties, 42 O118 (O118:H16 [n = 38] and O118:H− [n = 4]) strains were characterized. The strains displayed three different Stx combinations (Stx1 [36 of 42], Stx1 and Stx2 [2 of 42], and Stx2 [4 of 42]). A total of 41 strains (97.6%) harbored a large virulence-associated plasmid containing hly_EHEC (hly from enterohemorrhagic E. coli). The strains’ adhesive properties varied in relation to the eukaryotic cells tested. Only 28 of 42 strains (66.7%) showed localized adhesion (LA) in the human HEp-2 cell line. In contrast, in bovine fetal calf lung (FCL) cells, the number of LA-positive strains was much higher (37 of 42 [88.1%]). The locus of enterocyte effacement (LEE) was detected in 41 strains (97.6%). However, not all LEE-positive strains reacted positively in the fluorescence actin-staining (FAS) test, which indicated the attaching and effacing (AE) lesion. In HEp-2 cells, only 22 strains (52.4%) were FAS positive, while in FCL cells, the number of FAS-positive strains was significantly higher (38 of 42 [90.5%; P < 0.001]). In conclusion, the vast majority of the O118 STEC strains from calves (41 of 42 [97.6%]) have a high virulence potential (stx, hly_EHEC, and LEE). This virulence potential and the high prevalence of STEC O118 strains in calves suggest that these strains could be a major health threat for humans in the future. In addition, the poor association between results of the geno- and phenotypical tests to screen for the AE ability of STEC strains calls the diagnostic value of the FAS test into question.     

Two endoplasmic reticulum-associated degradation (ERAD) systems for the novel variant of the mutant dysferlin: ubiquitin/proteasome ERAD(I) and autophagy/lysosome ERAD(II)

Dysferlin is a type-II transmembrane protein and the causative gene of limb girdle muscular dystrophy type 2B and Miyoshi myopathy (LGMD2B/MM), in which specific loss of dysferlin labeling has been frequently observed. Recently, a novel mutant (L1341P) dysferlin has been shown to aggregate in the muscle of the patient. Little is known about the relationship between degradation of dysferlin and pathogenesis of LGMD2B/MM. Here, we examined the degradation of normal and mutant (L1341P) dysferlin. Wild-type (wt) dysferlin mainly localized to the ER/Golgi, associated with retrotranslocon, Sec61alpha, and VCP(p97), and was degraded by endoplasmic reticulum (ER)-associated degradation system (ERAD) composed of ubiquitin/proteasome. In contrast, mutant dysferlin spontaneously aggregated in the ER and induced eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation and LC3 conversion, a key step for autophagosome formation, and finally, ER stress cell death. Unlike proteasome inhibitor, E64d/pepstatin A, inhibitors of lysosomal proteases did not stimulate the accumulation of the wt-dysferlin, but stimulated aggregation of mutant dysferlin in the ER. Furthermore, deficiency of Atg5 and dephosphorylation of eIF2alpha, key molecules for LC3 conversion, also stimulated the mutant dysferlin aggregation in the ER. Rapamycin, which induces eIF2alpha phosphorylation-mediated LC3 conversion, inhibited mutant dysferlin aggregation in the ER. Thus, mutant dysferlin aggregates in the ER-stimulated autophagosome formation to engulf them via activation of ER stress-eIF2alpha phosphorylation pathway. We propose two ERAD models for dysferlin degradation, ubiquitin/proteasome ERAD(I) and autophagy/lysosome ERAD(II). Mutant dysferlin aggregates on the ER are degraded by the autophagy/lysosome ERAD(II), as an alternative to ERAD(I), when retrotranslocon/ERAD(I) system is impaired by these mutant aggregates.     

Vascular endothelial growth factor-C, a potential paracrine regulator of glomerular permeability, increases glomerular endothelial cell monolayer integrity and intracellular calcium

We have previously reported expression of vascular endothelial growth factor (VEGF)-A and -C in glomerular podocytes and actions of VEGF-A on glomerular endothelial cells (GEnC) that express VEGF receptor-2 (VEGFR-2). Here we define VEGFR-3 expression in GEnC and investigate the effects of the ligand VEGF-C. Renal cortex and cultured GEnC were examined by microscopy, and both cell and glomerular lysates were assessed by Western blotting. VEGF-C effects on trans-endothelial electrical resistance and albumin flux across GEnC monolayers were measured. The effects of VEGF-C156S, a VEGFR-3-specific agonist, and VEGF-A were also studied. VEGF-C effects on intracellular calcium ([Ca²⁺]_i) were measured using a fluorescence technique, receptor phosphorylation was examined by immunoprecipitation assays, and phosphorylation of myosin light chain-2 and VE-cadherin was assessed by blotting with phospho-specific antibodies. GEnC expressed VEGFR-3 in tissue sections and culture, and VEGF-C increased trans-endothelial electrical resistance in a dose-dependent manner with a maximal effect at 120 minutes of 6.8 Ω whereas VEGF-C156S had no effect. VEGF-C reduced labeled albumin flux by 32.8%. VEGF-C and VEGF-A increased [Ca²⁺]_i by 15% and 39%, respectively. VEGF-C phosphorylated VEGFR-2 but not VEGFR-3, myosin light chain-2, or VE-cadherin. VEGF-C increased GEnC monolayer integrity and increased [Ca²⁺]_i, which may be related to VEGF-C-S particular receptor binding and phosphorylation induction characteristics. These observations suggest that podocytes direct GEnC behavior through both VEGF-C and VEGF-A.