Role of 18F-FDG PET/CT Radiomics Features in the Differential Diagnosis of Solitary Pulmonary Nodules: Diagnostic Accuracy and Comparison between Two Different PET/CT Scanners.

The aim of this retrospective study was to investigate the ability of 18 fluorine-fluorodeoxyglucose positron emission tomography/CT (18F-FDG-PET/CT) metrics and radiomics features (RFs) in predicting the final diagnosis of solitary pulmonary nodules (SPN). We retrospectively recruited 202 patients who underwent a 18F-FDG-PET/CT before any treatment in two PET scanners. After volumetric segmentation of each lung nodule, 8 PET metrics and 42 RFs were extracted. All the features were tested for significant differences between the two PET scanners. The performances of all features in predicting the nature of SPN were analyzed by testing three classes of final logistic regression predictive models: two were built/trained through exploiting the separate data from the two scanners, and the other joined the data together. One hundred and twenty-seven patients had a final diagnosis of malignancy, while 64 were of a benign nature. Comparing the two PET scanners, we found that all metabolic features and most of RFs were significantly different, despite the cross correlation being quite similar. For scanner 1, a combination between grey level co-occurrence matrix (GLCM), histogram, and grey-level zone length matrix (GLZLM) related features presented the best performances to predict the diagnosis; for scanner 2, it was GLCM and histogram-related features and metabolic tumour volume (MTV); and for scanner 1 + 2, it was histogram features, standardized uptake value (SUV) metrics, and MTV. RFs had a significant role in predicting the diagnosis of SPN, but their accuracies were directly related to the scanner.

Acute Sarcomeric M-Line Disease Associated With ATP Synthase Subunit α Autoantibodies in Ankylosing Spondylitis.

M-line is the narrow transverse band located in the center of the sarcomeric A-band that is mainly responsible for the stabilization of myosin thick filaments. A 27-year-old male patient with a positive medical history for ankylosing spondylitis presented with one month of proximal upper limb muscle weakness associated with pain on both acromioclavicular joints. A biopsy of deltoid muscle documented the disappearance of M-line, the misalignment of myofilaments, and the loss of the distinction between the A and I bands. Complete resolution of muscle weakness occurred after one year of treatment with antiTNFα agent Etanercept. Because of the acute onset of symptoms and the recovery after immunosuppressive treatment we hypothesized that an immune-mediated mechanism was responsible for the muscle disorder. The serum IgG-mediated autoreactivity to skeletal muscle antigens resolved by bidimensional electrophoresis was assessed in the described patient and compared with that of control subjects. The comparative analysis of the immunoreactive spots revealed that ATP synthase subunit α is specifically recognized by patient's serum, suggesting that the protein might represent a putative antigenic target in the disease. This study reports an acute reversible myopathy pathologically characterized by M-line involvement and associated with serological antibodies to the subunit α of ATP synthase.

Bortezomib-Induced Muscle Toxicity in Multiple Myeloma.

Multiple myeloma (MM) accounts for ∼13% of all hematologic malignancies. Bortezomib treatment is effective in MM, but can be complicated with neurological side effects. We describe a patient with symptomatic MM who had a reversible metabolic myopathy associated with bortezomib administration and pathologically characterized by excessive storage of lipid droplets together with mitochondrial abnormalities. In a single-center prospective study, 14 out of 24 patients with symptomatic MM were treated with bortezomib and, among these, 7 developed muscular signs and/or symptoms. The myopathy was characterized by a proximal muscle weakness involving lower limbs and was an early complication. Complete resolution of muscle weakness occurred after treatment discontinuation. Conversely, none of the patients who received a treatment without bortezomib developed muscular symptoms. Experimental studies demonstrate that in primary human myoblasts bortezomib at low concentrations leads to excessive storage of lipid droplets together with structural mitochondrial abnormalities, recapitulating the pathologic findings observed in patient's muscle. Our data suggest that patients treated with bortezomib should be monitored for muscular signs and/or symptoms and muscle weakness should alert the clinician to the possibility of myopathy. Bortezomib-induced metabolic myopathy is a potentially reversible entity with important implications for management and treatment of patients with MM.

Evidence for caspase-dependent programmed cell death along with repair processes in affected skeletal muscle fibres in patients with mitochondrial disorders.

Mitochondrial disorders are heterogeneous multisystemic disorders due to impaired oxidative phosphorylation causing defective mitochondrial energy production. Common histological hallmarks of mitochondrial disorders are RRFs (ragged red fibres), muscle fibres with abnormal focal accumulations of mitochondria. In contrast with the growing understanding of the genetic basis of mitochondrial disorders, the fate of phenotypically affected muscle fibres remains largely unknown. We investigated PCD (programmed cell death) in muscle of 17 patients with mitochondrial respiratory chain dysfunction. We documented that in affected muscle fibres, nuclear chromatin is condensed in lumpy irregular masses and cytochrome c is released into the cytosol to activate, along with Apaf-1 (apoptotic protease-activating factor 1), caspase 9 that, in turn, activates effector caspase 3, caspase 6, and caspase 7, suggesting the execution of the intrinsic apoptotic pathway. Whereas active caspase 3 underwent nuclear translocation, AIF (apoptosis-inducing factor) mainly stayed within mitochondria, into which an up-regulated Bax is relocated. The significant increase in caspase 2, caspase 3 and caspase 6 activity strongly suggest that the cell death programme is caspase-dependent and the activation of caspase 2 together with PUMA (p53 up-regulated modulator of apoptosis) up-regulation point to a role for oxidative stress in triggering the intrinsic pathway. Concurrently, in muscle of patients, the number of satellite cells was significantly increased and myonuclei were detected at different stages of myogenic differentiation, indicating that a reparative programme is ongoing in muscle of patients with mitochondrial disorders. Together, these data suggest that, in patients with mitochondrial disorders, affected muscle fibres are trapped in a mitochondria-regulated caspase-dependent PCD while repairing events take place.

Immunoblot as a potential diagnostic tool for myofibrillar myopathies.

Myofibrillar myopathies (MFMs) are a group of inherited or sporadic neuromuscular disorders morphologically characterized by foci of myofibril dissolution, disintegration of the Z-disk, and insoluble protein aggregates within the muscle fibers. The diagnosis is based on muscle biopsy. Light and electron microscopy has a central role in the diagnostic work up, and immunohistochemistry shows abnormal deposition of several proteins including αB-crystallin, desmin, and myotilin. In contrast, immunoblotting does not have any diagnostic value because it does not highlight differences in the amount of involved proteins. We investigated the pattern and level expression of desmin, αB-crystallin, myotilin, and ZASP (Z-band alternatively spliced PDZ motif-containing protein) in muscle of seven patients with MFMs by immunoblotting after SDS-PAGE and 2D-PAGE using two different solubilizing solutions, one radioimmunoprecipitation assay (RIPA) buffer, and the other urea-containing buffer. Our data demonstrated that urea-containing buffer improves the solubilization and recovery of desmin, αB-crystallin, myotilin, and ZASP as compared with RIPA buffer and that the total content of these proteins is increased in muscles of patients. The present results provide evidence that immunoblotting is an additional tool for confirming diagnosis of MFMs.

Abnormal expression of RNA polymerase II-associated proteins in muscle of patients with myofibrillar myopathies.

AIMS: Myofibrillar myopathies (MFMs) are a group of inherited or sporadic neuromuscular disorders characterized morphologically by foci of myofibril dissolution, disintegration of the Z-disk and insoluble protein aggregates within the muscle fibres. The sequential events leading to muscle fibre damage remains largely unknown. METHODS AND RESULTS: We investigated the expression and the cellular localization of RNA polymerase II (RNAPII)-associated proteins (RPAPs) in muscle biopsies from patients with genetically proven and sporadic MFMs. Our data demonstrated that RPAP2, and to a lesser extent GPN1/RPAP4, are accumulated focally in the cytoplasm of MFM muscle fibres in which they co-localize with POLR2A/RPB1, the largest subunit of RNAPII, and correspond to αB-cystallin deposits in distribution and staining intensity. No abnormal staining for RPAP2 has been observed in muscle of patients with central cores, minicores and neurogenic target fibres. CONCLUSIONS: Together, these findings could provide new insights into the molecular pathogenesis of MFMs and suggest that RPAP2 immunostaining can be a useful diagnostic tool to depict protein aggregates in MFMs.

Differential regulation of TNF receptors in maternal leukocytes is associated with severe preterm preeclampsia.

We tested the hypothesis that maternal peripheral blood leukocytes contribute to elevated levels of soluble TNF receptors (sTNFR) in preeclampsia (PE) with concomitant intrauterine growth restriction (IUGR). TNFR1 and TNFR2 were evaluated in a cross-sectional study comparing preeclamptic (n = 15) with or without IUGR versus normotensive pregnant women (PREG, n = 30), and non-pregnant controls (Con; n = 20). Plasma levels of sTNFR1 were higher in PE (1675.0 ± 227.1 pg/mL) compared with PREG (1035.0 ± 101.1 pg/mL) and Con (589.3 ± 82.67 pg/mL), with the highest values observed in PE with IUGR (2624.0 ± 421.4 pg/mL; n = 6). Plasma sTNFR2 was higher during pregnancy (PE: 1836.0 ± 198.7 pg/mL; PREG: 1697.0 ± 95.0 pg/mL) compared with Con (598.3 ± 82.7 pg/mL). Urinary levels of sTNFR1 and sTNFR2 were higher in PE and PREG compared with the Con group. Abundance of TNFR1 mRNA in peripheral blood leukocytes was strongly correlated with plasma levels of sTNFR1 in PE. However, TNFR2 mRNA accumulation in leukocytes did not correlate with sTNFR2 plasma levels. The level of sTNFR1 in plasma was correlated with body weight of the newborn (r = -0.56). The data suggest that maternal leukocytes contribute to sTNFR1 levels in plasma in association with decreasing newborn weight and PE with concomitant IUGR.

Polymyositis in solid organ transplant recipients receiving tacrolimus.

Tacrolimus, also known as FK506, is an immunosuppressive agent widely used for the prevention of acute allograft rejection in organ transplantation and for the treatment of immunological diseases. This study reports two male patients who underwent solid organ transplantation (liver and kidney). After transplant, the patients received continuous immunosuppressive therapy with oral tacrolimus and later presented clinical manifestations and laboratory signs of myopathy. Muscle biopsies of both patients clearly documented an inflammatory myopathy with the histological features of polymyositis including CD8+ T cells which invaded healthy muscle fibers and expressed granzyme B and perforin, many CD68+ macrophages and MHC class I antigen upregulation on the surface of most fibers. Because of the temporal association while receiving tacrolimus and since other possible causes for myopathy were excluded, the most likely cause of polymyositis in our patients was tacrolimus toxicity. We suggest that patients on tacrolimus should be carefully monitored for serum CK levels and clinical signs of muscle disease.

SERCA1 protein expression in muscle of patients with Brody disease and Brody syndrome and in cultured human muscle fibers.

Brody disease is an inherited myopathy associated with a defective function of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 1 (SERCA1) protein. Mutations in the ATP2A1 gene have been reported only in some patients. Therefore it has been proposed to distinguish patients with ATP2A1 mutations, Brody disease (BD), from patients without mutations, Brody syndrome (BS). We performed a detailed study of SERCA1 protein expression in muscle of patients with BD and BS, and evaluated the alternative splicing of SERCA1 in primary cultures of normal human muscle and in infant muscle. SERCA1 reactivity was observed in type 2 muscle fibers of patients with and without ATP2A1 mutations and staining intensity was similar in patients and controls. Immunoblot analysis showed a significant reduction of SERCA1 band in muscle of BD patients. In addition we demonstrated that the wild type and mutated protein exhibits similar solubility properties and that RIPA buffer improves the recovery of the wild type and mutated SERCA1 protein. We found that SERCA1b, the SERCA1 neonatal form, is the main protein isoform expressed in cultured human muscle fibers and infant muscle. Finally, we identified two novel heterozygous mutations within exon 3 of the ATP2A1 gene from a previously described patient with BD.

Overexpression of TNF-α in mitochondrial diseases caused by mutations in mtDNA: evidence for signaling through its receptors on mitochondria.

Mitochondrial diseases (MDs) are heterogeneous disorders due to impaired respiratory chain function causing defective ATP production. Although the disruption of oxidative phosphorylation is central to the MD pathophysiology, other factors may contribute to these disorders. We investigated the expression and the cellular localization of TNF-α and its receptors, TNFR1 and TNFR2, in muscle biopsies from 15 patients with mitochondrial respiratory chain dysfunction. Our data unambiguously demonstrate that TNF-α is expressed in muscle fibers with abnormal focal accumulations of mitochondria, so-called ragged red fibers, and is delivered to mitochondria where both receptors are localized. Moreover TNF receptors are differentially regulated in patients' muscle in which the expression levels of TNFR1 mRNA are decreased and those of TNFR2 mRNA are increased compared with controls. These findings suggest for the first time that TNF-α could exert a direct effect on mitochondria via its receptors.

Selective pseudohypertrophy of vastus medialis muscles associated with calpain 3 deficiency.

INTRODUCTION: Calpain 3 deficiency causes limb girdle muscular dystrophy type 2A, which is one of the most common forms of limb girdle muscular dystrophy. Nevertheless, calpainopathy is not always associated with mutations in the specific gene and secondary reduction in protein expression has been described. CASE REPORT: We report a case of a 43-year-old man who complained of thigh muscle stiffness and had muscle hypertrophy of both vastus medialis with prolonged myotonic contraction by percussion. A muscle biopsy showed dystrophic features and calpain 3 deficiency was shown by immunoblot analysis although mutations in the specific gene were not found. Known cases of secondary calpain 3 protein deficiency were ruled out and mutations in MD1 and MD2 genes were excluded. CONCLUSIONS: This patient represents the first case of calpain 3 deficiency with selective pseudohypertrophy of vastus medialis muscles.

Increased protein nitration in mitochondrial diseases: evidence for vessel wall involvement.

Mitochondrial diseases (MD) are heterogeneous disorders because of impairment of respiratory chain function leading to oxidative stress. We hypothesized that in MD the vascular endothelium may be affected by increased oxidative/nitrative stress causing a reduction of nitric oxide availability. We therefore, investigated the pathobiology of vasculature in MD patients by assaying the presence of 3-nitrotyrosine in muscle biopsies followed by the proteomic identification of proteins which undergo tyrosine nitration. We then measured the flow-mediated vasodilatation as a proof of altered nitric oxide generation/bioactivity. Here, we show that 3-nitrotyrosine staining is specifically located in the small vessels of muscle tissue and that the reaction is stronger and more evident in a significant percentage of vessels from MD patients as compared with controls. Eleven specific proteins which are nitrated under pathological conditions were identified; most of them are involved in energy metabolism and are located mainly in mitochondria. In MD patients the flow-mediated vasodilatation was reduced whereas baseline arterial diameters, blood flow velocity and endothelium-independent vasodilatation were similar to controls. The present results provide evidence that in MD the vessel wall is a target of increased oxidative/nitrative stress.

A metabolic shift induced by a PPAR panagonist markedly reduces the effects of pathogenic mitochondrial tRNA mutations.

Mutations in mitochondrial DNA-encoded tRNA genes are associated with many human diseases. Activation of peroxisome proliferator-activated receptors (PPARs) by synthetic agonists stimulates oxidative metabolism, induces an increase in mitochondrial mass and partially compensates for oxidative phosphorylation system (OXPHOS) defects caused by single OXPHOS enzyme deficiencies in vitro and in vivo. Here, we analysed whether treatment with the PPAR panagonist bezafibrate in cybrids homoplasmic for different mitochondrial tRNA mutations could ameliorate the OXPHOS defect. We found that bezafibrate treatment increased mitochondrial mass, mitochondrial tRNA steady state levels and enhanced mitochondrial protein synthesis. This improvement resulted in increased OXPHOS activity and finally in enhanced mitochondrial ATP generating capacity. PPAR panagonists are known to increase the expression of PPAR gamma coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis. Accordingly, we found that clones of a line harbouring a mutated mitochondrial tRNA gene mutation selected for the ability to grow in a medium selective for OXPHOS function had a 3-fold increase in PGC-1α expression, an increase that was similar to the one observed after bezafibrate treatment. These findings show that increasing mitochondrial mass and thereby boosting residual OXPHOS capacity can be beneficial to an important class of mitochondrial defects reinforcing the potential therapeutic use of approaches stimulating mitochondrial proliferation for mitochondrial disorders.

Brody disease: insights into biochemical features of SERCA1 and identification of a novel mutation.

Brody disease is an inherited disorder of skeletal muscle function characterized by increasing impairment of relaxation during exercise. The autosomal recessive form can be caused by mutations in the ATP2A1 gene, which encodes for the sarcoplasmic/endoplasmic reticulum Ca-ATPase 1 (SERCA1) protein. We studied 2 siblings affected by Brody disease. The patients complained of exercise-induced delay of muscle relaxation and stiffness since childhood and had gene analysis of ATP2A1. Morphologic and biochemical studies were performed on a muscle biopsy from 1 patient. The biopsy showed fiber size variation and increased numbers of fibers with internal nuclei. Ultrastructural examination revealed dilatation of lateral cisternae and proliferation of tubular elements of the sarcoplasmic reticulum. By immunohistochemistry, SERCA1 was expressed in a normal pattern, but sarcoplasmic reticulum Ca-ATPase activity was significantly reduced. Immunoblotting after high-resolution 2-dimensional gel electrophoresis showed a significant difference in the amount of SERCA1 protein between the patient and controls. Both patients were found to have 2 previously unreported in-frame deletions in ATP2A1. Because SERCA1 protein has specific biochemical characteristics in our patient, these results underline the importance of a pathologic and biochemical analyses for the diagnosis. In addition, we describe 2 novel mutations in the ATP2A1 gene.

SERCA1 and calsequestrin storage myopathy: a new surplus protein myopathy.

We describe four patients, from four different families, affected by a mild myopathy or asymptomatic elevated serum creatine kinase levels, in whom toluidine blue-stained semithin sections of muscle specimens revealed inclusions of different size and shape. The inclusions did not stain by routine histochemical studies. The sarcoplasmic or endoplasmic reticulum calcium 1 (SERCA1) ATPase and/or calsequestrin reactivity of inclusions, by immunohistochemistry, and the SERCA1- and calsequestrin-increased expression, by immunoblot, suggested that inclusions were constituted by an excess of proteins normally present in the terminal cisternae of sarcoplasmic reticulum. Our cases, both sporadic and familial, represent a new type of surplus protein myopathy.

Circulating monocyte chemoattractant protein-1 and early development of nephropathy in type 1 diabetes.

OBJECTIVES: To investigate the possible role of hyperglycemia-dependent monocyte chemoattractant protein (MCP)-1 biosynthesis in the pathophysiology of early nephropathy in type 1 diabetes. RESEARCH DESIGN AND METHODS: We studied 30 patients with type 1 diabetes (15 with and 15 without microalbuminuria) compared with matched healthy control subjects. Plasma MCP-1 and plasma oxidant status (vitamin E, fluorescent products of lipid peroxidation [FPLPs], malondialdehyde [MDA]), HbA(1c), and albumin excretion rate [AER]) were evaluated at baseline. Furthermore, MCP-1, vitamin E, AER, and HbA(1c) were also analyzed in the microalbuminuric diabetic patients and in the healthy volunteers after 8 weeks of high-dose (600 mg b.i.d.) vitamin E treatment. RESULTS: FPLPs, MDA, and MCP-1 were significantly higher, whereas vitamin E was significantly lower in patients with microalbuminuria and poorer glycemic control as compared with normoalbuminuric patients and healthy control subjects. Plasma MCP-1 was positively correlated with HbA(1c), FPLPs, MDA, and AER, whereas plasma MCP-1 showed an inverse correlation with vitamin E. Interestingly, both MCP-1 and AER decreased significantly after vitamin E treatment, despite no changes in HbA(1c) values. CONCLUSIONS: This study suggests that prolonged hyperglycemia may lead to early renal complications in type 1 diabetes by inducing MCP-1 biosynthesis via enhanced oxidative stress. Long-term treatment of high-dose vitamin E significantly decreased MCP-1, thus providing a rationale basis for evaluating vitamin E supplementation as therapy adjuvant to conventional insulin treatment in type 1 diabetic patients in whom an acceptable glycemic control is difficult to achieve despite appropriate insulin treatment.