| Objective:Progressive muscular dystrophies(PMD) are a heterogeneous group of inherited disorders that share similar clinical features and dystrophic changes on muscle biopsy. They are characterized by progressive muscle weakness and atrophy that affects limb and facial muscles to a variable degree. In specific forms, other muscles, including respiratory muscles and cardiac smooth muscles, can also be affected. Serum creatase always elevates of varying degrees. Electromyography(EMG) mostly shows myogenic damage. Muscular pathology shows muscle fibers are in variable sizes, degeneration, necrosis and regeneration. Besides, an inflammatory response can be seen in some of the muscle biopsies. There is no effective treatment at present.At present, a large number of studies have shown that many mechanisms, such as mechanical damage, inflammation, oxidative stress, autophagy, fibrosis, apoptosis, participate in the pathophysiological process of PMD. While the specific mechanisms behind the loss of muscle function are largely unknown.There is no obvious feature of clinical manifestations and routine histopathological examination of PMD. When inflammatory cells infiltration can be seen in the muscle biopsies, it is very difficult to distinguish PMD from idiopathic inflammatory myopathy(IIM). Someone may be misdiagnosed with IIM, and given in appropriate treatments.In this study, immunohistochemical staining(IHC) is performed in order to characterize the types of the infiltrating cells, and the expression profiles of major histocompatibility complex(MHC) class I in dystrophinopathy and dysferlinopathy patients. Pathological characteristics were summarized. In addition, the mechanism of inflammation in PMD is studied preliminary. So as to provide basis for accurate diagnosis and differential diagnosis in the future, and to lay a foundation of seeking for an effective treatment. Besides, it could provide a direction for treatment and prognosis.Methods: In this study, we collected 36 muscle samples diagnosed with PMD from the second hospital of Hebei medical university between 2012 and 2014. All the samples were made into serial sections for routine histological staining and IHC staining of dystrophic associate antibodies. The further clinical classification was made due to the results of IHC.13 muscle samples of dystrophinopathy(DMD/BMD group) or dysferlinopathy(DYSF group) were collected for the following experiment, including 8 and 5 cases in each group. We summarized the clinical features of these patients. Polymyositis(PM group), dermatomyositis(DM group) and the patients pathological diagnosed of normal(CO group) were selected to serve as control samples, including 7, 7, 8 cases respectively. IHC staining were performed using monoclonal antibodies against several inflammatory cell types(CD4, CD8, CD20, CD68) and MHC class I in all the muscle samples. We observed the types, distribution of infiltrating inflammatory cells and the expression pattern of MHC class I molecules under light microscope.Results: 1 The routine pathological features of the 36 cases of PMDUnder light microscope, HE staining revealed that most muscle fibers arranged loosely, became disorganized and showed different alterations at diameter. All the muscle biopsies were visible of atrophic muscle fibers, a substantially circular appearance, most were visible of hypertrophic muscle fibers at the same time. In parts of the cases, there were moderate to severe degeneration, necrosis and autophagy. Regenerating fibers, splitting fibers and central nuclei could also been seen in several cases. An inflammatory response was seen in 6 cases of the muscle biopsies. MGT staining could see ragged red fiber(RRF) in only 5 cases. NADH and SDH staining showed that oxidase distributed unevenly in all of the muscle biopsies. Most of the muscle biopsies were visible of hyperchromatic muscle fibers in NSE staining. Mild increase of enzymatic activity was seen in a few muscle biopsies in ACP. Moreover, in ORO and PAS staining, most showed moderate to extensive replacement of muscle fibers by adipose cells(fat) or saccharides. ATPase staining showed most muscle fibers were mosaic arrangement, atrophy involving the two types. 2 The IHC staining results of 36 cases of PMDDystrophin and dysferlin deficiency(absence or reduced) were found in 14 and 11 cases respectively. While, at least one of the four kind of α/β/γ/δ-sarcoglycan deficiency could be found in 9 cases. 3 The clinical data of the patients diagnosed with dystrophinopathy ordysferlinopathyThere are numerous phenotypes of PMD. Because inflammatory cells infiltration can be observed in parts of dystrophinopathy or dysferlinopathy patients, it is difficult to distinguish with IIM. So that, the clinical data of the only two kinds mentioned above were retrospectively analyzed. 3.1 DystrophinopathyThere were 8 males diagnosed with dystrophinopathy, containing 3 cases for DMD and 5 for BMD. Only one person had a family history. The average age of onset was 12.81 years, duration ranged from 3 months to 5years. 6 cases onset with bilateral lower limb weakness; 1 only represented with high serum creatine kinase; another 1 came to cardiology department first with complain of heart palpitations and chest distress. 7 cases had muscle weakness when taking physical examination, and muscle atrophy and hypertrophy were seen in 3 and 5 cases respectively. Serum creatine kinase was increased in all cases. EMG showed myogenic damage in 6 cases; one’s showed mixed damage; and the rest one was almost normal. 2 patients’ electrocardiogram(ECG) and/or echocardiography showed abnormal. There were 2 patients once misdiagnosed as “IIM” and given corticoids. 3.2 DysferlinopathyThere were 5 cases diagnosed with d dysferlinopathy, including 3 males and 2 females. No one had a definite family history. The average age of onset was 23 years, an average duration was 4.02 years.4 cases onset with muscle weakness; 1 merely had a high serum creatine kinase. 2 cases had both muscle atrophy and hypertrophy. Serum creatine kinase increased in all cases. EMG showed myogenic damage in 6 cases. In the past, 3 patients were misdiagnosed as “IIM” by muscle biopsies in outer court and given corticoids and/or immunosuppressant. 4 Phenotypic characteristics of inflammatory cells and expression of MHCclass I 4.1 Normal controlsIn the CO group, inflammatory cells were merely visible in the vessels of perimysium and endomysium. MHC class I was expressed only in the endothelial cells of vessels. 4.2 Dystrophinopathy and dysferlinopathyIn DMD/BMD and DYSF group, inflammatory cells were mainly observed as clusters or scattered not only in perivascular sites and interstitial tissue, but also around degenerating, necrotic and atrophic fibers. Macrophages and CD4+ T cells were more abundant than CD8+T cells, no obvious B cells could be seen.In the two groups, there was only mild expression of MHC class I on the sarcolemma(±~+), mainly on the sarcolemma of degenerating, necrotic and atrophic fibers infiltrating with inflammatory cells. 4.3 IIMIn PM and DM group, inflammatory cells infiltration could been seen in all cases. In the PM group, inflammatory cells were mainly observed in endomysial spaces and around necrotic fibers. A number of non-necrotic fibers were focally surrounded and invaded by predominantly inflammatory cells. Abundant CD8+T cells and macrophages were observed, CD4+T cells were less abundant, and few B cells were detectable. In DM group, inflammatory cells were mainly found in perimysial and perivascular sites, and CD4+T cells and macrophages were more frequent, and B cells were more abundant than in PM group. CD8+T cells were less abundant.MHC class I was expressed on normal-looking fibers as well as degenerating fibers in PM and DM(++~+++). In some cases, pronounced cytoplasmic staining was present in fibers as well as inflammatory cells.Conclusions:1 PMD comprise many phenotypes characterized by different clinical manifestations, and there was no specific clinical and pathological features. Diagnosis simply relying on these has limitation. There is a need to perform IHC staining.2 IHC staining using dystrophic associate monoclonal antibodies is helpful to further diagnosis and classification, so as to provide a direction for genetic analysis, treatment and prognosis.3 We suspect that the inflammatory response in muscle biopsies of dystrophinopathy and dysferlinopathy patients may be secondary to degeneration and necrosis of muscle fibers.4 Inflammatory cells infiltration may be seen in partial muscle biopsies of patients with dystrophinopathy and dysferlinopathy, and they are easily misdiagnosed as IIM. Detection the phenotypic characteristics of inflammatory cells and expression of MHC class I can be used as an auxiliary method for differential diagnosis. |
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