What factor within the Japanese Association for Acute Medicine (JAAM) disseminated intravascular coagulation (DIC) criteria is most strongly correlated with trauma induced DIC? A retrospective study using thromboelastometry in a single center in Japan.

PURPOSE: The diagnostic criteria for disseminated intravascular coagulation (DIC) established by the Japanese Association for Acute Medicine (JAAM) is able to diagnose DIC accurately and promptly. The aim of this retrospective study is to evaluate the degree of association between each parameter of JAAM DIC criteria and the diagnosis of trauma induced DIC (T-DIC) utilizing thromboelastometry (ROTEM). METHODS: Trauma patients transported to our hospital with ROTEM performed in the emergency department between January 2013 and December 2015 were enrolled in this study. We evaluated (1) the characteristics of T-DIC, (2) the relationships between T-DIC and each parameter of the JAAM DIC criteria and (3) the diagnostic accuracies of each parameter for T-DIC by statistical measurement. RESULTS: All 72 patients (21 T-DIC and 51 control) were included in primary analysis. T-DIC was significantly related to younger age, more severe trauma scores, more cases of massive transfusions, and remarkable coagulation abnormality detected by standard coagulation tests. In the cases of T-DIC, ROTEM showed longer clotting time, lower acceleration, lower clot firmness, and inhibited fibrinolysis in EXTEM/INTEM. Within the JAAM DIC score, PT-INR ≥1.2 was the most accurate factor for T-DIC diagnosis; sensitivity 60.0%, specificity 100.0%, and accuracy 88.7%. PT-INR ≥1.2 was statistically correlated with the JAAM DIC score (p < 0.001, r = 0.709). The univariate analysis based on 1.2 of PT-INR indicated statistical differences in most categories of ROTEM, which is similar to analysis performed for the presence and absence of T-DIC. CONCLUSIONS: Among JAAM DIC criteria, the PT-INR ≥1.2 was the most accurate factor for both the diagnosis of T-DIC and the evaluation of its severity.

Calreticulin mutant mice develop essential thrombocythemia that is ameliorated by the JAK inhibitor ruxolitinib.

Mutations of calreticulin (CALR) are detected in 25-30% of patients with essential thrombocythemia (ET) or primary myelofibrosis and cause frameshifts that result in proteins with a novel C-terminal. We demonstrate that CALR mutations activated signal transducer and activator of transcription 5 (STAT5) in 293T cells in the presence of thrombopoietin receptor (MPL). Human megakaryocytic CMK11-5 cells and erythroleukemic F-36P-MPL cells with knocked-in CALR mutations showed increased growth and acquisition of cytokine-independent growth, respectively, accompanied by STAT5 phosphorylation. Transgenic mice expressing a human CALR mutation with a 52 bp deletion (CALRdel52-transgenic mice (TG)) developed ET, with an increase in platelet count, but not hemoglobin level or white blood cell count, in association with an increase in bone marrow (BM) mature megakaryocytes. CALRdel52 BM cells did not drive away wild-type (WT) BM cells in in vivo competitive serial transplantation assays, suggesting that the self-renewal capacity of CALRdel52 hematopoietic stem cells (HSCs) was comparable to that of WT HSCs. Therapy with the Janus kinase (JAK) inhibitor ruxolitinib ameliorated the thrombocytosis in TG mice and attenuated the increase in number of BM megakaryocytes and HSCs. Taken together, our study provides a model showing that the C-terminal of mutant CALR activated JAK-STAT signaling specifically downstream of MPL and may have a central role in CALR-induced myeloproliferative neoplasms.

Immobility reduces muscle fiber necrosis in dystrophin deficient muscular dystrophy.

Duchenne/Becker muscular dystrophy is a progressive muscle disease, which is caused by the abnormality of dystrophin. Spina bifida is characterized by paralysis of the feet, with most of the upper extremities not being affected. We report here on the first case of Becker muscular dystrophy coinciding with spina bifida. The muscle biopsy specimens of the patient showed dystrophic changes in upper extremities, but clearly less in lower extremities. The results show that the restriction of excessive exercise is important for dystrophin deficiency disease.

Disturbed circadian core body temperature rhythm and sleep disturbance in school refusal children and adolescents.

We examined the circadian rhythm of core body temperature (CBT) in 22 school refusal patients, ages between 12 and 18 years, who did not have any physical or psychiatric disorders, but had indefinite complaints, and were suspected to have a circadian rhythm disturbance. To obtain normal data for analysis, CBT in 9 healthy age-matched school attendants who did not have any sleep, psychiatric, or medical disturbance were monitored. Circadian variation of CBT in school refusal patients did not present a clear rhythm, and appearance time of their lowest CBT was markedly delayed compared to healthy subjects. Amplitude of circadian CBT changes, fitted to a cosinor curve by the least square method, was significantly smaller in school refusals than in healthy subjects. These findings suggest that in school refusal patients who do not have physical and psychiatric disorders, clinical psychosomatic symptoms (e.g., fatigue and memory disturbance) and school refusal could be closely related to the desynchronization of their biorhythms, particularly the circadian rhythm of body temperature and sleep-wake rhythm.

Childhood-type myositis and linear scleroderma.

A 5-year-old girl had linear scleroderma on the flexor surface of the right arm; muscle wasting included the shoulder girdle. IgM fluorescence on blood vessels and along dermal-epidermal junction was observed by direct immunofluorescence in biopsied skin. Biceps muscle underlying the plaque of the scleroderma showed atrophy of entire fascicles, perifascicular atrophy, and cellular infiltration around blood vessels that are quite similar to those found in childhood-type dermatomyositis. In addition, various abnormalities, including edema and thickening of basal lamina, were found on blood vessels in muscle tissue. The results suggested that the autoimmune collagen vascular disorder is responsible for this condition.

Infantile glycogen storage myopathy in a girl with phosphorylase kinase deficiency.

A 19-month-old girl with moderate hypotonia was studied. Histochemical and electronmicroscopic findings revealed that many skeletal muscle fibers contained an excess amount of glycogen. The phosphorylase reaction was normalized only after activation with 5' AMP. Biochemical studies showed an increased glycogen content and decreased activities of phosphorylase "a" and an active form of phosphorylase kinase, whereas activities of total phosphorylase, total phosphorylase kinase, and cyclic AMP-dependent protein kinase were all in the normal range. Thus, phosphorylase kinase in the patient's muscle seemed to be a variant form, which was activated partially under the physiologic condition. This condition may be inherited as an X-linked recessive trait.

Heterogeneous phenotypes of mitochondrial encephalomyopathy in a single kindred.

Five patients with mitochondrial disorders in a single family showed marked heterogeneity of clinical signs and symptoms. Two patients had the syndrome of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes; one had blepharoptosis, seizures, and diabetes insipidus; and two had a nonspecific encephalomyopathic disorder. This family supports the concept of a "mitochondrial cytopathy."

Atrophy of the cerebellum and brainstem in dentatorubral pallidoluysian atrophy. Influence of CAG repeat size on MRI findings.

To elucidate how the size of the expanded CAG repeat of the gene for dentatorubral pallidoluysian atrophy (DRPLA) and other factors affect the atrophy of the brainstem and cerebellum, and the appearance of high-intensity signals on T2-weighted MRI of the cerebral white matter of patients with DRPLA, we quantitatively analyzed the MRI findings of 26 patients with DRPLA, the diagnosis of which was confirmed by molecular analysis of the DRPLA gene. When we classified the patients into two groups based on the size of the expanded CAG repeat of the DRPLA gene (group 1, number of CAG repeat units > or = 66; group 2, number of CAG repeat units < or = 65), we found strong inverse correlations between the age at MRI and the areas of midsagittal structures of the cerebellum and brainstem in group 1 but not in group 2. Multiple regression analysis, however, revealed that both the patient's age at MRI and the size of the expanded CAG repeat correlated with the areas of midsagittal structures. Involvement of the cerebral white matter as detected on T2-weighted images was observed more frequently in patients belonging to group 2 than in group 1 patients. Furthermore it was demonstrated that high-intensity signals can be detected on T2-weighted images of the cerebral white matter of patients with a largely expanded CAG repeat (group 1) in their thirties. These results suggest that patient age as well as the size of the expanded CAG repeat are related to the degree of atrophy of the brainstem and cerebellum, and the white matter changes in patients with DRPLA.

Nerve growth factor receptor immunoreactivity on the tunica adventitia of intramuscular blood vessels in childhood muscular dystrophies.

Muscle tissues from cases of childhood neuromuscular disorders were examined immunohistochemically and immunoelectrophoretically using a monoclonal antibody against the human nerve growth factor receptor (NGFR). Strong NGFR immunoreactivity on the tunica adventitia of blood vessels and proliferating peripheral nerve endings in biopsied muscle specimens from muscular dystrophy patients was observed, but it was almost completely absent in specimens from non-diagnostic controls and cases of other neuromuscular disorders. This suggests a process in the sympathetic nervous system involving blood vessels in muscular dystrophies. Immunoblot analysis failed to show a band corresponding to 70-75 kd, the reported molecular size of the NGFR, but showed a clear band corresponding to 25 kd in muscular dystrophy patients, which is assumed to be a detached amino-terminal domain of the NGFR.

The lacZ gene under the control of the 7 kb of human dystrophin muscle specific promoter is expressed in cardiac muscle but not in adult skeletal muscle in transgenic mice.

In previous transgenic studies, we reported a 0.9 kb fragment from a mouse dystrophin muscle promoter that contains the regulatory elements required for expression of dystrophin only in the right heart. In this study, to further characterize the regulation of muscle type of promoter, we analyzed promoter activity and tissue specificity using a total 14 kb fragment around the human dystrophin muscular-specific exon 1 in vitro and in vivo. In vitro analysis showed that the lacZ construct of the 7 kb promoter and 7 kb intron 1 was expressed 2.5 times as strong as the lacZ construct of only the 7 kb promoter in C2/4 myotubes. In vivo analysis revealed expression of both constructs in the whole heart, skeletal muscle and vascular smooth muscle in embryos. However, in adults, the expression in skeletal muscle disappeared. We conclude that the 7 kb upstream region and the 7 kb intronic region included responsible elements for the expression in the heart, but not in skeletal muscle in vivo. It is possible that a strong enhancer element for skeletal muscle exists in some other region.

Transient expression of collagen type XIV during muscle development and its reappearance after denervation and degeneration.

In the formation of muscle pattern, the architectural arrangement is believed to be controlled by the local connective tissue cells. In this study we examined the immunohistological localization of Type XIV collagen recognized by a monoclonal antibody, MAb DBM, in embryonic chick hind limbs from stage (St.) 27 to 2 weeks post hatching. DBM staining was transiently observed in the epimysium from St. 30, in the perimysium of the dorsal region from St. 37, and in the entire perimysium from St. 39. After hatching, DBM staining was notably diminished in both epimysium and perimysium. In contrast, DBM staining and in situ hybridization signals for Type XIV collagen mRNA increased in the muscle connective tissues after denervation and around the regenerating muscle fibers. Therefore, Type XIV collagen expression appears to coincide with muscle activity and muscle regenerating conditions, and Type XIV collagen is considered to play roles in muscle development and regeneration.

Complex glycerol kinase deficiency: molecular-genetic, cytogenetic, and clinical studies of five Japanese patients.

Five male Japanese patients with complex glycerol kinase deficiency (CGKD) and their relatives were studied clinically, cytogenetically, and molecular-genetically. All patients had muscular dystrophy or muscle weakness, mental retardation, congenital adrenal hypoplasia, and glycerol kinase deficiency. High-resolution GTG-banded chromosomes showed a microdeletion in the Xp21 region in all four patients examined and in all five mothers. Southern hybridizations, after digestions by restriction endonucleases, with various cloned DNAs (D2, 99-6, B24, C7, L1-4, cDMD13-14, J66-HI, P20, J-Bir, ERT87-30, ERT87-15, ERT87-8, ERT87-1, XJ-1.1, 754, cx5.7, and OTC-1) that are located around Xp21 also showed a deletion in the genome of all patients and mothers. Although the deletion differed in size among patients, a segment commonly absent was located between the genomic sequences corresponding to L1-4 and cDMD13-14. This finding indicated that the gene coding for glycerol kinase (GK) is located within this segment. A comparison of the clinical manifestations of the present five patients and reported CGKD or Duchenne muscular dystrophy (DMD) patients with DNA deletion suggests the existence of a certain gene responsible for gonadotropin deficiency (GTD). The result of the present study and results of previous studies suggest that genes for ornithine transcarbamylase (OTC), DMD, and GK and putative genes responsible for congenital adrenal hypoplasia (AHC) and GTD are arranged from telomere to centromere as pter--GTD--AHC--GK--DMD--OTC--cen.

Molecular-genetic study of Duchenne and Becker muscular dystrophies: deletion analyses of 45 Japanese patients and segregation analyses in their families with RFLPs based on the data from normal Japanese females.

This study consisted of 1) molecular deletion analyses in patients with Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) using the entire cDNA for the DMD gene as hybridization probes, 2) RFLP analyses in a large number of Japanese normal women using 11 DMD-linked cloned DNAs as probes, and 3) segregation analyses with these RFLP data in 17 DMD families in which prenatal or carrier diagnosis was required. The deletion study showed that 18 (43%) of 42 male DMD patients had a deletion within the DMD gene, while no detectable deletion was found in 3 BMD patients. These deletions were preferentially observed at the 5' end of the DMD gene, while no deletion was found in the 3' portion of the gene. Of a total of 15 RFLPs detected with the 11 probes, one was a new RFLP (probe/enzyme: P20/MspI). In 6 RFLPs, the allele frequencies in the Japanese were statistically different from those in the Caucasian. Based on the RFLP data combined with the result of the deletion study, an estimated diagnostic rate for prenatal diagnosis and/or carrier detection in the Japanese DMD families was 63%. The real diagnostic rate obtained from the prenatal and carrier diagnoses, which were practically performed in 17 families, corresponded to the estimation. A protocol useful for the diagnosis in Japanese DMD families is presented.

Neuropathology of "spinning syndrome" induced by prenatal intoxication with a PCB in mice.

A striking motor dysfunction, "spinning syndrome," developed with a high frequency in weaning mice whose dams received oral 3,4,3',4'-tetrachlorobiphenyl (4-CB) during gestation (day 10 through day 16). The syndrome is permanent and is characterized by swift circling movements sustained in one direction at a minimal rate of 40 turns/min (usually 50 to 150 turns/min), restlessness, and hyperkinesia. Twenty-four spinners and 4-CB nonspinners and age-matched controls were subjected to histopathologic, histofluorescent, histochemical, and electron microscopic studies. The most reliable histopathologic marker for prenatal 4-CB injury to the CNS is the presence of cylindrical CNS peninsulas (CCPs) in the spinal and cranial nerve roots. The CCPs consist of either CNS-type myelinated fibers, unmyelinated fibers, or astroglial bundles in varying proportions, and are enclosed by a basement membrane. The CCPs are also observed in 4-CB nonspinners but in none of 12 controls studied. A selective defect in synaptogenesis induced prenatally by 4-CB is proposed as the primary event pursuant to the development of the CCPs, while interference with synaptogenesis may have occurred selectively in the striatonigral dopaminergic system. This is suggested by electron microscopy on the nucleus accumbens and also by the responses to administration of dopaminergic agonists and antagonist. The 4-CB induced clinico-pathologic anomaly may serve as a singular model for understanding human neurologic disorders, in particular, Werdnig-Hoffmann disease and minimal brain dysfunction syndrome.

Developmental studies of dystrophin-positive fibers in mdx, and DRP localization.

Dystrophin positive fibers (DPFs) were observed in about 1% of the total muscle fibers in 1-year-old mice. Some of these fibers were found to have positive staining with all six antibodies, while others showed a negative reaction with specific antibodies. These results suggest that the most likely mechanism giving rise to these DPFs is a second site mutation which prepares in-frame deletion. A study of the frequency of DPF during development showed single and scattered DPFs in younger mice, which gradually increased in number and began to form small groups with age. DRP was observed constantly on the neuromuscular junctions in both control and mdx muscle, and surface membrane of immature muscle fibers such as regenerating fibers in mdx and newborn muscle during 2 weeks of age in control and mdx.

Dystrophin isoforms and/or cross-reactive proteins on neurons and glial cells in control and mdx central nervous systems.

We studied the central nervous system (CNS) of control mice in comparison with that of mdx mice, immunohistochemically and immunoelectrophoretically, using 5 kinds of polyclonal antibodies against dystrophin (DMDP-II, 60-kDa, 30-kDa, P-20 and DMDP-IV) to determine whether or not and, if so, how dystrophin exists in the central nervous system. A positive dystrophin reaction was seen on the neurons and glial cells in both control and mdx tissue, without any immunohistochemical difference. In control mice, Western blot analysis showed two relatively clear bands corresponding to 400-kDa, with all 4 antibodies used (60-kDa, 30-kDa, P-20 and DMDP-IV), and 280-kDa, with 3 of them, the exception being 30-kDa, and 2 other faint bands corresponding to larger M(r) than 400-kDa, with 3 of them, the exception being P-20, respectively. In the mdx CNS, the 400-kDa band was absent, the other 3 bands being seen. The results suggest that dystrophin really exists in the control CNS, and some dystrophin isoforms or cross-reactive proteins exist on the neurons and glial cells in mdx as well as control mice. The localization of dystrophin in CNS also suggests its physiological function in the conduction system rather than a mechanical one, and a defect of it in CNS is a possible cause of the mental retardation in Duchenne muscular dystrophy.

Dystrophin isoforms expressed in the mouse retina.

The dystrophin gene is expressed in various tissues of the mouse. Previous immunohistochemical studies suggested the existence of dystrophin protein in the outer plexiform layer of the retina. We analyzed mRNAs from the retina and other tissues of mice and detected the dystrophin transcripts (DT) with the use of the reverse transcription and polymerase chain reaction (RT-PCR) method. The 5' sequences, corresponding to the first exon, of DT in the retina was mainly the brain type, whereas in the 3' region of DT that corresponds to the C-terminal domain of dystrophin, some additional RT-PCR products were detected. Base sequences in three of them showed homology to those for previously reported human dystrophin isoforms. The DT variations in mice were identical between the retina and the brain. It was thus concluded that dystrophin really expresses in the mouse retina and most of the retinal dystrophin proteins belong to the brain type isoform.

Vascular endothelial cell injury and platelet embolism in Duchenne muscular dystrophy at the preclinical stage.

Blood vessels in muscle biopsy specimens from 4 Duchenne muscular dystrophy (DMD) patients (including 3 at the preclinical stage) were examined by electron microscopy and compared with those in non-diagnostic biopsy specimens from age-matched controls and cases of other childhood neuromuscular disorders. The most striking feature was the blister-like swelling of vascular endothelial cells in the biopsied muscle specimens from the 3 preclinical stage DMD patients, which was observed in 23-39% of the small blood vessels examined. Other noticeable features in the preclinical DMD patients were: (1) replication of the basement membrane, there being more than 3 layers in 30% of the capillaries; (2) many degenerating and regenerating capillaries; and (3) platelet adhesion and aggregation in small blood vessels including small arteries and veins. Morphometric analysis showed that the capillary and endothelial cell areas were much greater in the preclinical DMD patients than in the controls or the cases of the other neuromuscular disorders. These phenomena strongly suggest an as yet undetermined process in blood vessels in preclinical DMD.

Behavior of sarcotubular system formation in experimentally induced regeneration of muscle fibers.

To examine the behavior of transverse (T)-tubule formation in experimentally-induced regenerating fibers, a local anesthetic, bupivacaine hydrochloride, was injected directly into the rat soleus muscle to cause myonecrosis. The regenerating fibers following necrosis were then examined by electron microscopy using lanthanum nitrate which clearly demonstrated the sarcotubular system. In the early stage of regeneration within 7 days after muscle necrosis, the T-tubules seemed to be composed of markedly proliferated subsarcolemmal caveolae with occasional honeycomb structure formation. Around 10 days, the T-tubules in regenerating fibers were tortuously and irregularly arranged with focal dilatation in diameter, and extended longitudinally along the axis of well organized myofibrils. As the regenerating fibers matured, the sarcotubular system, irregular in course and in shape, gradually became organized into a regular transverse position against the myofibrils, along with a marked decrease in longitudinally arranged tubular components. These morphological findings of the early T-tubule formation seen in the present study were similar to those found in early myogenesis, and in biopsied muscles from cases of polymyositis and progressive muscular dystrophy.

PCR and immunocytochemical analyses of dystrophin-positive fibers in Duchenne muscular dystrophy.

Immunocytochemical studies on serial sections of muscles from 19 patients with Duchenne muscular dystrophy (DMD) were done using seven kinds of antidystrophin antibodies that span dystrophin. Fifteen of the patients were screened for intragenic deletions by the multiplex polymerase chain reaction (PCR); 7 were also tested, using Southern blots. Dystrophin-positive fibers were detected in 10 of the 19 patients, occurring with a frequency of 0.06-75.7%, as clusters or as single fibers in certain muscle bundles. In 7 of the 10 patients, the fibers stained with all antibodies from the N- through the C-terminal region. However, in one patient, there were dystrophin-positive fibers that stained with an N-terminal antibody (DYS-3) and an antibody specific to the rod region (DYS-1), but not with C-terminal antibody (DYS-2). In 2 patients, there were two kinds of fibers: one that did not stain with DYS-1 or DYS-2 and another that stained with all the antibodies used. Single-fiber PCR analysis in 2 patients showed that the genotype of dystrophin-positive fibers differed from that of dystrophin-negative fibers. These results suggest that the majority of dystrophin-positive fibers are of the same clonal origin, but that some are derived from plural reverse mutations, each with a different translation-frame-repairing modality and somatic mosaicism.