Endogenous protein and enzyme fragments induce immunoglobulin E-independent activation of mast cells via a G protein-coupled receptor, MRGPRX2.

Mast cells play a central role in inflammatory and allergic reactions by releasing inflammatory mediators through 2 main pathways, immunoglobulin E-dependent and E-independent activation. In the latter pathway, mast cells are activated by a diverse range of basic molecules (collectively known as basic secretagogues) through Mas-related G protein-coupled receptors (MRGPRs). In addition to the known basic secretagogues, here, we discovered several endogenous protein and enzyme fragments (such as chaperonin-10 fragment) that act as bioactive peptides and induce immunoglobulin E-independent mast cell activation via MRGPRX2 (previously known as MrgX2), leading to the degranulation of mast cells. We discuss the possibility that MRGPRX2 responds various as-yet-unidentified endogenous ligands that have specific characteristics, and propose that MRGPRX2 plays an important role in regulating inflammatory responses to endogenous harmful stimuli, such as protein breakdown products released from damaged or dying cells.

The influence of cement thickness on stem subsidence and cement creep in a collarless polished tapered stem: When are thick cement mantles detrimental?

OBJECTIVES: Favourable results for collarless polished tapered stems have been reported, and cement creep due to taper slip may be a contributing factor. However, the ideal cement thickness around polished stems remains unknown. We investigated the influence of cement thickness on stem subsidence and cement creep. METHODS: We cemented six collarless polished tapered (CPT) stems (two stems each of small, medium and large sizes) into composite femurs that had been reamed with a large CPT rasp to achieve various thicknesses of the cement mantle. Two or three tantalum balls were implanted in the proximal cement in each femur. A cyclic loading test was then performed for each stem. The migration of the balls was measured three-dimensionally, using a micro-computed tomography (CT) scanner, before and after loading. A digital displacement gauge was positioned at the stem shoulder, and stem subsidence was measured continuously by the gauge. Final stem subsidence was measured at the balls at the end of each stem. RESULTS: A strong positive correlation was observed between mean cement thickness and stem subsidence in the CT slices on the balls. In the small stems, the balls moved downward to almost the same extent as the stem. There was a significant negative correlation between cement thickness and the horizontal:downward ratio of ball movement. CONCLUSION: Collarless polished tapered stems with thicker cement mantles resulted in greater subsidence of both stem and cement. This suggests that excessive thickness of the cement mantle may interfere with effective radial cement creep.Cite this article: E. Takahashi, A. Kaneuji, R. Tsuda, Y. Numata, T. Ichiseki, K. Fukui, N. Kawahara. The influence of cement thickness on stem subsidence and cement creep in a collarless polished tapered stem: When are thick cement mantles detrimental? Bone Joint Res 2017;6:-357. DOI: 10.1302/2046-3758.65.BJR-2017-0028.R1.

Induction of nitric oxide synthase by traumatic brain injury.

We investigated the dynamic induction/expression of inducible nitric oxide synthase (iNOS) using human brains made available through death by traumatic brain injury (TBI). Astrocytes, microglia, and neutrophils were identified in tissue using immunohistochemical staining with antibodies against glial fibrillary acidic protein (GFAP), MHC class II antigen, and neutrophil elastase, respectively. The localization of iNOS protein in each of these cell types was evaluated using immunohistochemistry. Within 2 days of injury, iNOS immunoreactivity was not detected. However, after 2 days, immunoreactivity was detected in the traumatized brain. The iNOS immunoreactivity was localized on neutrophils and microglia/macrophages in the areas around the tissue necrosis in the traumatized cortical hemisphere, in the deep part of the cortex and the dentate gyri of the hippocampi adjacent to the hemorrhage, and within the cytoplasm of vascular smooth muscle cell of a small artery or arteriole surrounding the injured region. This reactivity was absent after 8 days post-injury.These observations confirmed the prolonged induction of iNOS within various cells in the injured brain. These responses suggest that iNOS plays a crucial role in cerebrovascular damage and/or secondary brain damage subsequent to traumatic brain injury. Furthermore, the dense nitric oxide (NO) generated by iNOS may play a role in neuronal cell death after injury.

The 2.0-A crystal structure of tachylectin 5A provides evidence for the common origin of the innate immunity and the blood coagulation systems.

Because invertebrates lack an adaptive immune system, they had to evolve effective intrinsic defense strategies against a variety of microbial pathogens. This ancient form of host defense, the innate immunity, is present in all multicellular organisms including humans. The innate immune system of the Japanese horseshoe crab Tachypleus tridentatus, serving as a model organism, includes a hemolymph coagulation system, which participates both in defense against microbes and in hemostasis. Early work on the evolution of vertebrate fibrinogen suggested a common origin of the arthropod hemolymph coagulation and the vertebrate blood coagulation systems. However, this conjecture could not be verified by comparing the structures of coagulogen, the clotting protein of the horseshoe crab, and of mammalian fibrinogen. Here we report the crystal structure of tachylectin 5A (TL5A), a nonself-recognizing lectin from the hemolymph plasma of T. tridentatus. TL5A shares not only a common fold but also related functional sites with the gamma fragment of mammalian fibrinogen. Our observations provide the first structural evidence of a common ancestor for the innate immunity and the blood coagulation systems.

The expression of excitatory amino acid transporter 2 (EAAT2) in forensic autopsy cases.

Glutamate is the major excitatory neurotransmitter and the greater part of this amino acid is removed from the synaptic cleft by excitatory amino acid transporter 2 (EAAT2) located on perisynaptic astrocytes. Recently, it was reported that the EAAT2 protein content changed in rats following forebrain ischemia and administration of methamphetamine. We planned to demonstrate the immunohistochemical distribution of EAAT2 in the human brain and discuss the significance of its pathophysiological roles. Thirty-two cases were used from forensic autopsies. The tissues were sampled from the cerebral cortex, striatum and hippocampus. The distribution of EAAT2 was difficult to identify in cases of electrical fatalities. However, continuous and extensive staining of EAAT2 was observed in cases of death from hypothermia. In almost all asphyxia death, we were able to observe a weak stain of EAAT2. In case of solvent abuse, EAAT2 staining was continuous and extensive as in the cases of hypothermia, and patchy negative zones were mixed. This study clearly showed the differences in EAAT2 localization according to the cause of death. These findings suggested that the differences in EAAT2 staining depended on the cause and course (pathophysiological conditions) of death.

An autopsy case of neuroleptic malignant syndrome (NMS) and its immunohistochemical findings of muscle-associated proteins and mitochondria.

Neuroleptic malignant syndrome (NMS) is a rare but potentially fatal disorder. In forensic cases, post-mortem diagnosis of NMS is sometimes difficult if ante-mortem information, such as neuroleptic ingestion or signs and symptoms, cannot be obtained. A 39-year-old Japanese male on a neuroleptic treatment regimen suddenly became agitated and died. Autopsy revealed muscle rigidity and hyperthermia. Post-mortem examination of blood revealed elevation of creatine phosphokinase-MM (CK-MM) and lactate dehydrogenase-4 and dehydrogenase-5 (LDH-4 and LDH-5). In renal glomeruli and tubules, myoglobin was stained immunohistochemically. From these findings, the cause of death was considered to be NMS. To support the diagnosis of NMS, both skeletal and cardiac muscles were stained with actin, myoglobin, desmin and mitochondria antibodies immunohistochemically. Actin, myoglobin, desmin, and mitochondria had been lost from skeletal, but not from the cardiac muscle, which suggested that only the skeletal muscle was damaged. Moreover, because mitochondria had disappeared only from the skeletal muscle, it was considered that skeletal muscle degeneration was caused by mitochondrial damage. Therefore, it is suggested that immunostaining of skeletal muscle by antibodies for muscle-associated proteins and mitochondria is useful to corroborate a diagnosis of NMS.

HSP70 and c-Fos expression of brain stem hypoglossal nucleus in drowning.

The brain stem hypoglossal nucleus (HN) is the center of nerves innervating the upper respiratory tract and is related to control of mastication, deglutition, speech and respiration. To elucidate the relationship between asphyxia and the HN, we investigated the change of hypoglossal neurons in cases of hanging, strangulation, smothering, choking, drowning and respiratory failure. Using immunohistochemical techniques, we observed the brain stem HN with antibodies against microtubule-associated protein 2 (MAP2), muscarinic acetylcholine receptor (mAChR), c-fos gene product (c-Fos) and 72 kD heat-shock protein (HSP70). MAP2, a cytoskeletal protein of the neuron, is a marker of neuronal damage. Muscarinic AChR was used as a marker of neuronal membrane and ACh signaling. We employed both HSP70 and c-Fos as markers of stress- or damage-related events. We measured the percentage of immunopositive neurons in total neurons of HN. Drowning produced higher expression of HSP70 and c-Fos than other causes of asphyxia, suggesting that drowning induces more severe damage in HN neurons. Furthermore, it was suspected that neuronal changes in drowning might relate to functions of the HN. These observations indicate that immunohistochemical examination of the brain stem HN could provide useful information for determining the cause of asphyxia.

Horseshoe crab acetyl group-recognizing lectins involved in innate immunity are structurally related to fibrinogen.

We have characterized and cloned newly isolated lectins from hemolymph plasma of the horseshoe crab Tachypleus tridentatus, which we named tachylectins 5A and 5B (TLs-5). TLs-5 agglutinated all types of human erythrocytes and Gram-positive and Gram-negative bacteria. TLs-5 specifically recognize acetyl group-containing substances including noncarbohydrates; the acetyl group is required and is sufficient for recognition. TLs-5 enhanced the antimicrobial activity of a horseshoe crab-derived big defensin. cDNA sequences of TLs-5 indicated that they consist of a short N-terminal Cys-containing segment and a C-terminal fibrinogen-like domain with the highest sequence identity (51%) to that of mammalian ficolins. TLs-5, however, lack the collagenous domain found in a kind of "bouquet arrangement" of ficolins and collectins. Electron microscopy revealed that TLs-5 form two- to four-bladed propeller structures. The horseshoe crab is equipped with a unique functional homologue of vertebrate fibrinogen, coagulogen, as the target protein of the clotting cascade. Our observations clearly show that the horseshoe crab has fibrinogen-related molecules in hemolymph plasma and that they function as nonself-recognizing lectins. An ancestor of fibrinogen may have functioned as a nonself-recognizing protein.

Japanese population study of a Y-linked dinucleotide repeat DNA polymorphism.

A polymorphic CA repeats (YCA II) was previously reported on the human Y chromosome. We have used a simple technique based on polymerase chain reaction amplification followed by native polyacrylamide gel electrophoresis to study the inheritance, the genetic stability, and the allele frequency distribution of this polymorphism in the Japanese. We found seven haplotypes which were tentatively designated as: A[(CA)19/(CA)21], B[(CA)19/(CA)22], C[(CA)19/(CA)23], D[(CA)19/(CA)19], E[(CA)21/(CA)21], F[(CA)22/(CA)22], and G[(CA)23/(CA)23]. The frequencies of these haplotypes were: A, 0.21; B, 0.29; C, 0.37; D, 0.02; E, 0.02; F, 0.07; G, 0.01. There was complete concordance with each father-son pairs. The results indicate the dinucleotide system YCA II is very useful for investigation of forensic samples, especially mixed stains in sexual offence cases.

[Immunohistochemical studies on neuronal changes in brain stem nucleus of forensic autopsied cases. I. Various cases of asphyxia and respiratory disorder].

Several nuclei in brain stem are well known to play an important role in supporting human life. However, the connection between neural changes of brain stem and the cause of death is not yet fully understood. To investigate the correlation of brain stem damage with various cause of respiratory disorders, neural changes of the arcuate nucleus (ARC), the hypoglossal nucleus (HN) and the inferior olivary nucleus (IO) were examined using immunohistochemical technique. Based on the cause of death, the forensic autopsy cases were divided into 5 groups as follows. Group I: hanging, ligature strangulation and manual strangulation, Group II: smothering and choking, Group III: drowning, Group IV: respiratory failure, control group: heat stroke and sun stroke. Brain was fixed with phosphate-buffer formalin, and the brain stem was horizontally dissected at the level of apex, then embedded in paraffin. The sections were stained with the antibodies against microtubule-associated protein 2 (MAP2), muscalinic acetylcholine receptor (mAChR), c-fos gene product (c-Fos) and 72 kD heat-shock protein (HSP70). Three nuclei showed no obvious morphological changes in all examined groups. However, in case of asphyxia (Group I to III), neurons in HN were positively stained with both HSP70 and c-Fos antibodies. This may indicate that the occlusion of upper airway results in the neuronal damage of HN without their morphological changes. Positive staining of HSP70 and c-Fos in IO was more frequently observed in Group III than other 4 groups. Since IO is involved in maintaining body balance which is often disturbed by drowning, it seems possible that neuronal damage in IO observed in drowning may be related to the disturbance of body balance. These observations indicate that immunohistochemical study on the damage to neurons in brain stem nuclei can provide useful information for determining the cause of death.

[Immunohistochemical studies on neuronal changes in brain stem nucleus of forensic autopsied cases. II. Sudden infant death syndrome].

Several nuclei in brain stem are well known to play an important role in supporting human life. However, the connection between neural changes of brain stem and the cause of death is not yet fully understood. Previously, in sudden infant death syndrome (SIDS) it has been suggested that impaired cardioventilatory control might contribute to cause of death. So, to investigate the brain stem damage in SIDS, neural changes of the arcuate nucleus (ARC), the hypoglossal nucleus (HN) and the inferior olivary nucleus (IO) was examined using immunohistochemical technique. Brain was fixed with phosphate-buffer formalin, and the brain stem was horizontally dissected at the level of apex, then embedded in paraffin. The sections were stained with the antibodies against microtubule-associated protein 2 (MAP2), muscalinic acetylcholine receptor (mAChR), c-fos gene product (c-Fos) and 72 kD heat-shock protein (HSP70). Morphological changes of neurons in three nuclei were not evident. Moreover, because MAP2 degeneration and expression of HSP70 and c-Fos were not observed, neuronal damage in those nuclei was not suspected. However, although there was no abnormality of mAChR immunostaining in HN and IO, the rate of mAChR-immunopositive neurons in ARC was less than that in control. These observations indicate that immunohistochemical study on the neuronal changes in ARC can provide useful information for diagnosing SIDS.

Isolated adrenocorticotropic hormone deficiency: an autopsy case of adrenal crisis. A case report.

We present a case of fatal adrenal crisis due to isolated adrenocorticotropic hormone (ACTH) deficiency. Autopsy revealed each adrenal gland weighed 0.9 g and the adrenal cortexes were very thin and atrophic. Additionally, cortisol could not be observed in the adrenal cortex by immunohistochemical staining. Furthermore, urine cortisol and 17-OHCS concentration had decreased to a very low level, 20 mg/L and 0.8 mg/L respectively. The anterior pituitary gland was atrophic, and showed fibrosis and lymphocytosis was suspected. Immunohistochemically growth hormone (GH)-stained pituitary gland cells were observed, but there were no cells stained with anti-ACTH antibody. From the history and pathological findings, no other deficiencies of pituitary hormones were evident. Therefore, isolated ACTH deficiency was suspected. Furthermore, as the thyroid gland showed lymphocytic thyroiditis, is was considered that isolated ACTH deficiency was associated with an autoimmune cause. Generally, as patients of chronic adrenocortical insufficiency are exposed to stress and, therefore, have an increased requirement for glucocorticoids, the blood pressure falls, leading to hypovolemic shock called " an adrenal crisis." Without treatment, patients die in crisis within several hours. In our case, the deceased had drunk alcohol without sleep for 2 days. We believe that the stress of drinking and sleeplessness induced adrenal crisis and caused his death.

[An autopsy case of the Reye's syndrome].

A case of sudden unexpected natural death in a 6-year-old girl was reported. She was found in dead with vomiting on her bed in the morning on 24. December. Autopsy did not reveal any injuries and abnormalities on her appearance. Small amount of light yellowish mucus in her nose, trachea and bronchus was found. Brain was slightly edematous (1395 g in weight). Liver was 750 g in weight and focal yellowish lesions were observed on its cut surfaces. Microscopically fatty degeneration (granulous fatty deposits) was observed in hepatocytes. Immunohistochemical staining of mitochondria in heart showed no staining microscopically, and degeneration and breakdown of mitochondria were found electromicroscopically. From the results of autopsy and histopathological findings, her cause of death was diagnosed as the Reye's syndrome.

Perception of visual apparent movement and topographic analysis of related evoked potentials.

Most of the studies in long-range apparent movement have been concerned with the psychophysical properties in central vision. However, apparent movement in peripheral vision is not clearly understood. This study focuses on the properties of the perception of apparent movement between central and peripheral vision. The results show that judgements of apparent movement in central vision indicate high performance at less than 4 degrees of spatial separation, and that judgements in peripheral vision indicate high performance at greater than 4 degrees. Evoked potentials associated with long-range apparent movement were also measured, and topographic maps were constructed to evaluate the active areas corresponding to visual apparent movement. The topographic maps show that the central regions are activated above 300 msec after the onset of the second apparent-movement stimulation. The results suggest that visual apparent movement occurs in the visual association area, or at higher levels in the central nervous system.

[An autopsy case of traumatic subdural hematoma from arterio-venous malformation with diffuse axonal injury].

Diffuse axonal injury (DAI) is defined as widespread damage to axons in the white matter of the brain without focal injury such as contusion and acute subdural hematoma. A case of traumatic subdural hematoma from arterio-venous malformation accompanied by DAI is reported. A 58-year-old man was assaulted, and immediately lost consciousness, and remained unconscious during about 44 hours until his death. The autopsy revealed acute subdural hematoma (about 160 g) on left temporal lobe and left cingular, uncal and cerebellar tonsillar herniation, and tear and hemorrhage of the corpus callosum. Under this subdural hematoma, gray-whitish vascular lesion with subarachnoid hemorrhage was found. Histologically, this lesion was diagnosed as the arterio-venous malformation. Neuropathological examination of the corpus callosum, dorsolateral part of midbrain and superior cerebellar peduncle revealed DAI findings, such as swelling and ballooning of the myelin fibers, swelling and waving of axons, and retraction balls. Axon degenerations were also observed immunohistochemically by anti-200 kD neurofilament antibody. From the results, his unconsciousness from the moment of impact might be occurred from not only subdural hematoma but also DAI.

[An autopsy case of Sjögren syndrome with organized and fresh subdural hemorrhage (hematoma)].

An autopsy case of Sjögren syndrome with organized and fresh subdural hemorrhages (hematoma) is reported. A 49-year-old woman who had been suffering from Sjögren syndrome had gradually lost her consciousness and was taken to the hospital where she died several hours later. Subsequently a doctor found the subdural hematoma of unknown origin on her Brain CT. At autopsy, her skin was dry and all of her teeth were missing. There were sporadic cutaneous purpura and subcutaneous hemorrhages in her trunk and limbs. The histopathological examination revealed that the submandibular gland had no normal acini, and was replaced by fibrous and adipose tissues with numerous lymphocytes. There were signs of fibrosis with inflammation in her liver, kidneys and lungs. The thyroid gland showed thyroiditis. Serological findings showed a significant high level of antinuclear antibody, positive RA factor and high gamma-globulinemia. The autopsy revealed that her cause of death was acute subdural hematoma and uncal herniation. There were no external injuries on her head or face. It is suggested that her acute subdural hematoma according to the hemorrhagic tendency, affected by her Sjögren syndrome.

Immunohistological investigations of autopsied carotid bodies and their application to diagnosing strangulation.

Using immunohistochemical staining, the histological changes and the presence of neuropeptides (enkephalin and VIP) in the carotid body have been investigated in medico-legal autopsy cases, especially asphyxia cases. Only in cases of manual and/or ligature strangulation cases that sustained a force near the carotid body, were the chief cells mainly lightly stained, indicating that they had been "active" cells. Furthermore, these cells and their nuclei were enlarged in comparison to the chief carotid body cells in other autopsy groups. It was thus felt that these changes had resulted from the force that had directly affected the carotid body. Based on these findings, it was concluded that immunohistochemical investigation of the carotid body offers a useful possibility for diagnosing manual asphyxia, especially in autopsy cases involving strangulation.

[Paternity test by DNA fingerprinting in a sexual assault].

We have applied the method of DNA fingerprinting to resolve a paternity case. DNA samples were extracted from the blood of mother, child (4 months old fetus) and alleged father, digested with the restriction endonuclease Hinf I and Hae III, size separated by agarose gel electrophoresis, and hybridized with the multi-locus minisatellite probe B.E.S.T.-MZ1.3 digoxigenin. DNA fingerprinting patterns in the child and alleged father indicated possibly paternity. Following extraction of DNA from cord blood (infant of 26 to 39 week's gestation), the quantity of DNA isolated was determined on a spectrophotometer at 260 nm and its integrity by electrophoresis in agarose gels. We found that high-molecular-weight DNA could be recovered in large quantities from cord blood as well as from adult blood.