Self-sustaining oscillation of two axonemal microtubules based on a stochastic bonding model between microtubules and dynein.

The motility of cilia and flagella plays important physiological roles, and there has been a great deal of research on the mechanisms underlying the motility of molecular motors. Although recent molecular structural analyses have revealed the components of the ciliary axoneme, the mechanisms involved in the regulation of dynein activity are still unknown, and how multiple dyneins coordinate their movements remains unclear. In particular, the mode of binding for axonemal dynein has not been elucidated. In this study, we constructed a thermodynamic stochastic model of microtubule-dynein coupling and reproduced the experiments of Aoyama and Kamiya on the minimal component of axonemal microtubule-dynein. We then identified the binding mode of axonemal dynein and clarified the relationship between dynein activity distribution and axonemal movement. Based on our numerical results, the slip-bond mechanism agrees quantitatively with the experimental results in terms of amplitude, frequency, and propagation velocity, implying that axial microtubule-dynein coupling may follow a slip-bond mechanism. Moreover, the frequency and propagation velocity decayed in proportion to the fourth power of microtubule length, and the critical load of the trigger for the oscillation agreed well with Euler's critical load.

Practical usefulness of atypical endometrial cell categories within the new classification of endometrial cytology when applied to conventional smears.

OBJECTIVE: Endometrial cytology has been widely used as a screening tool in Japan. Traditionally, a three-tier reporting system, consisting of 'negative', 'suspicious' and 'positive' categories, has been used. However, a more descriptive system, the New Terminology in Endometrial Cytology (NTEMC), which is based on the Bethesda System for uterine cervical cytology, was introduced recently. The objective of this study was to validate the NTEMC criteria. METHODS: Endometrial cytology specimens that had been categorised as 'suspicious' were collected in our hospital between 2003 and 2013, and from these, 106 specimens with corresponding histological results, were re-evaluated according to the NTEMC criteria. Diagnostic categories were assigned based on that chosen by the majority of the examining members. RESULTS: Negative, atypical endometrial cells, of undetermined significance (ATEC-US), atypical endometrial cells for which atypical endometrial hyperplasia or worse cannot be excluded (ATEC-A), endometrial hyperplasia, atypical endometrial hyperplasia and malignancy were selected as the diagnostic categories for 9 (8.5%), 34 (32.1%), 17 (16%), 34 (32.1%), 5 (4.7%) and 7 (6.6%) specimens, respectively. Corresponding histological categories of benign, endometrial hyperplasia, atypical endometrial hyperplasia and malignancy were established in 28 (82.4%), 1 (2.9%), 2 (5.9%) and 3 (8.8%) ATEC-US specimens, respectively, and in 6 (35.3%), 3 (17.6%), 2 (11.8%) and 6 (35.3%) ATEC-A specimens, respectively. The histological category distribution differed significantly (P = 0.001), and there was a significant correlation between corresponding cytological and histological categories (P = 0.005). CONCLUSION: The ATEC category of NTEMC system works well in a practical setting and resembles the Bethesda reporting system ASC (atypical squamous cells) category for cervical cytology.

Inhibition of plasmin attenuates murine acute graft-versus-host disease mortality by suppressing the matrix metalloproteinase-9-dependent inflammatory cytokine storm and effector cell trafficking.

The systemic inflammatory response observed during acute graft-versus-host disease (aGVHD) is driven by proinflammatory cytokines, a 'cytokine storm'. The function of plasmin in regulating the inflammatory response is not fully understood, and its role in the development of aGVHD remains unresolved. Here we show that plasmin is activated during the early phase of aGVHD in mice, and its activation correlated with aGVHD severity in humans. Pharmacological plasmin inhibition protected against aGVHD-associated lethality in mice. Mechanistically, plasmin inhibition impaired the infiltration of inflammatory cells, the release of membrane-associated proinflammatory cytokines including tumor necrosis factor-α (TNF-α) and Fas-ligand directly, or indirectly via matrix metalloproteinases (MMPs) and alters monocyte chemoattractant protein-1 (MCP-1) signaling. We propose that plasmin and potentially MMP-9 inhibition offers a novel therapeutic strategy to control the deadly cytokine storm in patients with aGVHD, thereby preventing tissue destruction.

Insulin-like growth factor-1 and lipoprotein profile in cord blood of preterm small for gestational age infants.

Low birth weight was associated with cardiometabolic diseases in adult age. Insulin-like growth factor-1 (IGF-1) has a crucial role in fetal growth and also associates with cardiometabolic risks in adults. Therefore, we elucidated the association between IGF-1 level and serum lipids in cord blood of preterm infants. The subjects were 41 consecutive, healthy preterm neonates (27 male, 14 female) born at <37-week gestational age, including 10 small for gestational age (SGA) infants (<10th percentile). IGF-1 levels and serum lipids were measured in cord blood, and high-density lipoprotein cholesterol (HDLC), low-density lipoprotein cholesterol (LDLC) and very low-density lipoprotein triglyceride (VLDLTG) levels were determined by HPLC method. SGA infants had lower IGF-1 (13.1 ± 5.3 ng/ml), total cholesterol (TC) (55.0 ± 14.8), LDLC (21.6 ± 8.3) and HDLC (26.3 ± 11.3) levels, and higher VLDLTG levels (19.0 ± 12.7 mg/dl) than in appropriate for gestational age (AGA) infants (53.6 ± 25.6, 83.4 ± 18.9, 36.6 ± 11.1, 38.5 ± 11.6, 8.1 ± 7.0, respectively). In simple regression analyses, log IGF-1 correlated positively with birth weight (r = 0.721, P < 0.001), TC (r = 0.636, P < 0.001), LDLC (r = 0.453, P = 0.006), and HDLC levels (r = 0.648, P < 0.001), and negatively with log TG (r = -0.484, P = 0.002) and log VLDL-TG (r = -0.393, P = 0.018). Multiple regression analyses demonstrated that IGF-1 was an independent predictor of TC, HDLC and TG levels after the gestational age and birth weight were taken into account. In preterm SGA infants, cord blood lipids profile altered with the concomitant decrease in IGF-1 level.

Association between serum lipoprotein lipase mass concentration and subcutaneous fat accumulation during neonatal period.

BACKGROUND/OBJECTIVES: Subcutaneous adipose tissue grows rapidly during the first months of life. Lipoprotein lipase (LPL) has a quantitatively important function in adipose tissue fat accumulation and insulin-like growth factor-I (IGF-I) is a determinant of neonatal growth. Recent studies showed that LPL mass in non-heparinized serum (LPLm) was an index of LPL-mediated lipolysis of plasma triacylglycerol (TG). The objective was to know the influence of serum LPL and IGF-I on neonatal subcutaneous fat growth, especially on catch-up growth in low birth weight infants. SUBJECTS/METHODS: We included 47 healthy neonates (30 males, 17 females), including 7 small for gestational age. We measured serum LPLm and IGF-I concentrations at birth and 1 month, and analyzed those associations with subcutaneous fat accumulation. RESULTS: Serum LPLm and IGF-I concentrations increased markedly during the first month, and positively correlated with the sum of skinfold thicknesses both at birth (r=0.573, P=0.0001; r=0.457, P=0.0035) and at 1 month (r=0.614, P<0.0001; r=0.787, P<0.0001, respectively). In addition, serum LPLm concentrations correlated inversely to very low-density lipoprotein (VLDL)-TG levels (r=-0.692, P<0.0001 at birth; r=-0.429, P=0.0052 at 1 month). Moreover, the birth weight Z-score had an inverse association with the postnatal changes in individual serum LPLm concentrations (r=-0.639, P<0.0001). CONCLUSIONS: Both serum LPLm and IGF-I concentrations were the determinants of subcutaneous fat accumulation during the fetal and neonatal periods. During this time, LPL-mediated lipolysis of VLDL-TG may be one of the major mechanisms of rapid growth in subcutaneous fat tissue. Moreover, LPL, as well as IGF-I, may contribute to catch-up growth in smaller neonates.

Touch imprint cytology and immunohistochemistry for the assessment of sentinel lymph nodes in patients with breast cancer.

AIMS: Accurate evaluation of sentinel nodes is of clinical importance to avoid further surgery for axillary node dissection. A prospective study was carried out to investigate the feasibility and accuracy of touch imprint cytology (TIC) and touch imprint immunohistochemistry (TIHC). METHODS: Two hundred and five sentinel nodes from consecutive 118 patients with primary breast cancer were studied after successful identification of sentinel nodes. Sentinel nodes were sectioned at 2 mm intervals and imprint specimens prepared from all cut surfaces were subjected to Papanicolaou staining and immunohistochemical staining using anti-cytokeratin antibody. RESULTS: Forty-nine sentinel nodes from 40 patients were positive by permanent section. The sensitivity of TIC was 84% (41/49) per sentinel node and 83% (33/40) on a per patient basis. The sensitivity of TIHC was 86% (42/49) per sentinel node and 83% (33/40) on a per patient basis. When the results of TIC and TIHC were combined, the sensitivity was 88% (43/49) per sentinel node and 85% (34/40) on a per patient basis. Among the 156 negative sentinel nodes, four sentinel nodes from four different patients were consistently positive by TIC and TIHC, but only one patient out of 78 node-negative patients was upstaged. CONCLUSIONS: Touch imprint cytology is sufficiently sensitive for intraoperative evaluation of sentinel nodes. A slight improvement in the sensitivity is expected when immunohistochemistry is used. The combination of these methods provides better sensitivity than either method alone.

Sialolipoma: a report of seven cases of a new variant of salivary gland lipoma.

AIMS: We propose the designation 'sialolipoma' to establish and characterize a new category of benign lipomatous tumour occurring in salivary glands. Until now, these tumours have not been regarded as a distinct entity in the salivary glands. METHODS AND RESULTS: We evaluated the clinicopathological and immunohistochemical features of seven sialolipomas among 2051 surgically resected primary salivary gland tumours deposited in our files. The seven patients with sialolipoma were five men and two women, aged 20-75 years (mean: 54.4 years). Five tumours had arisen in the parotid gland, one in the soft palate, and one in the hard palate. The tumours ranged from 10 to 60 mm (mean: 38 mm) in maximum diameter. Histologically, the tumours were characterized by a well circumscribed mass composed of glandular tissue and mature adipose elements. The adipose elements in the tumours arising in the parotid gland were more abundant than those arising in the minor salivary gland. The glandular components consisted of ductal, acinar, basal and myoepithelial cells, and closely resembled the cellular and structural compositions of normal salivary gland tissues. These findings were confirmed by immunohistochemical and ultrastructural studies. These components had no atypia, except for the presence of some minor variations, e.g. ductal ectasia with fibrosis and focal oncocytic metaplasia. In all cases, cell proliferative activity, as assessed by Ki67 (MIB1) immunostaining, was low. From these findings, it is likely that our cases were lipomas with secondary entrapment of the salivary gland elements. No recurrence was seen in all cases after superficial parotidectomy, or after surgical excision in the patients with palatal tumours. CONCLUSIONS: We regard sialolipoma as a distinct variant of salivary gland lipoma that can occur in both the major and minor salivary glands. Superficial parotidectomy, or surgical resection in the case of palatal tumours, is an appropriate treatment for this benign tumour.

Epstein-Barr virus associated B-cell lymphoma of brain developing in myelodysplastic syndrome with c-kit mutation (Try-557 -->stop).

The first case of B-cell lymphoma of brain in a patient with myelodysplastic syndrome (MDS) was reported. A 68-year-old man was admitted because of anemia, fever, and thrombocytopenia and was diagnosed as having MDS (refractory anemia with excess of blasts) on the basis of the findings of bone marrow aspiration and chromosomal analysis. The patient was followed up without chemotherapy, but a brain tumor appeared after 3 years. Histologic and immunohistologic examinations revealed diffuse large B-cell lymphoma. Mutations of the c-kit proto-oncogene (stem cell factor receptor) and the p53 tumor-suppressor gene were examined in the MDS lesion and malignant lymphoma (ML) by the polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) method followed by direct sequencing. The p53 mutation was not found in either MDS or ML, but a nonsense mutation (Try-557 --> stop) in exon 11 of the c-kit, which might lead to dysfunction of tyrosine kinase activity, was detected in MDS. This is the first report of c-kit mutation in MDS. Epstein-Barr virus (EBV) genome was demonstrated in the nucleus of brain ML cells by in situ hybridization with EBV-encoded RNA-1 probe. Immunohistochemistry showed that the tumor cells expressed latent infection gene products, including EBV nuclear antigen-2 and latent membrane protein-1. This pattern of latent gene expression was Lat III, which is usually found in malignant lymphomas developing in immunocompromised hosts. These findings suggest that a profound pancytopenia in MDS resulted in an immunodeficient condition, after which EBV-positive B-cell lymphoma of brain developed.

[Beneficial effects of CHOP therapy in a case of intravascular large B-cell lymphoma diagnosed by skin biopsy].

We report a case of intravascular large B-cell lymphoma. A 52-year-old man gradually developed dementia and abnormal behaviors, which were later accompanied by spastic paraplegia and sensory disturbance in his lower limbs. MR imaging of his brain showed high signal intensity lesions on T2 imaging. IMP-SPECT images of the brain showed diffuse reduction of radioisotope uptake. Many skin rashes that looked like senile hemangioma were observed on his body. Several of those were biopsied, and the diagnosis of intravascular large B-cell lymphoma was made because of malignant B lymphocytes filling the vessel lumens in one of the seven biopsy specimens. CHOP therapy was performed and found to be effective for the neurological disorders such as dementia, paraplegia, and sensory disturbance. Our case suggests that skin biopsy for more than one sample of the skin rashes is very important for the diagnosis of intravascular large B-cell lymphoma. CHOP therapy might be effective in this case because of early diagnosis by skin biopsy.

Angiotropic lymphoma: proliferation of B cells in the capillaries of cutaneous angiomas.

We report a 52-year-old patient with cutaneous angiomas on the trunk in association with angiotropic lymphoma involving the nervous system. The lesional skin showed proliferation of atypical B cells in the capillaries. The diagnosis of angiotropic lymphoma with neurological deficits is often difficult before death. Thus, skin biopsy in this case proved to be diagnostic of the central nervous system disease without the risk of brain biopsy. The diagnosis of angiotropic lymphoma in the future may be achievable by biopsy of coincidental angiomas.

Induction of chromosomal aberrations and growth-transformation of lymphoblastoid cell lines by inhibition of reactive oxygen species-induced apoptosis with interleukin-6.

Etiological evidence, indicating the relationships between the onset of malignant lymphoma and pre-existing chronic inflammation, has been accumulated. For the autonomous growth of malignant tumor, genetic lesions, such as chromosomal aberrations, amplification of oncogenes, and mutations of genes involved in the cell cycle regulation, must be essential. However, how the inflammation promotes the accumulation of genetic lesions and induces the autonomous growth of lymphoid cells remains unclear. Reactive oxygen species released by polymorphonuclear leukocytes and macrophages are factors causing DNA damage in the foci of inflammation, and thus could play a role in lymphomagenesis. The xanthine/xanthine oxidase (X/XOD) system produces a mixture of hydrogen peroxide and superoxide anion extracellularly, and thus serves as an in vitro source of reactive oxygen species. Cell death of lymphoblastoid cell lines (LCLs) was induced with X/XOD treatment in a dose-dependent manner. DNA fragmentation, which is the characteristic feature of apoptosis, was observed in LCLs at 4-8 hours after X/XOD treatment. Among cytokines such as interleukin-6 (IL-6), IL-10, and interferon-gamma, only pretreatment with IL-6 gave LCLs the resistance to X/XOD-induced cell death in a dose-dependent manner. The proportion of apoptotic cells in X/XOD-treated LCL culture was decreased with IL-6 pretreatment by quantification with flow cytometric analysis. Treatment of LCLs with IL-6 for 48 hours up-regulated bcl-2 mRNA expression. Furthermore, the LCLs repeatedly treated with X/XOD and cultured with or without IL-6 showed many more structural abnormalities of chromosomes than those without X/XOD treatment. Colony forming efficiency of X/XOD-treated LCLs with IL-6 was significantly higher than those without IL-6, and even relatively higher than LCLs without X/XOD treatment. IL-6 could support the survival of non-neoplastic B cells and accelerate the malignant transformation of B lineage cells in inflammatory lesions.

Primary large-cell neuroendocrine carcinoma of the parotid gland: immunohistochemical and molecular analysis of two cases.

Pulmonary large-cell neuroendocrine carcinoma (LCNEC) is a newly proposed clinicopathologic entity; a few cases of LCNEC have been reported in other sites, such as the uterine cervix and the thymus. In the salivary glands, LCNEC is extremely rare and is not recognized as a specific entity in the World Health Organization classification. We retrospectively reviewed from our files 1675 cases of surgically resected primary parotid gland tumors and found 2 cases of LCNEC that fulfilled the criteria of pulmonary LCNEC. These cases occurred in 72- and 73-year-old men who had short histories of enlarging parotid gland tumors. The tumors were composed of large cells that exhibited organoid, solid, trabecular, and rosette-like growth patterns with a high mitotic rate and a conspicuous tendency for necrosis. The tumor cells were polygonal and characterized by a moderate nuclear:cytoplasmic ratio, coarse chromatin, and conspicuous nucleoli. Immunohistochemical examination revealed that the tumor cells were positive for six general neuroendocrine markers, cytokeratin, p53, bcl-2, epidermal growth factor receptor, and cyclin D1. Markedly reduced expressions of p21Waf1 and p27Kip1 were also noticed. The Ki-67 labeling index was more than 50% in both cases. One case showed loss of heterozygosity at TP53 accompanied by a p53 gene point mutation. Loss of heterozygosity at chromosome 9p21 was detected in both cases; one was accompanied by a p16 gene silent point mutation. Both patients died of the disease, with recurrence 5 months and 4 years after surgery, respectively. These findings indicate that LCNEC is a rare but distinct salivary gland tumor with highly aggressive biologic behavior. Multiple alterations of cell cycle regulators and tumor suppressor genes may play an important role in presenting the biologic characteristics of this rare parotid gland tumor.

Expressions of Fas ligand and other apoptosis-related genes and their prognostic significance in epithelial ovarian neoplasms.

Expression of apoptosis-related proteins, bcl-2, Bax, Fas and Fas ligand (L), in ovarian epithelial neoplasms together with its clinical relevance was examined by immunohistochemistry. They included 36 cases with adenoma, 33 with low potential malignancy (LPM) and 63 with carcinomas. bcl-2 expression was observed in 14 of 36 cases (39%) with adenoma, five of 33 (15%) with LPM (P< 0.05) and 12 of 63 (19%) with carcinoma (P < 0.05). Cases with bcl-2 expression showed more favourable prognosis than those without, but the difference was not statistically significant. There was no difference in frequency of Bax and Fas expression between each histologic category. Fas L expression was observed in one of 36 cases (3%) with adenoma, but in 12 of 33 (36%) with LPM (P < 0.001) and 42 of 63 (67%) with carcinoma (P < 0.0001). In carcinomas, cases expressing Fas L showed a less favourable prognosis than those without (P = 0.02). Density of CD8+ lymphocytes, possibly cytotoxic T-cells, was higher in serous carcinoma with negative Fas L expression than those with positive Fas L expression. These findings suggest that Fas L expressing carcinomas induce apoptosis in infiltrating CTL with Fas expression, and escape from immune surveillance.

Electron microscopic observation of intracellular expression of mRNA and its protein product: technical review on ultrastructural in situ hybridization and its combination with immunohistochemistry.

In situ hybridization (ISH) at the electron microscopic (EM) level is essential for elucidating the intracellular distribution and role of mRNA in protein synthesis. Three different approaches have been applied by the investigators in this EM-ISH study: preembedding method; non-embedding method using ultrathin frozen sections; and postembedding method. In order to obtain satisfactory morphological preservation and retain the messages, we routinely utilized 6 microns-thick frozen sections fixed in 4% paraformaldehyde for the preembedding method and tissues embedded in LR White resin for the postembedding method. The hybridization signal intensity by the postembedding method was lower, and non-specific signals were relatively frequent, in comparison with the preembedding method. The preembedding method thus appears to be easier and better than the postembedding method from the viewpoint of applicability and preservation of mRNA, although quantitative analysis of the expression of mRNA is rather difficult in the preembedding method. EM-ISH is considered to be an important tool for clarifying the intracellular localization of mRNA and the exact site of specific hormone synthesis on the rough endoplasmic reticulum. The simultaneous visualization of mRNA and encoded protein in the same cells using preembedding EM-ISH and subsequent postembedding immunoreaction with protein A colloidal gold complex is also described. This ultrastructural double-staining method for mRNA and encoded protein can be expected to provide an important clue for elucidating the intracellular correlation of mRNA translation and secretion of translated protein.

Collagen abnormalities in the spinal cord from patients with amyotrophic lateral sclerosis.

During the last 10 years, we have demonstrated morphological and biochemical abnormalities of skin extracellular matrices in amyotrophic lateral sclerosis (ALS). However, currently little is known concerning collagen of the spinal cord in ALS. We measured the amount of collagen and characterized collagen at light and electron microscopic levels in posterior funiculus, posterior half of lateral funiculus and anterior horn of cervical enlargement of the spinal cord obtained from ten patients with ALS, 11 patients with other neurologic diseases (control group A), and ten patients without neurologic ones (control group B). In posterior half of lateral funiculus and anterior horn, (1) by light microscopy, there was no significant difference in vessel wall area between ALS patients and control groups A and B; (2) ultrastructurally, collagen bundles were more fragmented and widely separated, and the fibrils were randomly oriented in the perivascular space of capillaries in ALS patients, which were not observed in any areas of control groups or in posterior funiculus of ALS patients; and (3) the collagen contents in ALS were significantly lower (P<0.001 and P<0.001, respectively) than those in control groups A and B. Fragmented and widely separated collagen bundles in the interstitial tissue surrounding capillaries and markedly decreased amount of collagen in posterior half of lateral funiculus and in anterior horn of ALS could be related to the degeneration of the upper and lower motor neurons in the spinal cord in ALS, that is, selective neuronal vulnerability in ALS.

Muscle pathology in the early stage of Guillain-Barré syndrome.

Although many studies have been performed on nervous tissue pathology in Guillain-Barré syndrome (GBS), much less is known about pathological changes in skeletal muscle in this disorder. We have studied muscle biopsies from 5 patients with GBS, 5 patients with amyotrophic lateral sclerosis (control A), and 5 patients with polyarteritis nodosa (control B). We also examined muscle obtained after death from 7 patients without neurologic or muscular diseases (control C). By light microscopy, all specimens from patients with GBS exhibited necrosis and/or phagocytosis, none of which was observed in the other three controls. Neither small angulated fibers nor small group atrophy was found in patients with GBS and in control C, by contrast with controls A and B. Ultrastructurally, filamentous bodies, subsarcolemmal aggregates of mitochondria, accumulation of glycogen particles, and concentric laminated bodies were present much more frequently in patients with GBS than in all controls. Only GBS patients showed cytoplasmic bodies. These observation suggests that there is muscle involvement in the early stage of GBS and that these muscle changes may have an intimate and important relationship to the pathogenesis of GBS.

An improved ultrastructural double-staining method for rat growth hormone and its mRNA using LR White resin: a technical note.

An improved new method for the simultaneous visualization of mRNA and encoded protein in LR White resin-embedded specimens is described. This pre-embedding electron microscopical in situ hybridization (procedure) localized rat growth hormone mRNA specifically as high electron-density products on the polysomes of the rough endoplasmic reticulum. A subsequent post-embedding immunoreaction, using protein A colloidal gold particles, identified growth hormone as gold particles both in the cisternae of the rough endoplasmic reticulum and on the secretory granules. In our previous report, we used Epon resin for tissue embedment, which required an etching process using hydrogen peroxide or sodium periodate for immunoreactivity retrieval. In general, osmification and embedment in Epon resin are reported to decrease the immunoreactivity of the targeted protein, and the etching process using hydrogen peroxide or sodium periodate results in deosmification and shades off the signals of mRNA. To resolve these problems, we have recently used LR White resin for tissue embedment. In LR White resin-embedded tissues, retrieval of immunoreactivity using hydrogen peroxide or sodium periodate is not required, and, therefore, the gradation of the signals of mRNA can be avoided.