A stress sensor, IRE1α, is required for bacterial-exotoxin-induced interleukin-1ß production in tissue-resident macrophages.

Cholera toxin (CT), a bacterial exotoxin composed of one A subunit (CTA) and five B subunits (CTB), functions as an immune adjuvant. CTB can induce production of interleukin-1ß (IL-1ß), a proinflammatory cytokine, in synergy with a lipopolysaccharide (LPS), from resident peritoneal macrophages (RPMs) through the pyrin and NLRP3 inflammasomes. However, how CTB or CT activates these inflammasomes in the macrophages has been unclear. Here, we clarify the roles of inositol-requiring enzyme 1 alpha (IRE1α), an endoplasmic reticulum (ER) stress sensor, in CT-induced IL-1ß production in RPMs. In RPMs, CTB is incorporated into the ER and induces ER stress responses, depending on GM1, a cell membrane ganglioside. IRE1α-deficient RPMs show a significant impairment of CT- or CTB-induced IL-1ß production, indicating that IRE1α is required for CT- or CTB-induced IL-1ß production in RPMs. This study demonstrates the critical roles of IRE1α in activation of both NLRP3 and pyrin inflammasomes in tissue-resident macrophages.

Loss of Purkinje cells in the developing cerebellum strengthens the cerebellothalamic synapses.

Cerebellar damage early in life often causes long-lasting motor, social, and cognitive impairments, suggesting the roles of the cerebellum in developing a broad spectrum of behaviors. This recent finding has promoted research on how cerebellar damage affects the development of the cerebral cortex, the brain region responsible for higher-order control of all behaviors. However, the cerebral cortex is not directly connected to the cerebellum. The thalamus is the direct postsynaptic target of the cerebellum, sending cerebellar outputs to the cerebral cortex. Despite its crucial position in cerebello-cerebral interaction, thalamic susceptibility to cerebellar damage remains largely unclear. Here, we studied the consequences of early cerebellar perturbation on thalamic development. Whole-cell patch-clamp recordings showed that the synaptic organization of the cerebellothlamic circuit is similar to that of the primary sensory thalamus, in which aberrant sensory activity alters synaptic circuit formation. The hemizygous deletion of the tuberous sclerosis complex-1 ( Tsc1 ) gene in the Purkinje cell-known to cause Purkinje cell hypoactivity and autistic behaviors-did not alter cerebellothalamic synapses or intrinsic membrane properties of thalamic neurons. However, the ablation of Purkinje cells in the developing cerebellum strengthened the cerebellothalamic synapses and enhanced thalamic suprathreshold activities. These results suggest that the cerebellothalamic circuit is resistant to moderate perturbation in the developing cerebellum, such as the reduced firing rate of Purkinje cells, and that autistic behaviors are not necessarily linked to thalamic abnormality. Still, Purkinje cell loss alters the thalamic circuit, suggesting the vulnerability of the thalamus to substantial disturbance in the developing cerebellum.

Conventional Type 1 Dendritic Cells in Intestinal Immune Homeostasis.

Dendritic cells (DC) play critical roles in linking innate and adaptive immunity. DC are heterogenous and there are subsets with various distinct functions. One DC subset, conventional type 1 DC (cDC1), can be defined by expression of CD8α/CD103 in mice and CD141 in humans, or by expression of a chemokine receptor, XCR1, which is a conserved marker in both mice and human. cDC1 are characterized by high ability to ingest dying cells and to cross-present antigens for generating cytotoxic CD8 T cell responses. Through these activities, cDC1 play crucial roles in immune responses against infectious pathogens or tumors. Meanwhile, cDC1 involvement in homeostatic situations is not fully understood. Analyses by using mutant mice, in which cDC1 are ablated in vivo, revealed that cDC1 are critical for maintaining intestinal immune homeostasis. Here, we review the homeostatic roles of cDC1, focusing upon intestinal immunity.

Granule Cells Constitute One of the Major Neuronal Subtypes in the Molecular Layer of the Posterior Cerebellum.

The migration of neurons from their birthplace to their correct destination is one of the most crucial steps in brain development. Incomplete or incorrect migration yields ectopic neurons, which cause neurologic deficits or are negligible at best. However, the granule cells (GCs) in the cerebellar cortex may challenge this traditional view of ectopic neurons. When animals are born, GCs proliferate near the pia mater and then migrate down to the GC layer located deep in the cerebellar cortex. However, some GC-like cells stay in the molecular layer, a layer between the pia mater and GC layer, even in normal adult animals. These cells were named ectopic GCs nearly 50 years ago, but their abundance and functional properties remain unclear. Here, we have examined GCs in the molecular layer (mGCs) with a specific marker for mature GCs and transgenic mice in which GCs are sparsely labeled with a fluorescent protein. Contrary to the previous assumption that mGCs are a minor neuronal population, we have found that mGCs are as prevalent as stellate or basket cells in the posterior cerebellum. They are produced during a similar period as regular GCs (rGCs), and in vivo time-lapse imaging has revealed that mGCs are stably present in the molecular layer. Whole-cell patch-clamp recordings have shown that mGCs discharge action potentials similarly to rGCs. Since axonal inputs differ between the molecular layer and GC layer, mGCs might be incorporated in different micro-circuits from rGCs and have a unique functional role in the cerebellum.

Heterozygous missense variant of the proteasome subunit ß-type 9 causes neonatal-onset autoinflammation and immunodeficiency.

Impaired proteasome activity due to genetic variants of certain subunits might lead to proteasome-associated autoinflammatory syndromes (PRAAS). Here we report a de novo heterozygous missense variant of the PSMB9 proteasome subunit gene in two unrelated Japanese infants resulting in amino acid substitution of the glycine (G) by aspartic acid (D) at position 156 of the encoded protein ß1i. In addition to PRAAS-like manifestations, these individuals suffer from pulmonary hypertension and immunodeficiency, which are distinct from typical PRAAS symptoms. The missense variant results in impaired immunoproteasome maturation and activity, yet ubiquitin accumulation is hardly detectable in the patients. A mouse model of the heterozygous human genetic variant (Psmb9G156D/+) recapitulates the proteasome defects and the immunodeficiency phenotype of patients. Structurally, PSMB9 G156D interferes with the ß-ring-ßring interaction of the wild type protein that is necessary for 20S proteasome formation. We propose the term, proteasome-associated autoinflammatory syndrome with immunodeficiency (PRAAS-ID), to indicate a separate category of autoinflammatory diseases, similar to, but distinct from PRAAS, that describes the patients in this study.

Morphometric and cytomorphologic characterization of EGFR-mutated cancer cells-comparison between cultured lung cancer cell lines and lung adenocarcinoma clinical samples.

BACKGROUND: Recently molecular targeting therapies such as inhibition of enzyme activities associated with gene mutations responsible for lung carcinogenesis have been demonstrating promising outcomes, increasing the importance of gene analysis using clinical samples. Cytomorphologic findings with predictive value toward specific gene mutation such as EGFR mutation could be a useful tool to select appropriate gene analyses using limited clinical samples. METHODS: Morphometrical and cytomorphological evaluations were performed in 7 cultured lung cancer cell lines and 51 lung adenocarcinoma clinical samples to identify specific cytomorphologic characterization of EGFR-mutated cancer cells compared to the wild type. RESULTS: Morphometry demonstrated that the EGFR mutated cell lines had significantly smaller nuclear area and perimeter and more circular nuclei compared to the wild type. In contrast, EGFR-mutated clinical samples had significantly greater nuclear area and perimeter compared to the wild type EGFR samples. There were no clear differences in cytomorphologic parameters assessing nuclear atypicality between EGFR mutated cells and wild type EGFR cells in either cultured cell lines or clinical samples. CONCLUSION: Although our study suggested that EGFR mutation may have specific effects on nuclear morphology, no consistent characteristics of EGFR-mutated cells were identified in the clinical samples, probably due to various factors such as different pathologic stages and various incidences of lepidic growth. Further assessment of morphological characterization of EGFR-mutated cells in lung adenocarcinoma is warranted, increasing the number of samples and considering the effects of polyploidy, other gene mutations, pathology stage and tumor subtypes such as lepidic growth. Diagn. Cytopathol. 2016;44:717-724. © 2016 Wiley Periodicals, Inc.

Prediction of histological types of endometrial cancer by endometrial cytology.

AIM: Few studies have examined the accuracy of preoperative endometrial cytology in diagnosing low- and high-risk histology in women with endometrial cancer (EC). This single-institutional retrospective study compared the accuracy of endometrial cytology and biopsy in preoperatively predicting low-risk and high-risk histology of EC. METHODS: Between January 2006 and March 2013, 198 women with EC were examined by endometrial cytology, endometrial biopsy and hysterectomy specimen in National Kyushu Cancer Center. Among these women, 110 had endometrial cytology samples available to compare with endometrial biopsy, and were enrolled in our study (mean age ± standard deviation: 59.57 ± 10.32 years). Single-use plastic endometrial suction curettes were used in 12 of the 110 cases and thin metallic curettes for the rest. RESULTS: For type 2 EC, which includes grade 3 endometrioid adenocarcinoma and non-endometrioid histology, biopsy was 67.6% sensitive (25/37) and 84.9% specific (62/73); whereas cytology was 70.3% sensitive (26/37) and 91.8% specific (67/73). Cytology precisely diagnosed only one of 14 cases of serous carcinoma, but it diagnosed 11 of the 14 cases as type 2 EC, and its accuracy in distinguishing EC types was not inferior to endometrial biopsy (10/14). For EC, 9.1% (10/110) were unevaluable using biopsy, significantly more than the 0% (0/110) by cytology (P = 0.002). CONCLUSION: Although preoperative prediction of serous carcinoma was difficult, endometrial cytology had a higher evaluable rate for EC types. Endometrial cytology may complement endometrial biopsy in preoperative women with EC.

Long-term in vivo time-lapse imaging of synapse development and plasticity in the cerebellum.

Synapses are continuously formed and eliminated throughout life in the mammalian brain, and emerging evidence suggests that this structural plasticity underlies experience-dependent changes of brain functions such as learning and long-term memory formation. However, it is generally difficult to understand how the rewiring of synaptic circuitry observed in vivo eventually relates to changes in animal's behavior. This is because afferent/efferent connections and local synaptic circuitries are very complicated in most brain regions, hence it is largely unclear how sensorimotor information is conveyed, integrated, and processed through a brain region that is imaged. The cerebellar cortex provides a particularly useful model to challenge this problem because of its simple and well-defined synaptic circuitry. However, owing to the technical difficulty of chronic in vivo imaging in the cerebellum, it remains unclear how cerebellar neurons dynamically change their structures over a long period of time. Here, we showed that the commonly used method for neocortical in vivo imaging was not ideal for long-term imaging of cerebellar neurons, but simple optimization of the procedure significantly improved the success rate and the maximum time window of chronic imaging. The optimized method can be used in both neonatal and adult mice and allows time-lapse imaging of cerebellar neurons for more than 5 mo in ∼80% of animals. This method allows vital observation of dynamic cellular processes such as developmental refinement of synaptic circuitry as well as long-term changes of neuronal structures in adult cerebellum under longitudinal behavioral manipulations.

Dendritic translocation establishes the winner in cerebellar climbing fiber synapse elimination.

In many regions of the developing mammalian nervous system, functional synaptic circuitry is formed by competitive elimination of early formed redundant synapses. However, how winning synapses emerge through competition remains unclear in the brain largely because of the technical difficulty of directly observing this dynamic cellular process in vivo. Here, we developed a method of two-photon multicolor vital imaging to observe competitive elimination of supernumerary climbing fibers (CFs) in the cerebellum of live mouse pups. At birth, each Purkinje cell (PC) in the cerebellar cortex is innervated by multiple CFs; an activity-dependent regression of supernumerary CFs ultimately yields a single innervation for most PCs by postnatal day 21. As supernumerary CFs are pruned, the terminal field of CFs translocates from the soma to the dendrites of PCs. In vivo time-lapse imaging of CF elimination revealed that (1) CF terminals were highly motile on the soma, but their motility was significantly reduced on dendrites; (2) only one CF could translocate to the dendrites whereas their competitors were restricted to perisomatic regions; and (3) the CF that began dendritic translocation became the winner. Moreover, selective photo-ablation of the winning CF (that undergoes dendritic translocation) reversed the fate of its losing competitor. These results indicate that dendritic translocation is a key cellular event that determines the winner during CF elimination. We propose that CF terminals are selectively stabilized on dendrites, providing irreversible competitive vigor to the first CF to form dendritic synapses.

Endometrial scraping cytology in women with extragenital malignancies.

OBJECTIVE: To clarify the usefulness of endometrial scraping smears in women with extragenital malignancies. STUDY DESIGN: A total of 4,335 endometrial scraping smears were obtained during the 5-year period 1995-1999 at the National Kyushu Cancer Center and were retrospectively analyzed regarding extragenital malignancies. RESULTS: There were 88 cases of extragenital malignancies. Extragenital malignant cells were detected in endometrial smears in 13 cases. The cases consisted of 4 gastric cancers, 4 breast cancers, 2 lung cancers, 1 rectal cancer, 1 gastrointestinal stromal tumor of the small intestine and 1 case of adenocarcinoma of unknown origin. The patients' average age was 52.5 years. The symptoms and signs included abnormal vaginal bleeding, abdominal and lumbar pain, lower limb edema, abdominal mass and neck lymph node swelling. Both ascites and peritoneal dissemination were found in 8 cases. Ten of the 13 cases were diagnosed as of extrauterine origin based on the characteristic cancer cell appearance, the absence of cellular detritus among the poorly differentiated adenocarcinomas and, above all, the morphologic difference between normal endometrial cells and cancer cells. CONCLUSION: Endometrial scraping smears are useful for detecting extragenital malignant cells that enter the uterine cavity.

Occurrence of periodontal bacteria in healthy children: a 2-year longitudinal study.

OBJECTIVES: To examine the occurrence of specific periodontal bacteria in children and adolescents. METHODS: Ten putative periodontal bacteria were longitudinally examined in plaque and saliva samples from 119 periodontally healthy children (2-15 years old) using a polymerase chain reaction method. RESULTS: Capnocytophaga ochracea, C. sputigena, and Actinobacillus actinomycetemcomitans were frequently found in saliva, and tended to persist in saliva for the remainder of the study, whereas Porphyromonas gingivalis, Treponema denticola, and Prevotella intermedia were rarely detected. P. nigrescens was more frequently detected in plaque and its prevalence increased with age. Eikenella corrodens and Campylobacter rectus were sometimes detected in both plaque and saliva, while Tannerella forsythensis was occasionally detected in saliva. CONCLUSION: A. actinomycetemcomitans, C. ochracea, C. sputigena, P. nigrescens, C. rectus, and E. corrodens are common members of the oral microbial flora of healthy children, whereas P. gingivalis, P. intermedia, and T. denticola appear to be transient organisms.

Clinical and microbiological changes in a child with rapid alveolar bone loss and refill.

A 10-year-old Japanese girl with severe tooth mobility in her lower permanent incisors was examined clinically, as well as radiographic and microbiological means. The incisors had severe alveolar bond loss and pocket depths exceeding 7 mm at the first visit, however, 10 periodontal bacteria were not detected in subgingival plaque samples taken from the lower central incisors using a 16S rRNA-based polymerase chain reaction method. Periodontal treatment consisting of mechanical debridement and antibiltic medication resulted in a significant improvement of the clinical parameters. Three months after the first examination, dental radiographs showed refilling of alveolar bone in the region. Further, microbiological examinations after remission detected only oral microflora commonly found in health children including A. actinomycetemcomitans. Based on the clinical, readiographic, and microbiological findings, the present case was diagnosed as acute periodontitis.