The Value of Additional Conventional Transbronchial Biopsy in the Negative Results of Rapid On-site Evaluation During Endobronchial Ultrasound With Guide Sheath to Diagnose Small Peripheral Lung Cancer.

Objective: The accuracy of rapid on-site evaluation (ROSE) during endobronchial ultrasonography with guide sheath (EBUS-GS) was reported to be approximately 90% for diagnosing small peripheral pulmonary lesions (PPLs). When ROSE during EBUS-GS for diagnosing small peripheral lung cancer is carried out and does not include malignant cells in a position whereby the probe was located within or adjacent to the lesion, the best technique for overcoming the lower diagnostic yield remains unknown. This study aimed to evaluate factors affecting positive results of ROSE during EBUS-GS in such a probe position. Moreover, when the results of ROSE were consistently negative, we evaluated the effectiveness of conventional transbronchial biopsy (TBB) in addition to EBUS-GS alone. Methods: We performed a retrospective analysis of consecutive patients who underwent EBUS-GS combined with ROSE for diagnosing small peripheral lung cancer (≤30 mm). We classified the results of ROSE into two groups based on the presence of malignant cells: the ROSE positive group (included malignant cells) and the ROSE negative group (did not include malignant cells). The significant predictors of positive ROSE results during EBUS-GS were analyzed using multivariate logistic regression analyses. Results: We identified 67 lesions (43 lesions in the ROSE positive group and 24 lesions in the ROSE negative group, respectively). Multivariate logistic analysis revealed that the significant factor affecting positive ROSE results was lesion size (>15 mm) (OR = 9.901). The diagnostic yield of additional conventional TBB to EBUS-GS was significantly higher than that of EBUS-GS alone (75.0% vs 33.3%, P = .041). Conclusion: The positive results of ROSE during EBUS-GS were significantly influenced by lesion size (>15 mm). When the results of ROSE during EBUS-GS were consistently negative in a position whereby the probe was located within or adjacent to the lesion, additional conventional TBB was effective to improve the diagnostic yield compared with EBUS-GS alone.

Endobronchial ultrasound-guided transbronchial needle aspiration under non-invasive positive pressure for the diagnosis of lung metastasis due to renal cell carcinoma in a patient with respiratory failure.

A 65-year-old man with chronic respiratory failure caused by chronic obstructive pulmonary disease, had a pulmonary nodule adjacent to the inlet of right B1 and B3. The patient had undergone a surgery for right renal cell carcinoma and colon cancer 6 years prior. We attempted endobronchial ultrasound-guided transbronchial needle aspiration under non-invasive positive pressure ventilation for diagnosis, with rapid on-site cytology, which was performed without complications. The histological findings revealed lung metastasis involving renal cell carcinoma. Endobronchial ultrasound-guided transbronchial needle aspiration under non-invasive positive pressure ventilation is useful for diagnosing lesions that require access up to the segmental bronchus in patients with respiratory failure.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma arising in patient with a history of EBV-positive mucocutaneous ulcer and EBV-positive nodal polymorphous B-lymphoproliferative disorder.

Elderly patients with Epstein-Barr virus (EBV) infection are at increased risk for developing B-cell lymphoproliferative disorder (B-LPD) due to immunosenescence. Here, we describe a case of a 75-year-old man who developed an EBV-positive (EBV+) mucocutaneous ulcer (EBVMCU) in the gingiva with spontaneous regression. Eighteen months after regression, he had a cervical lymph node enlargement that was diagnosed as EBV+ nodal polymorphous B-LPD, Ann Arbor stage IA. Clinicians decided to observe his clinical course without any treatment. Fourteen months later, the patient developed EBV-positive diffuse large B-cell lymphoma (DLBCL), Ann Arbor stage IIA, and received six courses of age-adjusted dose chemotherapy and achieved a complete remission. No evidence of a clonal relationship was found among these three lesions by standard polymerase chain reaction (PCR) analysis for immunoglobulin heavy chain. However, they all had expression of PD-L1 in the EBV+ large B-cells and Hodgkin Reed-Sternberg-like cells. This is the first case report of a PD-L1-positive (PD-L1+) EBVMCU and the development of multiple EBV-driven B-LPDs in the setting of immunosenescence within a 32-month period.

Localization of D-serine and serine racemase in neurons and neuroglias in mouse brain.

D-serine is a novel candidate for an intrinsic ligand for the glycine site of N-methyl-D-aspartate (NMDA) receptors in mammalian brain. D-serine and serine racemase, which produces D-serine from L-serine, have long been presumed to be localized in astrocytes. However, we have reported that D-serine immunoreactivity was observed in neurons in rats. In the present study, the distributions of D-serine and serine racemase were investigated in combination with marker proteins for neurons, astrocytes and oligodendrocytes in mice. Immunoreactivities for D-serine and serine racemase were found in neurons and oligodendrocytes. These results suggest that D-serine can be produced in neurons as well as glias and used as a neurotransmitter, which control the synaptic function of NMDA receptors.

Neuropathological alteration of aquaporin 1 immunoreactive enteric neurons in the streptozotocin-induced diabetic rats.

Long-term diabetic patients exhibit major clinical gastrointestinal problems, such as diarrhea and constipation. In recent years, water channel protein, aquaporin 1 (AQP1) has been identified in the enteric nervous system (ENS). We have examined the pathological changes in AQP1 immunoreactive (IR) neurons in streptozotocin-induced (STZ) diabetic rats. Eight-week-old Wistar rats were injected with streptozotocin, and artificial diabetes was induced. Sixteen-week-old STZ rats were then examined with double immunofluorescence staining and ABC immunohistochemical staining. AQP1-IR neurons in STZ rats were significantly increased compared with control rats (p<0.01). The ratio of AQP1 vs. HuC/D in STZ rats was also clearly increased as compared with control rats (p<0.05). It was apparent that thick AQP1-IR fibers were frequently observed in the secondary and tertiary myenteric plexus of STZ rats. The AQP1-IR fibers of STZ rats conspicuously showed many swollen varicosities. These swollen varicose fibers were also observed in the longitudinal and circular muscle layers. Streptozotocin-induced diabetic rats showed pathological changes in AQP1-IR neurons of the ENS. The alteration of AQP1-IR neurons may be possible contribute to diabetic gastrointestinal dysfunction in streptozotocin-induced diabetic rats.

iNOS-dependent DNA damage in patients with malignant fibrous histiocytoma in relation to prognosis.

Malignant fibrous histiocytoma (MFH) is one of the most common soft tissue sarcomas. MFH has been proposed to be a lesion accompanied with inflammatory responses. During chronic inflammation, reactive nitrogen and oxygen species generated from inflammatory cells are considered to participate in carcinogenesis by causing DNA damage. 8-nitroguanine is a mutagenic nitrative DNA lesion formed during chronic inflammation. We examined whether nitrative DNA damage is related to the prognosis of MFH patients. We performed immunohistochemical analyses to examine the distribution of DNA damage and the expression of inflammation-related molecules including inducible nitric oxide synthase (iNOS), nuclear factor-kappaB (NF-kappaB), and cyclooxygenase-2 (COX-2) in clinical specimens from 25 patients with MFH. We also analyzed the correlation of DNA damage or the expression of these genes with the prognosis of MFH patients. Immunohistochemical staining revealed that the formation of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), an oxidative DNA lesion, occurred to a much greater extent in MFH tissue specimens from deceased patients than in live patients. iNOS, NF-kappaB and COX-2 were colocalized with 8-nitroguanine in MFH tissues. It is noteworthy that the statistical analysis using the Kaplan-Meier method demonstrated strong 8-nitroguanine staining to be associated with a poor prognosis. In conclusion, 8-nitroguanine appears to participate in not only the initiation and promotion of MFH, but also in the progression of MFH, and could therefore be used as a promising biomarker to evaluate the prognosis of cancer patients.

Aquaporin 1 immunoreactive enteric neurons in the rat ileum.

Most neurons in the central nervous system and peripheral nervous system do not express water transporting protein, aquaporin (AQP). In the present study, we have demonstrated the presence of AQP1 immunoreactivity in a particular neuronal subtype in the enteric nervous system (ENS) of the rat ileum. AQP1-immunoreactive (IR) neurons simultaneously expressed a neuronal marker HuC/D. Moderate numbers of AQP1-IR neuronal somata were found in the myenteric plexus, and a very few were found in the submucosal plexus. AQP1-IR neurons can be classified as Dogiel type I cells, which have several short processes and a single long process. Many AQP1-IR fibers were found both in the myenteric and submucosal plexi. Many AQP1-IR varicose fibers were closely associated with neuronal somata in the ganglia, whereas other AQP1-IR fibers penetrated into the muscle layers. These results suggest that AQP1-IR neurons probably play a significant role within the ENS to control gut functions.

Discrimination of cell nuclei in early S-phase, mid-to-late S-phase, and G(2)/M-phase by sequential administration of 5-bromo-2'-deoxyuridine and 5-chloro-2'-deoxyuridine.

5-Bromo-2'-deoxyuridine (BrdU) and 5-chloro-2'-deoxyuridine (CldU) were sequentially administered intraperitoneally into mice at 1-hr intervals. After one additional hr, the small intestines were resected, fixed, and embedded in paraffin. In histological sections stained with monoclonal antibody Br-3 reactive to both BrdU and CldU, and CldU antibody reactive only to CldU, three types of staining could be identified in the proliferating zone. Cells with nuclei stained only with Br-3 antibody were estimated to have completed DNA replication during the first 1 hr and were fixed in G(2)/M-phase. Those nuclei were frequently found in apical areas of the simple columnar epithelium of the intestine, whereas other nuclei were located basally in the cells. This observation suggested intracellular movement of cell nuclei in G(2)/M-phase. Identification of cells in early S-phase became possible using these antibodies in combination with DAB and fluorescence stainings. Replication sites in early S-phase nuclei were found to be numerous, whereas in late S-phase they were larger in size and much smaller in number.

Inducible nitric oxide synthase-dependent DNA damage in mouse model of inflammatory bowel disease.

Increased cancer risk occurs in inflammatory bowel disease (IBD) undergoing long-term chronic inflammation. To evaluate whether inducible nitric oxide synthase (iNOS)-dependent DNA damage plays a role in the carcinogenic process triggered by IBD, we prepared a mouse model of IBD induced by transfer of CD45RBhighCD4+ T cells lacking regulatory T cells to female severe combined immunodeficiency (SCID) mice. CD45RBhighCD4+ T cells were isolated from mouse spleen after staining with fluorescein isothiocyanate (FITC)-conjugated anti-CD45RB monoclonal antibody, followed by anti-FITC-conjugated microbeads. This IBD mouse model showed that the bodyweight increased with aging to a lesser extent than non-treated controls, and that the intestine was shortened. Pathological findings of this mouse model, which showed severe inflammation in colon tissues, were similar to IBD patients. Double immunofluorescence technique revealed that both 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were formed mainly in epithelial cells of the IBD mouse model. 8-Nitroguanine was formed in most of 8-oxodG-immunoreactive nuclei of epithelial cells. iNOS, proliferating cell nuclear antigen and p53 protein were also expressed in the colon epithelium. These results indicate that nitrative DNA damage, as well as oxidative DNA damage, is induced in colon epithelial cells of the IBD mouse model followed by proliferation of these cells, which may contribute to colon carcinogenesis.

L-aspartate-immunoreactive neurons in the rat enteric nervous system.

L-aspartate (L-Asp) is an excitatory neurotransmitter in the central nervous system. In the present study, we demonstrate, for the first time, the presence of L-Asp in a particular neuronal cell class in the enteric nervous system (ENS). Scattered L-Asp-immunoreactive neuronal cell bodies and nerve fibers were found extensively in both the myenteric and submucosal plexus throughout the small and large intestines. Many L-Asp-immunoreactive nerve fibers, which originated from intrinsic nerve cell bodies, were found in the ganglia and interconnecting nerve bundles. Electron microscopy revealed that L-Asp-immunoreactive terminals frequently formed synaptic contacts with intrinsic nerve cells, suggesting that some L-Asp-immunoreactive neurons might function as interneurons. These results suggest that L-Asp-immunoreactive neurons play a significant role within the ENS to control intestinal functions. The presence of enteric L-Asp-immunoreactive neurons provides strong support for the proposal that L-Asp is a neuromodulator in the rat ENS.

Oxidative DNA damage induced by toluene is involved in its male reproductive toxicity.

Toluene is widely used as an organic solvent in various industries and commercial products. Recent investigations have shown that toluene may induce male reproductive dysfunctions and carcinogenicity. To clarify whether the toxicity results from the interference of endocrine systems or direct damage to reproductive organs, we examined the effects of toluene on the male reproductive system in rats, comparing to those of diethylstilbestrol (DES), a potent synthetic estrogen. Toluene (50, 500 mg/kg) or DES (2 mg/kg) injected subcutaneously to male Sprague-Dawley rats once a day for 10 days decreased the epididymal sperm counts and the serum concentrations of testosterone. The mRNA level for gonadotropin-releasing hormone receptor in the pituitary was decreased by DES, but not by toluene. On the contrary, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation in testes, the biological marker for oxidative DNA damage, was increased by toluene but not by DES. These results suggest that toluene induces reproductive toxicity via direct oxidative damage of spermatozoa, whereas DES affects endocrine systems via the hypothalamo-pituitary-gonadal axis. Morphological findings supported the idea. To determine the mechanism of 8-oxodG formation in vivo, we examined DNA damage induced by toluene metabolic products in vitro. Minor toluene metabolites, methylhydroquinone and methylcatechols, induced oxidative DNA damage, and the methylcatechols induced NADH-mediated 8-oxodG formation more efficiently than methylhydroquinone did. We propose that oxidative DNA damage in the testis plays a role in reproductive toxicity induced by toluene.