Migration of enhanced green fluorescent protein expressing bone marrow-derived microglia/macrophage into the mouse brain following permanent focal ischemia

Brain ischemia induces a marked response of resident microglia and hematopoietic cells including monocytes/macrophages. The present study was designed to assess the distribution of microglia/macrophages in cerebral ischemia using bone marrow chimera mice known to express enhanced green fluorescent protein (EGFP). At 24 h after middle cerebral artery occlusion (MCAO), many round-shaped EGFP-positive cells migrated to the ischemic core and peri-infarct area. At 48-72 h after MCAO, irregular round- or oval-shaped EGFP/ionized calcium-binding adapter molecule 1 (Iba 1)-positive cells increased in the transition zone, while many amoeboid-shaped or large-cell-body EGFP/Iba 1-positive cells were increased in number in the innermost area of ischemia. At 7 days after MCAO, many process-bearing ramified shaped EGFP/Iba 1-positive cells were detected in the transition to the peri-infarct area, while phagocytic cells were distributed in the transition to the core area of the infarction. The distribution of these morphologically variable EGFP/Iba 1-positive cells was similar up to 14 days from MCAO. The present study directly showed the migration and distribution of bone marrow-derived monocytes/macrophages and the relationship between resident microglia and infiltrated hematogenous element in ischemic mouse brain. It is important to study the distribution of intrinsic and extrinsic microglia/macrophage in ischemic brain, since such findings may allow the design of appropriate gene-delivery system using exogenous microglia/macrophages to the ischemic brain area.     

A promiscuous T cell hybridoma restricted to various I-A molecules

In a previous study, we identified T cell receptor and major histocompatibility complex (MHC) contact sites on the pigeon cytochrome c p43-58 peptide. Positions 46 and 54 of p43-58 were shown to be the MHC-binding sites. Specific amino acids were identified on the MHC-binding sites which bound to the relevant I-A molecule. In the present study, using NOD (I-A^g7) mice, we established a T cell hybridoma, NOE33-1-2, specific for a p43-58 analog 46R50E54A with arginine (R) and alanine (A) at positions 46 and 54, respectively. Interestingly, NOE 33-1-2 recognized 46R50E54A in the presence of not only I-A^g7, but also I-A^{d, s, u and v}. In contrast to previous reports that promiscuous T cells were able to recognize peptide antigens with various HLA-DR or I-E molecules consist of monomorphic α and polymorphic β chains, the promiscuous T cell clone NOE33-1-2 recognized peptides with various I-A molecules lacking the monomorphic chain.     

Past and current dermatological status of yusho patients

Skin symptoms of "yusho" and their change thereafter are described. In the early stage, skin eruptions were the most prominent and impressive features of yusho. Acneiform eruptions and pigmentation were most conspicuous. With the passage of time, there is some improvement and most patients now have few eruptions, except for the severely intoxicated group. As for blood PCB analysis, the severely intoxicated group who showed intensive skin symptoms showed "pattern A"--a typical specific yusho pattern. The blood PCB patterns of each patient were stable with no alteration after many years and, on the other hand, the blood PCB concentrations have diminished year by year.     

High HNRNPA3 expression is associated with lymph node metastasis and poor prognosis in patients treated with radical cystectomy

ObjectiveWe sought to identify heterogeneous nuclear ribonucleoprotein A3 (HNRNPA3) expression in bladder cancer and its relationship to clinicopathological findings and prognosis.MethodsImmunohistochemical staining for HNRNPA3 was performed on 122 archived radical cystectomy specimens, with immunoreactivity being stratified on a 0 to 3 scale. The percentage of HNRNPA3 expressing tumor cells was calculated and multiplied by the staining score over an average of 5 areas to obtain a semiquantitative H-score (maximum value: 300). HNRNPA3 expression was categorized as high (≥80) or low (<80).ResultsThe patients’ median age was 70 years, and the median follow-up period was 39.4 months. High HNRNPA3 expression was significantly associated with lymph node metastasis (P= 0.014) and S100A8, S100A9 and uroplakin III expression (P= 0.028, 0.002, and 0.047, respectively). Log-rank tests indicated that high HNRNPA3 expression was significantly associated with disease progression and cancer-specific death (P= 0.013 and 0.006, respectively). In the Cox proportional hazards regression analysis, only lymph node metastasis was associated with disease progression and cancer-specific survival.ConclusionHNRNPA3 may be a new biomarker to predict biologically aggressive cancers and determine the appropriate treatment modality in patients after radical cystectomy.     

Gene transfer into rat renal cells using adeno-associated virus vectors

Adeno-associated virus (AAV) vectors have a number of attractive features, including lack of cytotoxicity, ability to transduce nondividing cells, and long-term transgene expression. We investigated whether rat renal cells could be efficiently transduced with AAV vectors. Rat glomerular mesangial cells were transduced with AAV-lacZ vector containing beta-galactosidase gene in vitro, and the expression of beta-galactosidase was evaluated by X-gal staining and ELISA. For ex vivo experiments, sections of rat kidneys were incubated with AAV-lacZ, and then evaluated by X-gal histochemical staining. The level of beta-galactosidase expression in cultured rat mesangial cells increased in a dose-dependent manner (ranging from 1 x 10(5) to 5 x 10(6) particles/cell). When transduced with 5 x 10(6) vector particles/cell of AAV-lacZ, about 50% of mesangial cells were stained positively with X-gal, and the level of beta-galactosidase expression reached 9.9 +/- 1.5 ng/mg protein. Expression was detectable during the culture period for at least 7 days. X-gal histochemical examination of the ex vivo transduced renal tissue revealed tubular cell and interstitial tissue staining. However, gene transfer was not clearly observed in glomeruli. These findings suggest that AAV vectors have the potential for gene therapy of renal diseases.     

Highly sensitive detection of melanoma at an early stage based on the increased serum secreted protein acidic and rich in cysteine and glypican-3 levels.

PURPOSE: There are no available tumor markers detecting primary melanoma at an early stage. The identification of such serum markers would be of significant benefit for an early diagnosis of melanoma. We recently identified glypican-3 (GPC3) as a novel tumor marker but could diagnose only 40% of melanomas. Thereby, we focused out attention on secreted protein acidic and rich in cysteine (SPARC) overexpressed in melanoma as another candidate for tumor marker. EXPERIMENTAL DESIGN: Secreted SPARC protein was quantified using ELISA in the sera from 109 melanoma patients, five patients with large congenital melanocytic nevus, 61 age-matched healthy donors, and 13 disease-free patients after undergoing a surgical removal. We also quantified GPC3 and 5-S-cysteinyldopa in the same serum samples and compared these markers for their diagnostic value. RESULTS: The serum SPARC concentrations in melanoma patients were greater than those in healthy donors (P = 0.001). When we fixed a cutoff value at the mean concentration plus 2 SD of the healthy donors, the serum SPARC was found to have increased in the sera of 36 of the 109 (33%) melanoma patients, whereas there were three (4.9%) false-positive cases of 61 healthy donors. Surprisingly, 19 of 36 patients showing increased SPARC levels were in stages 0 to II. The serum SPARC level decreased under the cutoff level in 10 of 13 patients after surgical removal. Using SPARC and GPC3 in combination thus enabled us to diagnose 47 of 75 (66.2%) melanoma patients at an early stage (0-II). CONCLUSIONS: SPARC or its combination with GPC3 is thus considered a potentially useful tumor marker, especially for melanoma at an early stage.     

Noncalcified atherosclerotic lesions with vulnerable characteristics detected by coronary CT angiography and future coronary events

BackgroundThe ability of coronary CT angiography (CTA) findings such as plaque characteristics to predict future coronary events remains controversial.ObjectiveWe investigated whether noncalcified atherosclerotic lesions (NCALs) detected by coronary CTA were predictive of future coronary events.MethodsA total of 511 patients who underwent coronary CTA were followed for cardiovascular events over a period of 3.3 ± 1.2 years. The primary end point was defined as hard events, including cardiac death, nonfatal myocardial infarction, or unstable angina that required urgent hospitalization. Early elective coronary revascularizations (n = 58) were excluded. The relationship between features of NCALs and outcomes is described.ResultsA total of 15 hard events (2 cardiac deaths, 7 myocardial infarctions, 6 cases of unstable angina that required urgent hospitalization) were documented in the remaining 453 patients with modest risks during a follow-up period of 3.3 ± 1.2 years. For these hard events, a univariate Cox proportional hazard model showed that the hazard ratio for the presence of >50% stenosis was 7.27 (95% CI, 2.62–21.7; P = .0002). Although the presence of NCAL by itself was not statistically significant, NCALs with low attenuation and positive remodeling (low-attenuation plaque [LAP] and positive remodeling [PR]; plaque CT number ≤34 HU and remodeling index ≥1.20) showed an adjusted hazard ratio of 11.2 (95% CI, 3.71–36.7; P < .0001). With C-statistics analysis, when both LAP and PR and >50% stenosis were added, the C-statistic was significantly improved compared with the basal model adjusted for age, sex, and log₂ (Agatston score +1) (0.900 vs 0.704; P = .0018).ConclusionsIdentification of NCALs with LAP and PR characteristics by coronary CTA provides additional prognostic information to coronary stenosis for the prediction of future coronary events.     

Skip mediastinal nodal metastases in non-small cell lung cancer

Objective: To reveal the incidence and clinical significance of mediastinal nodal metastases without N1-station nodal metastases (‘skip-N2 metastases’) in non-small cell lung cancer (NSCLC). Methods: A total of 450 NSCLC patients who underwent tumor resection with a systemic mediastinal nodal dissection were retrospectively reviewed. p53 status and proliferative activity represented as proliferative index (PI) were also examined immunohistochemically. Results: Skip-N2 metastases were documented in 49 (13%) patients of all 450 patients; among 334 patients without N1-nodal involvement, 18% patients had skip-N2 metastases. The postoperative survival of skip-N2 patients was almost same as that for patients with metastases to both N1 and N2 nodes. Skip-N2 metastases were significantly more frequent in male patients and squamous cell carcinoma patients. In addition, the mean PI for tumor with skip-N2 metastases was significantly higher than that for any other pathologic nodal (pN)-status diseases. Combined with histologic type and PI, the incidences of skip-N2 metastases for adenocarcinoma showing lower PI were only 5% (7/137) of all patients and 7% (7/94) of patients without N1-nodal involvement. Conclusions: N1 nodal status is not a useful predictor of N2 nodal status in NSCLC, because skip-N2 metastases were documented in 18% patients showing no N1-nodal involvement. However, N1 node-guided dissection might be performed in patients with adenocarcinoma showing lower PI, because the incidence of skip-N2 metastases was extremely low.