Simplified Quantitative Assay System for Measuring Activities of Drugs against Intracellular Legionella pneumophila

We developed a new simple assay for the quantitation of the activities of drugs against intracellular Legionella pneumophila. The cells of a murine macrophage-like cell line (J774.1 cells) allowed the intracellular growth and replication of the bacteria, which ultimately resulted in cell death. The infected J774.1 cell monolayers in 96-well microplates were first treated with antibiotics and were further cultured for 72 h. The number of viable J774.1 cells in each well was quantified by a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and an enzyme-linked immunosorbent assay reader. The number of growing bacteria in each well was also determined by counting the numbers of CFU on buffered charcoal yeast extract-α agar plates. Viable J774.1 cell counts, determined by the colorimetric assay, were inversely proportional to the number of intracellular replicating bacteria. The minimum extracellular concentrations (MIECs) of 24 antibiotics causing inhibition of intracellular growth of L. pneumophila were determined by the colorimetric assay system. The MIECs of beta-lactams and aminoglycosides were markedly higher than the MICs in buffered yeast extract-α broth. The MIECs of macrolides, fluoroquinolones, rifampin, and minocycline were similar to the respective MICs. According to their intracellular activities, clarithromycin and sparfloxacin were the most potent among the macrolides or fluoroquinolones tested in this study. Our results indicated that the MTT assay system allows comparative and quantitative evaluations of the intracellular activities of antibiotics and efficient processing of a large number of samples.     

Liver metastasis from rectal cancer with prominent intrabile duct growth

Intrabiliary growth of liver metastases from colorectal cancer has rarely been studied. A surgically resected case of a metastatic liver tumor with prominent intrabiliary growth derived from rectal cancer is reported. The patient was a 62-year-old man who had received a low anterior resection for rectal cancer in March 2000. He was re-admitted due to obstructive jaundice in January 2003, and was diagnosed with hepatic malignancy in segment II of the liver with an intrabiliary tumor extending from the intrahepatic bile duct of segment II to the common hepatic duct. He underwent a left hepatectomy, a partial resection of segment VI, and an extrahepatic bile duct resection with reconstruction of the biliary tract. In the resected specimen, there were whitish tumors of 3 cm and 1.5 cm in diameter in segments II and VI, respectively, and an intrabiliary tumor originating from the main tumor in segment II extended to the common hepatic duct. Both the liver tumors and the intrabiliary tumor consisted of a well- to moderately differentiated adenocarcinoma, which showed the same histological features as the rectal cancer. The immunohistochemical findings strongly supported that these tumors, including the intrabiliary growth, were liver metastasis from the rectal cancer. The intrabiliary invasion and growth of metastatic liver tumors has generally been overlooked, notwithstanding their frequently observed biological behavior. The present case is informative, and further investigation into this type of metastatic liver tumor may be warranted.     

Development of murine hepatic sinusoidal endothelial cells characterized by the expression of hyaluronan receptors.

Endothelial cells (ECs) display distinct structural and functional characteristics depending on the tissue and developmental stage; however, the development of tissue-specific ECs remains poorly understood. Here, we describe the development of hepatic sinusoids in mice based on the expression of hyaluronan receptors Stab2 and Lyve-1. Flk-1(+) cells in and around the liver bud begin to express Stab2 at embryonic day (E) 9.5, before the formation of vascular lumen. Hepatic sinusoidal endothelial cells (HSECs) begin to express Lyve-1 at E10.5, and both markers continue to be expressed in HSECs thereafter. Although HSECs and lymphatic ECs (LECs) are known to share functional and phenotypic characteristics, we clearly show that HSECs can be distinguished from LECs by the expression of molecular markers and higher endocytotic activity. Our results provide new insight into the development of tissue-specific ECs and phenotypic criteria to distinguish HSECs from other types of ECs, including LECs.     

Two different roles of purified CD45+c-Kit+Sca-1+Lin- cells after transplantation in muscles

Recent studies have indicated that bone marrow cells can regenerate damaged muscles and that they can adopt phenotypes of other cells by cell fusion. Our direct visualization system gave evidence of massive muscle regeneration by green fluorescent protein (GFP)-labeled CD45+c-Kit+Sca-1+Lin- cells (KSL cells), and we investigated the role of KSL cells in muscle regeneration after transplantation with or without lethal irradiation. In the early phase, GFP signals were clearly observed in all the muscles of only irradiated mice. Transverse cryostat sections showed GFP+myosin+ muscle fibers, along with numerous GFP+ hematopoietic cells in damaged muscle. These phenomena were temporary, and GFP signals had dramatically reduced 30 days after transplantation. After 6 months, GFP+ fibers could hardly be detected, but GFP+c-Met+ mononuclear cells were located beneath the basal lamina where satellite cells usually exist in both conditioned mice. Immunostaining of isolated single fibers revealed GFP+PAX7+, GFP+MyoD+, and GFP+Myf5+ satellite-like cells on the fibers. Single-fiber cultures from these mice showed proliferation of GFP+ fibers. These results indicate two different roles of KSL cells: one leading to regeneration of damaged muscles in the early phase and the other to conversion into satellite cells in the late phase.     

Invasive fungal infection following reduced-intensity cord blood transplantation for adult patients with hematologic diseases.

Invasive fungal infection (IFI) is a significant complication after allogeneic hematopoietic stem cell transplantation (HSCT); however, we have little information on its clinical features after reduced intensity cord blood transplantation (RICBT) for adults. We reviewed medical records of 128 patients who underwent RICBT at Toranomon Hospital between March 2002 and November 2005. Most of the patients received purine-analogbased preparative regimens. Graft-versus-host disease (GVHD) prophylaxis was a continuous infusion of either tacrolimus 0.03 mg/kg or cyclosporine 3 mg/kg. IFI was diagnosed according to the established EORTC/NIH-MSG criteria. IFI was diagnosed in 14 patients. Thirteen of the 14 had probable invasive pulmonary aspergillosis and the other had fungemia resulting from Trichosporon spp. Median onset of IFI was day 20 (range: 1-82), and no patients developed IFI after day 100. Three-year cumulative incidence of IA was 10.2%. Four of the 13 patients with invasive aspergillosis (IA) developed grade II-IV acute GVHD, and their IA was diagnosed before the onset of acute GVHD. The mortality rate of IFI was 86%. Multivariate analysis revealed that the use of prednisolone >0.2 mg/kg (relative risk 7.97, 95% confidence interval 2.24-28.4, P = .0014) was a significant risk factor for IA. This study suggests that IFI is an important cause of deaths after RICBT, and effective strategies are warranted to prevent IFI.     

Insular component as a risk factor of thyroid carcinoma

Forty-four thyroid carcinomas with an Insular component (JC) were reviewed from 2457 tumors diagnosed as papillary (PC) or follicular carcinoma (FC). These tumors were classified as FC with an IC (FCIC; 30 cases) and PC with an IC (PCIC; 14 cases). Both tumors were composed of solid cell nests in some areas and had a tendency toward a characteristic nuclear size: FCIC had a small nucleus and PCIC contained a nucleus of an Intermediate type or a large nucleus similar to that of PC, although there were numerous tumors with an exceptional nuclear size. The mean age and tumor diameter were the highest and largest in FCIC, respectively, followed by PCIC. Among the 44 cases, 17 patients died of the disease, two were alive with the disease and 18 were alive without the disease. From 13 clinicopathological factors, the presence of an IC, age, non-encapsulation, tumor size, vascular invasion and necrosis were found to be independent variables for actual prognosis of FC and PC based on univariate analysis followed by multtvariate analysis. The results of the present study indicate that the presence of an IC is an independent aggressive prognostic factor for patients with PC and FC.     

Quantitative ultrastructure of synapses on functionally identified primary afferent neurons in the cat trigeminal mesencephalic nucleus

Though a number of studies have reported the presence of synapses on neurons in the trigeminal mesencephalic nucleus (Vmes), there have been no quantitative studies of either the density of innervation, or the ultrastructure, of the synapses on single, physiologically identified neurons in this nucleus. In this study we recorded from single neurons in the Vmes, identified them as being either muscle spindle afferents (MS) or periodontal ligament mechanoreceptor afferents (PL), and then labeled the neurons by intra-axonal injection of horseradish peroxidase (HRP). The material was first processed to reveal the HRP activity, following which ultrathin sections through the labeled somata were cut and examined under the electron microscope. Complete serial reconstructions were made through the soma of one MS neuron and one PL neuron, and the contacts on the neurons reconstructed. Boutons were found on the soma, spines, appendages and the axon hillock and the initial segment of the axon. The numbers of boutons terminating on the two neurons were 198 (PL) and 424 (MS), giving a packing density of 4.4 and 10.7 boutons respectively (i.e., number of boutons/100 micron 2 of the postsynaptic membrane). Boutons could be separated into two types on the basis of their vesicles: those containing clear, round vesicles (i.e., S-type) and those containing a mixture of round, oval and flattened vesicles (P-type). Ninety-five (PL neuron) and 99% (MS neuron) of terminals on the two neurons were P-type. All the S-type boutons and 80% of the P-type boutons formed asymmetric synaptic contacts while 10% of the P-type boutons made symmetric contacts. Quantitative measurements of the P-type boutons on the labeled neurons, in which the data of MS and PL neurons were pooled, revealed that bouton volume was highly correlated with bouton surface area, active zone number, total active zone area, vesicle number, and mitochondrial volume. However, comparing the quantitative measurements of the P-type boutons with those of previously reported vibrissa afferent terminals and their associated axon terminals revealed that all the parameters were smaller for the P-type boutons (on Vmes neurons) than those of the vibrissa afferent terminals but similar to those of axon terminals presynaptic to the vibrissa afferents. Taken together, our results emphasize the wide scope for synaptic interactions in the Vmes and suggest that it may be more fruitful to view the Vmes as an integrating center.     

Neurons in the rostral cingulate motor area monitor multiple phases of visuomotor behavior with modest parametric selectivity

We examined the cellular activity in the rostral cingulate motor area (CMAr) with respect to multiple behavioral factors that ranged from the retrieval and processing of associative visual signals to the planning and execution of instructed actions. We analyzed the neuronal activity in monkeys while they performed a behavioral task in which 2 visual instruction cues were given successively with an intervening delay. One cue instructed the location of the target to be reached; the other cue instructed which arm was to be used. After a second delay, the monkey received a motor-set cue to be prepared to initiate the motor task in accordance with instructions. Finally, after a GO signal, the monkey reached for the instructed target with the instructed arm. We found that the activity of neurons in the CMAr changed profoundly throughout the behavioral task, which suggested that the CMAr participated in each of the behavioral processing steps. However, the neuronal activity was only modestly selective for the spatial location of the visual signal. We also found that selectivity for the instructional information delivered with the signals (target location and arm use) was modest. Furthermore, during the motor-set and movement periods, few CMAr neurons exhibited selectivity for such motor parameters as the location of the target or the arm to be used. The abundance and robustness of the neuronal activity within the CMAr that reflected each step of the behavioral task and the modest selectivity of the same cells for sensorimotor parameters are strikingly different from the preponderance of selectivity that we have observed in other frontal areas. Based on these results, we propose that the CMAr participates in monitoring individual behavioral events to keep track of the progress of required behavioral tasks. On the other hand, CMAr activity during motor planning may reflect the emergence of a general intention for action.