Evaluation of a human monocytic cell line THP-1 model for assay of the intracellular activities of antimicrobial agents against Legionella pneumophila

We examined the intracellular activities of 11 antimicrobial agents against Legionella pneumophila using a human monocyte-derived cell line, THP-1. Colony counting and microscopic examination of L. pneumophila co-incubated with THP-1 cells (5 x 105 cells/well) were performed. Both extra- and intra-cellular multiplication of L. pneumophila were observed and were dependent on the inoculum of L. pneumophila in the culture; L. pneumophila did not grow in the cell culture medium alone. Light microscopic examination confirmed that extracellular L. pneumophila originated from THP-1 cells disrupted by bacterial multiplication. L. pneumophila multiplied by 3-4 logs after 24 h incubation with THP-1 cells and their number remained stable at 106-107 cfu/mL until 72 h. The results of viability studies using four antimicrobial agents-ciprofloxacin, erythromycin, minocycline and rifampicin-demonstrated that our system was suitable for the intracellular activity assay. We used a concept of 'minimum extracellular concentration inhibiting intracellular multiplication' (MIEC) to evaluate the intracellular activity of antimicrobial agents. The MIECs of three beta-lactams were markedly higher than their conventional MICs while those of macrolides, quinolones, rifampicin and minocycline were similar to their MICs. Our results suggest that evaluation of the clinical efficacy of drugs against L. pneumophila should include determination of their intracellular activity against the bacteria, which could be measured using our assay system in THP-1 cells.     

Electrophoretic and kinetic characterization of three variants of soluble cytoplasmic L-alanine:2-oxoglutarate aminotransferase in human liver tissue

Three common variants of soluble cytoplasmic L-alanine:2-oxoglutarate aminotransferase (ALT, EC 2.6.1.2), sALT 1, 2-1 and 2, were isolated from normal human liver, and characterized by electrophoretic and kinetic analyses. The isoelectric point of sALT 1 was pH 6.45. sALT 2-1 was focused into three bands with pl 6.1, 6.2 and 6.45; sALT was focused into one band with pl 6.1. The electrophoretic mobilities of sALTs altered to the fast beta-globulin fraction after aging or papain treatment. Ammonia was produced during the latter, and the altered migration was considered to be caused by deamidation of sALT. The relative molecular mass of each of the enzymes was 110,000. Minor differences in the apparent Km values among the multiple forms for both L-alanine and 2-oxoglutarate were observed after incubation with 100 mumol/L of pyridoxal phosphate (PALP). PALP stimulation of the enzyme activities was also different. sALT 1 was more stable than sALT 2-1 and 2 after heat and urea treatments. In human sera from 1065 adult Japanese, sALT 2-1, a heterozygote form of sALT 1 and 2, was dominant.     

Detection of bone marrow infiltration of lymphoma cells by fluorescence in situ hybridization

BACKGROUND: It is sometimes difficult to detect the bone marrow infiltration of lymphoma cells, because lymphoma cells are not distinguishable from normal lymphocytes due to the similarity of their phenotype. METHODS: Bone marrow involvement of 17 samples of 15 patients with follicular lymphoma, whose lymphoma cells were confirmed to harbor the translocation of chromosome14q32, were examined by microscopic analysis of bone marrow smear and biopsy, flow cytometorical analysis (FCM), chromosomal analysis of G-banding and fluorescence in situ hybridization (FISH). FISH was performed using a probe, which detects the split of IGH gene on 14q32. RESULTS: The positivity of FISH was highest among these methods and FISH was able to detect the bone marrow involvement in one case who was defined as negative by bone marrow biopsy. CONCLUSIONS: FISH can be used for detection of bone marrow involvement of malignant lymphoma that carries chromosomal rearrangement involving 14q32.     

Studies on immune adherence (C3b) receptor activity of human erythrocytes: relationship between receptor activity and membrane osmotic fragility

Although human erythrocytes (E) possess C3b receptors (C3b-R), their in vivo functions are unknown. We had observed that E from patients with immune complex diseases had defective or impaired C3b-R activity when circulating immune complexes (CIC) could be demonstrated. This phenomenon has been investigated in relation to membrane osmotic fragility of such E by a coil planet centrifugation (CPC) system. Osmotic fragility was defined by the hemolysis starting point (HSP), peak point (HPP) and end point (HEP) using NaCl osmotic gradient (150-30 mOsM) coiled tubes. It was observed that E with low C3b-R activity showed high osmotic fragility. Hemolysis of E with low C3b-R activity started at 104 +/- 8 mOsM (n = 133) compared to 97 +/- 3 mOsM (n = 18) of patients' E with normal C3b-R activity and 96 +/- 5 mOsM of E from normal healthy donors (n = 128). Furthermore, we observed that HSP shifted towards lower osmolarity with clinical and immunological improvement of disease activity after treatment with corticosteroids. When osmotic gradients were lower to 120-50 mOsM, 52 out of 116 E samples with low C3b-R activity separated into 2 E populations. In contrast, none of 18 E samples with normal C3b-R activity separated into 2 E populations. However, we observed broadened fragility patterns in these 18 E samples. Serial studies of C3b-R activity, osmotic fragility and the presence of CIC were performed in 7 patients. Improvement of disease activity was associated with increased C3b-R activity, decreased osmotic fragility and the disappearance of CIC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Small solid pseudopapillary tumor of the pancreas in a 32-year-old man: Report of a case

A solid pseudopapillary tumor (SPT) of the pancreas is a rare neoplasm that mainly occurs in young women. We herein report the case of a small SPT arising from the head of the pancreas in an asymptomatic 32-year-old man, plus a literature review of this tumor. A 32-year-old man was admitted to our department at Kumamoto University Hospital for the evaluation of a pancreatic mass. The tumor had central necrosis, which was poorly perfused on contrast-enhanced computed tomography (CT) and which had a high intensity on T2-weighted magnetic resonance imaging (MRI). Histology revealed the lesion to be a solid pseudopapillary tumor of the pancreas, with the characteristic pseudopapilla formation and central degeneration. However, no capsule formation was observed. The tumor was positive for CD56, CD10, α1-antitrypsin, α1-antichymotrypsin, β-catenin, and progesterone receptor. However, the tumor was negative for pancreatic hormones, chromogranin-A, carcinoembryonic antigen, and carbohydrate antigen 19–9. We diagnosed the patient to have an SPT based on these histological findings. Small-sized solid pseudopapillary tumors of the pancreas are being increasingly recognized because of the recent advances in CT and MRI. We should also consider SPT even if it occurs in a male when the tumor contains necrosis-suspected areas which are poorly perfused on contrast-enhanced CT with a high intensity on T2-weighted MRI.     

5-azacytidine, a chemotherapeutic drug, induces TRAIL-mediated apoptosis in mouse thymocytes in vivo

5-azacytidine (5AzC) is a cytidine analogue with two main effects on cellular conditions; DNA damage, resulting in apoptosis, and DNA hypomethylation, restoring normal growth control and differentiation. However, the molecular mechanism of 5AzC-induced apoptosis is not fully understood. The aim of the present study is to clarify this mechanism in mouse thymocytes in vivo. Ten-week-old, male C57BL/6J mice were injected with 5AzC (100 mg/kg) intraperitoneally, and thymuses were examined for apoptotic changes. In the 5AzC-treated thymus, increases of TUNEL-positive thymocytes and cleaved caspase-3 protein, both biochemical features of apoptosis, were detected. 5AzC-induced apoptosis was observed even in the thymuses of mice deficient in p53, a critical factor in the intrinsic apoptotic pathway, and mice with mutated Fas, a death receptor. Furthermore, levels of p53 and Fas proteins were unchanged in the thymus following 5AzC-treatment in wild-type mice. In the 5AzC-treated thymus, the level of cleaved caspase-8 protein, an initiator of the extrinsic apoptotic pathway, increased with the cleavage of its target protein, Bid. Moreover, the level of TRAIL protein, which induces apoptosis through the cleavage of caspase-8, robustly increased in the thymus treated with 5AzC. In conclusion, the 5AzC-induced apoptosis of thymocytes in vivo is implemented through the extrinsic pathway with the activation of TRAIL.     

Isolated tumor cell in lateral lymph node has no influences on the prognosis of rectal cancer patients

BACKGROUND AND AIMS: The aim of this study was to determine the incidence of isolated tumor cells (ITC) and micrometastasis in lateral lymph nodes of patients with rectal cancer and its possible correlation with prognosis. MATERIALS AND METHODS: One hundred seventy-seven rectal cancer patients who underwent curative resection with lateral lymph node dissection were enrolled. Dissected lymph nodes were examined using hematoxylin-eosin staining (HE) and immunohistochemistry (IHC) with anti-keratin antibody (AE1/AE3). States of lymph node metastasis were divisible into three groups: detectable with HE (HE+), detectable with only IHC (HE-/IHC+), and undetectable even with IHC (IHC-). Almost all the HE-/IHC+ group was classified as ITC consisting of a few tumor cells according to the UICC criteria (ITC+). Survival rates were compared among HE+, ITC+, and IHC-. RESULTS: ITC+ were detected in 24.1% of patients with HE-negative lateral lymph nodes. No significant difference in overall 5-year survival was observed between ITC+ and IHC- patients (76.1 and 82.9%, respectively, p = 0.25). Multivariate analysis showed that perirectal HE+ lymph nodes, but not ITC+ lateral lymph nodes, was an independent prognostic factor. CONCLUSIONS: ITC in lateral lymph nodes does not contribute to the prognosis of rectal cancer in patients who undergo extended lateral lymph node dissection, unlike HE+ lateral lymph node metastasis.     

Involvement of epithelial-mesenchymal transition in adenoid cystic carcinoma metastasis

The high frequencies of recurrence and distant metastasis of adenoid cystic carcinoma (AdCC) are significant obstacles for the long-term cure of patients with AdCC and emphasize the need for better understanding of the biological factors associated with these outcomes. To identify proteins that mediate AdCC metastasis, we established three AdCC cell lines expressing green fluorescent protein (GFP) from the ACCS cell line using orthotopic transplantation and in?vivo selection in nude mice: Parental ACCS-GFP, highly tumorigenic ACCS-T GFP and metastatic ACCS-M GFP. ACCS-GFP and ACCS-M GFP were subjected to DNA microarray analysis and the results were used for data mining studies. DNA microarray analysis revealed significantly altered biological processes in the ACC-M GFP cells, including events related to cell adhesion (three categories) and signaling (three categories). In particular, a significant down-regulation of cell adhesion molecules, such as cadherins and integrin subunits was observed. The loss of E-cadherin and integrins and the gain of vimentin in ACCS-M GFP cells were confirmed by immunoblotting. These results suggest that epithelial-mesenchymal transition (EMT) is a putative event in AdCC metastasis that induces tumor cell dissemination from the primary tumor site. In summary, in this study we established a useful nude mouse metastasis model which will enable further AdCC metastasis research and clinical treatment trials and we also provide evidence that EMT is significantly involved in the AdCC metastatic process.