A differential assay of NK-cell-mediated cytotoxicity in K562 cells revealing three sequential membrane impairment steps using three-color flow-cytometry

Annexin V and propidium iodide (PI) staining is a general technique for detecting apoptosis by flow-cytometry (FCM). The release of 2',7'-bis-(2-carboxyethyl)-5- (and-6)-carboxyfluorescein (BCECF), a non-lipophilic membrane-impermeable labeling dye, from the cytoplasm of target cells is an indicator of increased membrane permeability. This study aimed to devise a three-color FCM technique involving the BCECF-release parameter in addition to conventional Annexin V and PI staining for the analysis of target K562 cells undergoing cytotoxic/apoptotic processes mediated by natural killer (NK) cells. The results demonstrated the following step-wise process of membrane impairment: (1) initiation of Annexin V staining accompanied by increasing forward scatter (FSC) before BCECF-release, indicating membrane impairment without permeabilization by necrosis; (2) BCECF-release with decreasing FSC before PI influx; and (3) PI staining with the lowest FSC state. Therefore, the early stage of cytotoxicity/apoptosis conventionally defined by the flow-cytometric criteria of Annexin V staining before PI staining could be sub-divided into two stages before and after BCECF-release. Annexin-V staining in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis was also initiated without BCECF-release. Although the underlying mechanism of the transition process from stage 1 to stage 2 is still unknown, this FCM technique should be a useful tool for differential assays of target cells regarding the sequential processes of NK-induced cytotoxicity.     

Glyceraldehyde 3-phosphate dehydrogenase is a novel autoantigen leading autoimmune responses to proliferating cell nuclear antigen multiprotein complexes in lupus patients

Using 2-dimensional electrophoresis and ion-pair chromatography, we have identified elements of proliferating cell nuclear antigen (PCNA) multiprotein complexes that are reactive to antibodies in sera from patients with systemic lupus erythematosus. Among the various elements of the complexes, a 37 kDa protein (PI 8.5) that specifically reacted with SLE sera, but not with sera from patients with other connective tissue diseases, was identified as glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Immunoblot analysis showed that SLE sera reactive with the 37 kDa protein specifically reacted with GAPDH, as did anti-GAPDH mAbs. The purified autoantibodies to GAPDH from lupus serum showed both nuclear speckled and cytoplasmic staining patterns in immunofluorescence on Hep-2 cells. In addition, enzyme-linked immunosorbent assay (ELISA) revealed the presence of anti-GAPDH autoantibodies in 47% of lupus patients. Longitudinal analysis of the reactivity of lupus sera to PCNA complexes showed the autoimmune response to spread from GAPDH to other elements of PCNA complexes, and the presence of anti-GAPDH antibodies was significantly correlated with increased levels of serum PCNA. Taken together, these findings suggest that GAPDH interacting with PCNA in association with its cellular function is a novel autoantigen recognized by lupus sera, and that GAPDH thus plays an important role in the induction of autoimmune responses against the PCNA complex.     

Reconstitution of a functional interleukin (IL)-7 receptor demonstrates that the IL-2 receptor γ chain is required for IL-7 signal transduction

The interleukin (IL)-2 receptor γ chain has recently been shown to be a component of the IL-7 and IL-4 receptors. Using a transient transfection assay and the trans-activation of reporter gene constructs which are under the control of cytokine-responsive promoter elements, we have studied signal transduction through the IL-7 receptor (IL-7R). The reporter gene expression was not stimulated by receptors that contained the cytoplasmic domain of the IL-7R, either as intact IL-7R or as part of a chimeric receptor. However, co-expression of the IL-7R with the IL-2 receptor γ chain was able to stimulate gene activation. For maximal stimulation the intact cytoplasmic domains of each chain was required.     

A comparison of three bioelectrical impedance analyses for predicting lean body mass in a population with a large difference in muscularity

This study tested the hypothesis that, as compared to whole-body bioelectrical impedance (BI) analysis, segmental BI analysis can estimate lean body mass (LBM) more accurately in a population with a large difference in muscularity. In addition to whole-body BI, which determines impedance (Z) between the wrist and ankle, two segmental BI analyses which determine the Z value of every body segment in each of (1) the arms, legs and trunk (distal BI) and (2) the upper arms, upper legs and trunk (proximal BI) were applied to a group of 125 male athletes and 75 non-athletes. The subjects were divided into validation and cross-validation groups. Simple and multiple regression analyses were applied to (length)²/Z (BI index) values for the whole-body and each body segment, to develop the prediction equations of LBM measured using air-displacement plethysmography. In the validation group, the SE of estimation was similar in the whole-body (3.4 kg, 5.4%), distal (3.4 kg, 5.5%) and proximal BI (3.3 kg, 5.2%) analyses. However, the whole-body and distal BI analyses produced systematical errors in the estimates of LBM. Moreover, the residuals in the two methods significantly (P<0.05) correlated with the ratios of BI indices of the upper arms and upper legs to those of the arms and legs, respectively, calculated as variables approximating the relative development of lean tissues at the proximal area of limbs. On the other hand, the proximal BI analysis was validated and cross-validated. Thus, the accuracy of estimating LBM was similar in the whole-body and the two segmental BI analyses. However, the prediction equations derived from the use of the whole-body BI index and a combination of the arms, legs and trunk BI indices produced a systematical error relating to the difference between the limb segments in lean tissue development.     

Development of a PCR method for rapid identification of new Streptococcus mutans serotype k strains

In a previous study, we isolated and characterized a new serotype k of Streptococcus mutans from human blood and oral cavities. Analysis of the genes involved in biosynthesis of the serotype-specific polysaccharide of serotype k strains revealed that the serotype k-specific nucleotide alignment was commonly present in the 5' region of the rgpF gene (350 bp from the initial sequence) compared to the reference strains, and then a method for rapid identification of serotype k strains was developed by use of PCR with primers designed on the basis of the sequence of the variable region. PCR assays with primers specific for amplification of serotype k strains showed a negative reaction with serotype c, e, and f strains and a positive reaction with serotype k strains, with the sensitivity for identification of the serotype k strains shown to range from 5 to 50 cells. Next, the frequency of positive reactions for serotype k-specific primers was surveyed with DNA taken from saliva samples from 200 subjects (2 to 18 years of age), and 10 of those showed a positive reaction, which was higher than the frequency in our previous survey with a serological method. In addition, all saliva samples from subjects with serotype k strains in our previous study were shown to be positive with the serotype k-specific primers. These results indicate that this new PCR method is effective for identification of subjects with S. mutans serotype k.     

Dna ploidy of pheochromocytoma on cytology specimen by image analysis

Pheochromocytoma usually shows prominent nuclear atypia, but the presence of such atypical cells is known to be an unreliable predictor of malignancy. DNA ploidy of pheochromocytomas has been analyzed by flow cytometry or photospectrometry on paraffinem-bedded tissue, but the results were controversial. We performed DNA analysis on cytology specimens of 11 pheochromocytomas using an image analysis system. All tumors had a mixed pattern of a large population of diploid cells and a small population of polyploid cells. DNA content correlated with nuclear size, and larger cells had more DNA content. Such larger tumor cells had polyploid nuclei, such as 4 C, 8 C, 16 C, and 32 C, in both malignant and benign pheochromocytomas. The larger polyploid nuclei may result from difficulty of duplication at the mitotic phase of the cell cycle.     

Sinonasal teratocarcinosarcoma: ultrastructural and immunohistochemical evidence of neuroectodermal origin

The authors report a case of sinonasal teratocarcinosarcoma in a 37-year-old man, which was located in the anterior skull base and extended to the right nasal cavity and paranasal sinuses. The tumor was surgically resected twice, but it could not be removed completely. Microscopically, it was mainly composed of primitive cell nests within a moderately cellular stroma. The components of squamous cell epithelia with focal teratoid appearance and adenocarcinomatous differentiation were observed. There were many rhabdomyoblasts scattered in the nests and stroma. Ultrastructurally, the primitive cells had many neural processes with parallel microtubules, resembling olfactory neuroblastoma. Rhabdomyoblasts showed various degrees of skeletal muscle differentiation. Some of the stromal spindle cells had actin filaments with dense patches and dense core granules. Immunohistochemically, the primitive cells were positive for epithelial markers, neuron-specific enolase, synaptophysin, and myogenic regulatory proteins. The rhabdomyoblasts showed immunoreactivity for myoid markers, cytokeratin, epithelial membrane antigen, and synaptophysin. Most of the stromal spindle cells were positive for smooth muscle actin, neuron-specific enolase and synaptophysin. The immunohistochemical and ultrastructural findings suggest that primitive cells had the most primitive phenotype of placodes, and support the possibility that sinonasal teratocarcinosarcoma is essentially a neuroectodermal tumor with divergent differentiation.     

Molecular targeted treatment--new treatment strategy for patients with chronic myeloid leukemia

Imatinib mesylate is a new drug that can inhibit the tyrosine kinase activity of Bcr-Abl, the receptors for platelet-derived growth factor receptor(PDGF) and stem cell factor, or c-kit. Chronic myeloid leukemia (CML) is distinguished by the presence of a reciprocal translocation between chromosomes 9 and 22 that results in a shortened chromosome 22, termed the Philadelphia(Ph) chromosome. As a result of the translocation, a fusion gene called the Bcr-Abl gene is created from two normal cellular genes, encoding a chimeric Bcr-Abl protein with a deregulated tyrosine kinase activity. The expression of Bcr-Abl tyrosine kinase has been shown to be necessary and sufficient for the transformed phenotype of CML cells. Imatinib can block the kinase activity of Bcr-Abl, thus inhibiting the proliferation of Ph-positive progenitors, and has shown activity against all phases of CML, though responses are most substantial and durable in patients in the chronic phase. An international phase III study which compared the efficacy of imatinib with that of interferon alpha combined with low-dose cytarabine in newly diagnosed chronic-phase CML showed the rate of major cytogenetic response at 24 months was 90%, including 82% of complete cytogenetic response. These results indicated that imatinib was superior to interferon-containing treatment as a first-line therapy. More than 10,000 patients worldwide, including those in Japan, have been treated with imatinib in clinical trials, and a lot of information has been accumulated on the use of this drug. The aim of this article is to review the use of this drug and the practical management of patients with chronic myeloid leukemia.     

Application of personal computer to an analysis of smallde novo chromosomal insertion: A case ofde novo 3q2 trisomy with ins(8;3)

To identify the origin of a small inserted segment in ade novo 8p+ chromosome, an originally programmed computerized data-base for chromosomal aberration syndromes was utilized. The system selected 3q2 trisomy and 10q2 trisomy as candidates. As a result of a careful comparison of several high-resolution banding patterns among chromosomes 3, 10 and the inserted segment, her karyotype was disignated as: 46,XX,–8,+der(8), inv ins(8;3)(p21.1;q26.32q24)de novo. A small segment from 3q24 to 3q26.32 was trisomic, and invertedly inserted into the short arm of chromosome 8. This computerized database was considered to be useful for analyses of the smallde novo inserted chromosomal segment.