Clinical application of rapid assay of interleukin-6 in influenza-associated encephalopathy

The characteristics of influenza-associated encephalopathy is the high mortality and nimble progress with coma which appears in general cases within 48 hours. Most of patients show no abnormalities in the standard blood checks on admission or in early stage. In this study we investigated if a rapid assay of interleukin (IL)-6 is useful in influenza-associated encephalopathy in early stages. The levels of IL-6 in patients with influenza-associated encephalopathy did not show any significant difference compared with those in patients with febrile convulsion and rotavirus-associated convulsion. However the levels of IL-6 in severe cases were significantly higher than those of mild cases with influenza-associated encephalopathy. Consequently the rapid assay of serum IL-6 is useful to evaluate and decide the therapies.     

Establishment of an HIV cell-cell fusion assay by using two genetically modified HeLa cell lines and reporter gene

Infection of human cells with the human immunodeficiency virus type I (HIV-1) can be mimicked by a fusion process between cells expressing the HIV envelope protein (Env) and cells expressing both human CD4 together with the appropriate human chemokine receptors. In this study, a T-tropic HIV cell-cell fusion assay was established that utilized CD4, human CXCR4 and HIV NL4-3 gp160 as fusion components and a T7 polymerase-activated luciferase as a reporter system. The HeLa T4 cells used, expressed CD4 and CXCR4, and the applied HeLa KS386 cells expressed HIV NL4-3 gp160. By combining HeLa T4 cells with HeLa KS386 cells, an approximately about 100- to 300-fold increase in luciferase activity could be elicited relative to the control. The addition of anti-CD4 monoclonal antibody (Mab) (RPA-T4) or anti-CXCR4 Mab (12G5) in the assay significantly inhibited the fusion event; in contrast, an anti-CCR5 Mab (2D7) had no effect, indicating that the fusion assay was CD4 and CXCR4 dependent. In this report, fusion events could be monitored by both the luciferase reporter system and syncytia formation. Fusion events were monitored and compared using these two approaches. The luciferase reporter system was found to be more sensitive than syncytia formation. Moreover, compared with previous HIV fusion models, such as using recombinant vaccinia viruses, this system has several advantages, including simplicity and sensitivity. Finally, the system provides a powerful tool to study fusion mechanisms mediated by T-tropic HIV gp160, as well as to screen for fusion-blocking antibodies and antiviral agents.     

Immunofluorescence technique using HeLa cells expressing recombinant nucleoprotein for detection of immunoglobulin G antibodies to Crimean-Congo hemorrhagic fever virus

A HeLa cell line continuously expressing recombinant nucleoprotein (rNP) of the Crimean-Congo hemorrhagic fever virus (CCHFV) was established by transfection with an expression vector containing the cDNA of CCHFV NP (pKS336-CCHFV-NP). These cells were used as antigens for indirect immunofluorescence (IF) to detect immunoglobulin G antibodies to CCHFV. The sensitivity and specificity of this IF technique were examined by using serum samples and were compared to those of the IF technique using CCHFV-infected Vero E6 cells (authentic antigen). Staining of the CCHFV rNP expressed in HeLa cells showed a unique granular pattern similar to that of CCHFV-infected Vero E6 cells. Positive staining could easily be distinguished from a negative result. All 13 serum samples determined to be positive by using the authentic antigen were also determined to be positive by using CCHFV rNP-expressing HeLa cells (recombinant antigen). The 108 serum samples determined to be negative by using the authentic antigen were also determined to be negative by using the recombinant antigen. Thus, both the sensitivity and the specificity of this IF technique were 100% compared to the IF with authentic antigen. The novel IF technique using CCHFV rNP-expressing HeLa cells can be used not only for diagnosis of CCHF but also for epidemiological studies on CCHFV infections.     

Genomic alterations in primary cutaneous melanomas detected by metaphase comparative genomic hybridization with laser capture or manual microdissection: 6p gains may predict poor outcome

To clarify the correlation of genomic alterations with clinical and histological features, we performed metaphase comparative genomic hybridization analysis on 20 primary cutaneous melanomas, which were obtained by laser capture or manual microdissection, and 16 melanoma cell lines. There were no differences in the average number of aberrations between acral melanomas (AM) and non-AM, although gains of 5q and 11q13 were more frequent (P=0.05) and 10q loss was less frequent (P=0.01) in AM than in non-AM. Although tumor thickness is considered a measurable estimate of clinical expression, there were no differences in the average number of aberrations among 4 groups, classified by thickness of the tumor. While the majority of aberrations were equally distributed among the 4 groups, 6p gains were found only in the thickest tumors. Patients with 6p or 1q gains had a lower overall survival rate than those without them (P=0.0002 or P=0.013). While gains of 1q, 2q, 3p, 3q, 7q, 20p, and 20q were more frequent in the cell lines than in the primary tumors (P<0.01), losses of 6q, 9p, 10p, and 10q were equally found in both cell lines and primary tumors. The present study showed that chromosomal aberrations had already occurred in the thinner tumors, and that 6p and 1q gains may be a prognostic factor.     

Genetic susceptibility testing for hereditary breast and ovarian cancer--approach from a commercial clinical laboratory

Genetic testing for hereditary cancers and other common diseases are still considered as the research testing, not for the clinical testing in Japan. One of the major reason of this situation is related to the guidelines regarding the human genetic testing issued successively in the spring of 2001, one by joint work of the eight learned societies and the other by the Japan clinical laboratories association. Both of these guidelines warn the condition of the clinical application of genetic testing after research stage must have the evidence data for clinical utility. We describe the situation of the genetic testing in the U.S. focusing the social background of increasing breast cancer cases and the contribution of Myriad Genetic Laboratories, Inc. for the genetic testing industry. We also describe the Japanese situation of the genetic testing and problems to be solved before spreading widely.     

Expression of tumor necrosis factor ligand superfamily costimulatory molecules CD27L, CD30L, OX40L and 4-1BBL in the heart of patients with acute myocarditis and dilated cardiomyopathy.

BACKGROUND: T-cell-mediated myocardial damage is known to be involved in acute myocarditis and dilated cardiomyopathy. Recently, we found that tumor necrosis factor (TNF) ligand superfamily costimulatory molecules, especially 4-1BBL, played an important role in the myocardial damage of murine acute viral myocarditis. METHODS AND RESULTS: To investigate the roles for CD27L, CD30L, OX40L and 4-1BBL, which belong to TNF ligand superfamily, in the development of acute myocarditis and dilated cardiomyopathy, we analyzed the expression of these antigens in the myocardial tissues of patients with acute myocarditis and dilated cardiomyopathy. We also examined expression of the receptors for these molecules, CD27, CD30, OX40 and 4-1BB, which belong to TNF receptor superfamily, on the infiltrating cells. Strong expression of CD27L, CD30L and 4-1BBL and weak to moderate expression of OX40L was found in the cardiac myocytes of patients with acute myocarditis. Moderate expression of CD27L, CD30L and 4-1BBL and weak expression of OX40L was found on the cardiac myocytes of patients with dilated cardiomyopathy. Most of the infiltrating cells expressed CD27, CD30 and 4-1BB and a part of the infiltrating cells expressed OX40. CONCLUSIONS: Our findings suggest that expression of TNF ligand superfamily costimulatory molecules on cardiac myocytes may play a role in the cell-mediated myocardial damage in patients with acute myocarditis and dilated cardiomyopathy as in murine viral myocarditis.     

Postmortem Diagnosis of Acute Megakaryocytic Leukemia Usefulness of lmmunohistochemistry and Tissue Hemogram

An autopsy case of acute megakaryocytic leukemia (AMKL) is presented. The bone marrow was hypercellular with proliferation of three lineages, especially megakaryocytes. Immunohistochemical examination revealed many platelet glycoprotein IIb/IIIa (GP IIb/IIIa)- positive blast cells in bone marrow. The proportion of the blasts was 26.4% by tissue hemogram. GP IIb/IIIa-positive blasts and megakaryoblasts were deposited massively in lymph nodes. lmmunohistochemistry against GP IIb/IIIa and tissue hemograms by paraffin section are needed to diagnose AMKL by postmortem examination, since the identification of ultra-structural platelet peroxidase in autopsy materials is difficult.     

Glomerular expression of cell-cycle-regulatory proteins in human crescentic glomerulonephritis

To elucidate the mechanism underlying crescentic formation, we assessed the phenotypic characterization and cell-cycle protein expression in human crescentic glomerulonephritis (CRGN). Kidney tissue specimens taken from CRGN patients (10 patients with pauci-immune type rapidly progressive glomerulonephritis (RPGN), 2 patients with Henoch-Schönlein purpura nephritis, and 1 patient with IgA nephropathy) were examined immunohistochemically. Most of the cellular components of the crescents expressed cytokeratin, whereas few cells expressed PHM-5. CD68-positive cells were minor components of cellular crescents, indicating that the major principal cellular component of the crescents is made up of cells with the parietal glomerular epithelial cell (PEC) phenotype. Additionally, serial section analysis revealed that Ki-67-positive cells in the crescents were frequently cyclin-A positive and Bcl-2 positive, but seldom cyclin-B₁ positive. Moreover, the expression of cyclin-dependent kinase inhibitor p27^Kip1 was low in the cellular crescents, despite being exclusively positive in podocytes within the same section. We concluded that the major component of the cellular crescents is made up of PECs and that apparent expression of cyclins and Bcl-2 and restrained expression of p27^Kip1 may be synergistically associated with the development of cellular crescents in human CRGN.     

CUSHING'S SYNDROME WITH BILATERAL MULTINODULAR ADRENAL HYPERPLASIA

An unusual case of Cushing's syndrome of a 59-year-old man with bilateral multinodular adrenal hyperplasia and microadenoma of the pituitary gland is presented. Failure to suppress plasma Cortisol with large doses of dexamethasone may suggest autonomous growth of hyperplastic nodules of the adrenals, which were at first induced by prolonged stimuli of ACTH from the microadenoma of the pituitary gland. ACTH could not be detected in the microadenoma cells on paraffin sections, while Crooke's cells were strongly positive for ACTH. The interrelation between bilateral multinodular adrenal hyperplasia and pituitary microadenoma is discussed.     

Mixed growth hormone cell- prolactin cell pituitary adenoma with acromegaly: α-subunit most growth hormone cells

A 51 -year-old woman with mixed growth hormone (GH) cell-prolactin (PRL) cell pituitary adenoma is presented. She had clinical signs due to hypersecretion of GH and PRL. Resected tissue was studied immunohistochemically and morphologically. The serial sections revealed that GH and α-subunit were co-localized in most cells, while GH and PRL were localized in different cells.