Chemiluminescence of whole blood. II. Application to clinical examination of phagocytic functions of whole blood from various types of disease

Measurements of whole blood chemiluminescence (CL) were carried out by using 0.1 ml of blood to evaluate phagocytic functions from various kinds of pediatric patients. Patients with definite bacterial infections showed an elevated background CL and high peak CL per 1000 granulocytes after stimulation with zymosan. These results suggest that granulocytes of these patients had been preactivated in vivo, their phagocytic and/or metabolic activity being enhanced. Contrary to these results, blood from the patients with viral infections showed almost no abnormalities. Patients with chronic granulomatous disease showed no significant CL by this method similar to conventional ones. This method was also shown to be a useful technique for monitoring the phagocytic functions of blood after granulocyte transfusion.     

Effect of interferon treatment on serum 2',5'-oligoadenylate synthetase levels in hepatitis C-infected patients

Interferon (IFN) is widely used for patients with hepatitis C. Less than half of treated patients respond to IFN therapy, however, and increased resistance to IFN is particularly observed in genotype 1b patients. Recently, genotype 1b patients with the wild type sequence in the NS5A gene were shown to be resistant to therapy, suggesting that the NS5A protein may be involved to IFN resistance. Thus, we investigated the serum 2',5'-oligoadenylate synthetase (2',5'-OAS) levels before and during IFN treatment. In addition, other biochemical markers and NS5A mutations were also examined in 30 HCV genotype 1b-positive patients. Before IFN treatment, 2',5'-OAS activity in sera was significantly lower in wild type patients than in mutant type patients. All patients were subsequently enrolled in IFN therapy, and 2',5'-OAS activity was elevated both in wild and mutant type patients, irrespective of the number of mutations in NS5A. Logistic regression analysis revealed that clearance of serum HCV RNA was independently related to the pretreatment viral load and NS5A mutations, but not to serum 2',5'-OAS activity. We concluded that the NS5A protein, that is associated with the outcome of IFN therapy, affects the kinetics of IFN-induced molecules, such as 2', 5'-OAS. 2',5'-OAS activity does not, however, seem to be related to long-term virological response to IFN therapy.     

The Serum Factor from Patients with Ulcerative Colitis that Induces T Cell Proliferation in the Mouse Thymus Is Interleukin-7

The disturbance of immune regulatory T cells is related to the pathogenesis of ulcerative colitis. Here we demonstrated and characterized the serum factor from ulcerative colitis patients that induced proliferation of intrathymic T cells. The factor isolated from the patient sera by a combination of gel filtration and anion-exchange chromatography induced proliferation of CD4⁺CD8^– intrathymic T cells in the organ-cultured embryonic mouse thymus. Purification and amino acid sequence analysis of the serum factor demonstrated that the N-terminal 12 sequence was homologous to that of interleukin-7. SDS-PAGE and Western blot confirmed that purified serum factor was interleukin-7. Enzyme immunoassay demonstrated that the serum interleukin-7 concentration was significantly increased in the patients. PCR and Southern blot hybridization demonstrated that interleukin-7 mRNA expression was increased in the thymus tissues from patients but decreased in the colonic mucosa. Since interleukin-7 is a crucial cytokine for proliferation and differentiation of T cells in the thymus, the present study indicates that interleukin-7 may contribute to the disturbance of immune regulatory T cells in ulcerative colitis.     

Concentration quenching of photoluminescent Ru(bpy) dispersed in a polysiloxane film containing 2,2′-bipyridine pendant groups in dependence of molecular distribution

The photoluminescent Ru(bpy) complex was dispersed in a polysiloxane film containing 2,2′-bipyridine (bpy) pendant groups. The unusually long photoluminescence lifetime of the Ru(bpy) (1,94 μs at 25°C) and the blue-shifted photoluminescent wavelength suggest a rigid polymer matrix. The fluorescence yield becomes lower with higher probe concentration, indicating concentration quenching. According to the analysis based on Stern-Volmer plots, the quenching obeys a mechanism composed of both static and dynamic processes. A statistical intermolecular distance distribution between the probes was used to interpret the results in terms of static and dynamic quenching. It is shown that in the present system the dispersed complexes diffuse slightly during the excited state.     

Protection of hepatocytes from apoptosis by a novel substance from actinomycetes culture medium

A novel substance, #675, found from an Streptomyces sp. SM675 culture medium, dose-dependently stimulates the proliferation of human functional liver cell 4 (FLC4). When FLC4 cells were incubated under conditions without fetal bovine serum (FBS), typical features of apoptotic cell death such as shrinkage and nuclear condensation appeared; high molecular weight (HMW) DNA fragments were found; and caspase-3 and poly (ADP-ribose) polymerase (PARP) proteins were cleaved. When FLC4 cells were incubated with #675 and without FBS, the cells grew healthy, no HMW DNA fragments were found, and caspase-3 and PARP cleavage weakened, suggesting that #675 protects FLC4 cells from apoptosis induced by FBS-deprivation. The quantitative reverse-transcribed polymerase chain reaction did not show differences in PARP or Bcl-2 mRNA expression in FLC4 cells incubated with or without #675, indicating other genes may be involved in this anti-apoptosis effect. These results show that #675 enhances FLC4 proliferation via an apoptosis-inhibition pathway, implying potential pharmacological and clinical applications.     

An ultrastructural and immunohistochemical study of microcystic meningioma with emphasis on matrix proteins and connexin 26 type gap junctions

Although the histopathological subtypes of meningioma do not themselves appear to have prognostic significance, they are collectively important for defining the overall histopathological entity of microcystic meningioma (MCM) and allowing a distinction from other intracranial tumors, such as capillary hemangioblastoma, glioma, and metastatic renal cell carcinoma showing similar histology. Four cases of MCM were analyzed by conventional histology, immunohistochemistry, and electron microscopy. The present series of MCM was characterized by spindle- or cobweb-shaped tumor cells, characteristically associated small blood vessels, and a peculiar microcystic pattern. Among the microcystic meningeal tumor tissue, small areas of conventional subtypes were identified. Immunohistochemically, tumor cells showed the mesenchymal features of vimentin positivity and a rich distribution of matrix proteins around tumor cells. They lacked epithelial marker positivity but were faintly EMA positive. Ultrastructurally, primitive cellular junctions, desmosomes, and gap junctions were frequently seen between tumor cells. The gap junctions correlated with connexin 26 immunoreactivity. Although lacking an obvious epithelial nature, these features could be interpreted as showing an abortive differentiation mimicking meningothelial (arachnoidal) cells, which, physiologically, regulate cerebrospinal fluid between blood vessels and brain parenchyma.     

A possible role of two hydrophobic amino acids in antigen recognition by synovial T cells in rheumatoid arthritis

Synovial T cells play a crucial role in the pathogenesis of rheumatoid arthritis (RA) synovitis. We have quantitatively analyzed the T cell receptor (TcR) variable (V) region gene repertoire of freshly isolated synovial fluid (SF) T cells, comparing it with that of peripheral blood (PB) T cells in RA. The TcR V gene repertoire of PB and SF T cells in RA and osteoarthritis was heterogeneous. In contrast, Vail in SF was expressed to a greater degree in three of five RA patients, and increased levels of Vp6, 1-3 were found in the SF of four of six RA, compared with paired PB. Of note, Vβ6, 1–3 was universally used in four RA patients with a disease duration of less than 10 years, irrespective of their HLA-DR types. This was in contrast to two other RA patients, suffering for more than 20 years, who showed different Vα and Vβ usages. β-chain sequence analysis in RA patients with a preference for Vβ6, 1–3 has shown that a few clones dominated in SF, whereas polyclonality was observed in PB. These findings suggest oligoclonal expansion of T cells in response to specific antigen(s) in the SF of these patients with RA of relatively short duration. Concomitant use of two hydrophobic amino acids, leucine and valine, in the Dβ region was noticeable among the predominant SF clones. These two amino acids might directly contact a peptide specific for the induction of synovitis in RA patients. TcR-directed therapy may, therefore, be useful for the treatment of early RA synovitis.     

Reduced NR2A expression and prolonged decay of NMDA receptor-mediated synaptic current in rat vagal motoneurons following axotomy

To elucidate characteristic changes in the N -methyl- d -aspartate (NMDA) receptor on neurons following axotomy, subunit expressions and functional features of the NMDA receptor were examined in the dorsal motor nucleus of vagus (DMV) of rats receiving vagal axotomy at the neck. Western blotting analysis demonstrated that the expression of NR2A decreased 2–3 days after in vivo axotomy, while expression of NR1 and NR2B, NR2C and NR2D subunits did not change significantly. To examine the functional changes, patch clamp recordings in whole-cell mode were employed on the axotomized DMV neurons identified by retrograde labelling with fluorescent dye. The amplitude ratios of ifenprodil-sensitive components of NMDA response and d , l -2-amino-5-phosphovaleric acid (APV)-sensitive evoked postsynaptic current increased after axotomy. In addition, APV-sensitive postsynaptic currents exhibited a longer decay time in identified axotomized vagal motoneurons than in control neurons. No significant differences in the current density of the NMDA response and the peak amplitude of APV-sensitive synaptic currents were observed between axotomized and intact DMV neurons. In conclusion, a decrease in NR2A expression results in the appearance of functional characteristics of the NMDA receptor predominantly containing the NR2B subunit. This might lead to a long-term increase of the susceptibility of neurons to excitotoxicity.