Protective effect of recombinant neutrophil elastase inhibitor (R-020) on sepsis-induced organ injury in rat

Severe inflammatory responses after major surgeries, trauma, and infection develop multiple organ dysfunction. In the mechanisms of the pathogenesis of these responses, activated neutrophils are thought to be important in terms of their ability to produce various kinds of proteinases, which can degrade various proteins constructing human tissues. Among their proteinases, neutrophil elastase is the strongest serine proteinase secreted from activated neutrophils. Thus, we examined in this study the inhibitory effect and therapeutic efficacy of newly produced recombinant human Kunitz-type proteinase inhibitor (R-020), which coded the second domain of human urinary trypsin inhibitor. R-020 was effective in significantly improving the survival rate after induction of the rat lethal peritonitis model (cecal ligation and punctureinduced septic shock model). We suggest that various serine proteinases are implicated in the pathogenesis of neutrophil-related multiple organ failure and that recombinant human Kunitz-type proteinase inhibitor might be effective in the treatment of these kinds of organ dysfunction.     

Characterization of genomic rearrangements of the alpha1-acid glycoprotein/orosomucoid gene in Ghanaians

In this study, the structure of the α₁-acid glycoprotein (AGP), or orosomucoid (ORM), gene was investigated in a Ghanaian mother and her child, who shared an unusual variant, ORM1 S2(C), found by isoelectric focusing. Three remarkable changes of nucleotide sequence were observed: (1) The two ORM1 alleles, ORM1 ^* S and ORM1 ^* S2(C), had the AGP2 gene-specific sequence at one and three regions, respectively, in exon 5 to intron 5. The variant allele originating from ORM1 ^* S was characterized by a G-to-A transition, resulting in an amino acid change from valine to methionine, which is also detected in ORM1 F2, a form that is common in Europeans. (2) The AGP2 gene of the child, inherited from the father, was duplicated, as revealed by long-range polymerase chain reaction. (3) Three new mutations were observed in two exons of the AGP2 genes of the mother and child. All of these novel genomic rearrangements, which were not observed in Japanese subjects, may have arisen through point mutation, gene conversion, and unequal crossover events. It is likely that the rearrangement of the AGP gene has often occurred in Africans. Received: June 15, 2001 / Accepted: July 10, 2001     

Current source density analysis of ON/OFF channels in the frog optic tectum

In the vertebrate retina, it is well known that an ON/OFF dichotomy is present. In other words, ON-center and OFF-center cells participate in segregated pathways morphologically and physiologically. However, there is no doubt that integration of both channels is necessary to generate the complicated response properties of visual neurons in higher optic centers. So far, functional organization of the ON and OFF channels in the optic centers has not been demonstrated at the level of neuronal populations. In this review article, we summarize our experimental approaches to demonstrate functional organization of the ON and OFF channels using current source density (CSD) analysis in the frog optic tectum. First, we show that one-dimensional CSD analysis, assuming constant conductivity, is applicable in the tectal laminated structure. The CSD depth profile of a response to electrical stimulation of the optic tract is composed of three current sinks (A, B, and D) in the retinorecipient layers and two current sinks (C and E) below those layers. This result is in agreement with previous morphological and physiological findings, and shows that CSD analysis is very useful to demonstrate the flow of visual information processing. Second, CSD analysis of tectal responses evoked by diffuse light ON and OFF stimuli reveals obviously different distributions of synaptic activity in the laminar structure. Two or three current sinks (I, II and III) are generated in response to ON stimulation only in the retinorecipient layers, while up to six current sinks (IV, V, VI, VII, VIII and IX) to OFF stimulation throughout the tectal layers. Based on well known properties of retinal ganglion cells of the frog, possible neuronal mechanisms underlying each current sinks and their functional roles in visually guided behavior are considered.     

Proapoptotic effect of proteolytic activation of matrix metalloproteinases by Streptococcus pyogenes thiol proteinase (Streptococcus pyrogenic exotoxin B)

Streptococcus pyogenes thiol proteinase, also known as streptococcal pyrogenic exotoxin B (SpeB), has been suggested to be a major virulence factor in S. pyogenes infection. SpeB was reported to induce apoptosis of host cells, but its mechanism of action is not yet fully understood. In this study, we examined the involvement of matrix metalloproteinases (MMPs) in SpeB-induced apoptosis. We first developed a large-scale preparation of recombinant SpeB and precursors of human MMP-9 and -2 (proMMPs) by using Escherichia coli Rosetta (DE3)pLysS and baculovirus-insect cell expression systems, respectively. Treatment with SpeB induced effective proteolytic activation of both proMMP-9 and -2. When RAW264 murine macrophages were incubated with SpeB-activated proMMP-9, the level of tumor necrosis factor alpha (TNF-alpha) in conditioned medium (CM), assessed by an enzyme immunoassay, was elevated. This increase was completely inhibited by addition of the MMP inhibitor SI-27 to the cell culture. The CM also produced marked induction of apoptosis of U937 human monocytic cells. Similarly, soluble Fas ligand (sFasL) was detected in CM of cultures of SW480 cells expressing FasL after treatment with SpeB-activated proMMPs; this CM also induced apoptosis in U937 cells. SpeB had a direct effect as well and caused the release of TNF-alpha and sFasL from the cells. SpeB-dependent production of MMP-9 and -2 and proapoptotic molecules (TNF-alpha and sFasL) was evident in a murine model of severe invasive S. pyogenes infection. These results suggest that SpeB or SpeB-activated MMPs contribute to tissue damage and streptococcal invasion in the host via extracellular release of TNF-alpha and sFasL.     

Prediction of sensitivity of advanced non-small cell lung cancers to gefitinib (Iressa, ZD1839)

Gefitinib (Iressa, ZD1839), an inhibitor of epidermal growth factor receptor-tyrosine kinase, has shown potent anti-tumor effects and improved symptoms and quality-of-life of a subset of patients with advanced non-small cell lung cancer (NSCLC). However, a large portion of the patients showed no effect to this agent. To establish a method to predict the response of NSCLC patients to gefitinib, we used a genome-wide cDNA microarray to analyze 33 biopsy samples of advanced NSCLC from patients who had been treated with an identical protocol of second to seventh line gefitinib monotherapy. We identified 51 genes whose expression differed significantly between seven responders and 10 non-responders to the drug. We selected the 12 genes that showed the most significant differences to establish a numerical scoring system (GRS, gefitinib response score), for predicting response to gefitinib treatment. The GRS system clearly separated the two groups without any overlap, and accurately predicted responses to the drug in 16 additional NSCLC cases. The system was further validated by the semi-quantitative RT-PCR, immunohistochemistry and ELISA for serological test. Moreover, we proved that the anti-apoptotic activity of amphiregulin, a protein that was significantly over-expressed in non-responders but undetectable in responders, leads to resistance of NSCLC cells to gefitinib in vitro. Our results suggested that sensitivity of a given NSCLC to gefitinib can be predicted according to expression levels of a defined set of genes that may biologically affect drug sensitivity and survival of lung cancer cells. Our scoring system might eventually lead to achievement of personalized therapy for NSCLC patients.     

Haplotype structure,LD blocks,and uneven recombination within the LRP5 gene

Patterns of linkage disequilibrium (LD) in the human genome are beginning to be characterized, with a paucity of haplotype diversity in "LD blocks," interspersed by apparent "hot spots" of recombination. Previously, we cloned and physically characterized the low-density lipoprotein-receptor-related protein 5 (LRP5) gene. Here, we have extensively analysed both LRP5 and its flanking three genes, spanning 269 kb, for single nucleotide polymorphisms (SNPs), and we present a comprehensive SNP map comprising 95 polymorphisms. Analysis revealed high levels of recombination across LRP5, including a hot-spot region from intron 1 to intron 7 of LRP5, where there are 109 recombinants/Mb (4882 meioses), in contrast to flanking regions of 14.6 recombinants/Mb. This region of high recombination could be delineated into three to four hot spots, one within a 601-bp interval. For LRP5, three haplotype blocks were identified, flanked by the hot spots. Each LD block comprised over 80% common haplotypes, concurring with a previous study of 14 genes that showed that common haplotypes account for at least 80% of all haplotypes. The identification of hot spots in between these LD blocks provides additional evidence that LD blocks are separated by areas of higher recombination.     

Endotoxin contamination in wound dressings made of natural biomaterials

Contamination by endotoxin of nine kinds of wound dressings made of natural biomaterials (calcium alginate, collagen, chitin, and poly-L-leucine) was examined with the use of water extracts. By applying the Limulus amoebocyte lysate (LAL) test, high concentrations of endotoxin were detected in extracts from three kinds of products made of calcium alginate. These extracts evoked fever in rabbits and induced the release of a proinflammatory (pyrogenic) cytokine, interleukin-6 (IL-6), from human monocytic cells (MM6-CA8). The effects disappeared when the extracts were treated with endotoxin-removing gel column chromatography or with an endotoxin antagonist, B464, confirming that the contaminating pyrogen was endotoxin. A noteworthy finding was that one of the endotoxin-containing extracts showed very weak IL-6-inducibility in human monocytic cells in contrast to its high pyrogenicity to rabbits. The discrepancy could be explained based on differences between humans and rabbits in sensitivity to the endotoxin, because the extract showed higher proinflammatory-cytokine (TNF-alpha)-inducibility in rabbit whole-blood cells (WBCs) than human WBCs. The results suggest that the LAL test is a useful method of detecting endotoxin contamination in wound dressings and the MM6-CA8 assay is a good supplement to the LAL test for evaluating pyrogenicity in humans accurately.     

Neutrophil and endothelial cell activation in the vasa vasorum in vasculo-Behçet disease

AIM: The aim of this study was to analyse the immunopathological mechanisms of vasculo-Beh?et disease, which were also compared to cases of Takayasu's arteritis and inflammatory aneurysm to evaluate differences in inflammatory mechanisms. METHOD AND RESULTS: We reviewed six cases of vasculo-Beh?et disease, four of Takayasu's arteritis and seven inflammatory aneurysms which underwent surgical repair. Immunohistochemical studies were performed on paraffin-embedded tissue using a labelled streptavidin-biotin method, as was in-situ hybridization for Epstein-Barr virus. Microscopically, neutrophils and lymphocytes accumulated around the vasa vasorum. Neutrophils were prominent as compared to Takayasu's arteritis and inflammatory aneurysm. Elastic fibres were not severely destroyed. Endothelial cells (ECs) of most vasa vasorum expressed HLA-DR. The number of vasa vasorum around which inflammatory infiltrating cells were observed in vasculo-Beh?et disease was significantly greater than in inflammatory aneurysms and Takayasu's arteritis (P < 0.001). The cytokines IL-1alpha, TNF-beta and IFN-gamma were expressed in neutrophils and lymphocytes which were distributed around vasa vasorum, as well as neutrophils adherent to HLA-DR positive ECs. CONCLUSION: Our results suggest that vasculo-Beh?et disease should be classified as a neutrophilic vasculitis targeting the vasa vasorum. Aneurysm formation may be related to degeneration of arterial wall caused by inflammation of the vasa vasorum.     

Validation of ECRHS Questionnaire in Japanese to use for nation-wide prevalence study of adult asthma]

PURPOSE: To enable international comparison of prevalence in asthma, we translated and evaluated ECRHS Questionnaire, which is introduced in GINA. Considering COPD prevalence in elder people, we added two questions to the ECRHS Questionnaire. METHOD: The Japanese edition of ECRHS Questionnaire was responded by 366 patients who were diagnosed asthma without COPD, 61 patients who were diagnosed COPD without asthma, and 137 healthy persons who were not diagnosed asthma or COPD. We analyzed the answers of the each group and evaluated the validity of the questionnaire to use for the nation-wide prevalence study of adult asthma in future. RESULTS: The question of 'Wheezing at any time in the last 12 months' had the highest Youden's index and validity to pick up asthma patients. The questions of 'Waking up with a feeling of tightness in chest at any time in the last 12 months' and 'Waking up by an attack of shortness of breath at any time in the last 12 month' had the highest specificity to pick up asthma patients. Most of the questions which were related asthma were able to be answered by asthma patients properly, but some questions were improperly answered by patients and healthy persons in elderly. The results in this study showed the less recognition of their diseases in elderly patients than younger patients and the limitation of the study with written questionnaire for elderly people. Not a few COPD patients complained wheezing or whistling in the chests as same as asthma patients in this study. CONCLUSION: We concluded that we had almost enough reliability in the Japanese edition of the ECRHS questionnaire for screening survey of asthma prevalence in Japan.     

Auto-MHC class II-reactive T cell line obtained from MRL/+ mice suffering from lpr-GVHD. II. Analyses of functional characteristics of T cell line by in vivo administration.

Functional characteristics of an autoreactive (I-Ek-restricted) T cell line (l/+ T1), previously established from MRL/M(p-)+/+(MRL/+) mice with lpr-GVHD, were analyzed in vivo. Intravenous injection of l/+ T1 cells to non-irradiated H-2k (MRL/+ or AKR) mice (but not H-2d mice) induced enhanced spontaneous proliferation of recipient spleen cells; this was also I-Ek self-restricted. This augmented self-reactivity seemed to be mediated by recipient L3T4+ T cells, since few l/+ T1 cells were detected in the spleen cells of l/+ T1-injected AKR mice by cell surface marker analyses, and the treatment of the spleen cells with anti-Thy-1.1 antibody (Ab) or anti-L3T4 Ab plus complement abolished this enhanced spontaneous proliferation. The production of IgM rheumatoid factor (RF) in AKR mice and IgG RF in MRL/+ mice increased, although no enhancement of anti-ssDNA Ab production was observed. Judging from both spleen B cell proportion and serum Ig levels, autoantibody induction by the injection of l/+ T1 cells was not associated with polyclonal B cell activation. When lethally irradiated B10 congenic mice were used as recipients, B10. BR mice showed elevated levels of IgM anti-ssDNA and IgM RF 1 wk after l/+ T1 cell injection; it is likely that lethal irradiation causes autoantigens, particularly DNA, to be exposed. These findings suggest that the autoreactivity of l/+ T1 cells can be transferred to recipient L3T4+ T cells via T-T interaction or the immunological network, and that increased autoreactivity induces autoantibody production in the presence of autoantigen stimulation. In contrast to the stimulatory effects observed in AKR and MRL/+ mice, MRL/Mp-lpr/lpr(MRL/lpr) mice showed a different response to the injection of l/+ T1 cells; spontaneous proliferation of spleen cells and autoantibody production were not enhanced, and suppression of the mitogen responses was observed. It is discussed that lpr-GVHD may be due to these unusual features of MRL/lpr mice.