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.     

Role of macrophage migration inhibitory factor in otitis media with effusion in adults

Otitis media with effusion (OME) is one of the most common ear diseases. Bacterial endotoxins and several inflammatory cytokines appear to be involved in the pathogenesis of OME in children; however, little is known of the immunological aspects of the onset of OME in adults. We sought to determine the presence of macrophage migration inhibitory factor (MIF) as well as interleukin 1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), RANTES (regulated upon activation, normal T-cell expressed and presumably secreted), and endotoxin in middle ear effusions (MEEs) from adult patients with OME. In addition, the levels of MIF in MEEs from adults and children were compared. MEE was obtained from 95 adults and 11 children. The levels of MIF, IL-1beta, TNF-alpha, and RANTES were determined by enzyme-linked immunosorbent assay, and the concentrations of endotoxin and total protein were determined by the Endospec assay and bicinchoninic acid assay, respectively. MIF was detected in 97.9% of the MEEs from adults, while endotoxin, IL-1beta, TNF-alpha, and RANTES were detected in 96.8, 12.6, 5.3, and 43.9%, respectively. In addition, the level of MIF was significantly higher than those of endotoxin, IL-1beta, and TNF-alpha. A positive correlation between the levels of MIF and endotoxin was observed. MIF and endotoxin were detected in 81.8 and 72.7%, respectively, of the MEEs from the children. The level of MIF was significantly higher in the children, and conversely that of endotoxin was significantly higher in the adults. These results suggest that the interaction between MIF and endotoxin may promote fluid collection in the middle ear, particularly in adults.     

Increased bcl-2 expression in lymphocytes and its association with hepatocellular damage in patients with autoimmune hepatitis

The proto-oncogene product bcl-2 is known to inhibit apoptotic cell death, and its dysregulation might play a critical role in the development of autoimmune disease. To elucidate the role of bcl-2 in autoimmune hepatitis (AIH), its expression in peripheral blood mononuclear cells (PBMC) and in liver-infiltrating lymphocytes (LIL) was investigated. Increased bcl-2 expression in PBMC was found in AIH patients compared with that in chronic hepatitis C (CHC) patients and in healthy controls. The level of bcl-2 expression significantly correlated with serum ALT level. Further analysis showed that CD4+ T cells are enriched in bcl-2-expressing PBMC. To characterize the Th1/Th2 profile of bcl-2-expressing CD4+ T cells, intracellular interferon-gamma (IFN-gamma) and IL-4 were analysed. The results revealed that most of the bcl-2-expressing cells were found to be IFN-gamma-secreting Th1 cells. In three patients for whom their clinical courses could be followed, bcl-2 expression was decreased after the initiation of immunosuppressive therapy with corticosteroids. However, the level of IFN-gamma + cells was not altered. Immunohistochemical analysis also showed that large amounts of bcl-2+ cells were observed in periportal area in the liver. In conclusion, bcl-2-expressing cells were shown to be increased in peripheral blood and liver in AIH and the bcl-2 product was expressed mainly in CD4+ Th1-type cells, suggesting that these cells might promote the cellular immune response and contribute to the development of hepatitis and hepatocellular damage in AIH.     

Rabbit autoantibodies to actin induced by immunization with heterologous actins; a possible mechanism of smooth muscle antibody production

Antisera against actins from chicken gizzard smooth muscle and ascaris body wall were prepared in rabbits. Immunological cross-reactivity of the antisera with actins of several different species was demonstrated by precipitation reactions in agarose gels and by immunofluorescence studies. The antisera were also reactive with actin of rabbits, the homologous animal used for immunization. The latter finding indicated termination of natural tolerance to actin by immunization of cross-reactive actins. A possible mechanism operating in the induction of smooth muscle antibodies, autoantibodies which have a similar nature to the anti-actin antibody is discussed in relation to the termination of natural tolerance to actin.     

Mechanoenergetics characterizing oxygen wasting effect of caffeine in canine left ventricle

Caffeine causes a considerable O(2) waste for positive inotropism in myocardium by complex pharmacological mechanisms. However, no quantitative study has yet characterized the mechanoenergetics of caffeine, particularly its O(2) cost of contractility in the E(max)-PVA-VO(2) framework. Here, E(max) is an index of ventricular contractility, PVA is a measure of total mechanical energy generated by ventricular contraction, and VO(2) is O(2) consumption of ventricular contraction. The E(max)-PVA-VO(2) framework proved to be powerful in cardiac mechanoenergetics. We therefore studied the effects of intracoronary caffeine at concentrations lower than 1 mmol/l on left ventricular (LV) E(max) and VO(2) for excitation-contraction (E-C) coupling in the excised cross-circulated canine heart. We enhanced LV E(max) by intracoronary infusion of caffeine after beta-blockade with propranolol and compared this effect with that of calcium. We obtained the relation between LV VO(2) and PVA with E(max) as a parameter. We then calculated the VO(2) for the E-C coupling by subtracting VO(2) under KCl arrest from the PVA-independent (or zero-PVA) VO(2) and the O(2) cost of E(max) as the slope of the E-C coupling VO(2)-E(max) relation. We found that this cost was 40% greater on average for caffeine than for calcium. This result, for the first time, characterized integratively cardiac mechanoenergetics of the O(2) wasting effect of the complex inotropic mechanisms of intracoronary caffeine at concentrations lower than 1 mmol/l in a beating whole heart.     

Non-IgG1 nature of cutaneous basophil hypersensitivity factor in contact sensitivity. II. Demonstration of antigen-dependent basophil chemotactic activity of cutaneous basophil hypersensitivity factor

It was recently demonstrated that there was a specific activity to induce basophil-rich skin reaction in the sera of contact-sensitized guinea pigs (CBH factor, CBH-F). In the in vitro chemotactic assay system, CBH-F was shown to have weak basophil chemotactic activity but enhanced its activity in the presence of corresponding antigen(s). Furthermore, basophil chemotaxis in reaction to the antigen(s) was observed when the cells were preincubated with CBH-F. It worked mainly for Percoll-separated bone marrow basophils but not for oil-induced peritoneal macrophages. Immunological analysis revealed that CBH-F was a protein with a small molecular weight (MW 4-6 X 10(4] with an antigen binding site and isoelectric point of between 4.5 and 5.0. It did not show any characteristics of IgG1 or IgG2 on immunoadsorbent column and immunoelectrophoresis. Enzyme treatment with insoluble trypsin eliminated the chemotactic activity but this was not the case with neuraminidase treatment.     

Contact sensitivity in vitro: The production of macrophage inhibition factors from DNCB sensitized lymphocytes by subcellular organelles obtained from DNCB epidermal tissue

Actively allergized cells from guinea-pigs with contact sensitivity to DNCB were specifically triggered to produce the macrophage inhibition factors—antigen-dependent and non-antigen-dependent MIF by microsomal—DNCB complexes. The implication of this observation is that epidermal microsomal—DNCB complexes behave as complete antigens in a MIF assay system. It is therefore possible that such a combination acts as an immunogen in the induction of contact sensitivity to DNCB.     

Expression of Functional Molecules in Non-Hodgkin's Lymphoma

It is well known that non Hodgkin's lymphoma (NHL) cells express various antigens which are normally involved in a variety of functions. In addition, NHL is diverse in its proliferative capacity. To investigate the relation between these factors and the clinical picture, 45 cases of NHL were studied by immunohistochemistry using snap-frozen materials obtained before therapy. Reactivities with 27 monoclonal antibodies were examined and the results were correlated with clinical findings. The expression of surface μ and CAM-1 in B-NHLs and CD25 in T-NHLs were significantly associated with bone marrow involvement. B-NHLs without expression of CD21(B2) and T-NHLs with CD25 were seen more frequently in cases with a LDH value of over 500 units/ml. The positivity rate of Ki-67 on B-NHLs was correlated with serum LDH value, NHL histologic classification, and overall survival. These data indicate that immunophenotyping and determination of the proliferative capacity of NHL are of value not only for confirmation of the histopathologic classification of the tumor but also for assessment of clinical behavior.     

Effects of non-MHC background genes on the induction of CD4+ T cells that prevent rejection of a highly immunogenic tumor, FBL-3

This study showed that non-MHC genes common to (DBA/2 H-2^d)and (DBA/1 H-2^q) gave rise to suppressor T (T_a) cells in thehybrid F₁ mice between C57BL/6 (B6) strain in the antl-FBL-3tumor responses. FBL-3, a Friend virus-induced tumor cell lineof B6 mouse origin, is highly immunogenic as shown by findingsthat syngenelc and hybrid F₁ mice with several other inbredstrains rejected up to 3 x 10⁷ tumor cells inoculated s.c. andgenerated potent CTL responses after mixed lymphocyte tumorcell culture. In contrast to these mice, (B6 x DBA/2) and (B6x DBA/1)F1 mice did not reject the tumor as the tumor dosesincreased. Progressive tumor growth in these F₁ mice was blockedby an I.p. Injection of cyclophosphamlde (250 mg/kg) on day10, but not on day 5, after tumor cell inoculation. Antl-CD4(GK1.5) mAb exerted similar therapeutic effects against tumorwhen given twice, between day 0 and 10, whereas the additionalinjection of antl-CD8 mAb enhanced the tumor growth in micethat otherwise rejected the tumor. Thus, In the response of(B6 x DBA/2)F, mice to FBL-3 tumor cells, CD4⁺ T₈ seemed todown-regulate the immunologically mediated regression of thetumor produced by CD8⁺ CTL. This was evidenced by limiting dilutionculture analyses, which showed that the frequency of an FBL-3-speclflcCTL precursor in the (B6 x DBA/2)F1 mice that rejected the tumorwith antl-CD4 mAb was 7- to 9-fold higher than that in micein which the tumor regressed spontaneously. That more than onegene was involved in suppressor T cell induction was shown bythe tumor growth pattern in (B6 x DBA/2)F₁ x B6 backcross andB6D2F2 mice.