Endotoxin Binding and Elimination by Monocytes: Secretion of Soluble CD14 Represents an Inducible Mechanism Counteracting Reduced Expression of Membrane CD14 in Patients with Sepsis and in a Patient with Paroxysmal Nocturnal Hemoglobinuria

Little is known about the role of peripheral blood mononuclear cells (PBMCs) in lipopolysaccharide (LPS) elimination. We studied the endotoxin elimination capacities (EEC) of PBMCs of 15 healthy volunteers, 13 patients with sepsis, and 1 patient suffering from paroxysmal nocturnal hemoglobinuria (PNH). Although expression of CD14, the best-characterized receptor for LPS to date, was reduced from 93.6% ± 0.8% in healthy subjects to 50.5% ± 6.5% in patients with sepsis and was 0.3% in a patient with septic PNH, EEC were found to be unchanged. There was no difference in the amount of tumor necrosis factor alpha (TNF-α) released by PBMCs of healthy donors and patients with sepsis. Anti-CD14 antibodies (MEM-18) completely suppressed EEC, binding of fluorescein isothiocyanate-labeled LPS to monocytes as determined by FACScan analysis, and TNF-α release in all three groups studied. The concentrations of soluble CD14 (sCD14) secreted by endotoxin-stimulated PBMCs from healthy donors and patients with sepsis amounted to 4.5 ± 0.4 and 20.1 ± 1.8 ng/ml, respectively. Based on our results, we suggest that PBMCs eliminate LPS by at least two different mechanisms; in healthy subjects, the membrane CD14 (mCD14) receptor is the most important factor for LPS elimination, while in patients with sepsis (including the septic state of PNH), increased sCD14 participates in LPS elimination. Secretion of sCD14 is strongly enhanced under conditions of low expression of mCD14 in order to counteract the reduction of mCD14 and maintain the function of monocytes. This sCD14 may substitute the role of mCD14 in LPS elimination during sepsis. The elimination of LPS by PBMCs correlates with the binding reaction and the secretion of TNF-α.     

Confocal observation of senile plaques in Alzheimer's disease: Senile plaque morphology and relationship between senile plaques and astrocytes

Senile plaques In the brains of Alzheimer's disease (AD) were examined by confocal laser scanning microscopy (CLSM) with the following three findings. First, in sections stained with Congo red, the serial CLSM images of optical sections clearly revealed that a classic plaque is composed of a plaque core and a corona. Radially arranged process-like structures, corresponding to bundles of amyloid fibrils, formed amyloid cores and stronger signals were detected in the center of some cores. Second, in sections stained with Congo red and anti-gllal fibrillary acidic protein (GFAP), reactive astrocytes were found around the senile plaques and many astrocytlc processes surrounded the plaque cores and some processes had penetrated into them. Third, three-dimensional reconstruction on classic plaque revealed that the surface of classic plaque showed a 'coral-like' appearance.     

Immunolocalization of extracellular matrix proteins and integrins in sarcoid lymph nodes

 To improve our understanding of the role of extracellular matrix (ECM) proteins and integrins during the processes of granuloma formation in sarcoidosis, we examined the distribution of ECM proteins and the expression of integrins in sarcoid lymph nodes by immunohistochemical methods. We also examined the expression of transforming growth factor-β1 (TGF-β1), which is one of major regulators for synthesis of ECM proteins. Most ECM proteins were detected in the periphery of the granulomas in a concentric pattern, and fibronectin was diffusely detected from an early to a regressive stage. Compared with normal lymph nodes, most β1-integrin subfamilies (α1, α4, α5 and α6) were more strongly expressed on lymphocytes around the granulomas. Epithelioid cells exhibited strong expression of the α5 molecule. Fibroblasts exhibited the expression of the α2 and α5 molecules surrounding ECM proteins. The α5β1 molecule had a distribution similar to that of fibronectin. TGF-β1 was detected in epithelioid cells throughout the various evolutional stages and its expression was especially marked in mature granulomas. Interaction of fibronectin and the α5β1 molecule may have an important role in the process of formation of sarcoid granuloma. The expression of TGF-β1 may be involved in the regression of sarcoid granuloma by initiating fibrosis and atrophy of epithelioid cells. Received: 2 December 1997 / Accepted: 19 February 1998     

Inactivation of chemotactic activity of C5a by the serratial 56-kilodalton protease.

The effects of the 56-kilodalton protease (56K protease) from Serratia marcescens on complement-derived chemotactic activity were examined. Fresh human serum was incubated with zymosan to produce C5a. This activated serum was then incubated with various concentrations of 56K protease, and the chemotactic activity of mouse peritoneal exudate polymorphonuclear leukocytes (PMN) and macrophages was evaluated. A significant dose-dependent decrease of chemotactic activity was observed after protease treatment. Furthermore, treatment of human recombinant C5a with 56K protease at a dose of 1.0 microgram/ml resulted in a complete loss of chemotactic activity. When the living bacteria of the virulent strain, which produced about 10 times more protease than did the less virulent strain, were injected intraperitoneally into mice, the magnitude of infiltration of polymorphonuclear leukocytes into the peritoneal cavity was much lower than that caused by the less virulent strain. Because complement-dependent chemotactic activity is an initial response to bacterial infection, these results suggest indirect pathogenic functions of serratial proteases that suppress chemotactic activity.     

Inhibition of tumor invasion and extracellular matrix degradation by ubenimex (bestatin)

We have investigated the effect of the immunomodulator ubenimex (hereafter referred to as bestatin) on the enzymatic degradation of the extracellular matrix by human renal cell carcinoma SN12M cells during the invasive process. The invasion of SN12M cells into reconstituted basement membrane (Matrigel) was inhibited by the presence of bestatin in a concentration-dependent manner. However, bestatin did not have any effect on tumor cell adhesion and migration to the extracellular matrices which may be involved in tumor cell invasion. Bestatin inhibited the degradation of type IV collagen by tumor cells, but not by tumor-conditioned medium (TCM), in a concentration-dependent manner. We also found that bestatin inhibited hydrolysing activities towards substrates of aminopeptidases in SN12M cells. Since bestatin was found to inhibit aminopeptidase activity, the inhibition of tumor invasion by bestatin is likely to be associated with its action as an enzyme inhibitor. Bestatin only slightly inhibited tumor cell plasmin activity, which can lead to the conversion of the latent collagenase to the active form, but this slight effect was not significant. The zymography of TCM from SN12M cells showed that the treatment of tumor cells with bestatin resulted in the disappearance of the 68 kDa type IV collagenase-enzyme level (active form) and slight reduction of the 72 kDa type IV collagenase-enzyme level (latent form). These results indicated that bestatin may inhibit tumor cell invasion through a mechanism involving its inhibitory action on aminopeptidases in tumor cells, suggesting that the aminopeptidase may partly be associated with the conversion of a latent form of type IV procollagenase to an active form or the secretion of the collagenases from tumor cells.     

Hepatitis B surface antigen particles of subtypes adw and adr, and compound subtype (adwr) in symptom-free carriers in Japan.

Of sera from 1,878 Japanese blood donors who carried hepatitis B surface antigen (HBsAg), 420 were subtyped as adw (22.4%) and 1,443 as adr (76.8%); only 15 (0.8%) contained HBsAg of subtype ayw or ayr. Sera with HBsAg/adr had higher HBsAg titres than those with HBsAg/adw (geometric mean of haemagglutination titre: 10.1 +/- 2.4 vs. 9.7 +/- 2.4, p less than 0.01), and a higher prevalence of hepatitis B e antigen (24% vs. 13%, p less than 0.001). Carriers of HBsAg/adr progressively predominated over those of HBsAg/adw with increasing age. Of sera from 1,863 carriers of HBsAg/adw or HBsAg/adr, 182 (9.8%) contained HBsAg particles with both subtypic determinants in the w/r allele. The presence of w and r determinants on the same particles was ascertained by sandwiching them between monoclonal antibody with the specificity for w and that with the specificity for r. HBsAg particles of compound subtype (adwr) were found more often in sera with hepatitis B e antigen than those without it (145/403 [36.0%] vs. 37/1,460 [2.5%], p less than 0.001). Sera with HBsAg/adwr particles had HBsAg titres higher than those without them (12.4 +/- 1.9 vs. 9.7 +/- 2.3, p less than 0.001). HBsAg/adwr particles arise from phenotypic mixing of the S-gene product of wild-type virus and that of mutants with point mutations for subtypic changes. The results obtained indicated that HBV strains of subtype adr have a higher replicative activity than those of adw, and suggested that mutations in the S gene for subtypic changes would be associated with an active replication of hepatitis B virus.     

Adhesion polypeptides are useful for the prevention of peritoneal dissemination of gastric cancer

We examined the effect of adhesion polypeptides on the adhesion and invasiveness of gastric cancer cell lines. We previously reported the establishment of an extensively peritoneal-seeding cell line, OCUM-2MD3, from a poorly seeding human scirrhous gastric carcinoma cell line, OCUM-2M. Both 21 and 31 integrin expression was markedly increased on OCUM-2MD3 cells compared with OCUM-2M cells, and the ability of OCUM-2MD3 cells to bind to the extracellular matrix (ECM) was also significantly higher than that of OCUM-2M cells. The adhesion polypeptides, YIGSR and RGD, and two RGD derivatives significantly inhibited the adhesion of OCUM-2MD3 cells to the submesothelial ECM, while not inhibiting the adhesiveness of OCUM-2M cells and two well differentiated human gastric cell lines, MKN-28 and MKN-74. The YIGSR and RGD peptides also significantly inhibited the invasiveness of OCUM-2MD3 cells. The survival of nude mice with peritoneal dissemination given YIGSR sequenc e intraperitoneally was obviously longer than that of untreated mice. The survival of mice treated with RGD was also improved, and this effect was increased using the RGD derivatives, poly(CEMA-RGDS) and CM-chitin RGDS. These polypeptides appear to block the binding of integrins, which are expressed on OCUM-2MD3 cells, to the submesothelial ECM, and consequently inhibit peritoneal implantation. The peritoneal injection of adhe-sion polypeptides may be a new therapy against the dissemination of scirrhous gastric cancer, and may be useful for the prevention of dissemination in high-risk patients. © Rapid Science Ltd.     

Ultrastructural,immunohistochemical and biochemical studies on amylase and ACTH producing lung cancer

Summary Tumour tissue from a lung cancer patient who showed elevated serum amylase and adrenocorticotropin (ACTH) was studied ultrastructurally, immunohistochemically and biochemically. Histologically the tumour was a small cell carcinoma. On electron microscopic examination the tumour cells contained large zymogen-like granules within the cytoplasm. Furthermore, cells which possessed many small dense core granules of the endocrine type were also observed. It was of interest that the large zymogen-like granule-containing tumour cells had microvilli at the apical border, connected by desmosomes and forming lumina showing adenocarcinomatous differentiation. Electrophoretic analysis of the serum revealed that the major elevated amylase was of the salivary type with minor components. Immunostaining clearly demonstrated that most of the tumour cells possessed immunoreactive ACTH, whereas salivary amylase was only found in occasional clusters of the tumour cells. The results seem to indicate that the tumour showed both endocrine and exocrine characteristics - an amphicrine carcinoma, expressing amylase and ACTH simultaneously.     

LYMPHOMATOID GRANULOMATOSIS: Light Microscopic, Electron Microscopic and Immunohistochemical Study

A case of lymphomatoid granulomatosis (LYG) involving the lungs, skin, stomach, and possibly the left kidney in a 60-year-old man is presented. The infiltrates in the lungs, stomach, and skin showed a polymorphic appearance, and consisted predominantly of lymphocytes of mature and blastic form and of a few neutrophils, plasma cells, and histiocytes. Most lymphoid cells showed irregularly shaped nuclei and clustered dense bodies, characteristics indicative of T lymphocytes. An immunohistochemical study confirmed the T cell origin of the lymphocytes; i.e. they were positive for Leu-1, Leu-3a and la-like antigens but negative for Leu-2a antigen and the antibodies against light chains. The homogeniety of the major population of infiltrates in LYG indicates that at least some forms of LYG may be neoplastic or pre-neoplastic lymphocytic disorders which may ultimately progress to malignant lymphoma. ACTA PATHOL. JPN. 35 : 711–721, 1985.     

Membrane Fusion In Mitochondria Ii. An Ultrastructural Interpretation Of Cristae-Enriched Megamitochondria Induced By Cuprizone

Ultrastructure of megamitochondria in mouse hepatocytes induced by cuprizone was analyzed in the light of the fusion of inner mitochondrial membranes. Hepatic parenchymal cells of mice fed with a 0.5% cuprizone-diet for 7–8 days were characterized by megamitochondria with numerous cristae including disoriented, shorter ones than those of the control and those in vesicular configuration. After feeding the animals with cuprizone for 15–16 days, megamitochondria increased their sizes further and the number of cristae decreased drastically. Numbers of cristae based on a unit-mitochondrial area were 41.3$pL10.8 for the former type of cristae-enriched megamitochondria and 3.8 $pL 1.8 for the latter type of megamitochondria with the value of 23.9 $pL 6.2 for the control. Considering the content of cytochromes in megamitochondria previously reported, these results might suggest that the fusion and defusion of inner mitochondrial membrane take place along with the fusion of outer membranes during the megamitochondrial formation. ACTA PATHOL. JPN. 34: 481–488, 1984.