Induction of neutrophil infiltration by rat chemotactic cytokine (CINC) and its inhibition by dexamethasone in rats

In vivo effects of cytokine-induced neutrophil chemotactic factor (CINC) derived from rats on neutrophil infiltration were investigated using an air-pouch-type inflammation model in rats, and effects of dexamethasone on neutrophil infiltration induced by CINC was also examined in order to gain further insight into the mechanism of antiinflammutory activity of glucocorticoids. Injection of CINC into the air pouch made on the dorsum of rats induced a marked infiltration of neutrophils into the pouch fluid but not mononuclear cells and eosinophils during a 30-min interval after the injection. Maximum effect was induced at a dose of 1.4g/pouch. Treatment with dexamethasone 3 h before the injection of CINC suppressed the neutrophil infiltration in a dose-dependent manner, but no complete inhibition was observed. CINC injection into the air pouch of rats that had been sacrificed by bleeding in order to minimize neutroph il infiltration from blood stream also stimulated neutrophil infiltration into the pouch fluid when the carcass was incubated at 37C for 30 min, but the number of infiltrated neutrophils was about 35% of CINC-induced neutrophil infiltration in intact ruts. CINC-induced neutrophil infiltration in the carcass, which is supposed to be a reflection of neutrophil migration from extravascular space in subcutaneous tissues to pouch fluid, was not inhibited by dexamethasone treatment. Therefore, the inhibition of neutrophil infiltration by dexamethasone might be due to inhibition of the extravasation of peripheral neutrophils but not due to inhibition of neutrophil chemotaxis from subcutaneous extravascular space to pouch fluid. These findings suggest that clinical effects of steroidal antiinflammatory drugs on neutrophil infiltration in inflammatory disease is partly due to inhibition of neutrophil extravasation induced by preformed neutrophil chemotactic factors in the inflammatory site.     

Vinyl polymerization. 294. Decomposition of tetramethyltetrazene in the presence of p-substituted benzyl chlorides

A study was made of the polymerization of acrylonitrile in dimethylformamide (DMF) initiated by the binary systems of tetramethyltetrazene (TMT) and p-substituted benzyl chlorides. The polymerization rate increased linearly with the σ-constants of substituents as electron-releasing groups were introduced to the phenyl ring of benzyl chloride. In order to elucidate the initiation mechanism of the polymerization, a kinetic investigation was also undertaken of the decomposition of TMT in the presence of p-substituted benzyl chlorides in DMF. The decomposition rate was first-order in TMT and first-order in p-substituents benzyl chloride. The decomposition rate also increased with the σ constants of substituents as electron-releasing groups were introduced. On the basis of the results, the initiation mechanism for the polymerization was discussed.     

Lack of association between DNA base excision repair gene XRCC1 Gln399Arg polymorphism and risk of malignant lymphoma in Japan

Growing evidence suggests that the polymorphism of DNA base excision repair gene XRCC1 Arg399Gln is associated with altered DNA repair proficiency and subsequent cancer susceptibility; however, no evidence is available for malignant lymphoma. We therefore conducted a case-control study (372 cases, 500 controls) to evaluate links with malignant lymphoma risk in Japan. The risk was evaluated in terms of odds ratio (OR) and 95% confidence interval (CI) adjusted for age and sex in an unconditional logistic regression model. There was no statistical risk change with the Arg/Gln (adjusted OR 0.89; 0.65-1.23, P = 0.492) or the Gln/Gln (0.57; 0.27-1.17, P = 0.127) compared with the Arg/Arg of the XRCC1 Arg399Gln polymorphism. The results were unchanged in analyses according to histological subtype (diffuse large lymphoma, follicular lymphoma, low-grade lymphoma of mucosa-associated lymphoid tissue, and others). These data suggest that XRCC1 Gln399Arg polymorphism plays a limited role in lymphomagenesis. Further study on the interaction between the polymorphism and environmental exposure is required.     

Multiple epithelial cysts of the spleen and on the splenic capsule, and high serum levels of CA19-9, CA125 and soluble IL-2 receptor

An 18-year-old woman with abdominal pain was diagnosed as having splenic cysts by computed tomography scan. She had high serum levels of CA19-9 (2886.8 U/mL; normal value, <35 U/mL), CA125 (131.1 U/mL; normal value, <35 U/mL) and soluble IL-2 receptor (1490 U/mL; normal range, 220-530 U/mL). The resected spleen weighed 1050 g, was 14 x 28 cm, and had more than 10 macroscopic cysts up to 10.3 x 9.5 cm. There were numerous microscopic cysts in the spleen and several on the splenic capsule. The levels of CA19-9 and CA125 in the cyst fluid were 2165550 U/mL and 160400 U/mL, respectively. After the surgery, the serum levels of the tumor markers decreased gradually. The inside of the largest cyst was mainly covered by granulation tissue with a focal lining of epithelial cells, and the other macroscopic cysts had stratified squamous epithelium. The microscopic splenic cysts and cysts on the splenic capsule were lined by either attenuated single-layered or multilayered epithelial cells. The lining epithelial cells of these cysts were positive for epithelial membrane antigen and cytokeratins. CA19-9 and CA125 were detected in the lining cells of the splenic cysts. In the present case, it is suspected that the splenic cysts were derived from the capsular lining cells that showed migration from the capsule or formed microcysts on the splenic capsule, as in the case of ovarian inclusion cysts.     

The plasminogen activation system reduces fibrosis in the lung by a hepatocyte growth factor-dependent mechanism

Mice deficient in the plasminogen activator inhibitor-1 gene (PAI-1-/- mice) are relatively protected from developing pulmonary fibrosis from bleomycin administration. We hypothesized that one of the protective mechanisms may be the ability of the plasminogen system to enhance hepatocyte growth factor (HGF) effects, which have been reported to be anti-fibrotic in the lung. HGF is known to be sequestered in tissues by binding to extracellular matrix components. Following bleomycin administration, we found that HGF protein levels were higher in bronchoalveolar lavage fluid from PAI-1-/- mice compared to wild-type (PAI-1+/+) mice. This increase could be suppressed by administering tranexamic acid, which inhibits plasmin activity. Conversely, intratracheal instillation of urokinase into bleomycin-injured PAI-1+/+ mice to activate plasminogen caused a significant increase in HGF within bronchoalveolar lavage and caused less collagen accumulation in the lungs. Administration of an anti-HGF neutralizing antibody markedly increased collagen accumulation in the lungs of bleomycin-injured PAI-1-/- mice. These results support the hypothesis that increasing the availability of HGF, possibly by enhancing its release from extracellular matrix by a plasmin-dependent mechanism, is an important means by which activation of the plasminogen system can limit pulmonary fibrosis.     

Coordinated Movement of Bile Canalicular Networks Reconstructed by Rat Small Hepatocytes

Hepatocytes in vivo have a potential for liver regeneration, but it has been very difficult to reconstruct hepatic organoids in vitro. Recent studies have shown that small hepatocytes (SHs) can reconstruct hepatic organoids including functional bile canaliculi (BC). In the present study we analyzed the movement of BC formed in the hepatic organoids, focusing on the coordination of contraction and dilation among cells and the mechanism producing the coordination. Hepatic cells, including SHs, were isolated from an adult rat liver and cultured. Time-lapse images of BC movements were taken and analyzed in cells treated with or without cytochalasin B (CB). Time-lapse images revealed that all BC, regardless of region contracted in a coordinated manner. Actin filaments were observed along the BC even after the BC networks treated with CB dilated markedly. Microinjection of dye was also carried out to investigate the flow thorough BC. Secreted fluorescein from the injected cell flowed along BC, and gap junctional protein connexin 32 was expressed along BC networks, suggesting cell-to-cell communication. Thus, groups of hepatocytes in the hepatic organoids act in a coordinated manner through intercellular communication.     

Localization of [F]fluorodeoxyglucose in mouse brain neurons with micro-autoradiography

This is the first study of micro-autoradiography (micro-ARG) for [¹⁸F]2-fluoro-2-deoxy- -glucose ([¹⁸F]FDG). The localization of [¹⁸F]FDG was demonstrated in dendrites of neuron and also in the myelinated axon in mouse normal brain in vivo. The nucleolus was relatively free of label. The counted silver grain numbers in autoradiogram were linearly correlated to the ¹⁸F radioactivities in the specimen. The micro-ARG using positron emitting ¹⁸F is a very time-saving technique with 4 hours exposure compared with the conventional method using ³H- or ¹⁴C-labeled tracers.     

Implantation study of a novel hydroxyapatite/collagen (HAp/col) composite into weight-bearing sites of dogs

A hydroxyapatite/type I collagen (HAp/Col) composite, aligning hydroxyapatite nanocrystals along collagen molecules, has been prepared. The biocompatibility, osteoconductive activity, and efficacy as a carrier of rhBMP-2 of this novel biomaterial implanted in the weight-bearing site have been examined. The HAp/Col implants (15 mm in diameter and 20 mm in length) with a surface cross-linked layer containing rhBMP-2 (0 or 400 microg/ml) were implanted into bone defects of tibiae in three beagle dogs and fixed according to the Ilizarov method. As a control, bone defects of 20 mm in two beagle dogs did not receive implants, and the dogs were allowed to walk using an Ilizarov extraskeletal fixator. The specimens were removed from one dog in each group after 12 weeks. Also, the Ilizarov fixators in the rhBMP-treated dogs were removed after 12 weeks, after which full weight bearing started. The specimens were further taken out after 18 and 24 weeks in the rhBMP-treated and non-rhBMP-treated dogs, and after 24 weeks in the control group. The change of bone mineral density, as well as radiological and histological findings, suggest that the implants are able to induce bone remodeling units and are a superior carrier of rhBMP-2 due to the stimulation of early callus and new bone formation.     

The prevalence of TT virus (TTV) infection and its relationship to hepatitis in children

TT virus (TTV) is a newly discovered virus from a patient with post-transfusion hepatitis. We investigated the frequency and pathogenesis of TTV infection in children. A semi-nested PCR assay was used to amplify TTV-DNA in serum samples from 254 ambulatory children without liver disease, 20 with hepatitis of unknown etiology, and 18 transfusion recipients or hemophiliacs. In positive samples, TTV-DNA was quantified by real-time quantitative PCR using a fluorescent probe. We detected TTV-DNA in 20% of children with hepatitis of unknown etiology, which was not statistically different from the 23% prevalence in ambulatory children. In transfusion recipients or hemophiliacs, the frequency was higher (50%) than that in ambulatory children (P = 0.01). Among ambulatory children, TTV-DNA was frequently detected in children with acute gastroenteritis (36%). TTV-DNA was detected in 10% of the infants under 6 months old, and 20% of the children from 7 to 12  months old. The prevalence was constant after the age of 1 year; however, the copy number of TTV-DNA was significantly higher in children under 1 year of age (mean: 10^5.4 versus 10^3.8 copies/ml, P= 0.008). Finally, TTV-DNA was quantified serially in three children with chronic hepatitis who were positive for TTV-DNA. The presence or amount of TTV-DNA was unrelated to the serum alanine aminotransferase level. These results indicate that TTV infection is common in children. The larger quantity of TTV-DNA in infants and the high prevalence of TTV in children of all ages suggest that TTV may be transmitted in early childhood. Its relationship to hepatitis is doubtful in children. Received: 8 April 1999     

Proliferation Kinetics of Rat Kupffer Cells after Partial Hepatectomy Immunohistochemical and UItrastructural Analysis

In an effort to settle the conflicting views on the proliferation kinetics of Kupffer cells (Kc), we performed 2/3 partial hepatectomy on rats injected with Pelikan ink. Using an anti-rat macrophage monoclonal antibody, ED 2, we evaluated the numerical changes in total, carbon-positive ED 2⁺ cells and carbon-negative ED 2⁺ cells in the portal and central area. We also analyzed the ultrastructure and peroxidase cytochemistry of various types of cells observed during regeneration. The total numbers of ED 2⁺ cells in the remaining liver increased rapidly from day 2 to 5, and the number of dividing ED 2⁺ cells reached a maximum on day 2. Thus, the numerical increase in ED 2⁺ cells corresponded to the division phase. In contrast, the carbon-labeling experiment showed a continuous increase of carbon negative ED 2⁺ cells from day 2 to 7. In the central area where division was less frequent, the proportion of carbon-positive cells decreased markedly to 50% on day 7, as against 97% in control rats. These findings suggest the possibility of an influx of carbon-negative Kc in addition to cell division. Ultrastructurally, the presence of carbon-negative "small Kc" and "immature Kc" with morphological features different from those of carbon-positive Kc was demonstrated. Such carbon-negative Kc with a high nucleus-to-cytoplasm ratio and rather few phagosomes, were not observed in control rats. Furthermore, we demonstrated two types of possible precursor cell, i.e. "transitional" forms between monocytes and Kc, and "immature macrophages". The former showed peroxidase activity in some lysosomes as well as in the rough endoplasmic reticulum and nuclear envelope. Our result indicated that the proliferation kinetics of Kc depend upon both local proliferation and influx.