Exercise-induced stimulation of murine macrophage phagocytosis may be mediated by thyroxine

The present study was designed to test the hypothesis that changes in plasma concentrations of hormones may be responsible for the exercise-induced macrophage phagocytic stimulation. The effect of 30-min incubation of macrophages with plasma from mice previously exposed to swimming until exhaustion (with or without previous training) was studied, and the results showed a similar stimulation of the phagocytic capacity (attachment and ingestion) of these cells to that found in previous studies after exercise. Also, changes in plasma concentration of both thyroxine (T4) and 3,3',5-triiodo-L-thyronine (T3) after exercise were measured, and their effect on phagocytic capacity after in vitro incubation with peritoneal macrophages was investigated. Results indicated that, after exercise, plasma concentrations of T3 and T4 increased. Incubation of peritoneal macrophages for 30 min with a concentration of T3 similar to that observed in the plasma immediately after exercise (1.5 ng/ml) induced no modifications in the phagocytic capacity. However, a physiological concentration of T4 after exercise (75 ng/ml) stimulated the phagocytic capacity of peritoneal macrophages. In addition, a 10,000-fold greater concentration of these thyroid hormones did not modify the macrophage function. It is concluded that physiological concentration of T4 may be a mediator of the stimulation of the phagocytic function in macrophages induced by exercise.     

Two distinct patterns of peritoneal involvement shown by in vitro and in vivo ovarian cancer dissemination models

We established an in vitro peritoneal dissemination model using six ovarian cancer cell lines and cultured mesothelial cells. Ovarian cancer cells were classified into two types, invasive or adhesive, on the basis of their interaction with the mesothelial cell monolayer. The ovarian cancer cell lines derived from mucinous cystadenocarcinoma, poorly differentiated adenocarcinoma and undifferentiated carcinoma, which belonged to the invasive type, began to invade beneath the mesothelial monolayer from several hours after seeding in vitro, expelling the mesothelial cells at the periphery and forming colonies directly on the dish surface. On the other hand, cancer cell lines of clear cell carcinoma, which belonged to the adhesive type, showed colony formation with adhesion on the mesothelial monolayer even 18 h after seeding. Invasive-type cell lines invaded into the mesothelial monolayer at various rates in vitro, and the degree of invasiveness showed good correlation with the degree of peritoneal dissemination in vivo after intraperitoneal injection of cancer cells into nude mice. Adhesive-type cells showed rather higher dissemination rates in vivo. Microscopic observation of in vivo peritoneal dissemination at one day after inoculation also revealed two patterns of peritoneal involvement similar to those in vitro. In the in vitro model, anti-integrin alpha 2- and beta 1-antibodies inhibited the infiltration of invasive-type cells into the mesothelial monolayer, but did not affect colony formation by adhesive-type cells on the monolayer, indicating that invasion by both cell types was mediated by different molecules. This in vitro model is thought to be useful for analysis of the molecular mechanisms of peritoneal dissemination.     

Reovirus growth in cell culture does not require the full complement of viral proteins: identification of a sigma1s-null mutant

The reovirus sigma1s protein is a 14-kDa nonstructural protein encoded by the S1 gene segment. The S1 gene has been linked to many properties of reovirus, including virulence and induction of apoptosis. Although the function of sigma1s is not known, the sigma1s open reading frame is conserved in all S1 gene sequences determined to date. In this study, we identified and characterized a variant of type 3 reovirus, T3C84-MA, which does not express sigma1s. To facilitate these experiments, we generated two monoclonal antibodies (MAbs) that bind different epitopes of the sigma1s protein. Using these MAbs in immunoblot and immunofluorescence assays, we found that L929 (L) cells infected with T3C84-MA do not contain sigma1s. To determine whether sigma1s is required for reovirus infection of cultured cells, we compared the growth of T3C84-MA and its parental strain, T3C84, in L cells and Madin-Darby canine kidney (MDCK) cells. After 48 h of growth, yields of T3C84-MA were equivalent to yields of T3C84 in L cells and were fivefold lower than yields of T3C84 in MDCK cells. After 7 days of growth following adsorption at a low multiplicity of infection, yields of T3C84-MA and T3C84 did not differ significantly in either L cells or MDCK cells. To determine whether sigma1s is required for apoptosis induced by reovirus infection, T3C84-MA and T3C84 were tested for their capacity to induce apoptosis, using an acridine orange staining assay. In these experiments, the percentages of apoptotic cells following infection with T3C84-MA and T3C84 were equivalent. These findings indicate that nonstructural protein sigma1s is not required for reovirus growth in cell culture and does not influence the capacity of reovirus to induce apoptosis. Therefore, reovirus replication does not require the full complement of virally encoded proteins.     

Lipoarabinomannan inhibits nonopsonic binding of Mycobacterium tuberculosis to murine macrophages

The initial phagocytic interaction between Mycobacterium tuberculosis and macrophages (M phi) in the lung is probably nonopsonic, which would mean that M phi receptors will bind directly to bacterial ligands without the involvement of serum opsonins. Lipoarabinomannan (LAM) is a major component of the cell wall of mycobacteria. The possibility that LAM is involved in the nonopsonic binding of M. tuberculosis to M phi was investigated by using competitive inhibition assays. LAM inhibited binding of M. tuberculosis to murine peritoneal M phi in a dose-dependent manner. LAM also inhibited the binding of Mycobacterium avium and Mycobacterium bovis BCG to M phi. Phosphatidylinositol mannoside and lipomannan have the same phosphatidylinositol (PI) moiety as LAM, but differ in their glycosylation patterns. Both molecules inhibited binding of M. tuberculosis to M phi. Deacylation of LAM abrogated its capacity to inhibit binding of M. tuberculosis to M phi. These observations indicated that it was the PI moiety of LAM that was important in mediating its inhibitory properties. In support of this hypothesis, commercial PI was shown to inhibit the binding of M. tuberculosis to M phi. Our results suggest that cellfree LAM is able to inhibit the binding of mycobacteria to M phi, but that it does not do so by competing with any interaction between M phi receptors and cell-associated LAM, because the PI end of the molecule is believed to be anchored in the bacterial plasma membrane, and therefore not available as a ligand on the cell surface. However, LAM that is released from M. tuberculosis in the course of its active replication during infection may be able to interfere with the phagocytic clearance of mycobacteria.     

Value of peritoneal cytology after hysteroscopy in surgical stage I adenocarcinoma of the endometrium

Nineteen clinical stage I adenocarcinoma of the uterus with favourable histological prognosis factors (low grade, no myometrial extension, and no pelvic node involvement) were diagnosed using a pre-operative hysteroscopy. During the laparotomy, peritoneal cytology was performed systematically. The frequency of positive peritoneal washings was abnormally high (7 cases) with cytologic findings showing grouped cells in large clusters. However, these patients have not experienced peritoneal recurrences. The endoscopic procedures may have facilitated the transtubal malignant cell dissemination and are questionable in endometrial carcinoma.     

Value of peritoneal cytology after hysteroscopy in endometrial adenocarcinoma stage I

Nineteen clinical stage I adenocarcinoma of the uterus with favourable histological prognosis factors (low grade, no myometrial extension, and no pelvic node involvement) were diagnosed using a preoperative hysteroscopy. During the laparotomy, a peritoneal cytology was performed systematically. The frequency of positive peritoneal washings was abnormally high (7 cases) with cytologic findings showing grouped cells in large clusters. However, these patients have not experienced peritoneal recurrences. The endoscopic procedures may have facilitated the transtubal malignant cell dissemination and are questionable in front of endometrial carcinoma.     

Antirotaviral activity of milk proteins: lactoferrin prevents rotavirus infection in the enterocyte-like cell line HT-29

Different milk proteins were analyzed for their inhibitory effect on either rotavirus-mediated agglutination of human erythrocytes or rotavirus infection of the human enterocyte-like cell line HT-29. Proteins investigated were alpha-lactalbumin, beta-lactoglobulin, apo-lactoferrin, and Fe(3+)-lactoferrin, and their antiviral action was compared with the activity of mucin, a milk glycoprotein known to affect rotavirus infection. Results obtained demonstrated that beta-lactoglobulin, apo- and Fe(3+)-lactoferrin are able to inhibit the replication of rotavirus in a dose-dependent manner, apo-lactoferrin being the most active. It was shown that apo-lactoferrin hinders virus attachment to cell receptors since it is able to bind the viral particles and to prevent both rotavirus haemagglutination and viral binding to susceptible cells. Moreover, this protein markedly inhibited rotavirus antigen synthesis and yield in HT-29 cells when added during the viral adsorption step or when it was present in the first hours of infection, suggesting that this protein interferes with the early phases of rotavirus infection.     

Does hepatitis C virus co-infection accelerate clinical and immunological evolution of HIV-infected patients?

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 alpha2beta1 and alpha3beta1 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 sequence 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 adhesion 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.     

Hepatocyte growth factor (HGF) produced by peritoneal fibroblasts may affect mesothelial cell morphology and promote peritoneal dissemination

Mesothelial cell monolayers have been reported to prevent infiltration of cancer cells into the peritoneum. We have previously reported that peritoneal fibrosis induced by gastric cancer cells prior to metastatization may provide a congenial environment for peritoneal metastases. In this study, we investigated the effects of peritoneal fibroblasts on peritoneal mesothelial cell morphology. Human gastric cancer (OCUM-2MD3), peritoneal fibroblast (NF-2P) and mesothelial (MS-1) cell lines were established in our laboratory. Histology of the peritoneum was investigated following intraperitoneal inoculation of serum-free conditioned media (SF-CM) from OCUM-2MD3 cells into nude mice. SF-CM from peritoneal fibroblasts was added to monolayer-cultured mesothelial cells, and their morphology was examined by phase-contrast microscopy. This experiment was conducted in the presence and absence of neutralizing antibodies against various factors. Mesothelial cells exposed to fibroblasts proliferation became hemispherical and separated from each other, while unexposed mesothelium remained as a flat monolayer. Cultured-mesothelial cells rounded up or exhibited a fibroblast-like shape following the addition of peritoneal fibroblast SF-CM. Anti-hepatocyte growth factor (HGF) neutralizing antibody partly inhibited this effect. We suggest that soluble factors, such as HGF, produced by peritoneal fibroblasts affect the morphology of mesothelial cells in monolayers so that the resulting environment may become prone to the peritoneal dissemination of cancer cells.     

Promyelocytic blast crisis of chronic myelogenous leukaemia with translocations (9;22) and (15;17)

The murine monoclonal antibody A7 (MAb A7) is reactive against most human gastric cancer cell lines. Using a nude mouse peritoneal dissemination model of human gastric cancer, we investigated targeted chemotherapy using a conjugate of neocarzinostatin (NCS) with MAb A7 (A7-NCS). After demonstrating cytotoxicity of the complex against the human gastric cancer cell line MKN45 in vitro, we intraperitoneally injected A7-NCS, NCS or saline into nude mice bearing peritoneally disseminated human gastric cancer. A7-NCS inhibited peritoneal dissemination significantly more effectively than NCS. MAb A7 may prove to be an effective carrier for antineoplastic drugs in patients with peritoneal dissemination of gastric cancer.     

Histamine induces nuclear factor of activated T cell-mediated transcription and cyclosporin A-sensitive interleukin-8 mRNA expression in human umbilical vein endothelial cells

Strong evidence indicates that virions of mammalian reoviruses undergo proteolytic processing by acid-dependent cellular proteinases as an essential step in productive infection. Proteolytic processing takes the form of a series of cleavages of outer-capsid proteins final sigma3 and mu1/mu1C. Previous studies showed an effect of both NH4Cl and E-64 on these cleavages, indicating that one or more of the acid-dependent cysteine proteinases in mammalian cells (cathepsins B and L, for example) is required; however, these studies did not address whether acid-dependent aspartic proteinases in those cells (cathepsin D, for example) may also be required. To determine the role of aspartic proteinases in reovirus entry, studies with pepstatin A, a specific inhibitor of aspartic proteinases, were performed. The results showed that pepstatin A neither blocks nor slows reovirus infection of L or MDCK cells. Experiments using ribonuclease A and other proteins as cleavable substrates showed that cathepsin-D-like proteinases from these cells are inhibited within the tested range of pepstatin A concentrations both in vitro and within living cells. In other experiments, virion-bound final sigma3 protein was shown to be a poor substrate for cleavage by cathepsin D in vitro, consistent with the findings with inhibitors. In sum, the data indicate that cathepsin-D-like aspartic proteinases provide little or no activity toward proteolytic events required for infection of L or MDCK cells with reovirus virions.     

Continuous hyperthermic peritoneal perfusion for peritoneal dissemination of gastric cancer

BACKGROUND/AIMS: We investigated the histological and clinical effectiveness of continuous hyperthermic peritoneal perfusion (CHPP) for treating peritoneal dissemination (therapeutic CHPP) and for the prevention of peritoneal recurrence (prophylactic CHPP). METHODOLOGY: In 5 patients with gastric cancer and peritoneal dissemination, the apoptosis index of the cancer cells on in situ end-labeling for detection of apoptotic cells was 3.0+/-1.2% before CHPP, and had increased to 52.9+/-8.3% after CHPP. The survival curve of the therapeutic CHPP group was significantly better than that of the control group. The therapeutic CHPP group was classified as miliary type or nodular type. The survival curve in the miliary type was significantly better than that in the nodular type. RESULTS: In prophylactic CHPP, there was no improvement in prognosis, but a prophylactic effect against peritoneal recurrence was demonstrated in the patients who were n4 negative when the mean intraperitoneal temperature during CHPP (MIT) was maintained above 42 degrees C. CONCLUSIONS: These results indicated that an improved prognosis can be expected after therapeutic CHPP in patients with peritoneal spread. The beneficial effects are especially marked in patients with the miliary type. Moreover, prophylactic CHPP exerts a prophylactic effect against peritoneal recurrence in patients with n4 negative, providing that the MIT can be maintained above 42 degrees C.     

Effect of sodium thiopentone anesthesia on the phagocytic activity of rat peritoneal macrophages

To elucidate the effect of sodium thiopentone anesthesia on the function of phagocytic cells, albino rats were anesthetized with 60 mg/kg. of sodium thiopentone. After 90 min., peritoneal macrophages were harvested and their capacity for superoxide anion generation was detected. Following anesthesia for 90 min. latex particles were injected intraperitoneally, and after additional 30 min. the macrophages were derived, embedded in agar and the number of cells engaged in phagocytosis, as well as the number of latex particles engulfed by each individual cell were counted in semi-thick sections. Macrophages of anesthetized animals showed a statistically significant decrease of both superoxide anion generation and mean number of phagocytic cells, and engulfed fewer particles than those of the controls. Similar results were obtained following incubation of the cells with sodium thiopentone in vitro. The serum corticosterone level in anesthetized rats was significantly higher than that of the control animals. The results indicate that impaired phagocytosis following anesthesia induced by sodium thiopentone, in addition to alterations of the immune system caused by surgical trauma, may be one of the reasons for increased susceptibility to infections of surgical patients during the postoperative period.     

Accumulation of protein-coated liposomes in an extravascular site: influence of increasing carrier circulation lifetimes

The primary objective of this work was to test whether increased blood levels and circulation lifetimes result in increased passive targeting of protein-coated liposomal drug carriers. The system used to evaluate this was based on i.v. injection of 100 nm of distearoyl phosphatidylcholine/cholesterol liposomes with covalently bound streptavidin. The circulation lifetime of these liposomes was increased by procedures that involved blockade of liposome uptake by phagocytic cells in the liver and/or the incorporation of a poly(ethylene glycol)-modified phospholipid [poly(ethylene glycol)2000-modified distearoyl phosphatidylethanolamine]. Blockade of liver phagocytic cells with a low predose (2 mg/kg of drug) of liposomal doxorubicin increased the circulation half-life of the streptavidin liposomes from less than 1 hr to greater than 3 hr. A further 2-fold increase in circulating half-life (to approximately 7.5 hr) was achieved by using liposomes with 2 mole % of poly(ethylene glycol)2000-modified phosphatidylethanolamine. In combination with RES blockade, the circulation lifetimes of poly(ethylene glycol)phosphatidylethanolamine containing streptavidin liposomes could be increased to greater than 12 hr. The ability of these liposomes to move from the plasma compartment to an extravascular compartment was measured by using the peritoneal cavity as a convenient, accessible, extravascular site. The tendency for liposomes to accumulate in this site was not, however, clearly dependent on circulating blood levels. Comparable levels of liposomes in the peritoneal cavity were achieved when using systems that exhibited significantly different circulation lifetimes.     

Lysosomal exocytosis induced by hyperthermia: a new model of cancer cell death. II. Effect on peritoneal macrophages

Recently we described the "macrophagic lysosomal exocytosis" (MLE) as a possible new mode of cancer cell death induced by hyperthermia (HT) [1]. In order to confirm this first report, HT was applied at the peritoneum level with perfusional procedure, after catheter insertion under local anesthesia. We evaluated the subcellular changes of peritoneal macrophages in human gastric tumor, before and after hyperthermic treatment at 42 degrees C for 90 minutes. Transmission electron microscopy showed that treatment produced the disappearance of the cytoplasmic granules, with a consequent extracellular scavenger action of the phagocytic cells, the proliferation of some organelles such as mitochondria, endoplasmic reticulum and Golgi complex that may be addressed to a subsequent restoration of the granular pool in the degranulated macrophages. This phenomenon can enhance the already documented effect of hyperthermic perfusional chemotherapy in peritoneal tumors.     

Early after/depolarizations and triggered activity: mechanisms and autonomic regulation

Although the clearance and distribution of ligand molecules in circulation represent the function of hepatic sinusoidal cells, these mechanisms revealed a network that is more intricate than would at first seem, since several receptors are common to not only one type of cell, but also to two or three types of cells in the liver. In the case of latex particles in which their uptake by a particular cell type seems to be determined by their size, sinusoidal endothelial cells are able to internalize particles up to 0.23 microns under physiologic conditions, in vivo, and larger particles are taken up by Kupffer cells. However, when the phagocytic function of Kupffer cells is impaired by frog virus 3 or alcohol, endothelial cells have been found to take up particles larger than 1 micron in diameter after the injection of an excess amount of latex particles. Endothelial cells would thus constitute a second line of defense in the liver in that they remove foreign materials from the blood when Kupffer cell phagocytic function is totally disturbed. This potential role may not, however, be fully expressed under physiologic conditions when Kupffer cells are active in clearing foreign substance from the circulation. The functions of liver sinusoidal cells are varied and complex and these cells can be regarded as "a sinusoidal cell unit." This cellular interaction must be taken into account for any quantitative analysis.     

Mutations in reovirus outer-capsid protein sigma3 selected during persistent infections of L cells confer resistance to protease inhibitor E64

Mutations selected in reoviruses isolated from persistently infected cultures (PI viruses) affect viral entry into cells. Unlike wild-type (wt) viruses, PI viruses can grow in the presence of ammonium chloride, a weak base that blocks acid-dependent proteolysis of viral outer-capsid proteins in cellular endosomes during viral entry. In this study, we show that E64, an inhibitor of cysteine proteases such as those present in the endocytic compartment, blocks growth of wt reovirus by inhibiting viral disassembly. To determine whether PI viruses can grow in the presence of an inhibitor of endocytic proteases, we compared yields of wt and PI viruses in cells treated with E64. Prototype PI viruses L/C, PI 2A1, and PI 3-1 produced substantially greater yields than wt viruses type 1 Lang (T1L) and type 3 Dearing (T3D) in E64-treated cells. To identify viral genes that segregate with growth of PI viruses in the presence of E64, we tested reassortant viruses isolated from independent crosses of T1L and each of the prototype PI viruses for growth in cells treated with E64. Growth of reassortant viruses in the presence of E64 segregated exclusively with the S4 gene, which encodes viral outer-capsid protein sigma3. These results suggest that mutations in sigma3 protein selected during persistent infection alter its susceptibility to cleavage during viral disassembly. To determine the temporal relationship of acid-dependent and protease-dependent steps in reovirus disassembly, cells were infected with wt strain T1L or T3D, and medium containing either ammonium chloride or E64d, a membrane-permeable form of E64, was added at various times after adsorption. Susceptibility to inhibition by both ammonium chloride and E64 was abolished when either inhibitor was added at times greater than 60 min after adsorption. These findings indicate that acid-dependent and protease-dependent disassembly events occur with similar kinetics early in reovirus replication, which suggests that these events take place within the same compartment of the endocytic pathway.     

Macrophages and mesothelial cells in bacterial peritonitis

Research in recent years has examined the mechanisms underlying cellular host defence in the peritoneal cavity. These studies have established that the resident cells of the peritoneal cavity, the peritoneal macrophages (PM phi) and the mesothelial cells (HPMC) contribute to the initiation, amplification and resolution of peritoneal inflammation. Ex vivo measurements of intra-peritoneal inflammatory mediators during peritonitis has elucidated the time courses for the generation of proinflammatory, chemotactic and anti-inflammatory cytokines and have identified that their secretion occurs largely within the peritoneum. These studies provide evidence that both PM phi- and HPMC-derived mediators are directly involved in controlling inflammation. It has been widely accepted that resident PM phi form the first line of defence against peritoneal infection, a more contemporary view would suggest that the direct or indirect (via secreted pro-inflammatory cytokines) interaction between PM phi and HPMC is pivotal to the activation and subsequent amplification of the peritoneum's response to infection. Whilst the site of these interactions is unknown, considerable evidence suggests that it occurs on the surface of the mesothelium, where invading micro-organisms may colonize. In this respect Staphylococcal exoproducts can directly activate HPMC cytokine synthesis. Once the inflammatory response is initiated, recent evidence suggests, that mesothelial cells upon activation by PM phi-derived IL-1 beta and TNF-alpha, are capable of amplifying inflammation and generating signals (via the creation of a gradient of chemotactic cytokines, IL-8, MCP-1 and RANTES) for the recruitment of leukocytes into the peritoneum. This process is also facilitated via the cytokine driven up-regulation of adhesion molecule expression (ICAM-1 and VCAM-1) on HPMC. Much less is understood about the mechanisms by which inflammation is resolved, although the secretion of anti-inflammatory molecules (IL-6, IL-1ra and soluble TNF-p55/75) by receptors by PM phi and HPMC may be important in the process. The existence of a peritoneal cytokine network controlling inflammation is now well established, within this the interaction of PM phi and HPMC appears to play a pivotal role in the hosts response to peritoneal infection.