Glucan-induced modification of murine viral hepatitis.

Glucan, a macrophage stimulant, was evaluated for its ability to alter survival and phagocytic dysfunction in mice challenged with mouse hepatitis virus strain MHV-A59. Administration of glucan before the mice were challenged with the virus significantly prolonged median survival time but did not modify overall mortality compared with control mice given dextrose. Maximal effectiveness was achieved when glucan was administered both before and after the viral challenge. In contrast to the marked hepatic parenchymal cell necrosis observed in the control mice, glucan-treated mice exhibited reduced pathology. Intraperitoneal administration of MHV-A59 resulted in a significant depression of phagocytic activity compared with controls that were not exposed to the virus. The enhancement in phagocytic function in glucan-treated control mice was unaltered in virus-challenged, glucan-treated mice. Thus glucan is capable of increasing survival, inhibiting hepatic necrosis, and maintaining an activated state of phagocytic activity in mice challenged with MHV-A59. Macrophage stimulants may have a significant role in the modification of virally induced hepatic lesions.

Peroxidation of liver lipids in the pathogenesis of the ethanol-induced fatty liver.

Administration of an acutely intoxicating dose of ethanol produced significant increases in the concentration of liver triglyceride and enhanced the peroxidation of liver lipids in rats. Adipose triglyceride and lipid peroxide concentrations were unaltered. Coenzyme Q(4), an effective antioxidant, significantly inhibited accumulation of liver triglyceride following ethanol intoxication and prevented the peroxidation of liver lipids. These results, which demonstrate the selective ability of ethanol to induce peroxidation of liver lipids, together with the effectiveness of antioxidants, support the previously proposed hypothesis that peroxidation of liver lipids following ethanol intoxication is a factor in the pathogenesis of ethanol-induced liver injury.

Reticuloendothelial blockade: effect of puromycin on opsonin-dependent recovery.

Reticuloendothelial blockade induced by the administration of a gelatinized "reticuloendothelial test lipid emulsion" is due to a loss of opsonic activity in the plasma. Recovery from blockade, which is associated with restoration of plasma opsonins, was inhibited by the administration of puromycin. The effect of puromycin appears to be mediated by inhibition of opsonin formation rather than a puromycin-induced macrophage defect in phagocytosis.

Increased resistance to Staphylococcus aureus infection and enhancement in serum lysozyme activity by glucan.

Glucan is a potent reticuloendothelial stimulant whose immunobiological activity is mediated, in part, by an increase in the number and function of macrophages. In studying the role of glucan as a mediator of antibacterial activity, we attempted to ascertain the ability of glucan to modify the mortality of mice with experimentally induced Gram-positive bacteremia, and to enhance antibacterial defenses in rats as denoted by serum lysozyme and phagocytic activity. After intravenous administration of glucan, serum lysozyme concentrations were increased approximately sevenfold over control concentrations. The increase in serum lysozyme appeared to parallel the glucan-induced increase in phagocytosis and induced hyperplasia of macrophages. Prior treatment of mice with glucan significantly enhanced their survival when they were challenged systemically with Staphylococcus aureus. These studies indicate that glucan confers an enhanced state of host defense against bacterial infections.

Macrophage-mediated destruction of human malignant cells in vivo.

Macrophages require a plasma component, designated "recognition factor" (RF), for the expression of optimal function. The RF activity was profoundly depleted in plasma from patients with malignant disease, and the degree of depletion and the severity of the malignant state seemed to be related. Since experiments demonstrated that an active RF significantly inhibited tumor growth, clinical studies were initiated to investigate the influence of intratumor administration of an active RF fraction. Glucan, a potent macrophage activator, was also employed alone or combined with RF. These studies were undertaken to enhance the recognition of malignant cells by macrophages and to mobilize and activate macrophages intralesionally. The initial 9 patients studied had malignant melanoma, adenosquamous carcinoma of the lung, or carcinoma of the breast. Control and experimental lesions were injected; subsequently biopsies were performed at varying intervals for histologic evaluation. Always when glucan or glucan and RF fraction were administered intralesionally, the size of the lesion was strikingly reduced in as short a period as 5 days. This reduction was associated with necrosis of the tumor and a monocytic infiltrate. In small lesions, resolution was complete, whereas in large lesions, resolution was partial. The amount of glucan injected and the quantity of residual tumor appeared to be related. The induced necrosis of the tumor nodule was associated with an increase in plasma levels of circulating RF activity.

Preparation for hapten help by glucan, muramyl dipeptide, and its L-ala-Glycerol-mycolate derivative.

Previously, we reported that one of the factors that determines whether or not an animal will be prepared for hapten help after priming is the type of adjuvant used. The present work was undertaken, therefore, to determine which of a diverse variety of adjuvants or biological response modifiers would be effective. They included Freund's complete (CFA) and incomplete (FICA) adjuvants, particulate glucan, muramyl dipeptide (MDP), and its L-ala-glycerol-mycolate derivative. Help by the azobenzenearsonate (ABA) hapten was measured as the augmentation of the anti-bovine gamma-globulin (BGG) plaque-forming cell (PFC) response to ABA-BGG of mice that had been hapten-primed with ABA conjugated to ovalbumin (OVA). The results showed that FICA was ineffective. MDP was effective but only if administered with FICA during hapten-priming. MDP-L-ala-glycerol-mycolate was effective without any adjuvant but only within a narrow dose range. Particulate glucan was as effective as CFA in preparing mice for hapten help. As the macrophage is the primary cellular target of those biological response modifiers that were effective, we conclude that it plays an important role in the cellular interaction involved in the mediation of hapten help.

In vitro tumoricidal activity of resting and glucan-activated Kupffer cells.

Kupffer cells compose 80-90% of fixed tissue macrophages and have been suggested to play an important role in hepatic antitumor resistance. In the present study, the ability of resting and activated Kupffer cells to lyse syngeneic mammary adenocarcinoma BW10232 cells was evaluated. Activated Kupffer cells were isolated from C57Bl/6J mice following single of multiple intravenous (IV) injections of glucan (0.45 mg/mouse), a potent macrophage-activating agent. Mice receiving 5% (w/v) dextrose served as control. Resting Kupffer cells induced significant (P less than .05) 4% and 12% specific lysis of adenocarcinoma cells at target:effector ratios of 1:10 and 1:50, respectively. Kupffer-cell-mediated tumoricidal activity was depressed on day 1 following a single IV injection of glucan. By day 3 postglucan, the antitumor activity of Kupffer cells returned to control levels and was enhanced on days 5 and 10. Following multiple IV injections of glucan on days -5, -3, and -1, Kupffer-cell-mediated cytotoxicity was elevated on days 1 and 4. These observations demonstrate that resting Kupffer cells are significantly cytotoxic to adenocarcinoma cells at T:E ratios of 1:10 and 1:50 and following a transient inhibition of Kupffer-cell-mediated tumoricidal activity, glucan was effective in significantly enhancing the antitumor activity of Kupffer cells.

Immunotherapeutic modification of Escherichia coli--induced experimental peritonitis and bacteremia by glucan.

Previous data from our laboratory have demonstrated that glucan administration significantly alters the course of a variety of experimentally induced infectious diseases. In view of the increasing incidence of gram-negative infections, studies were initiated to evaluate the effect of intraperitoneal glucan therapy on Escherichia coli-induced peritonitis and sepsis. Male ICR/Tex mice were injected intraperitoneally with glucan or dextrose on days 5 and 3 prior to intraperitoneal challenge with 1.0 x 10(8) E. coli. Glucan administration resulted in a significant enhancement of survival. Evaluation of the mechanism of protective action of glucan revealed that both the glucan and dextrose control groups showed an equivalent level of blood-borne E. coli at early periods. At 6 hours after challenge the glucan group showed a significant decrease in blood-borne E. coli. In contrast, the dextrose control group demonstrated progressive bacteremia. A significant depression of phagocytic activity occurred in E. coli-infected mice as compared with control mice that were not exposed to the bacterial challenge. The enhancement in phagocytic function observed in glucan-treated control mice was unaltered in E. coli challenged, glucan-treated mice. The possible importance of hyperfunctional macrophages in reduction of mortality from E. coli sepsis was denoted by methyl palmitate-induced reversal of the glucan hyperfunctional state. Methyl palmitate-treated glucan injected mice were not protected against E. coli infection. These data denote that the intraperitoneal administration of glucan significantly modifies the course of E. coli-induced peritonitis and bacteremia due, in part, to glucan-induced enhancement of macrophage function.

A temporal relationship between reticuloendothelial system phagocytic alterations and antibody responses in mice infected with Plasmodium berghei (NYU-2 strain).

Malaria-induced immunosuppression has been demonstrated in humans and experimental animals. The suppressed immune response has been suggested to be primarily humoral and not cellular in nature, since classical lymphocytic cell-mediated responses have been reported to be normal. Since previous results have demonstrated that an impairment in macrophage antigen processing may be a contributing factor in malaria-induced immunosuppression, the present studies were conducted to determine if the macrophage/reticuloendothelial system (RES) alteration occurs parallel to the course of the malarial infection and if the impairment in antibody formation is temporally related to the RES alteration. The present study has demonstrated that a profound impairment in splenic direct plaque forming cell (PFC) formation occurs in malaria-infected Balb/c mice which had been immunized with sheep erythrocytes (SRBC) either 2 or 4 days after inoculation with Plasmodium berghei, NYU-2 strain. Serum hemagglutinin titers were significantly depressed in mice which received the SRBC 4 days post-inoculation; however, no alterations in antibody titers were observed in mice immunized with SRBC 2 days post-inoculation. Coincident with the depression of serum antibody titers at the day 4 immunization period was a profound increase in the vascular clearance of 51Cr-SRBC with an enhanced hepatic uptake of the 51Cr-SRBC and a decreased splenic localization of the labelled erythrocytes. It is suggested that a direct vascular exposure of the splenic lymphoid-macrophage elements to the parasite may be responsible for the initial early alterations in the PFC response while the impairment in serum antibody titers and splenic phagocytic activity may be a result of the pathological alterations occurring later in the infection, e.g., tissue anoxia, anemia, and hemolysis.

Comparative evaluation of the tumor inhibitory and antibacterial activity of solubilized and particulate glucan.

A soluble derivative of particulate glucan was prepared and evaluated for its antitumor and antibacterial activity. Intravenous administration of soluble or particulate glucan resulted in significant reduction in the growth of a syngeneic anaplastic mammary carcinoma and melanoma B16 and enhanced survival. Soluble and particulate glucan also significantly enhanced survival of mice infected with Staphylococcus aureus. Hepatosplenomegaly and granuloma formation observed in particulate glucan-treated mice were not observed in the soluble glucan group. It is evident that the soluble glucan initiates significant antitumor and antistaphylococcal activity. The active soluble fraction of particulate glucan may be preferable to particulate glucan in view of inherent facility of parenteral administration.

A method for the solubilization of a (1----3)-beta-D-glucan isolated from Saccharomyces cerevisiae.

This report describes a method for the solubilization of a micro-particulate beta-D-glucan. Insoluble glucan is dissolved in methyl sulfoxide and urea (8M) and partially phosphorylated at 100 degrees. The resulting water-soluble product is called glucan phosphate. The conversion rate is 70%, and the preparation is endotoxin free as determined by the Limulus lysate procedure. Glucan phosphate is composed of 34.66% C, 6.29% H, 42.83% O, and 2.23% P and has a repeating-unit empirical formula of (C6H10O5)7.PO3H2, indicating a phosphate group substitution on every seventh glucose subunit. Molecular-weight averages, polydispersity, and intrinsic viscosity were determined by aqueous high-performance size-exclusion chromatography (s.e.c.) with on-line, multi-angle laser light scattering (m.a.l.l.s.) photometry and differential viscometry (d.v.). Two polymer peaks were resolved. Peak 1 (Mw = 3.57 x 10(6) daltons), represents approximately 2% of the total polymers, while peak 2 (Mw = 1.10 x 10(5) daltons) comprises approximately 98% of polymers. 13C- and 31P-n.m.r. spectroscopy confirmed the beta-1,3 interchain linkage and the presence of a phosphate group. In solution, glucan phosphate polymers self-associate in a triple-helical arrangement. The ability to prepare a immunologically active, non-toxic, water-soluble beta-D-glucan preparation will greatly enhance the clinical utility of this class of compounds.

Glucan induced modification of experimental Staphylococcus aureus infection in normal, leukemic and immunosuppressed mice.

Glucan, a beta 1 leads to 3 polyglucosidic component of Saccharomyces cerevisiae, was evaluated for its ability to provide nonspecific resistance to S. aureus septicemia in AKR/J mice. Intravenous injection of glucan (0.45 mg/mouse) 7 and 4 days prior to intravenous challenge with S. aureus (1.0 x 10(9)) resulted in a significantly increased survival as compared to control mice. Histological examination of the kidneys revealed that glucan decreased tissue necrosis associated with systemic staphylococcal disease. A post-treatment regimen of glucan significantly enhanced survival of AKR/J mice with lymphocytic leukemia as well as leukemic mice with experimentally induced systemic staphylococcal infection. The effect of glucan on S. aureus septicemia was also evaluated in cyclophosphamide-treated mice. Glucan increased peripheral leukocyte counts as well as significantly enhanced survival of cyclophosphamide-treated mice with systemic S. aureus infection. Histopathological examination revealed that glucan administration markedly inhibited renal and hepatic pathology in cyclophosphamide-treated mice following intravenous challenge with S. aureus. These data denote that glucan provides nonspecific resistance to bacterial sepsis in normal, leukemic as well as immunosuppressed mice.

Comparison of the in vitro cytolytic effect of hepatic, splenic and peritoneal macrophages from glucan-treated mice on sarcoma M5076.

Our previous results have demonstrated that glucan will significantly modify the course of syngeneic murine tumors. Additionally, results denote that glucan increases macrophage-mediated lysis of syngeneic tumor cells. The present study was undertaken to more closely examine the effect of parenteral glucan administration on the tumoricidal activity of hepatic, splenic and peritoneal macrophage populations. C56B1/6J mice were injected intravenously with glucan (0.45 mg/mouse) on days 1,3,6,9,12 and 15. Hepatic, splenic and peritoneal macrophages were isolated on days 8, 12 and 16. The macrophages were co-incubated for 72 h with syngeneic reticulum cell sarcoma M5076 at a target: effector ratio of 1:50. A significant increase in the cytolytic activity of all three macrophage populations was observed. Kupffer cell tumoricidal activity was increased (p less than 0.01) on day 8, but was not significantly different from control on days 12 and 16. Peritoneal exudate macrophages showed increased (p less than .001) tumoricidal activity on days 12 and 16, respectively. Splenic macrophages showed an enhanced (p less than 0.01) increase in lytic activity only on day 16. The present results indicate that: 1) glucan will enhance macrophage-mediated tumoricidal activity of three distinct macrophage populations and 2) a temporal relationship exists between glucan administration and expression of lytic activity by the macrophage populations. These observations further define the mechanism by which glucan exerts its potent anti-tumor activity.