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PubMed1985

Effect of splenectomy on the efficiency of chemo-immunotherapy of melanoma-bearing mice.

Determination by the immune system of chemo- and immunotherapy of a murine tumor was investigated. The effect of cyclophosphamide and levan (polyfructose) in single and combined treatment on the growth of the F10 variant of B16 melanoma was tested in splenectomized and non-splenectomized mice. Splenectomy was found to increase markedly the efficiency of all treatments, particularly the combined treatment, as judged by the delay of appearance and size of the subcutaneous tumors at the site of inoculation. Nevertheless, mortality rate was not always in correlation with the rate of local tumor evolution: some of the treated splenectomized mice died earlier than expected, with relatively small local tumors, due to enhanced metastasis. The results indicate that elements in the spleen of C57BL mice decrease the therapeutic efficiency of the drugs tested. It can be deduced that splenectomy must not be considered to increase the risk of a tumor-bearing host in all cases. On the contrary, it may improve the efficiency of cancer treatment in certain tumor-host systems.

Animals↗
PubMed1986

In vitro effect of levan-activated macrophages on Lewis lung carcinoma cells.

The polysaccharide levan (polyfructose) has previously been shown to exert an inhibitory effect on the growth of several murine tumors. This activity is mediated by a host reaction, involving mainly macrophages but also other elements of the immune system. It was not clear, however, whether levan-activated macrophages act by a direct cytocidal effect on the tumor cells or via the activation of a specific immune response to the tumor. In the present study, the possibility of a direct cytotoxicity of levan-activated macrophages against Lewis lung carcinoma cells was tested by coculture in vitro. It was found that levan-induced (as well as paraffin oil induced) macrophages actually exert a direct cytotoxic effect on Lewis lung carcinoma cells. The tumor cell killing is mediated by cell to cell contact. A cytoplasmic bridge was often seen between the macrophage and the tumor cell. The remaining tumor cells in the lysed area appear slender, shrunken and non-dividing.

Animals↗
PubMed1986

Effect of tumor inhibitory and stimulatory doses of levan, alone and in combination with cyclophosphamide, on spleen and lymph nodes.

Immunotherapeutic agents are often reported to induce opposite effects -- inhibitory and stimulatory -- on tumor growth, depending on the dose, timing or route of administration of the drug. The reason underlying these opposite effects is not yet known. The immunomodulatory polysaccharide levan (polyfructose) has been found to exert such opposite effects on the growth of the F10 variant of B16 melanoma. Low doses inhibit and high doses enhance tumor growth. Cyclophosphamide (CY) augment the inhibitory effect of levan. In order to clarify the mechanism of this switch, we tried in the present study to determine the changes induced by levan at inhibitory and stimulatory treatments, alone or with CY, on the morphology of spleens and lymph nodes of the melanoma-bearing mice. The growth of the tumor in non-treated mice was found to induce a moderate splenomegaly. Microscopically, two main changes were observed: a mild extramedullary hematopoiesis and a sharp increase in the number of germinal centers. A parallel increase in germinal center number was found in the lymph nodes. The data presented suggest that the immune system plays a role in both the inhibition and stimulation of tumor growth by levan. Levan induced a dose dependent splenomegaly, even more pronounced in combination with CY, due to an extramedullary hematopoiesis. Levan reduced the B cell activity caused by the tumor, proportionally to its dose. In combination with CY, levan accelerated the recovery of the B cell activity at the low dose, while the high dose prevented it. A similar trend was found in the lymph nodes. The changes involved in the switch inhibition-stimulation could be either the degree of reduction in B cell activity or the degree of extramedullary hematopoiesis or some interplay between the myelocytic and lymphocytic series, which was found to change in an opposite fashion under the influence of various treatments. Since the immune system is a finely equilibrated system, it is possible that immunomodulation rather than immunostimulation should be aimed at in cancer immunotherapy. However, the conditions required for achieving this equilibrium have to be defined.

Animals↗
PubMed1989

Effect of cyclophosphamide and levan treatment on bone marrow and peripheral blood cells in B16-F10 melanoma-bearing mice.

Immunotherapeutic agents have often been found to provoke opposite effects on tumor growth--inhibitory or stimulatory--depending on dose, timing or route of administration. The reason for these opposite effects is not yet known. Levan (polyfructose), an immunomodulatory polysaccharide, has been found to exert opposite effects on the growth of the F10 variant of B16 melanoma. Low doses inhibit and high doses enhance the growth of this tumor. Cyclophosphamide (CY) augments the inhibitory effect of the polysaccharide. In order to elucidate the mechanism of these opposite effects, we tried to determine the changes induced by levan at inhibitory and stimulatory doses, alone or in conjunction with CY, on the lymphatic and hematopoietic systems of B16-F10 melanoma-bearing mice. In a previous study we reported the effect of these treatments on the morphology of spleen and lymph nodes (Leibovici, Kopel, Siegal & Gal-Mor (1986). Int. J. Immunopharmac., 8, 391). In the present study, we examined the effect of the treatments on bone marrow and peripheral blood composition. The growth of the tumor itself, as well as the various treatments, induced very marked changes in both bone marrow and blood. Tumor inoculation produced a sharp leukopenia and anemia followed by a restoration of both white and red blood cells. In the bone marrow, the tumor caused a gradual decrease in lymphocyte number. CY accentuated the severe leukopenia caused by the tumor. Lymphocyte depletion was prolonged, while restoration of granulocytes was achieved by day 7. A similar pattern of changes was observed in the bone marrow. With levan, opposite effects were observed in blood and bone marrow with the two doses in relation to the number of the cells of the lymphoid and myeloid lines: while 0.1 mg (tumor inhibitory) doses caused a more active restoration of lymphocytes as compared to 10 mg (tumor stimulatory) doses, an opposite effect was seen on the myeloid series--the high dose induced a more pronounced granulocytosis than the low dose. In the combined treatment, the low levan dose accelerated lymphocyte restoration in bone marrow compared to CY, while the high dose delayed the recovery of these cells. The results of the present study in conjunction with our previous study may explain the basis of the intriguing tumor inhibitory-stimulatory effects of some immunomodulators. Moderate increases in myeloid cell series appear to favor tumor inhibition and high increases favor tumor stimulation. In addition, the results of this study suggest that a regulatory relation might exist between the proliferation of the lymphoid and myeloid cell series.

Animals↗
PubMed1984

Different stages of tumor development are unequally affected by pretreatment of tumor cells with a polysaccharide.

Pretreatment with the polysaccharide levan of Lewis lung carcinoma and AKR lymphoma cells affected unequally different stages of tumor development. While levan pretreatment sharply reduced the evolution of tumors from subcutaneously inoculated cells, no such inhibition was observed with pretreated cells inoculated intravenously. Since levan is known to affect the cell membrane, it is concluded that different cell membrane properties may be involved in the various stages of tumor progression.

Animals↗
PubMed1995

Functional antibody single-chain fragments from the cytoplasm of Escherichia coli: influence of thioredoxin reductase (TrxB).

The cytoplasmic expression of a functional antibody (Ab) fragment, containing the correct intradomain disulfide bonds, was investigated in E. coli. We used a single-chain Fv (scFv) fragment of the levan-binding Ab ABPC48, which was shown to be functional only in the presence of the disulfide bonds. Significant amounts of functional, disulfide-containing scFv could be produced in the cytoplasm of E. coli in the absence of thioredoxin reductase (TrxB) activity. The amount of soluble protein remained largely unchanged by this null mutation. A stronger promoter did not result in further improved yields of functional Ab fragment, despite much higher protein production, suggesting that inefficient disulfide formation was still limiting the yield of active scFv. This method of expressing functional Ab fragments in the cytoplasm of E. coli may be important for screening and selection systems.

Antigen-Antibody Reactions↗
PubMed2005

Determination of Ophiopogon japonicus polysaccharide in plasma by HPLC with modified postcolumn fluorescence derivatization.

Interest in antimyocardial ischemic activity of a graminan-type fructosan with an average molecular weight of 5,000 Da from Ophiopogon japonicus (FOJ-5) has necessitated the development of a sensitive and specific method to study its pharmacokinetics. An HPLC method with modified postcolumn fluorescence derivatization to determine FOJ-5 in plasma was developed in this study. The Shodex Sugar KS-802 high-performance gel column was chosen for separating FOJ-5 from its degradation products and endogenous carbohydrates. The postcolumn procedure involved acid hydrolysis of the column eluate at 150 degrees C, which decreased the detection limit for FOJ-5 from 1 microg to 25 ng, followed by fluorometric reaction with guanidine in an alkaline medium at 90 degrees C. The clearance of FOJ-5 from the bodies of rats following intravenous injection displayed a complex type of kinetics involving at least two compartments, and the half-life of the elimination of FOJ-5 from plasma administered at 15 mg/kg (18.1 min) was quicker than that administered at 50mg/kg (28.9 min). This modified approach can also be used for microanalysis of both nonreducing oligosaccharides and other neutral polysaccharides.

Animals↗
PubMed2004

Production of fructo-oligosaccharides from molasses by Aureobasidium pullulans cells.

Three different strains of Aureobasidium pullulans were grown in batch cultures to compare their abilities for the production of fructo-oligosaccharides. Specific intracellular enzyme activity was the highest with strain KCCM 12017 and enzyme production was closely coupled to growth. Using A. pullulans cells, 166 g/l fructo-oligosaccharides was produced from 360 g/l molasses sugar as sucrose equivalent at 55 degrees C and pH 5.5 after 24 h incubation.

Agriculture↗
PubMed2004

Identification and characterization of a novel inulin binding module (IBM) from the CFTase of Bacillus macerans CFC1.

A novel inulin-binding module (IBM), which was identified from the N-terminal region of the cycloinulinooligosaccharide fructanotransferase (CFTase) in Bacillus macerans CFC1, was characterized using the discrete entity of IBM produced by the recombinant Escherichia coli strains. Deletion analyses located the inulin binding activity in the N-terminal region between 241 and 389 amino acid residues, which was removed from the mature enzyme by processing when secreted from the B. macerans CFC1 cells. IBM bound specifically to polyfructans such as inulin and levan but it did not interact with any of the glycan polymers tested in this study including cellulose, xylan, and starch. Binding studies on the IBM revealed that the equilibrium dissociation constant K(d) and the maximum amount of protein bound [(PC)(max)] were 4.7 microM and 22 microM g(-1), respectively. Together, these results indicate that the IBM of CFTase has a relatively high and specific affinity for inulin. Adsorption of the IBM to inulin was highest at pH 7.0 and lowered slowly with decreasing pH down to 3.0. At pH 7.0, the binding activity was enhanced about twofold by the presence of 1 M MgCl(2). Chemical modification experiments with the aromatic amino acid-specific modifiers implied that tryptophan and tyrosine residues in the IBM are likely to participate in the interaction with the inulin molecules.

Bacillus↗
PubMed2004

Development of PCR assay to identify Pseudomonas fluorescens and its biotype.

The paper provides a simple protocol that uses the polymerase chain reaction to amplify a specific portion of the 16S gene, allowing the recognition of Pseudomonas fluorescens from other group I Pseudomonas. The amplified DNA patterns of 16S rRNA and ITS1, from the restriction fragment length polymorphisms VspI, HaeIII and TaqI digestion, produced band patterns that distinguished the biotypes of Ps. fluorescens. In addition to distinguishing the biotypes C and 3 we used a phenotypical method for levan production.

Bacterial Typing Techniques↗
PubMed2006

Dietary microbial levan enhances cellular non-specific immunity and survival of common carp (Cyprinus carpio) juveniles.

A preliminary study with a 75days feeding trial was conducted to study the immunomodulatory effect of microbial levan on Cyprinus carpio juveniles. Five purified isonitrogenous and isocaloric diets with graded levels of levan, namely (T(1)) 0.1% levan, (T(2)) 0.2% levan, (T(3)) 0.5% levan, (T(4)) 1.0% levan, and a control group without levan were fed to five groups of fishes in triplicate. The total erythrocyte count and haemoglobin content was significantly (p<0.05) enhanced in the T(3) group, though the total leucocyte counts, total protein and A/G ratio did not show any significant difference (p>0.05) when compared with the control group. The respiratory burst activity (NBT) of blood phagocytes and lysozyme activity was also highest in T(3) group. The relative survival percentage after challenge with Aeromonas hydrophila was highest (100%) in the T(3) group followed by 83.33% and 66.67% in the T(2) and T(4) groups, respectively. This suggests that microbial levan at 0.5% can be used as a dietary immunostimulant for C. carpio juveniles. This is the first report on microbial levan having an immunomodulatory effect on C. carpio.

Adjuvants, Immunologic↗
PubMed2004

Antitumor activity of levan polysaccharides from selected microorganisms.

Levans were isolated from the cultures of Gluconoacetobacter xylinus (G-levan; Mw = 40,000), Microbacterium laevaniformans (M; Mw = 710,000), Rahnella aquatilis (R; Mw = 380,000), and Zymomonas mobilis (Z; Mw = 570,000). The levans were composed mainly of fructose residues when analyzed by TLC and HPLC, and their main backbones were beta-(2,6)-linkages with beta-(2,1)-branches by GC-MS and NMR. In the in vitro antitumor activity test of the levans against eight different tumor cell lines, relatively stronger activity was observed from the SNU-1 and HepG2. The M- (52.54-62.05%) and R-levan (52.15-58.58%) showed the significantly high activity against SNU-1, while M-levan showed the highest (49.93-61.82%) activity against HepG2. During the in vivo analysis of inhibitory activity of the levans against Sarcoma-180 growth, M-, R- and Z-levans showed strong antitumor activity (average 66%) but G-levan (42%) had significantly lower activity.

Actinomycetales↗
PubMed2004

Effect of levan's branching structure on antitumor activity.

Levan produced from Microbacterium laevaniformans KCTC 9732 (M-levan) was isolated and treated with an inulinase to modify its branching structure. The chemical structures of levans were characterized, and the modified levans were applied on animal tumor cells to evaluate their antitumor activity. The GC-MS analysis indicated that beta-(2,1)-linked branches of M-levan were specifically hydrolyzed. As the ratio of applied inulinase to levan increased, the branching degree decreased proportionally. Sequential degrees of branching were obtained from 12.3 to 4.2%. Strong levan-induced inhibition of cell growth was detected on SNU-1 and HepG2 tumor cell lines. As the branching degree of M-levan reduced, antitumor activity on SNU-1 linearly decreased (r2=0.96). In HepG2, the antitumor activity rapidly dropped when the branching reached up to 9.3%, then slightly increased as the branching degree of M-levan further decreased. These results suggested that the branch structure would play a crucial role in levan's antitumor activity.

Actinomycetales↗
PubMed2004

Synthesis of levan in water-miscible organic solvents.

The synthesis of levan using a levansucrase from a strain of Bacillus subtilis was studied in the presence of the water-miscible solvents: acetone, acetonitrile and 2-methyl-2-propanol (2M2P). It was found that while the enzyme activity is only slightly affected by acetone and acetonitrile, 2M2P has an activating effect increasing the total activity 35% in 40-50% (v/v) 2M2P solutions at 30 degrees C. The enzyme is highly stable in water at 30 degrees C; however, incubation in the presence of 15 and 50% (v/v) 2M2P reduced the half-life time to 23.6 and 1.8 days, respectively. This effect is reversed in 83% 2M2P, where a half-life time of 11.8 days is observed. The presence of 2M2P in the system increases the transfer/hydrolysis ratio of levansucrase. As the reaction proceeds with 10% (w/v) sucrose in 50/50 water/2M2P sucrose is converted to levan and an aqueous two-phase system (2M2P/Levan) is formed and more sucrose can be added in a fed batch mode. It is shown that high molecular weight levan is obtained as an hydrogel and may be easily recovered from the reaction medium. However, when high initial sucrose concentrations (40% (w/v) in 50/50 water/2M2P) are used, an aqueous two-phase system (2M2P/sucrose) is induce, where the synthesized levan has a similar molecular weight distribution as in water and remains in solution.

Acetone↗
PubMed2005

Altered mRNA expression of hepatic lipogenic enzyme and PPARalpha in rats fed dietary levan from Zymomonas mobilis.

Levan or high molecular beta-2,6-linked fructose polymer is produced extracellularly from sucrose-based substrates by bacterial levansucrase. In the present study, to investigate the effect of levan feeding on serum leptin, hepatic lipogenic enzyme and peroxisome proliferation-activated receptor (PPAR) alpha expression in high-fat diet-induced obese rats, 4-week-old Sprague-Dawley male rats were fed high-fat diet (beef tallow, 40% of calories as fat), and, 6 weeks later, the rats were fed 0%, 1%, 5% or 10% levan-supplemented diets for 4 weeks. Serum leptin and insulin level were dose dependently reduced in levan-supplemented diet-fed rats. The mRNA expressions of hepatic fatty acid synthase and acetyl CoA carboxylase, which are the key enzymes in fatty acid synthesis, were down-regulated by dietary levan. However, dietary levan did not affect the gene expression of hepatic malic enzyme, phosphatidate phosphohydrolase and HMG CoA reductase. Also, the lipogenic enzyme gene expression in the white adipose tissue (WAT) was not affected by the diet treatments. However, hepatic PPARalpha mRNA expression was dose dependently up-regulated by dietary levan, whereas PPARgamma in the WAT was not changed. The results suggest that the in vivo hypolipidemic effect of dietary levan, including anti-obesity and lipid-lowering, may result from the inhibition of lipogenesis and stimulation of lipolysis, accompanied with regulation of hepatic lipogenic enzyme and PPARalpha gene expression.

Acetyl-CoA Carboxylase↗
PubMed2003

Interaction forces between particles containing grafted or adsorbed polymer layers.

The interaction forces between particles containing grafted or adsorbed polymer layers have been investigated using rheological and surface force measurements. Polystyrene latex dispersions with grafted poly(ethylene oxide) (PEO) chains (M=2000) were used for the rheological measurements. Results were also obtained for latex dispersions stabilised with adsorbed graft copolymers of poly(methyl methacrylate-methacrylic acid) with methoxy capped PEO chains (M=750). The relative viscosity eta(r)-volume fraction phi curves for the latex dispersions with grafted PEO chains were established for three particle radii of 77.5, 306 and 502 nm. For comparison the eta(r)-phi curve was calculated using the Dougherty-Krieger equation. This allows one to obtain the adsorbed layer thickness delta as a function of phi. The results showed a decrease of delta with increase of phi, which was attributed to the interpenetration and/or compression of the PEO chains on increasing phi. Viscoelastic measurements as a function of phi showed a change from predominantly viscous to predominantly elastic response at a critical volume fraction, which indicated the onset of the strong steric repulsion when the polymer layers begin to overlap. A similar trend was obtained with the latex particles containing adsorbed graft copolymer layers. A scaling law was used to fit the elastic part of the logG'-log phi curve (where G' is the elastic modulus). This fit could be used to estimate the compressibility of the PEO chains. The correlation of the rheology of concentrated sterically stabilised dispersions with interparticle interactions was investigated by measuring the energy-distance curves for the graft copolymer that was adsorbed on smooth mica sheets. Using de Gennes scaling theory, it was possible to calculate the energy of interaction between the polymer layers. The high frequency modulus of the latex dispersions was obtained as a function of the volume fraction and the results were compared with those calculated from the potential of the mean force. The trends in the variation of the modulus with volume fraction were similar for the experimental rheological results and those calculated using the directly measured interaction forces. The above results demonstrated the powerful use of rheological measurements for studying the interaction between sterically stabilised dispersions in concentrated systems.

Adsorption↗
PubMed2000

Plasma clearance of polyfructosan and extracellular body fluid distribution in idiopathic dilated cardiomyopathy and after heart transplantation.

The total extracellular fluid volume and distribution in plasma and interstitial spaces, and the microvascular permeability properties were studied in 16 nonedematous patients with congestive heart failure (CHF) due to idiopathic dilated cardiomyopathy and 17 such patients who underwent heart transplantation (HT) by analyzing the 3-hour plasma disappearance curve of polyfructosan. Eighteen healthy subjects served as controls. Polyfructosan (3.5 kD) is an extracellular marker and inulin analog transported almost solely by diffusion. The initial capillary membrane plasma clearance (i.e., the permeability-surface area product), the interstitial plasma clearance determined at 10 minutes (clearance[10), and the extracellular volume were determined from the polyfructosan curves. I-131-albumin was used as a plasma volume reference. Permeability-surface area product was elevated in both patient groups (6.6 +/- 1.9 ml/ kg/min in the CHF group and 6.7 +/- 2.0 ml/kg/min in the HT group vs 5.1 +/- 1.3 ml/kg/min in controls, p <0.01 for both), whereas clearance(10) was normalized in the HT group (4.5 +/- 0.9 ml/kg/min in the HT group, 4.4 +/- 0.7 ml/kg/min in controls vs 5.0 +/- 0.9 ml/kg/ min in the CHF group, p <0.1 and p <0.05, respectively). The normalization of interstitial plasma clearance of polyfructosan was associated with time since HT (r = 0.49, p <0.05). Plasma volumes were similar in all 3 groups (41 +/- 8 ml/kg in controls, 44 +/- 13 in the CHF group and 39 +/- 8 in the HT group). In contrast, total extracellular volume was elevated in both patients groups (177 +/- 27 ml/kg in the CHF group and 173 +/-27 in the HT group vs 152 +/- 12 in controls, p <0.01). The results strongly suggest a microvascular permeability defect in both patient groups that perhaps plays a role in the extravascular distribution of the excess extracellular fluid volume.

Adult↗