An in vivo model of hyperacute rejection: characterization and evaluation of the effect of transgenic human complement inhibitors

Hyperacute rejection (HAR) occurring after transplantation within phylogenetically distant species is a severe reaction triggered by preexisting xenoreactive antibodies and complement activation, leading to the destruction of the donor organ. Expression of human complement inhibitors in transgenic pig organs prolongs the survival of xenograft in experimental models. Moreover, the extent of protection from hyperacute rejection is dependent on the level and site of expression of the transgenic molecules and, probably, on the combination of different molecules. In this regard a small animal model to test the efficacy of expression vectors and different human molecules could be very advantageous. A murine model developed in our laboratory was characterized by measurement of several parameters characteristic of HAR in the livers of control and transgenic mice expressing transgenic human DAF (CD55) or MCP (CD46) at the end of 2h of perfusion with human plasma and after 1 day. The parameters studied were heamatological values of hepatic functions (GOT and GPT), induction of pro-inflammatory molecules and histopathological evaluation. Cytokines (IL-1, IL-1, IL-6) induction and exposure of P-selectin on the endothelial cell surface, was only observed in control animals after 2h of perfusion, as an early event. GOT and GPT values increase drammatically after 2h perfusion and 1 day after the treatment according to the histopathological observation of liver damage. On the contrary, the livers of hDAF or hMCP transgenic mice, under the same treatment were significantly protected although the extent of this protection is dependent on the level of expression of transgenic human molecules.     

Lusitropic effects of α- and β-adrenergic stimulation in amphibian heart

The effects of and -adrenergic stimulation in amphibian superfused hearts and ventricular strips were studied. Superfusion with 3×10^–8 M isoproterenol produced a positive inotropic effect, as detected by a 92±24% increase in the maximal rate of contraction and a positive lusitropic effect characterized by a decrease in both the ratio (23±5%) and the half relaxation time (t_1/2) (19±4%). The mechanical behavior induced by the -agonist was associated with an increase in the intracellular cAMP levels from control values of 173±19 to 329±28 nmol/mg wet tissue. Hearts superfused with³²P in the presence of isoproterenol showed a significant increase in Tn 1 phosphorylation (from 151±13 to 240±44 pmol³²P/mg MF protein) without consistent changes in phosphorylation of C-protein. In sarcoplasmic reticulum membrane vesicles, no phospholamban phosphorylation was detected either by -adrenergic stimulation of superfused hearts or when phosphorylation conditions were optimized by direct treatment of the vesicles with cAMP-dependent protein kinase (PKA) and [y ³²P] ATP.The effect of -adrenergic stimulation on ventricular strips was studied at 30 and 22°C. At 30°C, the effects of 10^–5 to 10^–4M phenylephrine on myocardial contraction and relaxation were diminished to non significant levels by addition of propranolol. At 22°C, blockage with propranolol left a remanent positive inotropic effect (10% of the total effect of phenylephrine) and changed the phenylephrine-induced positive lusitropic effect into a negative lusitropic action. These propranolol-resistant effects were abolished by prazosin. Our results suggest that in amphibian heart, both the inotropic and lusitropic responses to catecholamines are mainly due to a -adrenergic stimulation which predominates over the -adrenergic response. Phospholamban phosphorylation seems not to be involved in mediating the positive lusitropic effect of -adrenergic agents whereas phosphorylation of troponin 1 may play a critical role.     

Homocysteine stimulates the expression of monocyte chemoattractant protein-1 in endothelial cells leading to enhanced monocyte chemotaxis

The effect of intraperitoneal administration of tocopherol (100 mg/kg wt/24 h) on ascorbate (0.4 mM) induced lipid peroxidation of mitochondria and microsomes isolated from rat liver and testis was studied. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids C20:4 n6 and C22:6 n3 in liver and C20:4 n6 and C22:5 n6 in testis. The lipid peroxidation of liver mitochondria or microsomes produced a significant decrease of C20:4 n6 and C22:6 n3 in the control group, whereas changes in the fatty acid composition of the tocopherol treated group were not observed. The light emission was significantly higher in the control than in the tocopherol treated group. The lipid peroxidation of testis microsomes isolated from the tocopherol group produced a significant decrease of C20:4 n6 , C22:5 n6 and C22:6 n3, these changes were not observed in testis mitochondria. The light emission of both groups was similar. The treatment with tocopherol at the dose and times indicated showed a protector effect on the polyunsaturated fatty acids of liver mitochondria, microsomes and testis mitochondria, whereas those fatty acids situated in testis microsomes were not protected during non enzymatic ascorbateFe²⁺ lipid peroxidation. The protector effect observed by tocopherol treatment in the fatty acid composition of rat testis mitochondria but not in microsomes could be explained if we consider that the sum of C20:4 n6 + C22:5 n6 in testis microsomes is 2-fold than that present in mitochondria.     

Immunological evaluation of patients with hematological malignancies receiving ambulatory cytokine-mediated immunotherapy with recombinant human interferon-α2a and interleukin-2

Summary Immunological parameters were evaluated in patients treated with cytokine-mediated immunotherapy (CMI) consisting of low doses of recombinant human interferon 2a (rIFN) and recombinant human interleukin-2 (rIL-2) administered either concomitantly or sequentially by subcutaneous self-injections in an outpatient setting. Twenty-six patients with hematological malignancies and 2 metastatic melanoma patients in a progressive stage were enrolled in this clinical trial. Of the 26 patients, 24 were at a stage of minimal residual disease, including 14 patients who had received autologous bone marrow transplantation (ABMT) 2–5 months previously, 7 chronic myelogenous leukemia (CML) and 3 acute myeloid leukemia (AML) patients. Two patients (1 CML and 1 mult. myeloma) were treated at a stage of progressive disease. Non-MHC-restricted cytotoxicity directed against natural-killer(NK)-resistant (Daudi) and NK-sensitive (K562) target cells was assessed before, during and after CMI, either in fresh peripheral blood samples (spontaneous activity) or after in vitro rIL-2 activation (induced activity). Spontaneous killing activity was low prior to treatment, but increased upon termination of treatment in 10/15 evaluated cycels. rIL-2-activated cytotoxicity in vitro was markedly elevated in 8/12 and 6/8 patients after one and two cycles, respectively, of sequential treatment, as well as in 3/8 CML and 5/6 patients after one and two cycles, respectively, of concomitant treatment Activation of the T cell mitogenic response was demonstrated in 6/9 patients after concomitant CMI, while no such effect was observed throughout a sequential treatment in lymphoma and leukemia patients after ABMT. Although a direct correlation between immune stimulation and the in vivo antitumor response cannot yet be determined, our clinical observations support a beneficial therapeutic effect in a substantial number of patients. These results indicated that the ambulatory CMI protocol of rIL-2 and rIFN could stimulate the host defense immune system and may be helpful in mediating the in vivo antitumor response in patients with minimal residual disease.     

β-Carotene production by Flavobacterium multivorum in the presence of inorganic salts and urea

Flavobacterium multivorum, a non-fermenting Gram-negative bacteria, normally produces zeaxanthin (3R, 3 R-, -carotene-3, 3 diol) as its main carotenoid. The effect of supplementation of various inorganic salts and urea on the growth, total carotenoid production, and proportion of -carotene (, -carotene), -cryptoxanthin (, -caroten-3-ol), and zeaxanthin produced by F. multivorum was investigated. Urea and several salts, such as calcium chloride, ammonium chloride, lithium chloride, and sodium carbonate, improved total carotenoid production by 1.5- to 2.0-fold. Urea and sodium carbonate had an unexpectedly strong positive effect on -carotene production at the expense of zeaxanthin formation. The effect was found to be independent of incubation time, and -carotene represented 70% (w/w) of the total carotenoid content. The cumulative effect of urea and sodium carbonate was further studied using response surface methodology. An optimum medium was found to contain 4,000 and 4,070 mg l^–1 urea and sodium carbonate, respectively. The maximum -carotene level was 7.85 g ml^–1 culture broth, which represented 80% (w/w) of the total carotenoid produced. Optimization resulted in 77- and 88-fold improvements in the volumetric and specific -carotene levels, respectively, accompanied by a simultaneous decrease in the zeaxanthin level as compared to the control medium. The carotenoid production profile in the optimized medium indicated that -carotene was produced maximally during the late exponential phase at 0.41 g ml^–1 h^–1. It is possible that this organism could be an excellent commercial source of either -carotene or zeaxanthin, depending on initial culture conditions.     

Isolation and separation of α-amylase inhibitors I-1 and I-2 from seeds of ragi (Indian finger millet, Eleusine coracana) by metal chelate affinity precipitation

The concept of immobilized metal affinity chromatography (IMAC) was integrated with affinity precipitation for the single step isolation of -amylase inhibitors I-1 and 1-2 from the seeds of ragi (Indian finger millet, Eleusine coracana). -Amylase inhibitor I-1 was purified 13-fold with a yield of 84%, using Cu(II) loaded thermosensitive metal chelate copolymer of N-isopropylacrylamide (NIPAM) and 1-vinyl imidazole (VI). The protein also showed trypsin inhibitory activity. The binding of the protein to the copolymer was strongly pH dependent. -Amylase inhibitor I-2 was recovered in the supernatant as unprecipitated protein with significant purification and constituted 27% of the total inhibitor power. The yield with respect to inhibitor I-2 was around 85%. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed significant purification of inhibitor I-1 and indicated evident separation of the two proteins on metal chelate affinity precipitation.     

Glutathione and glutathione conjugate efflux from cultured liver cells

Efflux of glutathione (GSH) and GSH-conjugates from cultured rat liver epithelial cell lines; the non-tumorigenic ARL-15C₁ and the -glutamyl transpeptidase containing, tumorigenic ARL-16T₂, has been assessed under basal condition and during chronic treatment with 75 and 150 M ethacrynic acid (EA). The intracellular level of GSH increased in proportion to EA concentration during chronic exposure. The rates of GSH and GSH-EA conjugate efflux increased with intracellular GSH in both ARL cell lines.Glutathione-S-transferase activity measured with EA as substrate increased over the experimental time course after treatment with 150, but not 75 M EA. When intracellular GSH content was increased by treatment with the cysteine pro-drug, 2-L-oxothiazolidine 4-carboxylic acid, the rate of GSH efflux was increased, but not the rate of GS-EA conjugate export. Inhibition of -glutamyl transpeptidase by acivicin (AT-125) increased the GSH and GS-EA conjugate efflux rate in ARL-16T₂ cells by factors of approximately 2 and 15, respectively. Acivicin treatment of ARL-16T₂ cells chronically treated with EA elevated GSH efflux rate by 10-fold and GS-EA efflux by 40-fold versus control samples. These studies show that GSH and GSH conjugate efflux are accomplished as independently regulated processes. Efflux of GSH is enhanced by increased in racellular GSH, but increase in the conjugate transport rate requires the presence of the GSH conjugate. The response of the efflux process to treatment with a chronic GSH depleting agent was identical in two cell lines in which the metabolic fate of glutathione is known to differ fundamentally.Abbreviations GSH reduced glutathione - GSSG oxidized glutathione - GS-EA the glutathione conjugate of ethacrynic acid - EA ethacrynic acid - CDNB 1-chloro 2,4-dinitrobenzene - HBS HEPES buffered saline - OTC 2-L-oxothiazolidine 4-carboxylic acid - CYSSG cysteinyl-glutathione mixed disulfide - FDNB 1-fluoro-2,4-dinitrobenzene - GCS -glutamyl cysteine synthetase - GST glutathione-S-transferase - BCA bicinchoninic acid - SDS sodium dodecyl sulfate - PCA perchloric acid     

Activities ofβ-glucanases andβ-glucosidases during blastospore formation inSaccharomycopsis fibuligera

Summary The activities of three glycosidases, -glucosidase and (1,3)- and (1,6)-glucanases have been monitored during growth and blastospore formation inSaccharomycopsis fibuligera. The assays were carried out on the cell-free culture and in a cell-free extract and a wall autolysate preparation from the growing cells. In complex medium containing 1% glucose an increase in the level of all three enzymes was associated with the transition from mycelium to blastospores. When the level of glucose was increased to 5% blastospore formation was repressed and the level of -glucanases only increased at the end of the fermentation. The -glucosidase activity increased during the growth phase. In a defined medium in which slow growth in a wholly yeast-like form was observed, growth was not associated with a high level of -glucanase activity.     

Na-butyrate increases the production and α2,6-sialylation of recombinant interferon-γ expressed by α2,6- sialyltransferase engineered CHO cells

A non-human like glycosylation pattern in human recombinant glycoproteins expressed by animal cells may compromise their use as therapeutic drugs. In order to correct the CHO glycosylation machinery, a CHO cell line producing recombinant human interferon- (IFN) was transformed to replace the endogenous pseudogene with a functional copy of the enzyme 2,6-sialyltransferase (2,6-ST). Both the parental and the modified CHO cell line were propagated in serum-free batch culture with or without 1 mM sodium butyrate. Although Na-butyrate inhibited cell growth, IFN concentration was increased twofold. The IFN sialylation status was determined using linkage specific sialidases and HPLC. Under non- induced conditions, IFN expressed by 2,6-engineered cells contained 68% of the total sialic acids in the 2,6- conformation and the overall molar ratio of sialic acids to IFN was 2.3. Sodium butyrate addition increased twofold the molar ratio of total sialic acids to IFN and 82% of total sialic acids on IFN were in the 2,6-conformation. In contrast, no effect of the sodium butyrate was noticed on the sialylation of the IFN secreted by the 2,6-ST deficient parental cell line. This study deals for the first time with the effect of Na-butyrate on CHO cells engineered to produce human like sialylation.     

Arginine specific endopeptidases modify the aggregation properties of a synthetic peptide derived from Alzheimer β/A4 amyloid

A synthetic peptide corresponding to the first 28 amino acids of the Alzheimer disease amyloid /A4 peptide (3.2 kDa) aggregated to a high molecular weight (15 kDa) on SDS/urea polyacrylamide gels. Proteinase K, V8 protease, trypsin, and endopeptidase Lys-C readily degraded the aggregate. By contrast, when digested by endopeptidase Arg-C, a new polypeptide aggregate of higher molecular weight (16 kDa) was observed on denaturing gels without degraded smaller products. The new aggregate was comprised of three peptides: an intact /A4(1–28) and partially degraded peptides /A4(1–5) plus /A4(6–28). The results were confirmed by treatment of /A4 with other arginine-specific proteases: the gamma subunit of nerve growth factor and clostripain. The results indicate that arginine-specific proteases, including a growth factor processing enzyme, can nick aggregated /A4(1–28) amyloid and alter the configuration to produce a complcomple aggregated form. If similar highly specific proteolytic mechanisms occur in the Alzheimer disease brain, the processing may promote the formation of high molecular weight aggregates that contribute to the development of relatively insoluble senile plaque core protein.     

Combination of induced autolysis and sodium hypochlorite oxidation for the production of Saccharomyces cerevisiae (1 → 3)-β-D-glucan

(1 3)--D-Glucans have received much attention with respect to their biological functions. A novel method to extract (1 3)--D-glucan from Saccharomyces cerevisiae cell wall is proposed in present work, which is based on the combination of induced autolysis and subsequent oxidation of the autolysed cell by sodium hypochlorite to remove undesirable substances. Influences of temperature, pH value and organic solvent on S. cerevisiae FL 1 autolysis were investigated. Results indicated that each factor had its significant effect on induced autolysis and the optimal conditions were 52 °C, pH 5.5 and 1.5% (v/v) ethyl acetate. The kinetic behaviour of the yeast autolytic process under the optimized conditions was further studied. After 36 h of autolysis, 42.0% (w/w) cellular substances were released while the cell wall nearly remained intact. Finally, an ideal glucan yield as high as 22.9% (w/w) was obtained when S. cerevisiae FL 1 was treated by the novel method.     

Protective specific immunity induced by cyclophosphamide plus tumor necrosis factor α combination treatment of EL4-lymphoma-bearing C57BL/6 mice

A combination treatment protocol initiated 12 days after tumor injection, when the tumor was large, by administering cyclophosphamide (CY, 150 or 250 mg/kg) intraperitoneally followed by intravenous tumor necrosis factor (TNF, 1000 units/injection) on days 13, 16, 18, 21, and 23, resulted in about 60% longterm survival (i.e., survival for at least 60 days) in the syngeneic C57BL/6 mouse/EL4 lymphoma model system. The establishment of a specific antitumor immune memory and its possible therapeutic relevance was verified by reinjecting 60-day survivors with EL4 cells; all 60-day survivors that had received the combination treatments rejected the implants and survived for a further 60 days. Thymic cellularity was reduced during treatment and its recovery appeared to correlate with long-term survival and immunity. Thymocytes from mice treated with the combination were found to express significant levels of specific anti-EL4 cytolytic activity following a 4-day stimulation culture with X-irradiated EL4 cells and low concentrations of interleukin-2. This response could not be generated with thymocytes from naive animals. In each case the effect seen with the combination of a moderate CY dose (150 mg/kg) with TNF was better than that seen with either dose of CY alone and equal to or better than that seen with the higher dose of CY combined with TNF. These results indicate that treatment with a single moderate dose of CY in combination with TNF is effective against a large, established tumor in this murine model. Furthermore, all the long-term survivors induced by this treatment developed protective immunity against reimplanted tumor and demonstrated a long-term specific immune memory in the thymus.This work was done in partial fulfillment of the M.S. degree requirements (C. M. K.) in the Program of Natural Sciences, Roswell Park Graduate Division, SUNY at Buffalo     

Effects of Aging and Amyloid-β Peptides on Choline Acetyltransferase Activity in Rat Brain

Choline acetyltransferase (ChAT, acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6), involved in the learning and memory processes is responsible for the synthesis of acetylcholine. There are many discrepancies in literature concerning ChAT activity during brain aging and the role of amyloid beta peptides in modulation of this enzyme. The aim of the study was to investigate the mechanism of ChAT regulation and age-related alteration of ChAT activity in different parts of the brain. Moreover the effect of A peptides on ChAT activity in adult and aged brain was investigated. The enzyme activity was determined in the brain cortex, hippocampus and striatum in adult (4-months-old), adult-aged (14-months-old) and aged (24-months-old) animals. The highest ChAT activity was observed in the striatum. We found that inhibitors of protein kinase C, A, G and phosphatase A2 have no effect on ChAT activity and that this enzyme is not dependent on calcium ions. About 70% of the total ChAT activity is present in the cytosol. Arachidonic acid significantly inhibited cytosolic form of this enzyme. In the brain cortex and striatum from aged brain ChAT activity is inhibited by 50% and 37%, respectively. The aggregated form of A 25-35 decreased significantly ChAT activity only in the aged striatum and exerted inhibitory effect on this enzyme in adult, however, statistically insignificant. ChAT activity in the striatum was diminished after exposure to 1 mM H₂O₂. The results from our study indicate that aging processes play a major role in inhibition of ChAT activity and that this enzyme in striatum is selectively sensitive for amyloid beta peptides.     

Uniform GFP-expression in transgenic medaka (Oryzias latipes) at the F0 generation

A green fluorescent protein (GFP) cDNA flanked by inverted terminal repeats (ITR) of adeno-associated virus was constructed. The construct sharply improved the efficiency and specificity of the transient expression of genes driven by two general promoters (cytomegalovirus and medaka -actin) and one muscle-specific promoter (zebrafish -actin) in transgenic medaka. In addition, treatment with ITR sequence-containing constructs resulted in a dramatic increase in the number of embryos showing uniform GFP-expression at F0. Of the GFP-positive embryos, 34.6% (81/234), 10% (10/60), and 18% (38/212) showed homogenous GFP-expression for the derivative constructs of the cytomegalovirus, -actin, and -actin promoters, respectively. As a result of uniform GFP-expression, green fluorescence in founders was (a) extended for an entire lifetime without degradation, and (b) transmitted as a genetic trait to F1 and F2 progeny of some transgenic lines via Mendelian inheritance. A Southern blot analysis revealed a random integration of the transgene into the genome of founders and progeny in both head-to-tail and tail-to-tail concatemerization patterns. Interestingly, some transgenic medaka with uniform and strong fluorescence could be visually noticeable to the unaided eye.     

Purification and characterization of β‐methylaspartase from Fusobacterium varium

Methylaspartase (EC 4.3.1.2) was purified 20fold in 35% yield from Fusobacterium varium, an obligate anaerobe. The purification steps included heat treatment, fractional precipitation with ammonium sulfate and ethanol, gel filtration, and ion exchange chromatography on DEAESepharose. The enzyme is dimeric, consisting of two identical 46 kDa subunits, and requires Mg²⁺ (K_m = 0.27 ± 0.01 mM) and K⁺ (K_m = 3.3 ± 0.8 mM) for maximum activity. Methylaspartasecatalyzed addition of ammonia to mesaconate yielded two diastereomeric amino acids, identified by HPLC as (2S,3S)3methylaspartate (major product) and (2S,3R)3methylaspartate (minor product). Optimal activity for the deamination of (2S,3S)3methylaspartate (K_m = 0.51 ± 0.04 mM) was observed at pH 9.7. The Nterminal protein sequence (30 residues) of the F. varium enzyme is 83% identical to the corresponding sequence of the clostridial enzyme.     

Effect of 17β-estradiol on calcium response to phytohaemagglutinin in human lymphocytes

Pre-treatment of human lymphocytes with 17-estradiol diminishes the increase in concentration of cytosolic free calcium after stimulation with phytohaemagglutinin. The effect is dependent on 17-estradiol concentration and on the preincubation time. The effect is not due to an interaction between 17-estradiol and phytohaemagglutinin, but appears to be a consequence of the binding of the hormone to the cell surface. The effect is specific for 17-estradiol, since the isomer and other steroid hormones (progesterone, testosterone, diethylstilbestrol and 5-androstan), have no effect. Since the effect of the 17-estradiol can be suppressed by treatment of lymphocytes with ouabain, it appears that the effect of estradiol on the rise of cytosolic calcium induced by phytohaemagglutinin is mediated by the (Na, K)-ATPase.     

Silver nitrate and aminoethoxyvinylglycine affect <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated apple transformation

In Fuji, the production of ethylene was increased with the addition of AgNO₃ and inhibited with the addition of 10 M aminoethoxyvinylglycine (AVG). The addition of 80 M AgNO₃ to transformed explants of Fuji cultured on selection medium resulted in increased ethylene production (20 l l^–1) at 3 weeks. Under examining the effect of AgNO₃ in Fuji, the 40 M AgNO₃ showed with higher 33.8% and 6.5% in the efficiency of regeneration and transformation. However, ethylene production in Gala explants treated with 10M AgNO₃ (3 l l^–1) decreased after 2 weeks compared with the control (5 l l^–1). Although the regeneration efficiency of Gala with 10 M AgNO₃ was higher (41.1%) than the control (20.1%), there was no significant difference in the transformation efficiency at the same concentration. Shoot regeneration of Fuji and Gala was completely inhibited with 10 M AVG. These results suggest that the addition of AgNO₃ affects the efficiency of Agrobacterium-mediated gene transfer in Fuji.Eun Soo Seong, Ill Min Chung- These two Authors Contributed equally to this work     

Partitioning and separation of α-lactalbumin and β-lactoglobulin in polyethylene glycol/ammonium sulphate aqueous two-phase systems

The partition behaviour of -lactalbumin (la) and -lactoglobulin (lg) on PEG/(NH₄)₂SO₄ system was studied. For purified proteins, a partition coefficient of 12.8 for la and 0.34 for lg, with mass recovery yields of 96.7% for la in the upper phase and 83.8% for lg in the lower phase was obtained, in 18% (w/w) PEG 900/14% (w/w) (NH₄)₂SO₄ system, at pH 7. PEG/(NH₄)₂SO₄ system was an economical alternative for the recovery and separation of the two proteins in cheese whey, allowing a 50% reduction in costs. An efficient and inexpensive separation of both proteins in cheese whey could be achieved, by using 16% (w/w) PEG 900/15% (w/w) (NH₄)₂SO₄, at pH 7.5.     

Bacillus Calmette-Guérin potentiates monocyte responses to lipopolysaccharide-induced tumor necrosis factor and interleukin-1, but not interleukin-6 in bladder cancer patients

During the past decade, particular attention has been focused on treatment of bladder cancer patients with the bacterial agent bacillus Calmette-Guérin (BCG). In these studies, bladder cancer patients were instilled with BCG (75mg/50ml) once per week for 6 weeks, 1–2 weeks following trans-urethreal resection of the bladder. Cystoscopy was performed after 6 weeks and, unless tumor progression was present, monthly treatments were given for 1 year. Blood was drawn 2 h after the last instillation, and monocytes were isolated (5×10⁶ cells/ml) and treated, or not, with lipopolysaccharide (LPS) (20 g/ml) for tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6) release. The levels of monokines were determined by a monokine-specific enzyme-linked immunosorbent assay. Out results clearly show that, after 18 h incubation, macrophages from BCG-treated bladder cancer patients produced from 2.8- to 1.9-fold and from 2.0- to 1.3-fold greater amounts of TNF and IL-1 respectively, compared to macrophages from healthy controls, 5-fold higher than bladder cancer patients not treated with BCG. IL-6 was not affected. In another set of experiments macrophages (5×10⁶ cells/ml) from healthy subjects were pretreated, or not, with BCG (100 g/ml) overnight and treated, or not, with LPS 20 g/ml alone and in combination with interleukin-1 receptor antagonist (IL-1ra) 250 ng/ml. Macrophages treated with BCG had a strong stimulatory effect on IL-1 release (9.45 ng/ml) while LPS was less effective (3.59 ng/ml). The combination of BCG plus LPS produced an additive effect on IL-1 release (13.71 ng/ml) compared to the effect of the compound alone. The addition of IL-1ra (250 ng/ml) to BCG was not effective, while when IL-1ra was added to BCG plus LPS only a partial inhibition of IL-1 release was found (9.83 ng/ml), compared to BCG plus LPS without IL-1ra (13.71 ng/ml). These effects seem to be related to the inhibition of IL-1 stimulated with LPS, but not BCG. The priming effect of BCG exerted on LPS-stimulated monocyte production of TNF and IL-1 from bladder cancer patients led us to study the possible modulation of fibrinogen and C-reactive protein in the serum of BCG-treated cancer patients. The plasma levels of fibrinogen and C-reactive protein were higher (approximately twice) in BCG-treated patients compared to values obtained in untreated patients or healthy controls. We conclude that the beneficial immunotherapeutic effects of BCG in bladder cancer patients are related to its capacity to prime macrophages to enhance the release of TNF and IL-1, but not IL-6 in response to physiological secondary stimuli, or through the direct stimulation of BCG on IL-1 or TNF, which are directly involved in the killing of cancer cells. Moreover, the increase of IL-1 or TNF in BCG bladder cancer patients may lead to high plasma levels of fibrinogen and C-reactive protein, two proteins responsible for the acute-phase response.     

Synthesis of disaccharide derivatives employing β-N-acetyl- d-hexosaminidase, β-d-galactosidase and β-d-glucuronidase

-N-Acetyl-d-hexosaminidase from Aspergillus oryzae catalysed the stereo- and regiospecific formation of the 6-O-benzylated disaccharide derivatives GalNAc1-3(6- OBn)Gal-SEt and GlcNAc1-3(6-OBn)Gal-SEt, which were obtained in transglycosylation reactions employing ethyl 6- O-benzyl-1-thio--d-galactopyranoside as acceptor. Preparative amounts of the chitobiose derivative GlcNAc1- 3GlcNAc-OPhNO2-p was prepared as well. - N-Acetyl-d-hexosaminidase from bovine testes catalysed the specific synthesis of GlcNAc1-3(6-OBn)GlcNH2-SEt and GalNAc1-3(6-OBn)GlcNH2-SEt, employing ethyl 2-amino-6-O-benzyl-2-deoxy-1-thio--d-glucopyranoside as acceptor. -d-Glucuronidase from E. coli was found to catalyse the formation of GlcA1-3(6-OBn)GlcNH2- SEt employing the same acceptor.