Modulation of telomerase activity by telomere DNA-binding proteins in Oxytricha

Telomere proteins protect the chromosomal terminus from nucleolytic degradation and end-to-end fusion, and they may contribute to telomere length control and the regulation of telomerase. The current studies investigate the effect of Oxytricha single-stranded telomere DNA-binding protein subunits α and β on telomerase elongation of telomeric DNA. A native agarose gel system was used to evaluate telomere DNA-binding protein complex composition, and the ability of telomerase to use these complexes as substrates was characterized. Efficient elongation occurred in the presence of the α subunit. Moreover, the α–DNA cross-linked complex was a substrate for telomerase. At higher α concentrations, two α subunits bound to the 16-nucleotide single-stranded DNA substrate and rendered it inaccessible to telomerase. The formation of this α·DNA·α complex may contribute to regulation of telomere length. The α·β·DNA ternary complex was not a substrate for telomerase. Even when telomerase was prebound to telomeric DNA, the addition of α and β inhibited elongation, suggesting that these telomere protein subunits have a greater affinity for the DNA and are able to displace telomerase. In addition, the ternary complex was not a substrate for terminal deoxynucleotidyltransferase. We conclude that the telomere protein inhibits telomerase by rendering the telomeric DNA inaccessible, thereby helping to maintain telomere length.     

Effects of Porcine Circovirus Type 2 (PCV2) Maternal Antibodies on Experimental Infection of Piglets with PCV2

To determine the effects of porcine circovirus type 2 (PCV2) maternal antibodies on and response to experimental PCV2 infection, 24 piglets were divided into four groups on the basis of the enzyme-linked immunosorbent assay titers of PCV2 maternal antibodies: group A (n = 6; sample/positive [S/P] ratio, <0.2), group B (n = 5; S/P ratio, >0.2 to <0.5), and groups C (n = 8) and D (n = 5) (S/P ratio, >0.5). Piglets in groups A, B, and C were inoculated with PCV2 at day 0 and challenged with PCV2 at day 42. Group D piglets were not exposed to PCV2 at day 0 but were challenged at day 42. Before challenge, seroconversion to PCV2 antibodies occurred in fiveofsix group A piglets, and the antibody level rose above the cutoff level in oneoffive group B piglets. Viremia was detected in fiveofsix, fouroffive, and twoofeight pigs in groups A, B, and C, respectively. After challenge, PCV2 DNA was detectable from 7 to 21 days postchallenge in the sera from sixofsix, fouroffive, threeofeight, and fiveoffive pigs in groups A, B, C, and D, respectively. The results indicated that protection against PCV2 infection conferred by maternal antibodies is titer dependent: higher titers are generally protective, but low titers are not.     

Caffeoylquinic Acids Generated In Vitro in a High-Anthocyanin-Accumulating Sweet potato Cell Line

Accumulation of phenolic compounds has beenmonitored in a suspension culture of anthocyanin-accumulatingsweet potato cell line grown under the conditions ofmodified Murashige and Skoog high-anthocyanin productionmedium (APM) over a period of 24 days. Tissue samplesextracted with 15% acetic acid were analysed using HPLC at adetection wavelength of 326nm. Among others, the followingderivatives of caffeoylquinic acids were detected:4,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid,3,4-dicaffeoylquinic acid, and 3,4,5-tricaffeoylquinic acid. Theirtotal amount reached a maximum of 110mg/gFW between the 4thand the 15th day of culture growth on APM. The major compound ofthe phenolic mixture was 3,5-dicaffeoylquinic acid with maximumaccumulation level of 80mg/100gFW. The potentialeffects of targeted phenolic compounds on the nutraceuticalqualities of in vitro produced anthocyanin-rich extracts arediscussed.     

Evaluation of the In Vitro Pyrogen Test System Based on Proinflammatory Cytokine Release from Human Monocytes: Comparison with a Human Whole Blood Culture Test System and with the Rabbit Pyrogen Test

The reliability of an in vitro pyrogen test system based on proinflammatory cytokine release from human monocytic cells was assessed by comparison with a test system based on a human whole blood culture as well as with the conventional rabbit pyrogen test. The human cells used as the pyrogen indicator cells were newly selected by subcloning of a human monocytic cell line, Mono-Mac-6. The selected cells, named MM6-CA8, responded to various pyrogens, including endotoxin, peptidoglycan (PG), Staphylococcus aureus Cowan 1 (SAC), and poly(I·C), with a high sensitivity and produced proinflammatory cytokines, such as interleukin 1 (IL-1), IL-6, and tumor necrosis factor alpha. Among these cytokines, IL-6 was produced most sensitively in response to traces of the pyrogens and detected in the largest quantities in the culture medium. The cytokine-producing responses of MM6-CA8 cells correlated significantly with the responses of cultured human whole blood, which represents an ex vivo culture test system reproducing pyrogen-induced cytokine production in the human body. In terms of cytokine inducibility, the pyrogens were ranked in the order endotoxin > PG > poly (I·C) > SAC in both culture systems, a ranking which almost agreed with the ranking of their pyrogenicity as assessed by the rabbit pyrogen test. These results suggest that the in vitro responsiveness of MM6-CA8 cells to various pyrogens is highly relevant for human pyrogenic reactions. Therefore, the in vitro test system is useful and reliable for detecting the presence of materials that are pyrogenic for humans.     

Fidelity and Mutational Spectrum of Pfu DNA Polymerase on a Human Mitochondrial DNA Sequence

The study of rare genetic changes in human tissues requires specialized techniques. Point mutations at fractions at or below 10⁻⁶ must be observed to discover even the most prominent features of the point mutational spectrum. PCR permits the increase in number of mutant copies but does so at the expense of creating many additional mutations or “PCR noise”. Thus, each DNA sequence studied must be characterized with regard to the DNA polymerase and conditions used to avoid interpreting a PCR-generated mutation as one arising in human tissue. The thermostable DNA polymerase derived from Pyrococcus furiosus designated Pfu has the highest fidelity of any DNA thermostable polymerase studied to date, and this property recommends it for analyses of tissue mutational spectra. Here, we apply constant denaturant capillary electrophoresis (CDCE) to separate and isolate the products of DNA amplification. This new strategy permitted direct enumeration and identification of point mutations created by Pfu DNA polymerase in a 96-bp low melting domain of a human mitochondrial sequence despite the very low mutant fractions generated in the PCR process. This sequence, containing part of the tRNA glycine and NADH dehydrogenase subunit 3 genes, is the target of our studies of mitochondrial mutagenesis in human cells and tissues. Incorrectly synthesized sequences were separated from the wild type as mutant/wild-type heteroduplexes by sequential enrichment on CDCE. An artificially constructed mutant was used as an internal standard to permit calculation of the mutant fraction. Our study found that the average error rate (mutations per base pair duplication) of Pfu was 6.5×10⁻⁷, and five of its more frequent mutations (hot spots) consisted of three transversions (GC→TA, AT→TA, and AT→CG), one transition (AT→GC), and one 1-bp deletion (in an AAAAAA sequence). To achieve an even higher sensitivity, the amount of Pfu-induced mutants must be reduced.     

CpG Islands of the Pig

We describe an analysis of the CpG islands (CGIs) of the pig. We have used both database survey and a porcine genomic library that is enriched for CGIs. Approximately half of 41 pig genomic database sequences had CGIs with an average G+C content of 65.3%, an average CpG observed/expected frequency of 0.85, and an average size of 978 bp. Of 27 CGI library clones, 16 were nonrepetitive, nonribosomal DNA and CGI-like. CGI library clones had similar average values for G+C and CpG frequency to CGIs of database genes, and an average size of 670 bp, as MseI cuts within some islands. Library clones were also shown to be low copy number and unmethylated in genomic DNA. The presence in the library of seven previously known CGI sequences was confirmed as was the absence of one nonisland sequence. The CGI library exhibits an R-band pattern for many chromosomes in FISH analysis. The pig chromosome arms that show the most dense CGI population are homologous to segments of human chromosomes that are known to be gene rich.     

Sour Cherry (Prunus cerasus L) Anthocyanins as Ingredients for Functional Foods

In the recent years many studies on anthocyaninshave revealed their strong antioxidant activity and their possibleuse as chemotherapeutics. The finding that sour cherries(Prunus cerasus L) (also called tart cherries) containhigh levels of anthocyanins that possess strong antioxidant andanti-inflammatory properties has attracted muchattention to this species. Here we report the preliminary resultsof the induction of anthocyanin biosynthesis in sour cherrycallus cell cultures. The evaluation and characterization of thein vitro produced pigments are compared to those of theanthocyanins found in vivo in fruits of several sour cherrycultivars. Interestingly, the anthocyanin profiles found in wholefruit extracts were similar in all tested genotypes but weredifferent with respect to the callus extract. The evaluation ofantioxidant activity, performed by ORAC and TEAC assays, revealeda relatively high antioxidant capacity for the fruitextracts (from 1145 to 2592μmol TE/100g FW) and alower one for the callus extract (688μmol TE/100g FW).     

Effect of Grape Seed Extract and Quercetin on Cardiovascular and Endothelial Parameters in High-Risk Subjects

Grape seed extract (GSE) has in vitro antioxidant activity but whether or notit works in vivo is not clear. In a fully randomised, crossovertrial with 4-week treatment periods on 36 men and womenwith above-average vascular risk, we aimed to demonstrate that2g/day of GSE (1g of polyphenols) alone,or with 1g/day of added quercetin in yoghurt, favourably altersvascular function, endothelial function, and degree of oxidative damagein comparison to a control yoghurt. GSE alone improved flow-mediateddilatation determined ultrasonically by an absolute 1.1% comparedwith control. There was no effect of the combination of GSE withquercetin. No other blood or urine measure was altered. Thussufficient polyphenols from GSE appear to be absorbed to influenceendothelial nitric oxide production, and GSE has the potential tofavourably influence vascular function.     

Overcoming cellular senescence in human cancer pathogenesis

Elevation of p16, the CDKN2/p16 tumor suppressor gene (TSG) product, occurs at senescence in normal human uroepithelial cells (HUC). Immortal HUCs and bladder cancer cell lines show either alteration of p16 or pRb, the product of the retinoblastoma (RB) TSG. In addition, many human cancers show p16 or pRb alteration along with other genetic alterations that we associated with immortalization, including +20q and −3p. These observations led us to hypothesize that p16 elevation plays a critical role in senescence cell cycle arrest and that overcoming this block is an important step in tumorigenesis in vivo, as well as immortalization in vitro. Using a novel approach, we tested these hypotheses in the present study by examining p16 and pRb status in primary culture (P0) and after passage in vitro of transitional cell carcinoma (TCC) biopsies that represented both superficial bladder tumors and invasive bladder cancers. We demonstrated that all superficial TCCs showed elevated p16 after limited passage in vitro and then senesced, like normal HUCs. In contrast, all muscle invasive TCCs contained either a p16 or a pRb alteration at P0 and all spontaneously bypassed senescence (P=0.001). Comparative genomic hybridization (CGH) was used to identify regions of chromosome loss or gain in all TCC samples. The application of a statistical model to the CGH data showed a high probability of elevated alteration rates of +20q11−q12 (0.99) and +8p22−pter (0.94) in the immortal muscle invasive TCCs, and of −9q (0.99) in the superficial TCCs. Three myoinvasive TCCs lost 3p13−p14. In this study, four of six myoinvasive TCCs also showed TP53 mutation that associated well with genome instability (P=0.001), as previously hypothesized. Notably, TP53 mutation, which has been used as a marker of tumor progression in many human cancers, was less significant in associating with progression in this study (P=0.04) than was p16 or pRb alteration (P=0.001). Thus, these data support a new model in which overcoming senescence plays a critical role in human cancer pathogenesis and requires at least two genetic changes that occur in several combinations that can include either p16 or pRb loss and at least one additional alteration, such as +20q11−q12, −3p13−p14, or −8p21−pter.     

Acute Plasmodium chabaudi chabaudi Malaria Infection Induces Antibodies Which Bind to the Surfaces of Parasitized Erythrocytes and Promote Their Phagocytosis by Macrophages In Vitro

CBA/Ca mice infected with 5 × 10⁴ Plasmodium chabaudi chabaudi AS-parasitized erythrocytes experience acute but self-limiting infections of relatively short duration. Parasitemia peaks (~40% infected erythrocytes) on day 10 or 11 and is then partially resolved over the ensuing 5 to 6 days, a period referred to as crisis. How humoral and cellular immune mechanisms contribute to parasite killing and/or clearance during crisis is controversial. Humoral immunity might be parasite variant, line, or species specific, while cellular immune responses would be relatively less specific. For P.c. chabaudi AS, parasite clearance is largely species and line specific during this time, which suggests a primary role for antibody activity. Accordingly, acute-phase plasma (APP; taken from P.c. chabaudi AS-infected mice at day 11 or 12 postinfection) was examined for the presence of parasite-specific antibody activity by enzyme-linked immunosorbent assay. Antibody binding to the surface of intact, live parasitized erythrocytes, particularly those containing mature (trophozoite and schizont) parasites, was demonstrated by immunofluorescence in APP and the immunoglobulin G (IgG)-containing fraction thereof. Unfractionated APP (from P.c. chabaudi AS-infected mice), as well as its IgG fraction, specifically mediated the opsonization and internalization of P.c. chabaudi AS-parasitized erythrocytes by macrophages in vitro. APP from another parasite line (P.c. chabaudi CB) did not mediate the same effect against P.c. chabaudi AS-parasitized erythrocytes. These results, which may represent one mechanism of parasite removal during crisis, are discussed in relation to the parasite variant, line, and species specificity of parasite clearance during this time.     

Effects of Dietary Copper Loading on Livers of Rats: I. Changes in Subcellular Acid Phosphatases and Detection of an Additional Acid p-Nitrophenylphosphatase in the Cellular Supernatant During Copper Loading

Significant changes occurred in lysosomal structure and function as copper was metabolized by rat livers. Hepatic total acid p-nitrophenylphosphatase (pNPase) activity was markedly increased in copper-loaded rats, and this increase was almost completely accounted for by heat- and formalin-stable (HFS) acid pNPase. Heat- and formalin-labile acid pNPase was essentially unchanged. On a subcellular level, the microsomal and supernatant fractions reflected the greatest relative increase in HFS acid pNPase. Increases in lysosomal, HFS acid pNPase in the large-granule fractions correlated with increase in solubilized large-granule enzymes, LG I, with mol wt > 200,000. LG II, representng solubilized large-granule enzymes with mol wt < 200,000, remained unchanged. Marked increases in supernatant acid pNPase were principally accounted for by a sevenfold increase in a supernatant lysosomal-like enzyme, DEAE Pk 1, separated by DEAE cellulose chromatography. An additional enzyme, DEAE Pk 2A′, that was hardly or not detectable in normal rats, was consistently demonstrated and increased in copper-loaded rats. Serum HFS acid pNPase increased in copper-loaded rats, suggesting that the increased hepatic supernatant acid pNPase in part escaped into the circulating fluid. Copper was principally associated with cytoplasmic organelles and was highest in mitochondrial and lysosomal fractions.     

Serum Cytokine Responses during Acute Human Granulocytic Ehrlichiosis

Human granulocytic ehrlichiosis (HGE) is caused by obligate intracellular bacteria in the Ehrlichia phagocytophila group. The disease ranges from subclinical to fatal. We speculated that cell-mediated immunity would be important for recovery from and potentially in the clinical manifestations of HGE; thus, serum tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), gamma interferon (IFN-γ), IL-10, and IL-4 concentrations were studied. IFN-γ (1,035 ± 235 pg/ml [mean ± standard error of the mean]) and IL-10 (118 ± 46 pg/ml) concentrations were elevated in acute-phase sera versus convalescent sera and normal subjects (P≤0.013 and P≤0.018, respectively). TNF-α, IL-1β, and IL-4 levels were not elevated. Cytokine levels in severely and mildly affected patients were not different. HGE leads to induction of IFN-γ-dominated cell-mediated immunity associated with clinical manifestations, recovery from infection, or both.     

Effect of Synthetic Truncated Apolipoprotein C-I Peptide on Plasma Lipoprotein Cholesterol in Nonhuman Primates

The present studies were conducted to determinewhether a synthetic truncated apoC-I peptide thatinhibits CETP activity in baboons would raise plasmaHDL cholesterol levels in nonhuman primates with lowHDL levels. We used 2 cynomolgus monkeys and 3baboons fed a cholesterol- and fat-enriched diet. Incynomolgus monkeys, we injected synthetic truncatedapoC-I inhibitor peptide at a dose of 20mg/kgand, in baboons, at doses of 10, 15, and 20mg/kgat weekly intervals. Blood samples were collected 3times a week and VLDL + LDL and HDL cholesterolconcentrations were measured. In cynomolgus monkeys,administration of the inhibitor peptide caused arapid decrease in VLDL + LDL cholesterolconcentrations (30%–60%) and an increase in HDLcholesterol concentrations (10%–20%). VLDL + LDLcholesterol concentrations returned to baselinelevels in approximately 15days. In baboons,administration of the synthetic inhibitor peptidecaused a decrease in VLDL + LDL cholesterol (20%–60%)and an increase in HDL cholesterol (10%–20%). VLDL+ LDL cholesterol returned to baseline levels byday 21, whereas HDL cholesterol concentrationsremained elevated for up to 26days. ApoA-Iconcentrations increased, whereas apoE andtriglyceride concentrations decreased. Subcutaneousand intravenous administrations of the inhibitorpeptide had similar effects on LDL and HDLcholesterol concentrations. There was no change inbody weight, food consumption, or plasma IgGlevels of any baboon during the study. Thesestudies suggest that the truncated apoC-I peptide canbe used to raise HDL in humans.     

Mechanical work and efficiency in level walking and running

1. The mechanical power spent to accelerate the limbs relative to the trunk in level walking and running, _int, has been measured at various `constant' speeds (3-33 km/hr) with the cinematographic procedure used by Fenn (1930a) at high speeds of running.

2. _int increases approximately as the square of the speed of walking and running. For a given speed _int is greater in walking than in running.

3. In walking above 3 km/hr, _int is greater than the power spent to accelerate and lift the centre of mass of the body at each step, _ext (measured by Cavagna, Thys & Zamboni, 1976b). In running _int < _ext up to about 20 km/hr, whereas at higher speeds _int > _ext.

4. The total work done by the muscles was calculated as W_tot = |W_int| + |W_ext|. Except that at the highest speeds of walking, the total work done per unit distance W_tot/km is greater in running than in walking.

5. The efficiency of positive work was measured from the ratio W_tot/Net energy expenditure: this is greater than 0·25 indicating that both in walking and in running the muscles utilize, during shortening, some energy stored during a previous phase of negative work (stretching).

6. In walking the efficiency reaches a maximum (0·35-0·40) at intermediate speeds, as may be expected from the properties of the contractile component of muscle. In running the efficiency increases steadily with speed (from 0·45 to 0·70-0·80) suggesting that positive work derives mainly from the passive recoil of muscle elastic elements and to a lesser extent from the active shortening of the contractile machinery. These findings are consistent with the different mechanics of the two exercises.

     

A Gene Encoding Sialic-Acid-Specific 9-O-Acetylesterase Found in Human Adult Testis

Using differential display RT-PCR, we identified a gene of2750bp from human adult testis, named H-Lse, whichencoded a putative protein of 523 amino acids and molecularweight of 58kd with structural characteristics similar tothat of mouse lysosome sialic-acid-specific 9-O-acetylesterase.Northern blot analysis showed a widespread distribution of H-Lsein various human tissues with high expression in the testis,prostate, and colon. In situ hybridization results showed thatwhile H-Lse was not detected in embryonic testis, positivesignals were found in spermatocytes but not spermatogonia inadult testis of human. The subcellular localization of H-Lse wasvisualized by green fluorescent protein (GFP) fused to the aminoterminus of H-Lse, showing compartmentalization of H-Lse in largedense-core vesicles, presumably lysosomes, in the cytoplasm. Thedevelopmentally regulated and spermatogenic stage-specificexpression of H-Lse suggests its possible involvement in thedevelopment of the testis and/or differentiation of germ cells.     

Possible role for Toll-like receptors in interaction of Fasciola hepatica excretory/secretory products with bovine macrophages

Alternative activation of macrophages (M) during helminth infection is a characteristic feature of the host immune response. Alternatively activated macrophages (AAM) are distinguished from others by high arginase 1 (Arg-1) activity, low nitric oxide (NO), and high interleukin 10 (IL-10) production. In murine models, these cells have been shown to possess anti-inflammatory properties. They have also been implicated in exacerbating a subsequent infection with a secondary pathogen. In this study we used cattle experimentally infected with Fasciola hepatica to monitor the kinetics of IL-4 and IL-10 over the course of infection. Using naïve M in vitro, we examined the effects of exposure to F. hepatica excretory/secretory products (FhepES) alone or in combination with IL-4. Our results suggest that FhepES may work in combination with IL-4 to produce AAM. The effects of FhepES on the subsequent responses to lipopolysaccharide (LPS) and purified protein derivative from Mycobacterium bovis (PPD-B), which are bovine Toll-like receptor 4 (TLR4) and TLR2 antagonists, respectively, were also examined. We found that M stimulated with FhepES together with LPS or PPD-B have reduced NO or gamma interferon production, respectively. The ability of FhepES to produce AAM was found to be heat labile and partially dependent on glycan residues. A possible role for TLR recognition is discussed.     

Perillyl Alcohol Inhibits Breast Cell Migration without Affecting Cell Adhesion

The monoterpene d-limonene exhibits chemotherapeutic andchemopreventive potential in breast cancer patients. D-limonene and its related compounds, perillyl alcohol and perillyl aldehyde, were chosen as candidate drugs for application in a screen for nontoxic inhibitors of cellmigration. Using the nontumorigenic human breast cell line MCF-10A, we delineated the toxicity as greatest for the perillyl aldehyde, intermediate for perillyl alcohol, and least for limonene. A noncytotoxic concentration of0.5mmol/L perillyl alcohol inhibited the migration, while the same concentration of limonene failed to do so. Adhesion of the MCF-10A cell line and the human breast cancer cell line MDA-MB 435 to fibronectin was unaffected by 1.5mmol/L perillyl alcohol. 0.4mmol/L perillyl alcohol inhibited the growth of MDA-MB 435 cells. All migration-inhibiting concentrations of perillyl alcohol for MDA-MB 435 cells proved to be toxic. These results suggest that subtoxic doses of perillyl alcohol may have prophylactic potential in the treatment of breast cancer.     

Production of Prednisolone by Pseudomonas oleovorans Cells Incorporated Into PVP/PEO Radiation Crosslinked Hydrogels

In order to rise the yield of prednisolone fromhydrocortisone, the Pseudomonasoleovorans cells were entrapped intoradiation crosslinked poly (vinylpyrrolidone)/poly(ethylene oxide) (PVP/PEO)hydrogel of different gel contents. The factors affecting the gel content andswelling behavior of the polymeric gel, such aspolymer composition, polymer blendconcentration, and irradiation doses, were investigated.The formation of gels having a good strength with theability to retain a desirable amount of water in theirthree-dimensional network can be achieved byusing PVP/PEO copolymer of composition (90 : 10) andconcentration of 15% prepared at 20kGyirradiation dose. At these conditions the preparedhydrogel is considered the most favorable one thatgave the highest hydrocortisone bioconversion andprednisolone yield, 81% and 62.8%, respectively. Theimprovement of prednisolone yield was also achieved byincreasing substrate concentration. Maximumhydrocortisone bioconversion (86.44) was obtained at18hours by using substrate concentration of 30mg.Reusability of immobilized Pseudomonasoleovorans entrapped into PVP/PEOcopolymer hydrogel was studied. The results indicatedthat the transformation capacity of hydrocortisone toprednisolone highly increased by the repeated use ofcopolymer for 4 times. This was accompanied by anincrease in prednisolone yield to 89% and thebioconversion of hydrocortisone was 98.8%.     

Three-Dimensional Culture of Hybridoma Cells Secreting Anti-Human Chorionic Gonadotropin by a New Rolling Culture System

Cell growth rate and production of monoclonal antibody (MAb) ofhybridoma cells producing anti-human chorionic gonadotropin (hCG)MAb have been used as investigation criteria in double-mouthedrolling bottle (DMRB). Compared with T-flask cell culture, bothof the cell number and MAb production increased by approximately42.5% when the medium was supplemented with 5% fetal calf serum(FCS) and DMRB rotated at 2 turns per minute. Yield of MAb wasexperimentally related to the number of viable cells.Interestingly, MAb yield was four times as high as that culturedin T-flask in the first 24hours, and about 75% yield oftotal MAb was secreted by 48hours during the culture. Itappears that the promoted cell growth and MAb yield are resultedfrom the three-dimensional growth of hybridoma cells under asuitably revolving condition.     

Activation of human monocytes by both human β1H and C3b

We tested whether highly purified human β1H and C3b, two proteins of the alternative pathway of complement activation, could exert an influence on the activity of human monocytes (M). The activation process of M was assessed by measurements of the respiratory burst in terms of nitro blue tetrazolium (NBT) reduction and by chemiluminescence (CL) tests. In NBT reduction experiments, we found a tendency for β1H to increase NBT reduction, while C3b was found to be rather inhibitory. In CL measurements, both β1H and C3b displayed a stimulatory effect on M, showing different time- and dose-dependency. For β1H, the maximum stimulation occurred after 15 min, whereas for C3b after 45 min. Zymosan particles which served as a positive control also showed the highest stimulation after 45 min. In dose—response experiments, β1H reached a plateau ranging from 30 to 80 μg/ml. In contrast, using C3b up to 170 μg/ml, no plateau was reached. M-depletion and enrichment studies suggested at M as being responsible for the stimulatory effects found.

The differences between NBT reduction and CL could possibly be explained by the measurement of only cell-bound reductive potentials by NBT reduction, while in CL measurements, products of the extracellular space are also assessed. Our results suggest that both human β1H and C3b are appropriate stimuli for human monocytes.