In vitro studies on the effects of flubendazole against Toxocara canis and Ascaris suum

Adult Toxocara canis and Ascaris suum were incubated in vitro in media containing 0.1, 1, 10 or 100 micro g/ml flubendazole in order to study drug-derived effects. This incubation was done for 8 h and repeated (in some groups) after 24 h for another 8 h. The onset and intensity of flubendazole-derived effects were dosage-dependent and time-dependent, i.e. the same grade of damage was reached when incubating for a longer period at a low dosage or for a shorter period in medium containing a high amount (10 or 100 micro g/ml) of flubendazole. A repeated incubation in drug-containing medium was superior to a single exposure. Flubendazole is apparently able to penetrate into the worm's interior via the cuticle. This became evident in worms with sealed orifices, which showed identical damage to worms which were not sealed. The type of tissue damage due to flubendazole was identical in both worm species when exposed to any of the drug dosages used. The principal mode of action of flubendazole was based on the complete reduction of microtubuli-polymerisation inside the parasite's cells. This apparently led to the complete destruction of the hypodermis, muscle layer and intestine. Flubendazole also stopped the formation of gametes. Summarising, even low concentrations of flubendazole (0.1 micro g/ml) led to significant and irreversible damage in all worms studied.     

Flubendazole in human Echinococcus granulosus hydatidosis. Preoperative care: parasit-pharmacologic study

The effect of flubendazole in human hydatic disease due to E. granulosus has been studied in 22 patients with one or several hydatic cysts of different sites. Flubendazole has been given orally before surgery for a 10 days mean period, at 4 g daily in adults and 1 g daily in children. Flubendazole has been titrated by radioimmuno-assay in sera, urines and bile taken 12 to 24 hours after the last ingestion and in the hydatic cyst (membranes and liquid). The scolex vitality was determined by direct optic microscopy and by intra-peritoneal mouse inoculation. Seven non-treated patients, without hydatic disease, were considered as controls. The flubendazole concentrations in sera (3.01 +/- 3.73 ng/ml) and in urines (7.6 +/- 7.5 ng ml) are low. They are also low in membranes (3.21 +/- ng/mg) and in hydatic liquid, but significantly more elevated in the hepatic localisation than in the pulmonary localisation. The bile concentrations are high (167.9 +/- 133.2 ng/ml). There is no correlation between these concentrations and the amounts of flubendazole administered. The scolex vitality was correlated neither with the amounts of flubendazole administered, nor with the concentrations of flubendazole in the hydatic cyst. Further studies are necessary before judging of the efficacy of flubendazole in the human hydatic disease.     

In vitro effects of flubendazole on Echinococcus granulosus protoscoleces

The aim of the present work was to determine the in vitro protoscolicidal effect of flubendazole (FLBZ) against Echinococcus granulosus. Protoscoleces of E. granulosus were incubated with FLBZ at concentrations of 10, 5 and 1 μg/ml. The first signs of FLBZ-induced damage were observed 3 days post-incubation. A clear protoscolicidal effect, reducing the vitality of protoscoleces to 35.6±0.7%, was observed after 18 days of incubation. After 25 days of FLBZ incubation (5 μg/ml), the percentage of vital protoscoleces was 13.9±5.9%. Protoscolex mortality was 100% (10 and 1 μg/ml) and 0.7±0.7% (5 μg/ml) after FLBZ incubation for 30 days. Results of vitality tests were consistent with the tissue damage observed at the ultrastructural level. The primary site of damage was the tegument of the parasite. The morphological changes included contraction of the soma region, formation of blebs on the tegument, rostellar disorganization, loss of hooks and destruction of microtriches. The data reported in this article demonstrate a clear in vitro effect of FLBZ against E. granulosus protoscoleces. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina     

Effects of an asymmetric triazine derivative,HOE 092 V,on Glugea anomala,Moniez, 1887 (Microsporidia) parasitic in the three-spined stickleback Gasterosteus aculeatus

 An asymmetric triazine derivative, HOE 092 V, 2-[3,5-α-dichloro-4-(4-methyl-sulfonylphenoxy)-phenyl]-1-methyl-hexahydro-1,2,4-triazine-3,5-dion, was tested in vivo against Glugea anomala parasitizing the connective tissue of sticklebacks (Gasterosteus aculeatus). Naturally infected sticklebacks were incubated in 10-l plastic aquaria in water containing 0, 2.5, 5, and 10 μg HOE 092 V/ml for 2, 3, and 4 h at 22°C. As seen at the ultrastructural level, the drug caused severe damage to all developmental stages of G. anomala except the mature spores. Starting with a dose of 2.5 μg/ml, the drug caused significant damage on uni- and multinucleate meronts, sporogonial plasmodia, and sporoblasts. The damage mainly consisted in a decrease in the number of ribosomes, an enlargement of the smooth endoplasmic reticulum and a vacuolization of the cytoplasma. When treatment was done with 5 μg for 2 h, multinucleate meronts and sporogonial plasmodia were no longer detectable, and the sporoblasts and the prespore stages except the mature spores had shrunk. After incubation of the infected fish with 10 μg HOE 092 V/ml and 4 h exposure, uninucleate meronts were no longer detectable by means of transmission electron microscopy. In the sporoblast mother cells, vacuolization of the cytoplasma and lysis of the nuclei occurred. However, mature spores were not affected. It seems likely that HOE 092 V can be successfully applied in medicinal baths against Microsporidia in fish. The infected fish should be incubated in separate, aerated containers. Received: 15 July 1995 / Accepted: 15 August 1995     

The effects of in vitro incubation with acetylsalicylic acid on thrombocyte aggregation in whole blood from normal ostriches

Avian thrombocytes are circulating nucleated blood cells which play an active role in both haemostasis and phagocytosis. To better define their role in haemostasis, thrombocyte aggregation was measured in whole blood from healthy adult ostriches using an electrical impedance aggregometer. Thrombocytes were stimulated with collagen (5μg/ml or 1μg/ml), platelet-activating factor (PAF (0.1μ m, 0.01μ m, or 0.001μ m), or ADP (25μ m). Irreversible aggregation occurred consistently in response to collagen or PAF but not to ADP. Aggregation in response to high concentrations of PAF or collagen was not affected by in vitro incubation with 500μ m aspirin. Aggregation in response to low concentrations of PAF or collagen was partially inhibited by in vitro incubation with 500μ m aspirin, suggesting the presence of a prostaglandin-dependent mechanism for regulation of thrombocyte activation similar to that in mammalian platelets.     

Treatment of fish parasites

In several experiments it was shown that praziquantel is effective against Monogenea (Dactylogyrus vastator, Dactylogyrus extensus) parasitizing the gills of pond-reared carp (Cyprinus carpio). Also, the effects of praziquantel onDiplozoon paradoxum from chub (Squalius cephalus) were investigated. Infested carp were incubated for 30, 60, 90, 120 or 180 min at 22° C in water containing 0 (control), 1, 5, 10, 100 g praziquantel/ml. It was found that the drug caused irreversible alterations in the tegument of Monogenea within 30 min after exposure to at least 1 g praziquantel/ml. The damage found in vitro resulted in vacuolisation of the parasites' teguments. The three species showed little differences in the grade of tegumental alternation:Dactylogyrus vastator showed vacuolisation of its tegument in all regions of its body, which was very marked in the opisthaptor and in the peduncle region. Parasites did not drop from the gills even when killed by treatment with praziquantel. InDactylogyrus extensus the opisthaptor region was especially affected. The worms usually lost their marginal hooklets as well as the large hooks. In contrast toDactylogyrus vastator, treated worms often dropped from gills.Diplozoon paradoxum was lesser sensitive to the drug. After incubation in 1 g praziquantel/ml for 7 h the worms had a reduced motility. Doses of 10g/ml and 150 g/ml for 1.5 h led to severe damage of the tegument, especially in the region of the oral sucker. However, the drug is effective in treatment of this parasite.It is suggested that therapy againstDactylogyrus vastator andDactylogyrus extensus in carp fingerlings may be done with 10 mg praziquantel/l for 3 h at 22° C during storage in smaller tanks.     

Cellular mechanisms involved in the increased contraction of portal veins from <Emphasis Type="Italic">Schistosoma mansoni</Emphasis>-infected mice

We previously reported that portal veins from mice infected with male Schistosoma mansoni exhibited an increased reactivity to 5-hydroxytryptamine (5-HT). Here, we extended our observations to mice infected by both male and female worms and we further investigated another constrictor agent and the mechanism(s) responsible for the enhanced maximal contraction (E_max). Bisexual infection increased the E_max of 5-HT (from 0.66ǂ.06 mN.s to 1.56ǂ.38 mN.s), in a similar way to the unisexual (male) infection. Infection with male worms increased portal vein reactivity to acetylcholine, as revealed by a higher E_max (1.03ǂ.2 mN.s) in relation to non-infected control animals (E_max = 0.54ǂ.08 mN.s). Sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibition with 100 nM thapsigargin reduced the E_max of 5-HT by 35% in both tissues, discharging a deficiency of SERCA pump in infected animals. In contrast, the number of voltage-dependent Ca²⁺ channels (L-type) was higher in portal veins from infected than non-infected control mice. Inhibition of Ca²⁺-activated chloride channels (Cl_Ca) with 10 µM niflumic acid reduced the E_max of 5-HT in portal veins more from infected than non-infected animals (remaining tension = 60.9DŽ.2% and 70.4DŽ.3%, respectively). Histopathological analysis revealed an increased content of collagen and elastin in portal veins from male S. mansoni-infected mice, compatible with an increased intraluminal pressure. In conclusion, male S. mansoni altered portal vein physiology, increasing the E_max of two vasoconstrictors, possibly by increasing membrane depolarisation through a more effective opening of Cl_Ca channels, with calcium entering through L-type Ca²⁺ channels.     

Light and scanning electron microscopy studies on the effects of the enantiomers of praziquantel and its main metabolite onSchistosoma mansoni in vitro

In the present study, the effects of the enantiomers of the anthelmintic drug praziquantel (PZQ) and its main metabolitetrans-4-hydroxypraziquantel (TRANS) on pairs ofSchistosoma mansoni worms were examined in vitro. Highly purified enantiomers (optical purity, >99.9% for PZQ and 99.0% for TRANS) were used. Paired worms were incubated for 4 h in RPMI medium containing 0.01, 0.02, 0.075, 0.1, 2.5, 10, and 100 g PZQ or TRANS enantiomers/ml, respectively, before being transferred to drug-free medium for another 20 h. PZQ is used as a racementa in the therapy, and its effect is attributed to the R(–)-enantiomer. R(–)-PZQ and R(–)-TRANS proved to be at least 10⁵ times more effective than the respective S(+)enantiomers, causing tegumental damage and surface blebbing onS. mansoni. As judged from the effective doses in 50% of the worms (ED₅₀ values); R(–)-PZQ and R(–)-TRANS showed nearly the same efficacy against adultS. mansoni. Male worms reacted more sensitively than did females. As determined by scanning electron microscopy, alterations in lethally damaged worms depended on the drug used, even following incubation at the lowest concentration tested (0.01 g/ml). Worms exposed to R(–)-TRANS were elongated, whereas treatment with R(–)-PZQ led to contractions and twisted parasites. Both compounds caused excessive surface blebbing along the dorsal side of the worms' tegument.Abbreviations B boss - CG canalis gynaecophorus - F female - IT invagination of the tegument - M male - OS oral sucker - SC sensory cilium - SP spine - V vacuole - VS ventral sucker     

Genotoxic and immunotoxic effects of cellulose nanocrystals in vitro

Nanocellulosics are among the most promising innovations for a wide‐variety of applications in materials science. Although nanocellulose is presently produced only on a small scale, its possible toxic effects should be investigated at this early stage. The aim of the present study was to examine the potential genotoxicity and immunotoxicity of two celluloses in vitro ‐ cellulose nanocrystals (CNC; mean fibril length 135 nm, mean width 7.3 nm) and a commercially available microcrystalline (non‐nanoscale) cellulose (MCC; particle size ～50 µm). Both celluloses showed 55% cytotoxicity at approximately 100 µg/ml after 4‐h, 24‐h, and 48‐h treatment of human bronchial epithelial BEAS 2B cells, as determined by luminometric detection of ATP and cell count (dead cells identified by propidium iodide). Neither of the materials was able to induce micronuclei (MN) in binucleate or mononucleate BEAS 2B cells after a 48‐h treatment (2.5–100 µg/ml). In human monocyte‐derived macrophages, MCC induced a release (measured by enzyme‐linked immunosorbent assay; ELISA) of the pro‐inflammatory cytokines tumor necrosis factor α (TNF‐α) and (after lipopolysaccharide‐priming) interleukin 1β (IL‐1β) after a 6‐h exposure to a dose of 300 µg/ml, but CNC (30–300 µg/ml) did not. In conclusion, our results show that nanosized CNC is neither genotoxic nor immunotoxic under the conditions tested, whereas non‐nanosized MCC is able to induce an inflammatory response. More studies are needed, especially in vivo, to further assess if CNC and other nanocelluloses induce secondary genotoxic effects mediated by inflammation. Environ. Mol. Mutagen. 56:171–182, 2015. © 2014 Wiley Periodicals, Inc.     

Further studies on mefloquine and praziquantel alone or interaction of both drugs against Schistosoma japonicum in vitro

The aim of the present study is to further understand and analyze the interaction of mefloquine with praziquantel against adult Schistosoma japonicum in vitro. Mice infected with S. japonicum cercariae for 35–37 days were sacrificed, and adult schistosomes were collected by perfusion. Schistosomes were placed to each of 12 wells of a Falcon plate and maintained in RPMI 1640 supplemented by 10% calf serum. For determination of 50% and 95% lethal concentration (LC50 and LC95) of the two drugs in vitro, schistosomes were exposed to mefloquine at concentrations of 1, 2, 3, 4, 5, 6, 7, and 10 μg/mL or praziquantel at concentrations of 0.001, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 10, and 30 μg/mL. The plate was incubated at 37°C in 95% air + 5% CO₂ for 72 h. According to the half-life of oral mefloquine and praziquantel in mice, mefloquine combined with praziquantel simultaneously, mefloquine administered within 1 h after praziquantel and praziquantel administered within 17 h after mefloquine were used to evaluate the effect of mefloquine in combination with praziquantel against S. japonicum in vitro. The results showed that the LC50 and LC95 of mefloquine calculated by the Bliss method were 6.17 μg/mL (95% confidence limits, 5.84–6.517 μg/mL) and 8.703 μg/mL (95% confidence limits, 7.632–9.797 μg/mL), respectively. As to praziquantel, no worm death was seen when schistosomes were exposed to praziquantel at concentrations of 0.005–0.2 μg/mL for 72 h. While in the worms exposed to praziquantel 1, 10, and 30 μg/mL, strong spasmodic contractions of the worm body and vesiculation along the worm surface were observed, but 48–75% of the schistosomes survived the exposure in 72-h incubation. Meanwhile, the number of dead worms that emerged in each group was not proportion to the increasing concentrations. Therefore, it is not appropriate to calculate the LC50 and LC95 of praziquantel. For evaluation of the interaction with the two drugs, praziquantel 0.1 or 0.2 μg/mL, which may induce moderate or strong spasmodic contractions of the worm body and vesiculation along the worm surface, was combined with mefloquine 5, 6, or 7 μg/mL. It was found that when mefloquine combined with praziquantel simultaneously or administered 1 h after addition of praziquantel, the spasmodic contraction of the male worm body was antagonized by mefloquine in various degrees according to the concentrations of mefloquine used. Meanwhile, praziquantel-induced weakened motor activity could be reversed by mefloquine. In female worms, morphological alterations and stimulated motor activity induced by mefloquine still developed. Interestingly, using these two regimens to combine mefloquine with praziquantel resulted in no impact or a decrease in worm mortality. On the other hand, praziquantel 0.2 μg/mL administered within 17 h after mefloquine 5 or 6 μg/mL promoted the damage to the tegument of the worms, which led to enhance the worm mortality compared with that of worms exposed to mefloquine alone. The results indicate that in vitro higher concentrations of praziquantel administered within 17 h after mefloquine may increase the effect against adult schistosomes, while praziquantel combined with mefloquine simultaneously or administered 1 h before addition of mefloquine exhibits no impact or decrease in the effect against schistosomes.     

Con A-induced suppressor cells in man. I. Induction and characterization of suppressor cells

Con A-induced suppressor cells were studied in autologous co-cultures of normal human lymphocytes. Lymphocytes pretreated with 5 µg/ml, 20 µg/ml, or 50 µg/ml of Con A were cultured for 24, 48 and 72 hours. Subsequently, the whole mononuclear (MN) cell subpopulation or T-enriched, B-enriched, and monocyte-enriched fractions were added to freshly obtained lymphocytes, stimulated with 5 µg/ml of Con A and cultured for a further 96 hours. MN cells pretreated with 5 µg/ml of Con A did not significantly affect the DNA synthesis in co-cultures with unfractionated MN cells, while cells pretreated with 20 µg/ml or 50µg/ml for 48 and 72, but not for 24 hours, possessed the suppressive activity. The suppressor cells were found in T-, but not B-enriched subpopulations. Moreover, suppression was induced by T-enriched fraction from MN cells pretreated with 5 µg/ml of Con A, while unfractionated cells failed to exhibit inhibitory activity. The degree of T-cell-induced suppression was dose-dependent. Irradiation with 3000 R diminished the suppressive activity of cells derived from 48-hour cultures and abrogated the activity of cells from 72-hour cultures.The present data indirectly prove that Con A-induced suppressor cells are radiosensitive T lymphocytes. The observation that induction of suppressor cells by Con A is dose- and time-dependent provides the further insight into regulatory immunological mechanism in humans.     

The effect of human eosinophils on cultured human nasal epithelial cell activity and the influence of nedocromil sodium in vitro.

Although there is increasing evidence of a pathogenic role for eosinophils in the airway epithelium, there is little direct evidence which demonstrates that eosinophils influence epithelial cell activity in humans. We have cultured human nasal epithelial cells in vitro and studied the effect of isolated human eosinophils on the ciliary beat frequency (CBF) and cell membrane integrity of these cells after incubation in the absence or presence of 0.1 microM phorbol 12-myristate 13-acetate (PMA) or 0.1 mg/ml opsonized latex beads and the absence or presence of 10(-5) M nedocromil sodium. CBF was monitored by an analogue contrast-enhancement technique, and cell damage was assessed by release of 51Cr from the cells. Cell cultures were also assessed for the percentage of eosinophil cationic protein (ECP) released into the medium at the end of incubation. Neither 0.1 microM PMA, 0.1 mg/ml opsonized latex beads, 10(-5) M nedocromil sodium, nor eosinophils alone altered the CBF of the epithelial cells. PMA-stimulated eosinophils, however, attenuated the CBF significantly, from 10.2 +/- 0.3 to 8.8 +/- 0.4 Hz (P less than 0.05) after 15 h of incubation. Similarly, opsonized latex bead-stimulated eosinophils led to a significant attenuation of CBF from 9.2 +/- 0.3 to 8.4 +/- 0.3 Hz (P less than 0.05), 6.9 +/- 0.5 Hz (P less than 0.001), and 7.5 +/- 0.3 Hz (P less than 0.001) after 2, 15, and 24 h of incubation, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mesangial matrix-activated mononuclear cells express functional scavenger receptors and accumulate intracellular lipid

Introduction Monocyte recruitment into the mesangium and foam cell formation are recognized features of glomerular injury. External signals encountered by these infiltrating cells may determine their behaviour and thereby potentially influence disease outcomes. Our previous studies indicate that activation of monocytes by mesangial matrix stimulates the production of a variety of mediators including inflammatory cytokines and matrix degrading enzymes. Methods Using expression of peroxisome proliferator activator receptor‐γ (PPAR‐γ) and scavenger receptor (ScR) as differentiation markers, we examined whether matrix activation was associated with the expression of monocyte characteristics usually associated with a macrophage phenotype. THP‐1 mononuclear cells were incubated for 7 days with 500 µg/ml solublized matrix extracted from cultured human mesangial cells. Results Using phorbol methyl ester (PMA) (125 nm) and albumin (500 µg/ml) as positive and negative controls, respectively, we demonstrated that matrix activation of monocytes led to intracellular lipid accumulation as demonstrated by oil red O staining. Matrix activation was also associated with a concentration‐dependent increase in the expression of both ScR and PPAR‐γ mRNA and a corresponding increase in PPAR‐γ protein expression on Western blotting. The presence of functional ScR was confirmed using FACS analysis in which incubation of matrix‐activated monocytes with Dil‐labelled acetylated low‐density lipoprotein (LDL) led to an increase in mean fluorescent intensity of 373% (P < 0.001) as compared to albumin (100%) and PMA (423%). This could be inhibited by addition of excess unlabelled ligand, suggesting specific binding to the ScR. Furthermore, incubation of LDL with mesangial matrix in the absence of cells led to enhanced electrophoretic mobility of recovered lipoprotein on agarose gel. A similar shift was seen when LDL was incubated with Cu²⁺, a powerful lipoprotein oxidant. Discussion These results demonstrate that interactions with mesangial matrix induce expression of monocyte characteristics associated with a macrophage phenotype and promote oxidation of LDL, thereby converting it to an ScR ligand. Such observations may help to explain foam cell formation in the mesangium in the context of glomerular disease.     

In vitro efficacy of the essential oil of Piper cubeba L. (Piperaceae) against Schistosoma mansoni

In this paper, cercariae, schistosomula, and adult Schistosoma mansoni worms were incubated in vitro with the essential oil of Piper cubeba (PC-EO) at concentrations from 12.5 to 200 μg/mL, and the viability was evaluated using an inverted microscopy. The effects of PC-EO at 100 and 200 μg/mL on the stages of S. mansoni were similar to those of the positive control (PZQ at 12.5 μg/mL), with total absence of mobility after 120 h. However, at concentrations from 12.5 to 50 μg/mL, PC-EO caused a reduction in the viability of cercariae and schistosomula when compared with the negative control groups (RPMI 1640 or dechlorinated water) or (RPMI 1640 + 0.1% DMSO or dechlorinated water + 0.1% DMSO). On the other hand, adult S. mansoni worms remained normally active when incubated with PC-EO at concentrations of 12.5 and 25 μg/mL, and their viabilities were similar to those of the negative control groups. In addition, at concentrations ranging from 50 to 200 μg/mL, separation of all the coupled adult worms was observed after 24 h of incubation, which is related to the fact of the reduction in egg production at this concentration. The main chemical constituents of PC-EO were identified by gas chromatography-mass spectrometry as being sabinene (19.99%), eucalyptol (11.87%), 4-terpineol (6.36%), β-pinene (5.81%), camphor (5.61%), and δ-3-carene (5.34%). The cytotoxicity of the PC-EO was determined, and a significant cytotoxicity was only obtained in the concentration of 200 μg/mL after 24 h treatment. The results suggest that PC-EO possesses an effect against cercariae, schistosomula, and adult worms of the S. mansoni.     

In vitro immunomodulatory effects of extracts from three plants of the Labiatae family and isolation of the active compound(s)

Plants may have the ability to modulate immune responses. In the present study, the effects of three plants belonging to Labiatae family, each traditionally used for the treatments of infections and inflammatory diseases, as well as the role of thymol (as one the major components of these plants), were investigated for their potential to affect the activation of lymphocytes. Four organic extracts of Thymus vulgaris and two other plants (i.e., T. daenensis and Zataria multiflora) were prepared. The effect of the extracts on mitogen (PHA)-stimulated peripheral blood lymphocytes was determined using a cell proliferation assay. The hexane extracts obtained from the three plants showed the strongest inhibitory effects on PHA-induced proliferation. Use of preparative thin layer and gas chromatographies in conjunction with the proliferation assay confirmed that thymol was the major component responsible for the observed effects from the three plants. Thymol inhibited inducible lymphocyte proliferation in a concentration-dependent manner, with reductions ranging from 62.8% at 50 µg/ml to 89.8% at 200 µg/ml (> 0.1 µg/ml (p?<?0.01). Flow cytometric analysis using propidium iodide staining showed that the inhibitory effect of thymol at 200 µg/ml was due to a cytotoxic activity. In conclusion, the three Labiatae plants studied here each showed immunosuppressive effects against lymphocytes and it was most likely that thymol was the compound in these plants responsible for this effect.     

Granulocyte colony-stimulating factor (G-CSF) increases histone-complexed DNA plasma levels in healthy volunteers

Granulocyte colony-stimulating factor (G-CSF) is an activator of neutrophil granulocytes. Neutrophil extracellular traps are a defensive mechanism consisting of neutrophils, platelets, DNA, histones and antimicrobial proteins. This study was performed to determine whether G-CSF increases histone-complexed DNA in the plasma of healthy volunteers. In total, 51 healthy volunteers (25 males and 26 females) were treated with G-CSF (18 with 300 µg single dose i.v., 27 with 5 µg/kg s.c. for 4 days) and six participants received a placebo. Histone-complexed DNA was measured by enzyme immunoassay in plasma samples at predefined time points (0, 2, 4, 6, 24 h after single dose, day 1, day 2 and day 5 after repeated doses). Histone levels were quantified by Western blotting. A single dose of G-CSF rapidly increased hc-DNA by about 50 % (p < 0.05 for 2–24 h). After repeated doses the increase was even more pronounced: hc-DNA increased by about 50 % (3.0 ± 0.9, p < 0.001 after 24 h and about fourfold after 96 h (p < 0.001)). A statistical significant increase in histone levels was detected as early as 4 h after G-CSF injection (0.43 ± 0.2 vs. 1.08 ± 0.3 µg/ml; p = 0.034). In the placebo group no significant changes occurred. Moreover, significantly higher levels of hc-DNA were measured in male compared to female subjects (226 ± 43 vs. 84 ± 19, p < 0.001). G-CSF injection substantially increases hc-DNA levels in healthy volunteers. There is a significant gender difference in hc-DNA at the baseline.     

Comparison of in vitro activity of metronidazole and garlic-based product (Tomex®) on Trichomonas vaginalis

Trichomonas vaginalis is a common sexually transmitted parasite in humans. Metronidazole has been the gold standard for treatment of trichomoniasis. The prevalence of metronidazole resistance and its unpleasant adverse effects drew the attention to the investigation of other lines of treatment, as that of herbal medicine. Garlic has been proven to have antibacterial, antiprotozoal, and antihelminthic activity. The current study was carried out to evaluate the efficacy of commercially available garlic (Tomex®) on T. vaginalis in vitro. The effect of different concentrations of garlic (12.5, 25, 50, and 100 μg/ml) was determined on multiplication and motility of trophozoites at different time points (after 24, 48, 72, and 96 h) in comparison to the same concentrations of metronidazole at the same different time points. The results showed that parasite multiplication inhibition was noticed in proportion of concentration of Tomex and incubation time. The minimal lethal concentration of Tomex was 100 μg/ml after 24 h, 50 μg/ml after 48 h, 25 μg/ml after 72 h, and 12.5 μg/ml after 96 h. These results were similar to that of metronidazole as its minimal lethal concentration was 50 μg/ml after 24 and 48 h and 12.5 μg/ml after 72 and 96 h. Garlic had completely inhibited the motility of trophozoites with concentration of 100 μg/ml after 24 h, 50 μg/ml after 48 h, 25 μg/ml after 72 h, and 12.5 μg/ml after 96 h while metronidazole had completely inhibited the motility of trophozoites with concentration of 50 μg/ml after 24 h, 25 μg/ml after 48 h, and 12.5 μg/ml after 72 and 96 h. This suggests that commercially available garlic (Tomex®) may be a promising phytotherapeutic agent for trichomoniasis.     

Synergistic protection against cartilage destruction by low dose prednisolone and interleukin-10 in established murine collagen arthritis

Objective: To investigate potential synergy of low dosages glucocorticosteroids (GC's) and interleukin-10 (IL-10), using established murine collagen-induced arthritis (CIA) as a model.¶Methods: DBA-1J/BOM mice were immunized with bovine type II collagen and boosted at day 21. Mice with established CIA were selected and treated for at least 7 days with either prednisolone (0.05-5 mg/kg), IL-10 (0.1-5 7g/day) or the combination of prednisolone/IL-10 (0.05/1 and 0.05/5). Arthritis score was monitored visually, and joint pathology was examined by histology, and serum cartilage oligomeric matrix protein (COMP) measured.¶Results: Amelioration of CIA was found with dosages of 1 and 5 mg/kg prednisolone, while a dose of 0.05 mg/kg prednisolone was ineffective. Treatment of CIA with 5 7g/day IL-10 resulted in a mild, but significant suppression. Synergistic effects were seen with the combination of low dose prednisolone and IL-10 (0.05 mg/kg, 1 7g/day). Both arthritis score and joint pathology were significantly reduced. Moreover, COMP levels were significantly decreased after IL-10/prednisolone treatment, confirming decreased cartilage involvement. Of great interest, treatment of CIA with prednisolone/IL-10 markedly reduced IL-1# and enhanced IL-10 production by synovial tissue. In addition, synovial mRNA levels for IL-1# were decreased, while mRNA levels for IL-10 and IL-1Ra were upregulated by combined treatment.¶Conclusion: This study demonstrates synergistic effects of combined treatment with prednisolone and IL-10 on suppressing disease activity of CIA as well as reducing cartilage.     

In vitro synergistic effect of minocycline combined with antifungals against Cryptococcus neoformans

BackgroundCryptococcus neoformans infections occur in immunocompromised patients, especially those with HIV infection, chemoradiotherapy after cancer, and organ transplantation. Infection can cause pneumonia and meningoencephalitis in severe cases with a high mortality rate if not treated. Although fluconazole and amphotericin B are the first-line treatments for cryptococcosis, the rate of fluconazole resistance has increased significantly due to long-term use. Minocycline is a derivative of tetracycline that exerts its antibacterial effect through inhibition of bacterial protein synthesis. It is also able to pass the blood-brain barrier to act on the central nervous system. The present study investigates the effects of minocycline in combination with antifungals in treating C. neoformans.ObjectiveTo determine in vitro interactions of minocycline combined with itraconazole, voriconazole, posaconazole, fluconazole and amphotericin B against C. neoformans.MethodsThe minimum inhibitory concentrations (MIC) of the antifungals were determined by the CLSI Clinical and Laboratory Standards Institute M27-A3 microdilution method. The in vitro synergistic effects of minocycline combined with itraconazole, voriconazole, posaconazole, fluconazole, and amphotericin B on C. neoformans were detected by the broth microdilution checkerboard technique and disk diffusion testing.Results and ConclusionThe working concentration ranges were 0.125–4 µg/mL for itraconazole, 0.03–0.125 µg/ml for voriconazole, 0.03–1 µg/ml for posaconazole, 0.25–16 µg/ml for fluconazole, and 0.125–2 µg/ml for amphotericin B. The synergistic rates of minocycline combinations against C. neoformans were 55% with itraconazole, 10% with voriconazole, 85% with posaconazole, 20% with fluconazole, and 70% with amphotericin B. The effective MIC value of minocycline in the synergistic combination decreased to 2–32 µg/ml, while the MIC of itraconazole decreased to 0.03–0.125 µg/ml, voriconazole 0.03–0.125 µg/ml, posaconazole 0.03–0.125 µg/ml, 0.125–4 µg/ml fluconazole, and 0.06–0.50 µg/ml amphotericin B. The disk diffusion assay showed that the plates containing minocycline and antifungal drugs produced inhibition zones with diameters larger than the single drug plates. Minocycline showed no antagonistic effect in the combinations. In conclusion, the combination of minocycline and azoles or amphotericin B has synergistic effects against C. neoformans in vitro.     

Tracking of myelin‐reactive T cells in experimental autoimmune encephalomyelitis (EAE) animals using small particles of iron oxide and MRI

Myelin‐reactive T cells are responsible for initiating the cascade of autoreactive immune responses leading to the development of multiple sclerosis. For better insights into the disease mechanism, it is of major importance to have knowledge on the sites at which these cells are active during disease progression. Herein, we investigated the feasibility of tracking myelin‐reactive T cells, upon labelled with SPIO particles, in the central nervous system (CNS) of experimental autoimmune encephalomyelitis (EAE) animals by MRI. First, we determined the optimal labelling condition leading to a high particle uptake and minimal SPIO–Poly‐l‐lysine (PLL) aggregate formation using Prussian blue staining and inductively coupled plasma spectroscopy measurements. Results from labelling of myelin reactive T cells with low concentrations of SPIO particles (i.e. 25 µg/ml) combined with different concentrations of PLL (0–1.5 µg/ml) showed that increasing amounts of PLL led to augmented levels of free remnant SPIO‐PLL aggregates. In contrast, a low PLL concentration (i.e. 0.5 µg/ml) combined with high concentrations of SPIO (i.e. 400 µg Fe/ml) led to a high labelling efficiency with minimal amounts of aggregates. Second, the labelled myelin‐reactive T cells were transferred to control rats to induce EAE. At the occurrence of hindlimb paralysis, the SPIO labelled myelin‐reactive T cells were detected in the sacral part of the spinal cord and shown to be highly confined to this region. However, upon transfer in already primed rats, T cells were more widely distributed in the CNS and shown present in the spinal cord as well as in the brain. Our study demonstrates the feasibility of tracking SPIO labelled myelin‐reactive T cells in the spinal cord as well as the brain of EAE rats upon systemic administration. Furthermore, we provide data on the optimal labelling conditions for T cells leading to a high particle uptake and minimal aggregate formation. Copyright © 2010 John Wiley & Sons, Ltd.