Interleukin-21 expanded NKDC in vitro reduces the B16F10 tumor growth in vivo

Innate immunity to tumors is mediated mainly by natural killer cells (NKs) and dendritic cells (DCs). The function of these cells is coordinated by cytokines produced during the inflammatory process. NK cells are highly active against tumors, being an important source of IFN-γ. Natural killer dendritic cells (NKDCs) were recently identified as a group of hybrid cells; some studies claim that they have lytic activity, produce IFN-γ and can also stimulate antigen-specific T cells. Interleukin 21 (IL-21) regulates the proliferation capacity and cytotoxicity of NK and T cells. The main objective of this study was to investigate if IL-21 influences the frequency of NKDCs in vitro as well as IFN-γ production and also to verify if these cells could enhance the antitumor activity against B16F10 tumor model in vivo. Splenocytes from C57BL/6 mice were isolated and the DC were enriched by immunomagnetic beads and cultured for four days with recombinant IL-21 (10, 20, 40 or 100 ng/ml). NKDC population was characterized as CD11c^low/medB220⁺NK1.1⁺. Expanded cells were used to treat B16F10 tumor bearing mice and tumor growth was compared between the doses of IL-21 10 ng/ml and 20 ng/ml. The results indicate that IL-21 increases the expansion of splenic NKDCs in vitro in doses of 10 ng/ml and 20 ng/ml and these cells produce IFN-γ. In vivo, cells expanded with IL-21 and injected directly into the growing tumor efficiently reduced the tumor size. Together, these results showed for the first time that IL-21 influences the biology and the effector activity of NKDCs.     

High serum osteopontin levels in pediatric patients with high risk Langerhans cell histiocytosis

Osteopontin (OPN) acts as an osteoclast activator, a proinflammatory cytokine, and a chemokine attracting histiocytes/monocytes and is abundantly expressed in Langerhans cell histiocytosis (LCH). We investigated whether serum OPN levels are related to disease types in LCH. Fifty-eight newly diagnosed LCH patients were studied; eight with risk organ (liver, spleen and/or hematopoietic) involvements positive multisystem (MS+) disease, 27 with risk organ involvement negative multisystem (MS−) disease and 23 with single system (SS) disease. Pediatric patients with non-inflammatory disease (n = 27) were used as controls. All of patients with MS+ disease were younger than 3 years. Serum OPN levels and 44 kinds of humoral factors were measured by ELISA and Bio-Plex suspension array system, respectively. In the patients younger than 3 years, the median serum OPN level (interquartile range) was 240.3 ng/ml (137.6–456.0) in MS+ (n = 8); 92.7 ng/ml (62.0–213.8) in MS− (n = 14) and 72.5 ng/ml (55.6–94.0) in SS (n = 9) and 74.4 ng/ml (42.2–100.0) in control (n = 12). The OPN values were significantly higher in the MS+ group than the MS−, SS and control groups (p = 0.044, p = 0.001 and p = 0.002, respectively), but not different between the MS−, SS and control groups. In the patients older than 3 years, the median level of serum OPN (IQR) was 56.2 ng/ml (22.9–77.5) in MS− (n = 13), 58.9 ng/ml (31.0–78.7) in SS (n = 14) and 41.9 (28.9–54.1) in control (n = 15). These values did not differ significantly between each group. The serum OPN levels were positively correlated with the serum IL-6, CCL2, IL-18, IL-8 and IL-2 receptor concentration. OPN may be involved in risk organ dissemination and poor prognosis of LCH through the function as inflammatory cytokine/chemokine.     

The plasma levels of CST and BCKDK in patients with sepsis

ObjectivesCST has been recently identified as a mediator of various beneficial effects in animal models of sepsis. At present, no data are available concerning the levels of CST in sepsis patients. In sepsis the plasma amino acid pattern is characterized by decreased branced chain amino acids (BCAAs). We investigated the levels of plasma CST or branched-chain α-ketoacid dehydrogenase kinase (BCKDK) and their relationship to component traits in patients with sepsis.Design and methodsWe studied 228 patients and divided them into two groups based on severity of infection. Blood samples were taken at study entry, and CST, BCKDK were measured.ResultsCST and BCKDK levels were significantly higher in patients with sepsis than in controls: the median plasma CST concentration was 103.1 ng/ml (range, <83.13–189.7 ng/ml) in patients with sepsis and 49.69 ng/ml (range, <19.38–100.8 ng/ml) in controls (p = 0.0022); the median plasma BCKDK concentration was 801.7 ng/ml in sepsis group and 745 ng/ml in controls (p = 0.0292). Additionally, there was correlation between the plasma concentrations of CST and BCKDK in sepsis patients (r² = 0.6357, p < 0.01).ConclusionsWe conclude that the plasma levels of CST in sepsis patients were higher than in controls, and there is a relationship between CST and BCKDK in sepsis patients. Future experimental and clinical studies are needed to evaluate CST as a novel prognostic tool in sepsis patients and its potential therapeutic use in sepsis.     

Green tea polyphenol suppresses tumor invasion and angiogenesis in N-butyl-(-4-hydroxybutyl) nitrosamine-induced bladder cancer

Background: Green tea polyphenol (GTP) suppresses malignancy in bladder cancer cell lines. However, the detail of its anti-carcinogenic effect in vivo is not fully understood. This study investigated the effect of GTP on bladder tumor size and angiogenesis in mice given N-butyl-(-4-hydroxybutyl) nitrosamine (BBN), with and without GTP. Methods: Eight-week-old female C3H/He mice were treated with and without 0.05% BBN solution for 14 or 24 weeks. In addition, they were also treated with and without 0.5% GTP solution for the same periods. Histopathological diagnosis was established using hematoxylin and eosin staining, and microvessel density (MVD) was estimated by counting CD34- and von Willebrand factor-positive vessels in the tumor area. Results: At 14 weeks, cancer cells were detected in BBN and BBN + GTP mice [5/14 (35.7%) and 3/14 (21.4%), respectively, p = 0.678]. At 24 weeks, the incidence of cancer cells was also similar between the groups (BBN + GTP: 61.9% vs. BBN: 82.6%; p = 0.179). However, the frequency of invasive tumors in BBN + GTP mice was significantly lower (23.8%; p = 0.030) than in those given BBN alone (65.2%). Tumor volume and MVD of intratumoral and stromal region in the BBN + GTP group were also significantly lower than in BBN mice. Conclusion: The results showed that GTP had no anti-carcinogenic effect, but inhibited tumor growth and invasion in mice with established bladder cancer, at least in part through the regulation of angiogenesis. Our data suggest that GTP seems to suppress tumor development in bladder cancer.     

A quantitative HPLC–MS method for the simultaneous determination of testosterone, 11-ketotestosterone and 11-β hydroxyandrostenedione in fish serum

A simple and novel HPLC–MS method for the simultaneous quantification of testosterone, 11-ketotestosterone, and 11β-hydroxyandrostenedione in fish serum was developed and validated. Separation was achieved on a C-18 column using a water–acetonitrile mobile-phase with a cycle time of 12 min. Ion detection was performed using ESI positive SIM at [M+H] (m/z 303, 303, 289). The linear ranges (0.2–50 ng/ml), limits of detection (0.1–0.2 ng/ml) and quantification (0.2–0.5 ng/ml) were established. The method was validated by measuring the three androgens in goldfish sera, displaying comparable values to those reported by other analytical techniques (RIA, EIA).     

Application of S-thanatin,an antimicrobial peptide derived from thanatin,in mouse model of Klebsiella pneumoniae infection

Thanatin was first discovered from the hemipteran insect Podisus maculiventris and showed a promising antimicrobial activity. Multidrug-resistant (MDR) clinical isolates of Klebsiella pneumoniae have developed resistance to current therapies. As an attempt to resolve this problem, the efficacy of thanatin and its analogues against clinical isolates of K. pneumoniae was studied in vitro and in vivo. S-thanatin showed an improved antimicrobial activity with the tested MIC values was 2–8-fold lower than those of other thanatin analogs. Antimicrobial assay indicated a high activity of S-thanatin against K. pneumoniae in vitro with MIC between 4 and 8 μg/ml. Its in vivo activity was evaluated using a K. pneumoniae-infected mice model. Adult male ICR mice were randomly grouped and given an intraperitoneal (i.p.) administration of 2 × 10¹⁰ colony-forming units of K. pneumoniae (CI 120204205). Afterwards, mouse groups were subjected to i.p. administration of saline or S-thanatin (5, 10, or 15 mg/kg). After an inspection of 72 h, the mice were finally sacrificed for analysis of in vivo bacterial growth and plasma endotoxin level. The results showed that S-thanatin administration apparently improved the survival rate and reduced the bacterial CFU from intra-abdominal fluid in mice. The plasma endotoxin level was improved as well. All above implied that S-thanatin, as an alternative, may provide a novel strategy for treating K. pneumoniae infection and other infections due to multidrug-resistant bacteria.     

Anti-rheumatoid arthritic activity of flavonoids from Daphne genkwa

The aim of the study was to investigate the anti-rheumatoid arthritic activity of four flavonoids from Daphne genkwa (FFD) in vivo and in vitro. Flavonoids of D. genkwa were extracted by refluxing with ethanol and purified by polyamide resin. An in vivo carrageenan-induced paw edema model, tampon-granuloma model and Freund's complete adjuvant (FCA)-induced arthritis mouse model were used to evaluate the anti-rheumatoid arthritic activities of FFD. Moreover, nitric oxide (NO) release and neutral red uptake (NRU) in lipopolysaccharide (LPS)-induced murine macrophage RAW264.7 cells were used to evaluate the anti-inflammatory effect in vitro. In addition, antioxidant effect of FFD was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. A high dose of FFD significantly reduced the degree of acute inflammatory paw edema in mice as a response to carrageenan administration (p < 0.01). FFD displayed a dose-dependent inhibition of granuloma formation in mice (p < 0.05). FFD also inhibited chronic inflammation in adjuvant-induced arthritis rats when administered orally at the dose of 50 mg/kg/day (p < 0.001). In addition, FFD suppressed the production of NO and exhibited immunoregulatory function in LPS-activated RAW264.7 cells in a dose-related manner. Simultaneously, FFD revealed conspicuous antioxidant activity with IC₅₀ values of 18.20 μg/ml. FFD possesses significant anti-inflammatory and antioxidant activity, which could be a potential therapeutic agent for chronic inflammatory disorders such as rheumatoid arthritis.     

Simultaneous quantification of rosiglitazone and its two major metabolites,N-desmethyl and p-hydroxy rosiglitazone in human plasma by liquid chromatography/tandem mass spectrometry: Application to a pharmacokinetic study

We present a simple, rapid, and sensitive liquid chromatography (LC)–tandem mass spectrometry (MS/MS) method for the simultaneous quantification of rosiglitazone and its two major metabolites via CYP2C8/9, N-desmethyl and p-hydroxy rosiglitazone, in human plasma. The procedure was developed and validated using rosiglitazone-d₃ as the internal standard. Plasma samples (0.1 ml) were prepared using a simple deproteinization procedure with 0.2 ml of acetonitrile containing 40 ng/ml of rosiglitazone-d₃. Chromatographic separation was carried out on a Luna C₁₈ column (100 mm × 2.0 mm, 3-μm particle size) using an isocratic mobile phase consisting of a 60:40 (v/v) mixture of acetonitrile and 0.1% formic acid_(aq). Each sample was run at 0.2 ml/min for a total run time of 2.5 min per sample. Detection and quantification were performed using a mass spectrometer in selected reaction-monitoring mode with positive electrospray ionization at m/z 358.1 → 135.1 for rosiglitazone, m/z 344.2 → 121.1 for N-desmethyl rosiglitazone, m/z 374.1 → 151.1 for p-hydroxy rosiglitazone, and m/z 361.1 → 138.1 for rosiglitazone-d₃. The linear ranges of concentration for rosiglitazone, N-desmethyl rosiglitazone, and p-hydroxy rosiglitazone were 1–500, 1–150, and 1–25 ng/ml, respectively, with a lower limit of quantification of 1 ng/ml for all analytes. The coefficient of variation for assay precision was less than 14.4%, and the accuracy was 93.3–112.3%. No relevant cross-talk and matrix effect were observed. This method was successfully applied to a pharmacokinetic study after oral administration of a 4-mg rosiglitazone tablet to healthy male Korean volunteers.     

Clinical significance of serum transforming growth factor-β1 in lung cancer

Background: Transforming growth factor-β1 (TGF-β1) plays a critical role in human cancer development. Present study aimed to explore the clinical significance of serum TGF-β1 levels in patients with lung cancer and analyze the relationship between TGF-β1 and existing tumor markers for lung cancer. Methods: Serum was collected from 118 patients with lung cancer and 40 healthy volunteers. Serum TGF-β1 levels were measured by enzyme-linked immunosorbent assay (ELISA), and the association with various clinical characteristics was analyzed. The diagnostic value of TGF-β1 was assessed alone and in combination with existing tumor markers for lung cancer. Results: Serum TGF-β1 levels were significantly higher in patients with lung cancer compared to healthy volunteers [0.6 × 10⁵ (0.4 × 10⁵, 0.9 × 10⁵) pg/ml vs 0.5 × 10⁵ (0.3 × 10⁵, 0.7 × 10⁵) pg/ml, P = 0.040]. Although there was a positive correlation between serum TGF-β1 levels and advanced stages, the significant difference was not found between early stages and advanced stages (P = 0.116). The ability of serum TGF-β1 to discriminate lung cancer at a cutoff value of 79,168 pg/ml exhibited sensitivity of 30.6% and specificity of 97.5%. Serum TGF-β1 levels were correlated to cytokeratin fragment 21-1 (CYFRA21-1; R = 0.308, P = 0.020) and neuron-specific enolase (NSE; R = 0.558, P = 0.003). The diagnostic accuracy rates for the existing lung-tumor markers, as SCC, CYFRA21-1, and NSE, were increased from 20.0%, 34.6%, and 45.9% to 48.9%, 51.7%, and 54.5%, respectively by the inclusion of serum TGF-β1 levels. Conclusion: Quantification of serum TGF-β1 levels by ELISA may provide a novel complementary tool for the clinical diagnosis of lung cancer.     

A microsphere-based duplex competitive immunoassay for the simultaneous measurements of aldosterone and testosterone in small sample volumes: Validation in human and mouse plasma

BackgroundThe small blood volumes available in rodent studies often limit adequate quantification of all hormones of interest. We report here the development of two new assays combining an extraction step with multiplex immunoassay (MIA) technology for the simultaneous determination of aldosterone and testosterone in 50 μl sample volume.MethodsFollowing solvent extraction, aldosterone and testosterone competitive immunoassays are performed incorporating biotinylated tracers and antibody-coated beads each having a unique fluorescence. Quantification is via addition of streptavidin–R–phycoerythrin (SA–PE). The assays were validated and compared to established methods. Baseline hormone levels in mice from four different strains, and changes after ACTH and HCG stimulation in CD-1 mice are shown.ResultsThe assays are sensitive (aldosterone 15 pg/ml, testosterone 12 pg/ml), reproducible (intra-/inter-assay imprecision aldosterone 5.1–15.6%/9.9–15.8% and testosterone 9.7–10.9%/7.7–11.4%) and correlate significantly to established assays (r = 0.94–0.95). Baseline aldosterone levels varied between strains, but not between the genders. Testosterone was significantly higher in male of all strains except in C57BL/6× NMRI mice. After ACTH injection, aldosterone (median, interquartile range) rose from 354 (261–396) pg/ml to 2008 (875–2467) in male and from 260 (210–576) to 1120 (734–1528) in female CD-1 mice. HCG injection in the same strain increased testosterone in male mice only (3.5 (0.4–8.3) ng/ml to 31.8 (30.4–33.9) ng/ml, P < 0.01).ConclusionsWe describe a MIA for the simultaneous measurement of aldosterone and testosterone in small volumes after extraction. In addition to presenting a new tool for steroid research in rodent models, our data show strain-dependent differences in steroid hormone metabolism in rodents.     

Cellular and nephrotoxicity of selenium species

ProjectBeside its useful functions at very low concentrations, selenium including supplementary Se sources pose a potential toxicological risk. The toxicity of selenium species was tested in HaCaT cell culture and related nephrotoxicity in mice.ProcedureThe apoptotic shrinkage and necrotic expansion of cells were measured by time-lapse image microscopy. Acute nephrotoxicity was estimated upon administration of various selenium species to mice for two weeks. To confirm or to refute the accumulation of Se in the kidney and its potential chronic effect, Se concentration in kidney tissue and histopathlology were tested.ResultsThe comparison of selenium species showed that organic lactomicroSe did not affect cell growth at 5 ppm, but inorganic nanoSe severely hampered it at lower concentration (1 ppm). The in vivo Se treatment (0.5, 5, 50 ppm, corresponding to 4, 40 and 400 μg/kg) was misleading as it did neither affect the outward appearance nor the weight of the kidney. Se accumulation was observed after selenate, selenite, SelPlex, selenite and nanoSe administration, while lactomicroSe caused no traceable accumulation. In vivo, ex vivo and in vitro experiments reflected this order of selenium toxicity: selenate > selenite > SelPlex = nanoSe > lactomicroSe.ConclusionWithin the tested species lactomicroSe was the only non-nephrotoxic selenium source recommended for nutritional Se supplementation.     

Protection of renal function by green tea extract during Plasmodium berghei infection

Impairment of renal function from oxidative stress during malaria infection is one of the leading causes of death in endemic areas. Since blood urea nitrogen and creatinine levels in plasma can be used as markers for monitoring renal damage, this study investigated the effect of green tea extract on reduction of blood urea nitrogen and creatinine levels during malaria infection using Plasmodium berghei ANKA infected mice as in vivo model. For in vivo testing, ICR mice were infected with 1 × 10⁷ parasitized erythrocytes and green tea extract was subsequently administered orally twice a day for 10 consecutive days. Parasitemia was estimated by standard microscopy, and blood urea nitrogen and creatinine levels in plasma were also measured. It was found that parasitemia kept increasing until animal death, and is strongly correlated with high blood urea nitrogen and creatinine. The highest levels of blood urea nitrogen and creatinine in plasma were found on day 10 after infection. However, blood urea nitrogen and creatinine levels in plasma were reduced and decreased significantly (p < 0.01) in green tea extract treated mice, compared with untreated group. It can be concluded that green tea extract can protect and maintain renal function during malaria infection, and this extract can be developed for use as a supplement and combination therapy.     

Microbial cell components induced tolerance to flagellin-stimulated inflammation through Toll-like receptor pathways in intestinal epithelial cells

In the intestine, bacterial components activate innate responses that protect the host. We hypothesize that bacterial components reduce Interleukin-8 (IL-8) production in intestinal epithelial cells stimulated by flagellin via the Toll-like receptor (TLR) signaling pathway. Caco-2 cells were pretreated with various doses of lipopolysaccharide (LPS), lipoteichoic acid (LTA), or low-dose flagellin (LDFL) for 24 h. Cells were then treated with flagellin (FL) 500 ng/ml (HDFL) for another 48 h. IL-8 production was measured in the cell culture medium by ELISA. Eighty-four genes in the TLR pathway were evaluated by RT Profiler PCR Array. Pathway Studio 8.0 software was used for altered pathway analysis. HDFL induced IL-8 production by 19-fold (p < 0.01). Pretreatment with LDFL at 20, 10 or 1 ng/ml reduced HDFL-induced IL-8 production by 61%, 52% and 40%, respectively (p < 0.05). LPS at 50 μg/ml decreased HDFL–induced IL-8 production by 38% (p < 0.05). HDFL up-regulated CXCL10, IL1B, IL-8, IRAK2, NF-κB1 and I-κB (all p < 0.05). Pathway Studio analysis showed that HDFL induced cell processes including inflammation, cell death and apoptosis. Pretreatment with LDFL at 10 ng/ml down-regulated FADD, FOS, MAP4K4, MyD88, TLR2, TLR3 and TNFERSF1A compared to HDFL (all p < 0.05). These down-regulated genes are integral for numerous cell functions including inflammatory response, cell death, apoptosis and infection. These results demonstrate that LPS and LDFL provoke tolerance to HDFL-induced IL-8 production. This tolerance effect was accompanied by a complex interaction of multiple genes related to inflammatory as well as other responses in the TLR pathway rather than a single gene alteration.     

Efficacy of biogenic selenium nanoparticles against Leishmania major: In vitro and in vivo studies

Project: This study investigated the in vitro and in vivo effectiveness of biogenic selenium nanoparticles (Se NPs), biosynthesized by Bacillus sp. MSh-1, against Leishmania major (MRHO/IR/75/ER). Procedure: The 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to evaluate the cytotoxicity effects of the biogenic Se NPs against both promastigote and amastigote forms of L. major. In a separate in vivo experiment, we also determined the preventive and therapeutic effects of biogenic Se NPs in BALB/c mice following subcutaneous infected with L. major. Results: The MTT assays showed that the highest toxicity occurred after 72 h against both promastigote and amastigote forms of L. major. The cytotoxicity of Se NPs was higher at all incubation times (24, 48, and 72 h) against the promastigote than the amastigote form (p < 0.05). The 50% inhibitory concentrations (IC₅₀) of the Se NPs were 1.62 ± 0.6 and 4.4 ± 0.6 μg ml^?1 against the promastigote and amastigote forms, respectively, after a 72-h incubation period. Apoptosis assays showed DNA fragmentation in promastigotes treated with Se NPs. In an animal challenge, prophylactic doses of biogenic Se NPs delayed the development of localized cutaneous lesions. Moreover, daily administration of Se NPs (5 or 10 mg kg^?1 day^?1) in similarly infected BALB/c mice that had not received prophylactic doses of Se NPs also abolished the localized lesions after 14 days. Conclusion: Based on these in vitro and in vivo studies, biogenic Se NPs can be considered as a novel therapeutic agent for treatment of the localized lesions typical of cutaneous leishmaniasis.     

Acute severe male hypo-testosteronemia affects central motor command in humans

Purpose: To indirectly evaluate the effect of androgens on neuromuscular system in humans we analyzed if an induced short-term hypogonadal state (serum total testosterone-TT < 2.3 ng/ml) may affect central drive to skeletal muscle and/or muscle neuro-mechanical performance. Methods: We compared voluntary and electrically evoked muscle sEMG signals from biceps brachii in nine hypogonadal male volunteers (Hypo) and in ten healthy controls (Cont). Serum TT and dihydrotestosterone (DHT) were assayed. Results: With respect to Hypo, Cont exhibited significantly higher median frequency content (MDF) at any angular velocity; normalized MDF [95.9% (SD = 23.3) vs 73.8% (SD = 9.3)]; muscle fiber conduction velocity (CV) from lowest to highest angular velocities; initial MDF at fatigue test [91.78 Hz (SD = 22.03) vs 70.94 Hz (SD = 11.06)] as well as was the normalized slope [−0.64 (SD = 0.14 vs −0.5 (SD = 0.11)]. In the non-fatigued state, Hypo showed a slower single twitches time to peak (TTP). In Cont, half relaxation time (HRT) decreased after fatigue while increased in Hypo (p < 0.05 between groups). A significant correlation between both TT and dihydrotestosterone with MDF and CV was found during voluntary contractions only. Conclusions: A brief exposure to very low serum TT concentration in males seem to determine a reduced excitability of the NM system which, in turn, would favor a predominant recruitment of slow twitch MUs.     

Simultaneous quantification of the organophosphorus pesticides dimethoate and omethoate in porcine plasma and urine by LC–ESI-MS/MS and flow-injection-ESI-MS/MS

Dimethoate is an organophosphorus toxicant used in agri- and horticulture as a systemic broad-spectrum insecticide. It also exhibits toxic activity towards mammalian organism provoked by catalytic desulfuration in the liver producing its oxon-derivative omethoate thus inhibiting acetylcholinesterase, initiating cholinergic crisis and ultimately leading to death by respiratory paralysis and cardiovascular collapse. Pharmaco- and toxicokinetic studies in animal models help to broaden basic understanding of medical intervention by antidotes and supportive care. Therefore, we developed and validated a LC–ESI-MS/MS method suitable for the simultaneous, selective, precise (RSD_intra-day 1–8%; RSD_inter-day 5–14%), accurate (intra-day: 95–107%; inter-day: 90–115%), and robust quantification of both pesticides from porcine urine and plasma after deproteinization by precipitation and extensive dilution (1:11,250 for plasma and 1:40,000 for urine). Accordingly, lower limits of quantification (0.24–0.49 μg/ml plasma and 0.78–1.56 μg/ml urine) and lower limits of detection (0.12–0.24 μg/ml plasma and 0.39–0.78 μg/ml urine) were equivalent to quite low absolute on-column amounts (1.1–2.1 pg for plasma and 2.0–3.9 pg for urine). The calibration range (0.24–250 μg/ml plasma and 0.78–200 μg/ml urine) was subdivided into two linear ranges (r² ≥ 0.998) each covering nearly two orders of magnitude. The lack of any interfering peak in 6 individual blank specimens from plasma and urine demonstrated the high selectivity of the method. Furthermore, extensive sample dilution causing lowest concentration of potentially interfering matrix ingredients prompted us to develop and validate an additional flow-injection method (FI-ESI-MS/MS). Validation characteristics were as good as for the chromatographic method but sample throughput was enhanced by a factor of 6. Effects on ionization provoked by plasma and urine matrix from 6 individuals as well as in the presence of therapeutics (antidotes) administered in an animal study were investigated systematically underling in the reliability of the presented methods. Both methods were applied to porcine samples derived from an in vivo animal study.     

Growth inhibition and pro-apoptotic activity of violacein in Ehrlich ascites tumor

The continuing threat to biodiversity lends urgency to the need of identification of sustainable source of natural products. This is not so much trouble if there is a microbial source of the compound. Herein, violacein, a natural indolic pigment extracted from Chromobacterium violaceum, was evaluated for its antitumoral potential against the Ehrlich ascites tumor (EAT) in vivo and in vitro. Evaluation of violacein cytotoxicity using different endpoints indicated that EAT cells were twofold (IC₅₀ = 5.0 μM) more sensitive to the compound than normal human peripheral blood lymphocytes. In vitro studies indicated that violacein cytotoxicity to EAT cells is mediated by a rapid (8–12 h) production of reactive oxygen species (ROS) and a decrease in intracellular GSH levels, probably due to oxidative stress. Additionally, apoptosis was primarily induced, as demonstrated by an increase in Annexin-V positive cells, concurrently with increased levels of DNA fragmentation and increased caspase-2, caspase-9 and caspase-3 activities up to 4.5-, 6.0- and 5.5-fold, respectively, after 72 h of treatment. Moreover, doses of 0.1 and 1.0 μg kg^?1 violacein, administered intraperitoneally (i.p.) to EAT-bearing mice throughout the lifespan of the animals significantly inhibited tumor growth and increased survival of mice. In view of these results, a 35-day toxicity study was conducted in vivo. Complete hematology, biochemistry (ALT, AST and creatinine levels) and histopathological analysis of liver and kidney indicated that daily doses of violacein up to 1000 μg kg^?1 for 35 days are well tolerated and did not cause hematotoxicity nor renal or hepatotoxicity when administered i.p. to mice. Altogether, these results indicate that violacein causes oxidative stress and an imbalance in the antioxidant defense machinery of cells culminating in apoptotic cell death. Furthermore, this is the first report of its antitumor activity in vivo, which occurs in the absence of toxicity to major organs.     

Safety of Prunus africana and Warburgia ugandensis in asthma treatment

The aim of the study was to determine the possible cytotoxicity of the aqueous stem bark extracts of Prunus africana and Warburgia ugandensis to Vero E6 cells and acute toxicity in BALB/c mice. Despite being some of the most popular medicinal plants used in Africa, little is known about the safety. In-vitro cytotoxicity tests on Vero E6 cells were investigated using MTT assay to assess the safety of the two plant extracts. Vero E6 cells on growing to confluence were incubated with different drug concentrations for 48 h for the drug to take effect. Viability of the cells was measured by a scanning multiwell spectrophotometer, color intensity being equivalent to viable cells which reduce MTT to soluble formazan crystals. This was done by determining the CC₅₀ of the extracts, CC₅₀ being the concentration of the dose of the compound/extract that kills 50% of the cells. In acute toxicity a total of 55 mice were used. Mice were divided into eleven groups of 5 mice, one group served as negative control and ten groups received oral gavage doses at 500, 889.56, 1581.6, 2812.15 or 5000 mg/kg body weight once. Mortality and other signs of toxicity were recorded within 24 h and the weights of the surviving mice taken for 14 days thereafter. P. africana had CC₅₀ of 104.08 μg/ml while W. ugandensis had CC₅₀ > 250 μg/ml and both were classified as not cytotoxic. There was no mortality observed in groups of mice that received P. africana extracts at 500 and 889.56 mg/kg body weight. There was 20%, 60% and 100% mortality observed within 24 h for mice that received P. africana extracts at 1581.64, 2812.15 and 5000 mg/kg body weight respectively. Lethal dose (LD₅₀) for P. africana was 2201.207 mg/kg body weight. W. ugandensis extracts had no mortality recorded in all dose levels and the LD₅₀ was > 5000 mg/kg body weight. The weights of mice that survived the entire 14 days in all groups increased and were not significantly different from that of controls p > 0.05. From the in vitro and in vivo studies, the two extracts were safe to use. Though with their customary value among many Kenyan communities in management of asthma among other ailments there is a need for further validation of any anti-asthmatic properties and responsible chemical compounds to augment the findings.     

Effect of serum albumin supplementation on in vitro capacitation and fertilization of caprine oocytes

The aim of the investigation was to study the effect of purity and the type of serum albumin on in vitro fertilization (IVF) and cleavage rate of in vitro matured goat oocytes. Ovaries were collected from the local abattoir and transported within 4 h to the laboratory in warm saline (37 °C) containing 100 IU penicillin-G and 100 μg streptomycin sulfate per ml. A total of 2509 cumulus oocyte complexes (COCs) were collected from 1313 ovaries. Oocytes were matured in TCM-199 medium containing FSH (5 μg/ml), LH (5 μg/ml) and estradiol-17β (1 μg/ml), supplemented with 20% fetal bovine serum at 38.5 °C and 5% CO₂ in an incubator under humidified air for 27 h. After 27 h of in vitro maturation (IVM), oocytes were denuded, washed and randomly divided into 4 groups. Group 1 consisted of in vitro matured oocytes (n = 627) co-incubated with sperm in a 50 μl drop of TALP medium containing a 6 mg/ml crystalline bovine serum albumin (BSA) fraction V and 10 μg/ml heparin. Group 2 was comprised of in vitro matured oocytes (n = 470), co-incubated with sperm in a 50 μl drop of TALP medium containing 3 mg/ml crystalline BSA fraction V, 10% estrous goat serum and 10 μg/ml heparin. Group 3 was comprised of in vitro matured oocytes (n = 489) co-incubated with sperm in a 50 μl drop of TALP medium containing a 6 mg/ml fatty acid free BSA and 10 μg/ml heparin. Group 4 consisted of in vitro matured oocytes (n = 422) co-incubated with sperm in a 50 μl drop of TALP medium containing 20% estrous goat serum and 10 μg/ml heparin. After 18 h of co-incubation, the oocyte–sperm mixture was washed in the culture medium 15–20 times and cultured in 50 μl EDM. Cleavage of the in vitro fertilized oocytes were recorded 48 h post-insemination under an inverted phase contrast microscope. The average oocyte recovery rate/ovary and maturation rate was 1.91% and 80.03%, respectively. The cleavage rate in Group 1, Group 2, Group 3 and Group 4 was 1.59%, 8.93%, 11.86% and 35.30%, respectively. It could be concluded that the use of fatty acid free albumin resulted in a significantly higher (P < 0.05) cleavage rate, compared to unmodified albumin, and the supplementation of 20% estrous goat serum in the fertilization medium, significantly (P < 0.05) increased the cleavage rate of in vitro matured goat oocytes, compared to defatted albumin.