Gene expression profiling in male B6C3F1 mouse livers exposed to kava identifies – Changes in drug metabolizing genes and potential mechanisms linked to kava toxicity

The association of kava products with liver-related health risks has prompted regulatory action in many countries. We used a genome-wide gene expression approach to generate global gene expression profiles from the livers of male B6C3F1 mice administered kava extract by gavage for 14 weeks, and identified the differentially expressed drug metabolizing genes in response to kava treatments. Analyses of gene functions and pathways reveal that the levels of significant numbers of genes involving drug metabolism were changed and that the pathways involving xenobiotics metabolism, Nrf2-mediated oxidative stress response, mitochondrial functions and others, were altered. Our results indicate that kava extract can significantly modulate drug metabolizing enzymes, potentially leading to herb–drug interactions and hepatotoxicity.     

Effects of carbamazepine and metabolites on IL-2, IL-5, IL-6, IL-10 and IFN-γ secretion in epileptic patients: the influence of co-medication

Carbamazepine is a widely used anticonvulsive agent. Its metabolic pathway leads not only to the major active metabolite, carbamazepine-10,11-epoxide, but also to minor terminal metabolites such as iminostilbene and acridine. Carbamazepine is usually well-tolerated, but it may lead to rare, but serious, hypersensitive reactions associated with hypereosinophilia. The mechanisms of hypersensitivity reactions to carbamazepine are still largely unknown, and the implications of the cell-mediated immune response (Th1 pathway) or the humoral immune response (Th2 pathway) are still not understood in these reactions. It is also unclear whether the parent drug or its subsequent metabolites are the primary trigger agent. In our study, we performed ex vivo experiments to evaluate the stimulation of cytokine secretion by carbamazepine, carbamazepine-10,11-epoxide, iminostilbene and acridine. IL-5, IL-6 and IL-10 were quantified as markers of the Th2 pathway, and IL-2 and IFN-γ were used as markers of the Th1 pathway. Blood samples (n=24) were obtained from epileptic patients routinely treated with carbamazepine alone or co-treated with lamotrigine or valproate. The concentrations of cytokines in the plasma were determined before and after 3 h stimulation with drugs. We found a significantly positive effect of co-treatment with valproate on the basal level of IL-5 (p<0.01) and IL-10 (p<0.05). IL-5 production increased only in blood stimulated with a high level of acridine (33 μM), whereas IL-6 production was less specifically stimulated (p<0.05). Because IL-5 is the most potent stimulating factor of the eosinophils, we suggest that the potential helper effect of valproate and acridine can lead to hypersensitive reactions to carbamazepine in the context of the humoral immune response.     

Changes and their significances of IFN-γ, TNF-α and IL-2 level in mouse peripheral blood following intraperitoneal injection with influenza viruses

Objective To observe changes of IFN-γ, TNF-α and IL-2 levels in mouse peripheral blood following intraperitoneal injection with influenza viruses. Methods Mice were divided into 3 groups randomly and injected with human H3N2 influenza virus, avian H9N2 influenza virus and sterilized virus-free allantoic fluid, respectively. Sera in different groups were collected at several time points after virus injection, and the levels of IFN-γ, TNF-α and IL-2 in peripheral blood were tested by sandwich ELISA. Statistical analysis was made using analysis of variance and LSD-t test. Results The levels of IFN-γ and IL-2 in peripheral blood in influenza viruses injection groups were significantly higher than those in negative control group, and no significant difference in TNF-α level was found between influenza viruses injection groups and negative control group. Conclusion Intraperitoneal injection with human or avian influenza viruses can promote the production of IFN-γ and IL-2 in mouse peripheral blood, but it has little effect on the production of TNF-α.     

Changes and their significances of IFN-γ, TNF-α and IL-2 level in mouse peripheral blood following intraperitoneal injection with influenza viruses

Objective To observe changes of IFN-γ, TNF-α and IL-2 levels in mouse peripheral blood following intraperitoneal injection with influenza viruses. Methods Mice were divided into 3 groups randomly and injected with human H3N2 influenza virus, avian H9N2 influenza virus and sterilized virus-free allantoic fluid, respectively. Sera in different groups were collected at several time points after virus injection, and the levels of IFN-γ, TNF-α and IL-2 in peripheral blood were tested by sandwich ELISA. Statistical analysis was made using analysis of variance and LSD-t test. Results The levels of IFN-γ and IL-2 in peripheral blood in influenza viruses injection groups were significantly higher than those in negative control group, and no significant difference in TNF-α level was found between influenza viruses injection groups and negative control group. Conclusion Intraperitoneal injection with human or avian influenza viruses can promote the production of IFN-γ and IL-2 in mouse peripheral blood, but it has little effect on the production of TNF-α.     

Antioxidant enzyme activities and lipid peroxidation in earthworm Eisenia fetida exposed to 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethyl-cyclopenta-γ-2-benzopyran

Polycyclic musks have been indicated to cause lethal and sublethal effects on exposed biota. However, knowledge about the effect of polycyclic musks on the antioxidant defense system in earthworms is vague. In this work, the activities of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and malondialdehyde (MDA) exposed to 1,3,4,6,7,8‐hexahydro‐4,6,6,7,8,8‐hexamethyl‐cyclopenta‐γ‐2‐benzopyran (HHCB) were systematically investigated. The investigation shows that their activities are closely related to the exposed dose and time of HHCB. For SOD and CAT, the activities increased monotonically with increased exposed dose of HHCB, which indicates a dose‐dependent change pattern. POD exhibited its peak activity in 0.0157 μg cm^?2 HHCB treatment and decreased at higher concentrations. These two changing patterns were complementary, which reveals the cooperation of enzymes in response to oxidative stress. MDA content in earthworms was basically unaffected with a 1‐day exposure and significantly increased after 2‐day and 3‐day exposures, correlating with changes in the activities of SOD and CAT when the concentration of HHCB was high. It was also found that the sensitivity of Eisenia fetida to HHCB increased over time. These results may support the theoretical hypothesis that oxidative stress is an important component for the response of earthworms to the toxicity of HHCB in environment. Among the studied enzymes, SOD and CAT appeared to be the most responsive biomarkers of oxidative stress caused by HHCB. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

Relationships between specific serum IgE,IgG, IFN-γ level and IFN-γ, IFNR1 polymorphisms in patients with penicillin allergy

Objective  The findings of numerous studies have suggested that both genetic and environmental influences are involved in the pathogenesis of allergic disease and atopy. We studied the polymorphisms in the interferon (IFN)-gamma (γ) and IFN-γ receptor 1 (IFNR1) gene with the aim of clarifying the relationships among these polymorphisms, penicillin allergy and anti-penicillin antibodies. Methods  A restriction endonuclease fragment length polymorphism (RFLP)-PCR analysis and sequencing were used to study the IFNR1 and IFN-γ polymorphisms. The presence and level of eight specific immunoglobulin (Ig)E and IgG antibodies were determined by the radioallergosorbent test (RAST) and enzyme-linked immunosorbent assay (ELISA), respectively. Results  The positive rates of specific IgE and IgG were 61.11 and 53.92%, respectively. There was no significant difference in the whole-allele of IFN-γ distribution between patients with a penicillin allergy and control subjects. Allele 7 (18CA repeat) was significantly less frequent in the urticaria group (3.19 vs. 11.93%) than in the controls. There was no difference in IFN-γ production among different alleles in IFN-γ. The frequency of G/A (Val/Met) in the IFNR1 gene in allergic patients was significantly less than that in the controls (P < 0.05). There were no significant differences in the positive rate of IgE among different alleles of IFN-γ. The same was true for the positive rate of IgG. Conclusions  The Met/Val allele in IFNR1 gene may have a protective role in the non-penicillin allergic population. The allele 18CA repeat in IFN-γ gene may be associated with urticaria.     

Plasma binding of betamethasone3H,dexamethasone-3H,and cortisol-14C— A comparative study

The binding of betamethasone (16β-methyl-9α-fluoroprednisolone) and dexamethasone (16α-methyl-9α-fluoroprednisolone) to the proteins of cow, dog, rat and human plasma has been studied in vitro by an equilibrium dialysis technique. These synthetic steroids were appreciably bound to the plasma proteins of all species, and the degree of binding did not change substantially over a wide range of concentration. Neither of the synthetic steroids was detectably bound to corticosteroid-binding globulin (CBG) in human plasma, nor did it compete with cortisol for its binding protein.In human and cow plasma, betamethasone was bound to protein to a lesser extent than was dexamethasone; while this condition held for dog plasma, the difference in binding was not as pronounced. In contrast to the other species, betamethasone was bound to a greater extent than was dexamethasone in the rat.The synthetic steroids were bound mainly to the albumin fraction of human plasma. Chromatography on Sephadex gel demonstrated that, although albumin has a high capacity for binding dexamethasone, the affinity constant of the steroid-protein complex is of a low order.     

Influenza A virus replication is inhibited in IFN-λ2 and IFN-λ3 transfected or stimulated cells

Interferons lambda (IFN-λ) are the most recently defined members of the class III cytokine family. To investigate whether IFN-λ2 and IFN-λ3 displayed antiviral activity against influenza A virus (IAV), a number of cell lines induced with IFNs - as well as two established cell lines (A549-IFN-λ2 and A549-IFN-λ3) - were infected with IAV. Our results indicate that IFN-λ2 has statistically significant antiviral activity in A549-IFN-λ2 (P=0.0028) although less so than IFN-λ3, which reduced viral titer to 10% (P<0.0001). The reverse was observed for cells treated with IFNs, with IFN-λ2-treated A549 cells inhibiting IAV infection more efficiently than IFN-λ3-treated A549 cells. The antiviral effect on IFN-stimulated cells was most apparent on Vero cells (compared with MDCK and HeLa). Both IFNs significantly inhibited IAV replication and inhibition was observed in a dose-dependent manner, with an optimal IFN concentration of 20 ng/ml. IFN-λ2 was more potent than IFN-λ3 on Vero cells while IFN-λ3 appeared more efficient than IFN-λ2 on MDCK and HeLa cells.     

Influenza A virus replication is inhibited in IFN-λ2 and IFN-λ3 transfected or stimulated cells

Interferons lambda (IFN-λ) are the most recently defined members of the class III cytokine family. To investigate whether IFN-λ2 and IFN-λ3 displayed antiviral activity against influenza A virus (IAV), a number of cell lines induced with IFNs – as well as two established cell lines (A549-IFN-λ2 and A549-IFN-λ3) – were infected with IAV. Our results indicate that IFN-λ2 has statistically significant antiviral activity in A549-IFN-λ2 (P = 0.0028) although less so than IFN-λ3, which reduced viral titer to 10% (P < 0.0001). The reverse was observed for cells treated with IFNs, with IFN-λ2-treated A549 cells inhibiting IAV infection more efficiently than IFN-λ3-treated A549 cells. The antiviral effect on IFN-stimulated cells was most apparent on Vero cells (compared with MDCK and HeLa). Both IFNs significantly inhibited IAV replication and inhibition was observed in a dose-dependent manner, with an optimal IFN concentration of 20 ng/ml. IFN-λ2 was more potent than IFN-λ3 on Vero cells while IFN-λ3 appeared more efficient than IFN-λ2 on MDCK and HeLa cells.     

Pegylated IFN-α-2a and ribavirin in the treatment of hepatitis C infection in children

The epidemiology, natural history and efficacy of treatment for chronic hepatitis C in children are presented. An increase in the number of vertical infections of this etiology is suggested. In children, especially in those vertically infected, spontaneous elimination of hepatitis C virus (HCV) is observed more often than it is in adults. The most common HCV genotype detected in children is genotype 1, but Italian researchers have described an increase of infection with genotypes 3 and 4 HCV in children in recent years. In the context of recent opinions suggesting a more rapid progression of HCV 3 genotype infection, treatment of these children should begin immediately. The high efficacy (sustained viral response > 50%), safety (few adverse events with less intensity as compared to adults) and good tolerance of therapy with pegylated IFN α-2a and ribavirin have been revealed in children. The differences in the efficacy and tolerability of HCV treatment between children and adults are described. A recommendation for inclusion and monitoring parameters of children’s physical and mental development during HCV treatment is presented. Regarding new anti-HCV therapies with very high efficacy, including IFN-free treatment, the introduction of these therapies to children is recommended.     

Anti-apoptotic,anti-inflammatory,and immunomodulatory activities of 3,3′-diselenodipropionic acid in mice exposed to whole body γ-radiation

The present study was designed to evaluate the possible protective effects of 3,3′-diselenodipropionic acid (DSePA), a potent radioprotector, against oxidative organ damage induced by whole body γ-irradiation and explore its mechanistic effects. The mice were subjected to whole body γ-irradiation at 5 Gy for the detection of oxidative stress, apoptosis, and proliferation in the intestinal (jejunum) tissue and at 7 Gy for the examination of intestinal inflammation and immune imbalance. Groups of mice received intraperitoneal injections of DSePA (2 mg/kg/day) or vehicle (phosphate-buffered saline) for 5 consecutive days prior to irradiation. The whole body γ-irradiation of mice led to the induction of oxidative stress and apoptosis in the intestinal tissue, and pretreatment with DSePA significantly reduced both these parameters. It was also found to abrogate the radiation-induced intestinal inflammatory response and augment the proliferation of intestinal cells. Additionally, irradiation-induced polarization of Th1/Th2 immune balance toward the Th2-dominant direction and pretreatment with DSePA ameliorated this shift, which may be beneficial for the recovery from radiation injury. In conclusion, pretreatment with DSePA prevented radiation-induced oxidative damage in small intestine and the underlying mechanisms responsible for this could be attributed to inhibition of oxidative stress, apoptosis, and inflammation.     

The influence of antibodies to TNF-α and IL-1β on haemodynamic responses to the cytokines,and to lipopolysaccharide,in conscious rats

1. Male, Long Evans rats (350–450 g) were anaesthetized and had pulsed Doppler probes and intravascular catheters implanted to allow monitoring of regional (renal, mesenteric and hindquarters) haemodynamics in the conscious state. Our main objectives were to:- assess the effects of administering human recombinant tumour necrosis factor (TNF)-α and human recombinant interleukin-1 (IL-1)β, alone and together; determine the influence of pretreatment with a mixture of antibodies to TNF-α and IL-1β on responses to co-administration of the cytokines; ascertain if pretreatment with a mixture of the antibodies to TNF-α and IL-1β had any influence on the responses to lipopolysaccharide (LPS).
2. TNF-α (10, 100 and 250 μg kg⁻¹, in separate groups, n=3, 9 and 8, respectively) caused tachycardia (maximum Δ, +101±9 beats min⁻¹) and modest hypotension (maximum Δ, −10±2 mmHg), accompanied by variable changes in renal and mesenteric vascular conductance, but clear increases in hindquarters vascular conductance; only the latter were dose-related (maximum Δ, +6±6, +27±9, and +61±12% at 10, 100 and 250 μg kg⁻¹, respectively).
3. IL-1β (1, 10, and 100 μg kg⁻¹ in separate groups, n=8, 8 and 9, respectively) evoked changes similar to those of TNF-α (maximum Δ heart rate, +69±15 beats min⁻¹; maximum Δ mean blood pressure, −14±2 mmHg; maximum Δ hindquarters vascular conductance, +49±17%), but with no clear dose-dependency.
4. TNF-α (250 μg kg⁻¹) and IL-1β (10 μg kg⁻¹) together caused tachycardia (maximum Δ, +76±15 beats min⁻¹) and hypotension (maximum Δ, −24±2 mmHg) accompanied by increases in renal, mesenteric and hindquarters vascular conductances (+52±6%, +23±8%, and +52±11%, respectively). Thereafter, blood pressure recovered, in association with marked reductions in mesenteric and hindquarters vascular conductances (maximum Δ, −50±3% and −58±3%, respectively). Although bolus injection of LPS (3.5 mg kg⁻¹) caused an initial hypotension (maximum Δ, −27±11 mmHg) similar to that seen with co-administration of the cytokines, it did not cause mesenteric or hindquarters vasodilatation, and there was only a slow onset renal vasodilatation. The recovery in blood pressure following LPS was less than after the cytokines, and in the former condition there was no mesenteric vasoconstriction. By 24 h after co-administration of TNF-α and IL-1β or after bolus injection of LPS, the secondary reduction in blood pressure was similar (−16±2 and −13±3 mmHg, respectively), but in the former group the tachycardia (+117±14 beats min⁻¹) and increase in hindquarters vascular conductance (+99±21%) were greater than after bolus injection of LPS (+54±16 beats min⁻¹ and +43±9%, respectively).
5. Pretreatment with antibodies to TNF-α and IL-1β (300 mg kg⁻¹) blocked the initial hypotensive and mesenteric and hindquarters vasodilator responses to co-administration of the cytokines subsequently. However, tachycardia and renal vasodilatation were still apparent. Premixing antibodies and cytokines before administration prevented most of the effects of the latter, but tachycardia was still present at 24 h.
6. Pretreatment with antibodies to TNF-α and IL-1β before infusion of LPS (150 μg kg⁻¹ h⁻¹ for 24 h) did not affect the initial fall in blood pressure, but suppressed the hindquarters vasodilatation and caused a slight improvement in the recovery of blood pressure. However, pretreatment with the antibodies had no effect on the subsequent cardiovascular sequelae of LPS infusion.
7. The results indicate that although co-administration of TNF-α and IL-1β can evoke cardiovascular responses which, in some respects, mimic those of LPS, and although antibodies to the cytokines can suppress most of the cardiovascular effects of the cytokines, the antibodies have little influence on the haemodynamic responses to LPS, possibly because, during infusion of LPS, the sites of production and local action of endogenous cytokines, are not accessible to exogenous antibodies.

     

Generation of TNFα, IFNγ, IL-4, IL-6 and IL-10 in Trichinella spiralis infected mice: effect of the anti-inflammatory compound mimosine

The plant amino acid mimosine has been demonstrated to arrest cell cycle progression in the late G1-phase, and inhibits [3H] thymidine incorporation in cultured fibroblasts. In this study, 10 mice were infected with Trichinella spiralis, a nematode parasite, and treated with the antiinflammatory compound L-mimosine to determine if any alteration in the chronic inflammatory state occurred by investigating the host's immunological response. Mimosine was used at 250 g/bolus for 25 days starting five days before the infection and continuing daily for 35 days then TNF, IFN-, IL-4, IL-6, and IL-10 were determined by ELISA method, after 0, 1, 7, 14, 21, 28, 35 days post-infection, in the serum of treated or untreated animals. When animals with T. spiralis were treated with L-mimosine, inhibition of TNF was observed within 21 days post-infection, compared with the controls (untreated mice). IFN was inhibited only up to the 21^st day, while IL-6 was inhibited up to the 7^th day post-infection and the inhibition of IL-4 was seen mainly at 21^st and 35^th day p.i. Mimosine-treated mice did not statistically affect the secretion of IL-10 (p > 0.05). In healthy animals, the production of cytokines were within the same limits compared with those of non-infected animals treated with L-mimosine. Our studies suggest that mimosine proved to be more effective in inhibiting TNF and IL-6, which are mainly produced by macrophages and less effective in inhibiting IL-4, which is produced by T-cells.     

High-Performance Liquid Chromatographic Determination of 2′, 3′ -Didehydro-3′ -deoxythymidine (d4T) in Human and Rabbit Plasma and Urine and Its Application to Pharmacokinetic Studies in the Rabbit

A rapid and sensitive liquid chromatographic assay for 2,3-didehydro-3-deoxythymidine (d4T) in plasma and urine is described. This assay uses thymidine oxetane (TO), a synthetic precursor of d4T, as internal standard. Sample preparation involves a simple extraction of plasma or urine with 5% isopropyl alcohol in methylene chloride. The method is specific and sensitive, allowing a linear response over a 2000-fold range of concentrations in human plasma (5 ng/ml to 10 µg/ml) and urine (50 ng/ml to 100 µg/ml). This assay, developed for human plasma and urine, is also applicable to rabbit samples with minor modification. Intravenous bolus doses of 10 mg/kg d4T to rabbits showed that the plasma concentration–time profile followed a biexponential decay. Estimates of the distribution and elimination half-lives were 6.7 ± 0.9 and 51 ± 6 min, respectively. The total-body and renal clearances were 23.4 ± 3.6 and 8.82 ± 3.9 ml/min · kg, respectively. That the renal clearance exceeds the glomerular filtration rate in the rabbit suggests that d4T is actively secreted in the renal tubule. The fraction excreted unchanged in the urine was 36 ± 8%. Similar results were obtained in the same rabbits at steady state during constant-rate intravenous infusion. Noncompartmental analysis estimates of the MRT and V _dss were 46 ± 5 min and 1.08 ± 0.13 L/kg, respectively.     

PPAR-γ2 and PTPRD gene polymorphisms influence type 2 diabetes patients' response to pioglitazone in China

Aim:

To investigate the influence of peroxisome proliferator-activated receptor γ2 (PPAR-γ2) gene polymorphism rs1801282 and protein tyrosine phosphatase receptor type D (PTPRD) gene polymorphism rs17584499 on the occurrence of type 2 diabetes and pioglitazone efficacy in a Chinese Han population.

Methods:

One hundred ninety seven type 2 diabetes patients and 212 healthy controls were enrolled. Among them, 67 type 2 diabetes patients were administered pioglitazone (30 mg/d, po) for 3 months. All the subjects were genotyped for genetic variants in PPAR-γ2 and PTPRD using MALDI-TOF mass spectrometry. Fasting plasma glucose, postprandial plasma glucose, glycated hemoglobin, serum triglyceride, total cholesterol, low-density and high-density lipoprotein-cholesterol were determined.

Results:

The PPAR-γ2 gene rs1801282 polymorphism was significantly associated with type 2 diabetes susceptibility (OR=0.515, 95% CI 0.268–0.990) and the PTPRD gene rs17584499 polymorphism was also significantly associated with type 2 diabetes (OR=1.984, 95% CI 1.135–3.469) in a dominant model adjusted for age, gender and BMI. After pioglitazone treatment for 3 months, the type 2 diabetes patients with PPAR-γ2 rs1801282 CG genotypes significantly showed higher differential values of postprandial plasma glucose and serum triglyceride compared with those with rs1801282 CC genotype. The patients with PTPRD rs17584499 CT+TT genotypes showed significantly lower differential value of postprandial plasma glucose compared to those with rs17584499 CC genotype.

Conclusion:

Diabetes risk alleles in PPAR-γ2 (rs1801282) and PTPRD (rs17584499) are associated with pioglitazone therapeutic efficacy.