Interleukin-10 of Red Nucleus Plays Anti-Allodynia Effect in Neuropathic Pain Rats with Spared Nerve Injury

Our previous studies have shown that pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) in red nucleus (RN) are involved in the development of neuropathic pain and play facilitated roles on the mechanical allodynia induced by peripheral nerve injury. The current study was designed to evaluate the expression and effect of IL-10, an anti-inflammatory cytokine, in the RN of rats with spared nerve injury (SNI). Immunohistochemical staining results demonstrated when 3 weeks after SNI, the expression level of IL-10 in the contralateral RN of SNI rats was apparently higher than those of sham-operated and normal rats. To further study the effect of IL-10 in the development of neuropathic pain, different doses of IL-10 (1.0, 0.5 and 0.1 μg/μl) were microinjected respectively into the RN contralateral to the nerve injury side of SNI rats. Results demonstrated that higher doses of IL-10 (1.0 and 0.5 μg/μl) significantly attenuated the mechanical allodynia of neuropathic rats, while 0.1 μg/μl of IL-10 did not show any analgesic effect. These results suggest that IL-10 of RN participates in the development of neuropathic pain and plays inhibitory roles on the mechanical allodynia induced by SNI.     

Correlation between strong adjuvanticity of Klebsiella O3 lipopolysaccharide and its ability to induce lnterleukin-1 secretion

Adjuvant activity of Klebsiella O3 lipopolysaccharide (KO3 LPS) in augmenting antibody response and delayed-type hypersensitivity to protein antigens in SMA mice was much stronger than that of LPS from Escherichia coli O55 and O127 (EO55 LPS and EO127 LPS). Relationship between strength of the adjuvant activity and that of the ability to induce interleukin-1 (IL-1) secretion by peritoneal macrophages from C3H/HeN or SMA mice was investigated using these three kinds of LPS. When supernatant samples of macrophages cultured at 37 °C for 24 hr in the presence of 5 μg/ml LPS were assayed by their mitogenic effect on thymocytes from C3H/HeJ mice, KO3 LPS induced the secretion of about four to six times greater amounts of IL-1 activity than did EO127 LPS. When concentration of LPS used for stimulation of macrophages was varied from 0.1 to 50 μg/ml, KO3 LPS induced the secretion of definitely greater amounts of IL-1 activity than did EO55 LPS and EO127 LPS throughout the LPS concentrations tested. Nearly the same amount of IL-1 activity as that produced by 10 μg/ml EO55 LPS or 50 μg/ml EO127 LPS could be produced by 1.0 μg/ml or lower concentrations of KO3 LPS.     

In Vitro Susceptibility of Chlamydia pecorum to Macrolides,Tetracyclines, Quinolones and β-Lactam

The in vitro susceptibility of Chlamydia pecorum to two macrolides (clarithromycin and erythromycin), two tetracyclines (doxycycline and minocycline), two quinolones (ofloxacin and ciprofloxacin) and one β-lactam (ampicillin) was determined. The MICs were 0.004 to 0.008 μg/ml for clarithromycin, 0.008 to 0.031 μg/ml for doxycycline and minocycline, 0.063 to 0.125 μg/ml for erythromycin, 0.25 to 0.5 μg/ml for ofloxacin and 0.25 to 1.0 μg/ml for ciprofloxacin. The MIC for ampicillin was greater than 1,024 μg/ml. The results show clarithromycin and doxycycline are the two most effective drugs against C. pecorum.     

Flame atomic emission spectrometric determination of aluminium in a bovine blood plasma matrix

A flame atomic emission spectrometric method, is described for the determination of aluminium in bovine blood plasma matrices. Plasma samples are wet-digested and solutions are aspirated into a conventional nitrous oxide-acetylene flame. Analyte emission is monitored at 396.15 nm with corrections for background emission being obtained from measurements several tenths nm on both sides of the aluminium line. The mean recovery of 0.3–5 μg/ml aluminium added to model solutions containing 500–5000 μg Na/ml, 50–1000 μg Ca/ml, 2000–5000 μg K/ml, or simulated plasma digests containing Na, K, and Ca was 100,6% (SD = 10.9, df = 60); the mean recovery of 0.3, 0.5, and 1.0 μg/ml aluminium added to blood plasma before digestion was 94.3% (SD = 9.8, df = 33) indicating no serious interferences. For standard solutions, the detection limit (signal: peak-to-peak noise = 1) was 0.02 μg/ml by flame emission, and 0.12 μg/ml by atomic absorption measurements with the same instrument. A sample taken through the analytical procedure, gave a detection limit of 0.05 μg/ml suggesting the submicrogram per milliliter region as the lower practical limit of the method.     

A protein fraction from aged garlic extract enhances cytotoxicity and proliferation of human lymphocytes mediated by interleukin-2 and concanavalin A

Fraction 4 (F4), a protein fraction isolated from aged garlic extract, enhanced cytotoxicity of human peripheral blood lymphocytes (PBL) against both naturalkiller (NK)-sensitive K562 and NK-resistant M14 cell lines. Although F4 treatment alone increased cytotoxicity, the effect was more remarkable when F4 was administered together with suboptimal doses of interleukin-2 (IL-2); combination treatment of 5 g/ml F4 plus 10 U/ml IL-2 for 72 h generated lymphokine-activated killer activity equivalent to that produced by 100 U/ml IL-2 alone against M14. F4 enhanced IL-2-induced proliferation and IL-2 receptor (Tac) expression of PBL without significant increase of IL-2 production. The enhancement of cytotoxicity both by F4 alone and by F4 plus IL-2 was abolished by anti-IL-2 antibody. F4 also enhanced concanavalin-A(ConA)-induced proliferation of PBL. Radiolabeled-ConA binding assays revealed that F4 treatment greatly augmented the affinity and slightly increased the number of ConA binding sites in PBL. F4 also enhanced ConA-induced IL-2 receptor (Tac) expression and IL-2 production of PBL. Anti-IL-2 antibody inhibited the effect of F4 on ConA-induced proliferation. These data suggest that IL-2 is involved in augmentative effects of F4. Our results indicate that F4 is a very efficient immunopotentiator and may be used for immunotherapy.     

Antigen recognition by T lymphocytes. 3. Evidence for two populations of thymus-dependent rosette-forming cells in spleen and lymph nodes

Rosette-forming cells, present in normal spleen, are composed of 70% theta-positive cells with “high” in vitro sensitivity to Azathioprine (AZ) (1 μg/ml) and 30% theta-negative cells with “low” sensitivity to AZ (500 μg/ml). Theta-positive RFC are also found in the thymus (with “high” sensitivity to AZ) and in lymph nodes and peripheral blood (with “intermediate” sensitivity to AZ:50 μg/ml). RFC with “high” sensitivity to AZ (T1) are eliminated from the spleen by adult thymectomy in less than 6 days; it takes more than 48 hours exposure to antilymphocyte serum (ALS) in vivo to suppress them, whereas RFC with “intermediate” sensitivity to AZ (T2), present in lymph nodes and peripheral blood, disappear 6 hours after in vivo ALS treatment but not after adult thymectomy.     

Cytotoxic and suppressor activity of human peripheral blood lymphocytes stimulated by interleukin-2 and phytohemagglutinin before and after fractionation in the percoll gradient

The cultivation of peripheral blood lymphocytes (PBL) obtained from patients with colorectal or bladder carcinoma and melanoma and from healthy donors in the presence of interleukin-2 (IL-2) and PHA resulted in the induction of cytotoxic activity against autologous and/or allogeneic tumour cells in 12 out of 13 patients and in 10 out of 10 donors. A higher level of cytolytic activity was achieved when PBL were separated by means of Percoll density gradient (1.077; 1.067 and 1.056 g/ml) centrifugation and the cells of fraction II (1.077-1.067 g/ml) were employed in the experiment, the level of cytotoxicity being elevated in all cases (1.7-fold elevation in donors and 2-fold elevation in patients on the average). The addition of fraction I (1.067-1.056 g/ml) to fraction II prevented (PHA + IL-2)-mediated induction of cytotoxic activity in all the patients, but in 4 out of 10 donors, i.e. cells of fraction I expressed a suppressor activity. The immunofluorescent analysis has shown that fraction II was enriched by T cells (92%) and depleted of monocytes (7%), as compared to unseparated PBL (66% and 27%, respectively). On the contrary, fraction I was characterized by a decreased T cell ratio (36%) and an increased monocyte level (up to 69%).     

高效氯氰菊酯对草鱼肝胰脏和肾脏溶菌酶活性的影响

研究了暴露在不同高效氯氰菊酯浓度下的草鱼Ctenopharyngodon idella肝胰脏和肾脏溶菌酶(LSZ)的活性变化.实验中高效氯氰菊酯浓度设5组,分别为0 μg/L、0.5μg/L、1.0 μg/L、3.0μg/L和5.0 μg/L,每组分别于1d、5d和12 d取样,测定肝胰脏和肾脏溶菌酶活性.结果显示,肝胰脏LSZ活性在暴露1d、5d、12 d时,各处理组均显著下降(P ＜0.05,P ＜0.01).肾脏LSZ活性在暴露1d、5d时,0.5μg/L、1.0 μg/L和3.0 μg/L组显著上升(P＜0.05,P＜0.01),5.0 μg/L组显著下降(P＜0.01);暴露12d时,0.5μg/L、1.0 μg/L组显著上升(P＜0.05),3.0μg/L和5.0 μg/L组显著下降(P＜0.05,P＜0.01).表明高效氯氰菊酯对草鱼肝胰脏和肾脏具有明显的毒害作用.     

A comparison of peritoneal exudate cells and peripheral blood leukocytes in direct and indirect migration inhibition tests as in vitro assays for tuberculin hypersensitivity in guinea pigs.

Peritoneal exudate cells (PEC) and peripheral blood leukocytes (PBL) are most frequently used in the migration inhibition test. The aim o this work was to compare the ability of these two types of cells to reflect tuberculin hypersensitivity in the migration inhibition test. We sensitized 36 guinea pigs with complete Freund's adjuvant and 20 controls were injected with incomplete Freund's adjuvant. Migration of PEC in medium containing 5, 15, or 75 μg of PPD/ml was assessed after 30 min, and 1, 2, 4, 18, 24, and 48 hr of incubation. The migration of PEC from sensitized animals was inhibited, the inhibition being dose dependent and, with lower concentrations of the antigen, becoming significant only after 4 hr or later. With both PEC and PBL from the same sensitized animal we observed virtually identical migration inhibition in the presence of 75 μg of PPD/ml. A correlation was found between the migration inhibition indices of PEC and PBL. In the indirect test, active supernatants containing lymphokines caused nearly identical migration inhibition of PEC and PBL from normal animals. It follows that in the guinea pig PEC and PBL behave alike both in the direct and in the indirect migration inhibition tests. Thus, PEC and PBL appear to be equally valuable sources of cells for migration inhibition tests.     

First‐derivative synchronous spectrofluorimetric method for estimation of losartan potassium and atorvastatin in their pure forms and in tablets

Losartan potassium (LOS) and atorvastatin (ATR) are used in combination for long‐term treatment of stroke and for treatment of hypertension with high‐level cholesterol. Both drugs were simultaneously determined and validated using a novel, easy, fast, and economical first‐derivative synchronous fluorescence spectroscopic method. Methanol was used as the solvent for both drugs at a Δλ 80 nm and with a scanning rate of 600 nm/min. Peaks were determined as at 288.1 nm and 263.6 nm for LOS and ATR, respectively. The proposed method was validated according to International Conference on Harmonization guidelines and, subsequently, the developed method was applicable to the analysis of the two compounds in their different formulations without interference from each other. Amplitude–concentration plots were rectilinear over the concentration ranges 1.0–10.0 μg/ml and 0.5–5.0 μg/ml for LOS and ATR, respectively. Detection limits were found to be 0.096 μg/ml and 0.030 μg/ml and quantitation limits were 0.291 μg/ml and 0.093 μg/ml for LOS and ATR, respectively. The proposed method was successfully applied to the analysis of both compounds in synthetic mixtures and in laboratory‐prepared tablets. These results were in accordance with the results acquired using the comparison method, high‐performance liquid chromatography.     

Circulating levels of vitamin E in captive Asian elephants (Elephas maximus)

Circulating levels of α-tocopherol (vitamin E) were examined via high-performance liquid chromatography in four female Asian elephants (Elephas maximus) at the New York Zoological Park between 1983 and 1987. Plasma vitamin E averaged 0.08 μg/ml in 1983, and was considered deficient. Over a four-year period of dietary supplementation ranging from 0.7 to 3.7 IU vitamin E/kg body mass (approximately 50 to 250 IU/kg diet as fed), mean plasma α-tocopherol increased to 0.6 μg/ml. Plasma and dietary vitamin E were found to be significantly correlated (p < 0.025) in these animals. Serum or plasma vitamin E measured in an additional 20 elephants from eight other zoological institutions in the United States and Canada averaged 0.5 μg/ml, but values were not significantly correlated (p > 0.05) with calculated dietary levels of the vitamin. To achieve the mean value for circulating α-tocopherol in captive elephants (0.5 μg/ml), feed must provide at least 1.0, and more likely 2.0 to 2.5 IU vitamin E/kg body mass (approximately 130 to 167 IU/kg diet).     

Determination of ibuprofen in serum by high-performance liquid chromatography and application to ibuprofen disposition

A high-performance liquid chromatographic method for quantitation of ibuprofen from serum and application of this method to ibuprofen disposition in the dog is described. The drug was extracted from acidified plasma with dichloromethane. The internal standard used was a methanolic solution of 4-n-butylphenylacetic acid. A μBondapak C₁ column was used for analysis; the mobile phase was methanol—water—glacial acetic acid (pH 3.4) (75:24:1, v/v). A wavelength of 272 nm was used to monitor ibuprofen and the internal standard.Method sensitivity was 0.5 μg/ml serum using either 0.5 or 1.0 ml of sample, and no interference was found from endogenous compounds or other commonly used anti-inflammatory agents. The coefficients of variation of the method were 4.2% and 6.0% for samples containing 50.0 and 6.25 μg/ml of ibuprofen, respectively, and the calibration curve was linear for the range of 0.5 to 100 μg/ml. This method was demonstrated to be suitable for pharmacokinetic and/or biopharmaceutical studies of ibuprofen in man and the dog.     

Chiral Assay of Atenolol Present in Microdialysis and Plasma Samples of Rats Using Chiral CBH as Stationary Phase

Two different enantioselective chiral chromatographic methods were developed and validated to investigate the disposition of the β₁-receptor antagonist atenolol in blood and in brain extracellular fluid of rats (tissue dialysates). System A for the plasma samples was a one-column chromatographic system with a Chiral CBH column with an aqueous buffer as mobile phase into which cellobiose was added for selective regulation of the retention of the internal standard, (S)-metoprolol. The plasma samples were analysed after a simple extraction procedure. The limit of quantitation was 0.2 μg/ml for the atenolol enantiomers. The repeatability of the medium concentration quality control plasma sample (6.0 μg rac-atenolol/ml) was 11–18% for the enantiomers. The dynamic linear range of the plasma samples was 0.5–20 μg/ml. For system B, since atenolol is an extremely hydrophilic drug, the tissue dialysate sample required a much more sensitive system as compared to the plasma samples. A coupled column system was used for peak compression of the enantiomers in the eluate after the separation on the Chiral CBH column, hence increasing the detection sensitivity. The limit of quantification was 0.045 μg/ml for the atenolol enantiomers in artificial CSF. The repeatability of the medium concentration quality control samples (0.1 and 4.0 μg rac-atenolol/ml in artificial CSF and Hepes Ringer, respectively) was 2.8–9.3% for the two enantiomers. The dynamic linear range of the brain samples was 0.05–1.0 and 0.5–20 μg/ml in artificial CSF and Hepes Ringer, respectively. Chirality 9:329–334, 1997. © 1997 Wiley-Liss, Inc.     

Ability of intestinal lactic bacteria to bind or/and metabolise phenol and p-cresol

Intestinal microflora can contribute to colon cancer by the production of substances playing a role in carcinogenesis. Metabolites of protein fermentation in the colon, such as ammonia, H₂S, indole, phenol, skatole are toxic. Lactic bacteria existing in the colon may exert an anti-carcinogenic action, but the mechanism is poorly understood. In the present study the ability of intestin|al lactobacilli to bind or metabolise phenol and p-cresolin vitro was determined.Lactobacillus strains were cultivated in MRS and in a modified MRS broth with reduced concentrations of carbon source. Phenol and p-cresol content in the media were from 2 to 10 μg/ml. In MRS medium lactobacilli could decrease the concentration of phenol and p-cresol and it was 0.2-5.8 μg/ml for phenol and 0.2-1.4 μg/ml for p-cresol. After cultivation in a modified MRS broth, the decrease was 0.5-2.0 μg/ml for phenol and 0.5-2.4 μg/ml for p-cresol. The binding capacity of bacterial cells was rather low. After incubation of non-growing bacteria the decrease of phenol concentration was 0.1-0.5 μg/ml and p-cresol 0.1-2.8 μg/ml. But the ability of growing lactobacilli to metabolise the compounds cannot be excluded. After interaction of lactobacilli with 10 μg/ml of phenol they displayed a lower genotoxicity, as evaluated by the alkaline comet assay. The phenomenon not always depended on the decrease of phenol concentration, but on the medium, the strain of bacteria and for phenol it ranged from 32 to 48%.Lactobacillus strains tested did not lower the genotoxicity of p-cresol.     

Determination of a novel α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor antagonist (LY300164) and its N-acetyl metabolite in mouse,rat, dog,and monkey plasma using high-performance liquid chromatography with ultraviolet detection

A method for the analysis of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) receptor antagonist LY300164 (compound I) and its N-acetyl metabolite (compound II) in plasma was developed. The assay utilized solid-phase extraction on a C₁₈ Bond Elut cartridge followed by reversed-phase HPLC with UV detection at 310 nm. The method exhibited a large linear range from 0.05 μg/ml to 50 μg/ml with an intra-sassay accuracy for compound I and compound II ranging from 89.0% to 114.5% and intra-assay precision ranging from 0.5 to 15.3% in mouse, rat, dog, and monkey plasma. The inter-assay accuracy of compound I and compound II was 93.3% to 101.8% and the inter-assay precision was 1.6% to 11.2% in dog plasma. The lower limit of quantitation was 0.05 μg/ml for compound I in plasma from all species tested. The lower limit of quantitation for compound II was 0.05 μg/ml in dog and monkey plasma and 0.1 μg/ml in mouse and rat plasma. Extracts of compound I and II from dog plasma were shown to be stable for 24 h at room temperature, and both compounds were stable when spiked into rat and monkey plasma frozen at −70°C for 27 days. The method has shown to be useful in the investigation of the pharmacokinetics of the parent compound (I) and metabolite (II) in preclinical studies.     

金黄色葡萄球菌肠毒素A对H22小鼠移植瘤的抑制作用

旨在探讨金黄色葡萄球菌肠毒素A(SEA)在KM鼠体内的抑瘤效果.建立H22荷瘤小鼠模型,将20只小鼠随机分为对照组(生理盐水)、低剂量组(15 μg/ml)、中剂量组(25 μg/ml)和高剂量组(50 μg/ml),观察肿瘤生长趋势,计算抑瘤率,通过ELISA检测IL-2的含量.结果显示,给药组肿瘤生长趋势均较对照组缓慢;对照组、低剂量组、中剂量组、高剂量组的抑瘤率分别为0,28.9%,34.0%,51.0%(P<0.05);血清中IL-2含量分别为58.9 pg/ml、83.6 pg/ml、91.8 pg/ml、118.1 pg/ml.金黄色葡萄球菌肠毒素A(SEA)可抑制H22肿瘤的生长,并上调IL-2的分泌.     

The effects of cyclosporin and hydrocortisone on human T lymphocyte colony formation

The immunosuppressive activities of two compounds, cyclosporin A and hydrocortisone, were evaluated using a human T lymphocyte colony assay. Both compounds produced a dose-related reduction in colony formation. With cyclosporin, concentrations of 1.0–100 μg/ml virtually abolished PHA-induced lymphocyte growth; as little as 0.01 μg/ml decreased clonal growth by 48%. Suppression was observed as late as 4 days after the addition of PHA. The addition of exogenous IL-2 did not completely restore clonal growth to normal. Similarly, hydrocortisone, in concentrations of 0.4 μg/ml, produced a 96% inhibition in clonal growth. Partial suppression could also be obtained if the drug was added as late as 4 days after PHA. Exogenous IL-2 completely reversed the inhibitory effects of hydrocortisone. By contrast, IL-1 was able to only partially restore growth potential. Parallel studies using TPA indicated that both immunosup-pressants inhibited responses to this mitogen. However, in plates containing both TPA and PHA, hydrocortisone failed to impair clonal growth.     

Aphidicolin induces alterations in Golgi Complex and disorganization of microtubules of HeLa cells upon long-term administration

Treatment of HeLa cells with aphidicolin at 5 or 0.5 μg/ml induced cell cycle arrest at G₁/S or G₂/M phase, respectively, and was accompanied by unbalanced cell growth. Long-term administration of aphidicolin (more than 48 h) resulted in noticeable loss of reproductive capacity though cells were viable at the time of treatment. Immunofluorescence with anti-Golgi membrane protein monoclonal antibody (mAbG3A5) showed disfigurement of the characteristic mesh-like configuration when cells were treated for more than 48 h. Interestingly, we found that the fragmented Golgi complex formed a ring around the nucleus in more than 20% of the cells. Immunoelectron microscopy using mAbG3A5 antibody demonstrated that the stack structure of the fragmented Golgi complex in aphidicolin-arrested cells appeared partially broken up and seemed to have converted to a vesicle-like structure. Analysis using an antibody to tubulin and anticentrosome human autoimmune serum showed that alterations in the Golgi complex were induced even by the lower 0.5 μg/ml dose. These alterations were accompanied by both changes in the distribution of microtubules and an increase in the number of centrosomes. These cells lost their distinct perinuclear microtubule organiz-ing center (MTOC). On the other hand, treatment with aphidicolin at 5 μg/ml did not induce multiplication of the centrosome although the loss of distinct MTOC was still evident. No changes took place in the Golgi complex, microtubule, or centrosome of cells treated with 0.5 μg/ml aphidicolin when cycloheximide was added simultaneously to the culture. J. Cell. Physiol. 176:602–611, 1998. © 1998 Wiley-Liss, Inc.     

In vitro activities of antimicrobials against Brucella abortus isolates from cattle in Korea during 1998-2006

In vitro activities of 13 antibiotics were assessed against 85 Brucella abortus isolates from naturally infected cattle in the Republic of Korea during 1998-2006, using broth microdilution test. Tetracyclines showed the most excellent activity against B. abortus, displaying MIC values of 0.5 μg/ml or below. In particular, minocycline showed the lowest MIC??/?? values (0.125/0.125 μg/ml) in this study. Among four fluoroquinolones tested, ciprofloxacin (MIC??/??, 0.5/1 μg/ml) and norfloxacin (MIC??/??, 8/8 μg/ml) had the most and the least activities, respectively. Gentamicin (MIC??/??, 1/1 μg/ml) was more effective than streptomycin, erythromycin, rifampin, and chloramphenicol (MIC??/??, 2/2 μg/ml).