[Study on the method of detection of benzene exposure biomarkers by solid phase extraction-gas chromatography-tandem mass spectrometry].

Objective: A gas chromatography-tandem mass spectrometry detection method for benzene and its metabolites was established to provide methodological support and theoretical basis for the study of benzene toxicity mechanism. Methods: In August 2019 to March 2020, the animal model of containing high concentration of benzene by inhalation of poison through the respiratory tract of mice was established, taken the blood of mice after dyeing the poison, and the HLB solid phase extraction method was used to extract and purify the samples. The gas chromatography-tandem mass spectrometry detection method was used to qualitative and quantitative analysis of the target substances. After separated by HP-17MS capillary chromatographic column, the compounds were ionized with EI ion source, mass spectrometry detection was carried out by selective ion scanning method (SIM) , and quantification was carried out by external standard curve method. Results: Benzene and its metabolites (phenol, catechol, hydroquinone and m-trihydroxybenzene) in blood could be effectively separated and quasi deterministic and quantitative by this method. The regression equations and correlation coefficients of this method for detecting benzene and its metabolites were: benzene: y=3252.1x+1540, r=0.9993; phenol: y=2046.5x+1423, r=0.9991; catechol: y=1853.9x+945, r=0.9993; hydroquinone: y=1891.5x+840, r=0.9992; m-trihydroxybenzene: y=1052.4x+655, r=0.9991. The detection limits for benzene, phenol, catechol, hydroquinone and m-trihydroxybenzene were 0.03, 0.03, 0.05, 0.05 and 0.10 µg/g, respectively. And the lower limits of quantification were 0.10, 0.10, 0.15, 0.15 and 0.30 µg/g, respectively. The intra-assay precision interval was 2.64%-10.06%, the inter-assay precision interval was 1.37%-10.17%, and the spike recovery rate was 89.8%-102.3%. This method could be used to quantitatively detect benzene, phenol, catechol, hydroquinone and m-trihydroxybenzene in the blood of benzene-infected mice. Conclusion: Solid phase extraction-gas chromatography-mass spectrometry can be used for qualitative and quantitative detection of benzene and its metabolites (phenol, catechol, hydroquinone and m-trihydroxybenzene) accurately.

[The value of serum abnormal prothrombin in clinical application of hepatocellular carcinoma].

Objective: To examine the value of serum protein induced by vitamin K absence or antagonist-Ⅱ (PIVKA-Ⅱ) detection in the early diagnosis and surveillance of hepatocellular carcinoma (HCC). Methods: The clinical data of 215 patients with HCC admitted to Department of Hepatobiliary-Pancreatic Surgery of China-Japan Union Hospital of Jilin University from October 2017 to May 2018 were analyzed retrospectively. There were 172 males and 43 females, aged of (59.0±9.3) years old (range 34 to 86 years old). In addition, there were 85 non HCC patients were enrolled in the control group, 42 males and 43 females, aged (54.2±11.3) years old (range 22 to 80 years old). The blood sample of 3 ml was drawn from the elbow vein at 6∶00 am on the next day of admission, and then was kept in low temperature away from light, and sent for PIVKA-Ⅱ detection on the same day. The positive value of AFP was ≥20 µg/L and PIVKA-Ⅱ was ≥32 AU/L. The data were analyzed statistically by χ(2) test, t test or rank sum test. The correlation between AFP, PIVKA-Ⅱ and tumor maximum diameter was analyzed by linear regression. Results: The sensitivity of PIVKA-Ⅱ detection only for the diagnosis of HCC in all stages was significantly higher than AFP or equivalent to AFP, the overall sensitivity of PIVKA-Ⅱ and AFP was 85.1% and 52.1%, respectively. But the specificity of PIVKA-Ⅱ was lower than that of AFP, they were 78.8% and 96.5%, respectively. In particularly, in the earlier stage of HCC (Ⅰa) , the sensitivity of PIVAK-Ⅱ to HCC was 64.5%, while the AFP was only 26.3%. Combined detection of PIVKA-Ⅱ and AFP significantly improved the diagnostic rate of HCC to 88.4%, and the specificity to 76.5%. Moreover, there was a positive correlation between PIVKA-Ⅱ level and the maximum tumor diameter (r(2)=0.587, P<0.05), but there was no correlation between the AFP level and the maximum tumor diameter (r(2)=0.296, P>0.05). The positive rate of PIVKA-Ⅱ in the diagnosis of HCC with vascular invasion was also significantly higher than that of AFP (P<0.01) . Conclusions: PIVKA-Ⅱ can be used as a serological marker for HCC screening and diagnosis. In particular, PIVKA-Ⅱ detection was significantly sensitive than AFP in the earlier stage of HCC. Combined detection of PIVKA-Ⅱ and AFP can effectively improve the diagnostic rate of HCC in all stages. The significant elevation of PIVKA-Ⅱ is also helpful to determine the tumor aggressiveness, vascular invasion and prognosis of HCC patients.

[The expressions of intercellular adhesion molecule-1 in renal tissue of rats with paraquat poisoning and the effect of melatonin].

OBJECTIVE: To study the mechanism of paraquat (PQ) -induced renal injury in rats, the expression changes of ICAM-1 to assess the protective effect of Melatonin in PQ poisoning. METHODS: Ninety adult healthy Sprague-Dawley (SD) rats were divided into three groups at random. CONTROL GROUP: 30 rats; Poisoned group: 30 rats; Melatonin group: 30 rats. Control group and Poisoned group were treated intragastrically with 1 ml of PQ (50 mg/kg) diluted with normal saline. Control groupwere treated with the same dose of normal saline as Poisoned group and Melatonin group. Melatonin group were given 1 ml of Melatonin at a dose of 10 mg/kg diluted with normal saline (once daily, intraperitoneally) Control and Poisoned group were treated with the same dose of normal saline (once daily, intraperitoneally) as Melatonin group. Pathology of renal tissue were oberserved by HE staining, and electron microscope. The histopathological changes and the expression of ICAM-1 were observed with mmunohistochemistry (IHC). RESULTS: (1) There were no obvious pathological changes in Control group. Poisoned group Renal glomerulus had hyperemia and distension.Renal tubule epith elial cell had edema and vacuolar degeneration and renal tubule lumina was narrowing on day 1, There were serious edema exudation and necrosis on day 5,which gradually lessened furthermore; Compared with Poisoned group, the aforementioned pathological lesion was more palliative in Melatonin group. (2) No obvious abnomal changes in ultrastructure of renal tissues in Control group. There were swelling of mitochondrion and rupture of renal tubule epithelial cell and endoplasmic reticulum had extension, lysosome was mult and had much phagocytosis in Poisoned group. (3) There was a very weak expression of ICAM-1 in Control group. while in Poisoned group, there was already a significant higher expression of ICAM-1 of renal tubule on day 1 after PQ poisoning, Immunohistochemistry score (IHS) of Poisoned group on day 1, 3, 5, 7, 14 were (0.1561±0.0295、0.2572±0.0259、0.3028±0.0153、0.2083±0.0227、0.9309±0.0059) , compared with Control group (P<0.01) ; Melatonin group were (0.1259±0.0061、0.2109±0.0280、0.2679±0.0233、0.1771±0.0186、0.0791±0.0135) , compared with Control group (P<0.01) , compared with Poisoned group (P<0.05) ; CONCLUSION: ICAM-1 was involved in the procedures of renal injury; MT surely had a protective effect, which might be mediated by ICAM-1 in the paraquat-induced renal injury, but its regulation path still need a further exploration.

An update on acupuncture point injection.

This overview reports the global research advances in acupuncture point injection in the last 5 years. Acupuncture point injection can be applied to a wide range of curable diseases, predominantly those involving pain, but it has poor clinical evidence. Progress has been attained in the mechanism research on acupuncture point injection, but further studies remain necessary. With the reported adverse effects of acupuncture point injection, the need to standardize its clinical procedure has become urgent.

Geometric Parameter Analysis of Ruptured and Unruptured Aneurysms in Patients with Symmetric Bilateral Intracranial Aneurysms: A Multicenter CT Angiography Study.

BACKGROUND AND PURPOSE: Previous studies of geometric and morphologic parameters of intracranial aneurysms have been conducted to determine rupture risk, which remains incompletely defined due to patient-specific risk factors, such as sex, hypertension, and age. To this end, we compared characteristics of ruptured and unruptured aneurysms in the same patients with symmetric bilateral intracranial aneurysms. MATERIALS AND METHODS: Between January 2008 and March 2014, 2361 patients with 2674 aneurysms were diagnosed by CT angiography or surgical findings at 4 medical centers. Geometric and morphologic parameters examined for symmetric bilateral intracranial aneurysms comprised aneurysm wall regularity, size, neck width, aspect ratio, size ratio, neck-to-parent artery ratio, and area ratio. Univariate and multivariate statistical analyses were performed to determine independent risk factors for rupture. RESULTS: Sixty-three patients (48 women, 15 men; mean age, 62.5 ± 9.8 years) with symmetric bilateral aneurysms were eligible for the study and were included. The most frequent aneurysm location was the posterior communicating artery. Univariate analysis disclosed that aneurysm size, aspect ratio, size ratio, area ratio, and irregular wall differed between patients with ruptured and unruptured aneurysms. Multivariate analysis indicated that aspect ratio of ≥1.6 (adjusted OR, 9.521; 95% CI, 2.182-41.535), area ratio of ≥1.5 (adjusted OR, 4.089; 95% CI, 1.247-13.406), and irregular shape (adjusted OR, 10.443; 95% CI 3.394-32.135) were significant predictive factors for aneurysm rupture after adjustment for aneurysm size. CONCLUSIONS: An aspect ratio of ≥1.6, area ratio of ≥1.5, and irregular wall are associated with aneurysm rupture independent of aneurysm size and patient characteristics. These characteristics alone can help in distinguishing ruptured bilateral intracranial aneurysms from unruptured ones.

The status of occupational blood and infectious body fluids exposures in five blood centres in China: a 5-year review.

BACKGROUND: Little information about the occupational exposures to blood and body fluid (BBF) among blood service workers (BSWs) in blood stations in China is available currently. OBJECTIVES: To assess current status of occupational exposure to BBF and assess the knowledge about occupational blood-borne pathogen exposures and universal precaution among BSWs in blood donations in China. To understand the incidence of occupational exposure in five blood centres in China. METHODS: A cross-sectional study was conducted from January 2008 to December 2013. RESULTS: There were a total of 99 BBF exposures reported during the study period. The total incidence of BBF exposures was 4.4 per 100 person-years. Higher rates were observed for persons employed less than five years and persons less than 45 years old. Nurses have the highest percentage (49.5%) of BBF exposures. BBF exposures occurred most commonly during the afternoon (62.7%). Percutaneous injuries were the most common BBF exposures. Most incidents occurred during sharps use (73.4%). The major cause of occupational exposure was that there was no continuous training (48.4%) and improper use of equipment (23.2%). Only 56.6% of BBF exposures had appropriate first aid measures. During this research work, one staff member was reported to have seroconverted to syphilis after BBF exposure. CONCLUSIONS: To reduce BBF exposures, it is urgent to take several effective actions in China, including improved occupational health systems, adequate education, administrative support, increased use of standard precautions, better safety devices/products and work practices.

[Establishment of a FVB/N mouse model of cardiac hypertrophy by isoprenaline].

OBJECTIVE: To investigate the condition of isoprenaline (ISO)-induced cardiac hypertrophy in the FVB/N mouse. METHODS: ISO (30 mg/kg/d) was administered either by daily subcutaneous injection, or by continuous infusion via an implanted osmotic minipump. The mice in each mode of administration were randomly divided into two groups. For subcutaneous injection: the mice received ISO or saline through daily subcutaneous injection for 2 weeks. The mice for minipump: the mice received continuous infusion of ISO via an implanted osmotic minipump for 2 weeks, or received sham operation as the control to mimipump. The ratio of heart weight to tibia length (HW/TI), the diastolic left ventricular posterior wall thickness (dLVPW) were used to indicate cardiac hypertrophy. Interstitial fibrosis was examined with picrosirius red staining. RESULTS: ISO (30 mg/kg/d) administered by daily subcutaneous injection did not lead to cardiac hypertrophy or fibrosis in the FVB/N mice, and 50% of the mice died before the end point. The mice receiving ISO via minipumps showed significant increase in HW/TI [(10.60±0.40 ) mg/mm vs. (7.93±0.19) mg/mm,P<0.001] and dLVPW [(0.87±0.03) mm vs. (0.68±0.06)mm,P=0.0116]. ISO administered via minipumps did not induce cardiac fibrosis. All the mice in this group survived to the end point. CONCLUSION: ISO (30 mg/kg/d) administered by continuous infusion via a minipump for 2 weeks can lead to significant cardiac hypertrophy.

A huge malignant peripheral nerve sheath tumor with hepatic metastasis arising from retroperitoneal ganglioneuroma.

Ganglioneuromas (GNs) are the rarest and most benign of the neuroblastic tumors. We experienced a case of huge retroperitoneal GN which differentiated into malignant peripheral nerve sheath tumors (MPNST) with hepatic metastasis. The tumor was located in the upper right quarter of the abdomen and pressed the right lobe of the liver, which was initially misdiagnosed as a liver carcinoma. The tumor shared blood supply with the right liver lob and had rich blood supplies from the abdominal aorta, renal artery and hepatic artery. It was also associated with skin pigment and recurrence shortly following resection. Our finding demonstrated that MPNST is a potent invasive malignant tumor and metastasis earlier with very poor prognosis.

Mucocutaneous manifestations of HIV-infected patients in the era of HAART in Guangxi Zhuang Autonomous Region, China.

BACKGROUND: Human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS) are frequently associated with diverse mucocutaneous manifestations. However, few studies of HIV/AIDS-related mucocutaneous manifestations have been reported in China. OBJECTIVE: To investigate the occurrence of mucocutaneous disorders and their relationship with the degree of immunosuppression in 348 HIV-infected Chinese patients. The influence of highly active antiretroviral therapy (HAART) on the spectrum of mucocutaneous manifestations was also evaluated. METHODS: A cross-sectional study was performed on 348 HIV-infected Chinese patients seen at the Guangxi Longtan Hospital from August 2010 to November 2010. Collected information included demographic data, HIV-associated mucocutaneous disorders, CD4 cell count, AIDS-defining illness and antiretroviral therapy. RESULTS: In this study, 62.9% of all patients had mucocutaneous disorders. The prevalence of mucocutaneous disorders in the patients who had received HAART was lower than in those without HAART (54.05% vs. 88.76%, P < 0.001). The prevalence of mucocutaneous disorders was higher in the patients with CD4 < 200 cells/mm(3) in comparison to those with CD4 ≥ 200 cells/mm(3) (P < 0.05). The most common mucocutaneous disorders were oral candidiasis (28.47%), Penicillium marneffei infection (11.49%), drug eruptions (10.06%) and pruritic papular eruption (PPE 5.75%). Oral candidiasis, P. marneffei infection and PPE were significantly more prevalent in patients with a CD4 cell count below 200 cells/mm(3) , but frequency of drug eruptions was not related to the level of CD4 cell counts. Patients treated with HAART had decreased rates of herpes simplex, oral candidiasis and P. marneffei infection, but increased rates of drug eruptions. CONCLUSIONS: A wide range of mucocutaneous disorders were observed in HIV-infected Chinese patients. Oral candidiasis, P. marneffei infection and PPE may be the signs of advanced HIV infection. HAART had an impact on the spectrum of HIV-associated mucocutaneous disorders.

Acute ethanol-induced adenosine diphosphate ribosylation regulates the functional activity of rat striatal pertussis toxin-sensitive g proteins.

BACKGROUND: We demonstrated previously that striatal adenosine modulates ethanol-induced motor incoordination (EIMI) via adenosine A1 receptors coupled to pertussis toxin (PT)-sensitive G protein and adenylyl cyclase-cyclic adenosine monophosphate (cAMP). Additionally, intrastriatal (IST) PT antagonizes EIMI and its potentiation by the adenosine A1 agonist N-cyclohexyladenosine; it also inhibits cAMP concentration. METHODS: Guide cannulas were stereotaxically implanted for IST pretreatment with PT followed 5 days later by IST of N-cyclohexyladenosine and intraperitoneal ethanol. The adenosine diphosphate (ADP) ribosylation reaction involved PT-catalyzed [P]nicotinamide adenine dinucleotide (NAD) labeling of rat striatal membranes. Antagonism of EIMI (Rotorod method) after IST microinfusion of PT was investigated to determine whether it was due to a decrease in the functional activity of G proteins due to ADP ribosylation of the Gialpha subunit caused it. RESULTS: Striatal membranes from IST PT (0.5 microg)-treated animals exhibited significantly attenuated (up to 90%) in vitro ADP ribosylation with [P]NAD. Striatal membranes from animals injected with ethanol (1.5 g/kg intraperitoneally) exhibited statistically significant increase (11%) in in vitro ADP ribosylation. Similarly, ethanol (50 mM) added to striatal membranes from untreated animals produced significant stimulation of in vitro ADP ribosylation. The decrease in the functional activity of G proteins due to ADP ribosylation of the Gialpha subunit after IST PT was functionally correlated with marked attenuation in EIMI, as observed previously. This finding suggests a blockade of functional activity of PT-sensitive striatal Gi/Go proteins (i.e., fewer available sites for labeled NAD incorporation). The in vivo ethanol results indicate that it must have caused an increase in the ribosylation capacity of Gialpha in vivo (i.e., increased Gi activity). Increased ADP ribosylation by in vitro ethanol increases Gi/Go activity, consistent with EIMI, as previously reported. CONCLUSIONS: The results provide biochemical evidence of an ethanol-induced increase in ADP ribosylation of Gialpha causing a decrease in the functional activity of G proteins coupled via Gi/Go to adenylyl cyclase-cAMP. These results confirm the previously observed antagonism of EIMI by PT (IST).

Essential role for IL-6 in postresuscitation inflammation in hemorrhagic shock.

Interleukin-6 (IL-6) is produced within multiple tissues and can be readily detected in the circulation in resuscitated hemorrhagic shock (HS). Instillation of IL-6 into lungs of normal rats induces polymorphonuclear neutrophilic granulocyte (PMN) infiltration and lung damage, while infusion of IL-6 into the systemic circulation of rats during resuscitation from HS reduces PMN recruitment and lung injury. The current study was designed to determine whether or not IL-6 makes an essential contribution to postresuscitation inflammation and which of the two effects of IL-6, its local proinflammatory effect or its systemic anti-inflammatory effect, is dominant in HS. Wild-type and IL-6-deficient mice were subjected to HS followed by resuscitation and death 4 h later. IL-6-deficient mice subjected to HS did not demonstrate any features of postresuscitation inflammation observed in wild-type mice, including increased PMN infiltration into the lungs, increased alveolar cross-sectional surface area, increased PMN infiltration into the liver, increased liver necrosis, increased signal transducer and activator of transcription 3 activation, and increased nuclear factor-kappaB activity. These findings indicate that IL-6 is an essential component of the postresuscitation inflammatory cascade in HS and that the local proinflammatory effects of IL-6 on PMN infiltration and organ damage in HS dominate over the anti-inflammatory effects of systemic IL-6.

Distinct effects of systemic infusion of G-CSF vs. IL-6 on lung and liver inflammation and injury in hemorrhagic shock.

Production of pro-inflammatory cytokines, such as granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6) occurs at multiple tissue sites in hemorrhagic shock (HS), resulting in elevated circulating plasma levels. The current study was designed to test the hypothesis that circulating G-CSF and IL-6 contribute to polymorphonuclear neutrophilic granulocyte (PMN)-mediated inflammation and organ injury in HS. Sprague-Dawley rats were subjected to decompensated HS (mean arterial blood pressure = 40 mm Hg for 2.5 h), followed by resuscitation with lactated Ringer's solution with or without G-CSF (3 microg/kg) or IL-6 (3 microg/kg). Animals were killed 4 h after resuscitation, and their lungs and livers were assessed quantitatively for PMN infiltration, organ injury, and activation of NF-kappaB and signal transducer and activator or transcription (STAT) 3. Infusion of G-CSF during resuscitation increased PMN infiltration into the lungs by 2.4-fold (P < 0.01) compared with animals resuscitated with lactated Ringer's solution alone. Increased PMN infiltration was accompanied by interstitial edema and pneumocyte swelling, resulting in a 42% increase in lung alveolar wall cross-sectional surface area (P < 0.01) and a 3.7-fold increase in Stat3 activity (P < 0.01). G-CSF infusion did not affect PMN infiltration into the liver and was accompanied by a 68% decrease in focal hepatocellular necrosis (P < 0.01). Infusion of IL-6, in contrast, dramatically decreased inflammation and injury in both the lung and liver; the anti-inflammatory effects of IL-6 may be mediated, in part, by down-modulation of nuclear factor (NF)-kappaB activity. Thus, circulating G-CSF and IL-6 have opposing effects on PMN recruitment and injury in the lung in HS while both protect against hepatic necrosis. The beneficial effect of these cytokines on liver injury in HS appears to be independent of PMN recruitment.

Propagation of genetically altered tumor cells derived from fine-needle aspirates of primary breast carcinoma.

Because primary breast tumors are diagnosed earlier in the clinic, procurement of sufficient amounts of tumor tissue for in-depth biological characterization is becoming increasingly difficult. We demonstrate here that relatively small numbers of tumor cells within samples of fine-needle aspirates (FNA) can be propagated in culture. Of 25 cases attempted, 12 were passageable, resulting in up to 10(7) viable cells. FNA-derived cultures were evaluated for anchorage-independence, c-erb-B2 overexpression, aneusomy, and pattern of allelic loss. In every case examined, the cultured cells closely resembled the original tumor tissue and displayed one or more tumor phenotypes. The incidence of erb-B2 overexpressing tumors was similar in passageable and nonpassageable cases (33% versus 31%, respectively). FNAs that are expanded from a wide range of clinical breast material could be useful for functional studies presently limited to rare established cell lines, such as aberrant signal transduction and gene regulation, and for testing potential anticancer vaccines and drugs.

Clozapine and haloperidol block the induction of behavioral sensitization to amphetamine and associated genomic responses in rats.

Behavioral sensitization resulting from repeated, intermittent exposure to psychostimulants such as amphetamine (Amp) is hypothesized to model pathophysiology of psychotic disorders. The present study was designed to characterize the effects of a typical and an atypical antipsychotic drug, haloperidol and clozapine, respectively, on the induction of context-independent sensitization to Amp. Peripheral Amp treatment for five days (2 mg/kg/day, s.c.) produced an augmented stimulant response to an acute Amp challenge (2 mg/kg, s.c.) given seven days after the last pretreatment injection. Interestingly, preexposure to high doses of either clozapine (20 mg/kg) or haloperidol (0.5 mg/kg) alone also led to a sensitized behavioral response to an acute Amp challenge. The cross-sensitization between Amp and high doses of the haloperidol and clozapine may have occluded any blockade of Amp behavioral sensitization by the antipsychotics. Indeed, administration of a lower dose of clozapine (4 mg/kg) or haloperidol (0.1 mg/kg) with Amp during the preexposure phase clearly blocked the induction of behavioral sensitization. In addition to the behavioral sensitization, Amp-pretreated rats showed a reduction in the ability of the acute Amp challenge to induce c-fos mRNA in the medial prefrontal cortex and neurotensin/neuromedin N (NT/N) mRNA in the nucleus accumbens-shell. At doses that blocked the initiation of behavioral sensitization to Amp, clozapine fully and haloperidol partially restored the capacity of acute Amp to induce c-fos and NT/N gene expression. These data lend support to the psychostimulant-sensitization model of psychosis and a role of dopamine D2-like receptors in the phenomenon.

Pulmonary responses to oil fly ash particles in the rat differ by virtue of their specific soluble metals.

Occupational exposure to residual oil fly ash (ROFA) particulate has been associated with adverse respiratory health effects in humans. We hypothesized that ROFA collected at different sites within an oil burning power plant, by virtue of its differing metal and sulfate composition, will induce differential lung injury. Ten ROFA samples collected at various sites within a power plant were analyzed for water- and 1.0 M HCl-leachable arsenic (As), beryllium (Be), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), manganese (Mn), nickel (Ni), lead (Pb), vanadium (V), zinc (Zn), and sulfur by inductively coupled plasma-atomic emission spectroscopy. All ROFA samples contained variable amounts of leachable (water-extractable) and 1.0 M HCl-extractable Fe, V, and/or Ni. All other metals, except Zn (ROFA No. 1 contained 3.43 and No. 3, 6.35 micrograms/mg Zn), were present in negligible quantities (< 1.0 microgram/mg) in the water extract. In vivo pulmonary injury from exposure to whole saline suspensions of these ROFA was evaluated. Male, SD rats (60 days old) were intratracheally instilled with either saline or saline suspension of whole ROFA (< 3.0 mass median aerodynamic diameter) at three concentrations (0.833, 3.33, or 8.33 mg/kg). After 24 h, lungs were lavaged and bronchoalveolar lavage fluid (BALF) was analyzed for cellular influx and protein content as well as lactate dehydrogenase (LDH) and N-acetyl glucosaminidase (NAG) activity and total hemoglobin as indicators of lung injury. ROFA-induced increases in BALF protein and LDH, but not neutrophilic inflammation, were associated with its water-leachable total metal, Ni, Fe, and sulfate content. However, the neutrophilic response following ROFA exposure was positively correlated with its water-leachable V content. Modest lung injury was observed with the ROFA samples which contained the smallest amounts of water-leachable metals. The ability of ROFA to induce oxidative burst in alveolar macrophage (AM) was determined in vitro using a chemiluminescence (CL) assay. AM CL signals in vitro were greatest with ROFA containing primarily soluble V and were less with ROFA containing Ni plus V. In summary, ROFA-induced in vivo acute pulmonary inflammation appears to be associated with its water-leachable V content; however, protein leakage appears to be associated with its water-leachable Ni content. ROFA-induced in vitro activation of AM was highest with ROFA containing leachable V but not with Ni plus V, suggesting that the potency and the mechanism of pulmonary injury will differ between emissions containing V and Ni.

Rat striatal adenosinergic modulation of ethanol-induced motor impairment: possible role of striatal cyclic AMP.

We have previously reported the involvement of the striatum in acute ethanol-induced motor incoordination and the striatal adenosinergic modulation of ethanol-induced motor incoordination through A1 receptor-mediated mechanism(s). The present study, a continuation of our previous work, was carried out to investigate the possible functional correlation between striatal cyclic AMP and ethanol-induced motor incoordination, and its modulation by striatal adenosine in Sprague-Dawley rats. Forskolin (0.1, 0.5 and 1.0 pmol), a known activator of adenylate cyclase, significantly attenuated ethanol-induced motor incoordination in a dose-dependent manner following its direct intrastriatal microinfusion. Forskolin also antagonized the accentuating effect of intrastriatal N6-cyclohexyladenosine on ethanol-induced motor incoordination. These results suggested that ethanol-induced motor incoordination might be functionally correlated to a decrease in the striatal cyclic AMP levels and that the striatal adenosine A1 receptors might modulate ethanol-induced motor incoordination through cyclic AMP signaling mechanism(s). Further support to this hypothesis was obtained by the actual measurement of the striatal cyclic AMP levels in the same experimental conditions as in motor coordination studies using high-performance liquid chromatography with fluoroscence detection. Regardless of the method (focused microwave irradiation, cervical dislocation or decapitation into a dry ice-ethanol mixture) used to kill the animals, a significant decrease in the striatal cyclic AMP levels was observed due to ethanol. Intrastriatal adenosine A1-selective agonist, N6-cyclohexyladenosine (24 ng), caused a further significant decrease in the striatal cyclic AMP levels in the ethanol- but not in the vehicle-treated animals. The further enhancement in the ethanol-induced decrease in the striatal cyclic AMP levels by intrastriatal N6-cyclohexyladenosine, therefore, functionally correlated with the observed potentiating effect of intrastriatal N6-cyclohexyladenosine on ethanol-induced motor incoordination. The effects of intrastriatal N6-cyclohexyladenosine+ethanol and of ethanol alone on the striatal cyclic AMP levels were blocked by intrastriatal pertussis toxin (500 ng) pretreatment, indicating the involvement of pertussis toxin-sensitive G-proteins (Gi, Go) and possibly of the adenosine A1 receptor coupled to the G-proteins in the striatum. Furthermore, ethanol alone significantly decreased the basal as well as the cyclic AMP-stimulated catalytic activities of the striatal cyclic AMP protein kinase, which were further reduced by intrastriatal N6-cyclohexyladenosine. The results of the present study therefore support an involvement of a cyclic AMP signaling pathway in the striatal adenosinergic modulation of ethanol-induced motor incoordination at the post-adenosine A1 receptor level.

Production and characterization of monoclonal antibodies against fumitremorgin B.

This paper reports the preparation and identification of two monoclonal antibodies against FTB, and the establishment of an indirect competitive ELISA methods for FTB determination in buckwheat, rice, and corn. Two of the hybridoma cell lines (1C9 and 2D10), which could produce specific antibodies against fumitremorgin B(FTB), were selected and developed. The affinity Kaff constants of the monoclonal antibodies with the coating antigen, FTBS-IgG, were found to be 6 x 10(8) M-1 and 9.8 x 10 M-1, respectively. The isotypes of the monoclonal antibodies are of two isotypes, IgG1 and IgM, respectively. The antibody titers were found around 1 x 10(6) and 1.5 x 10(6). The standard curves showed that as little as 5 pg of FTB in 50 mL could be detected, and the linear range of standard curve was from 10 pg to 1000 pg of standard FTB. There were no cross-reaction for McAbs in the assay system with some mycotoxins tested. The mean recovery rate from buckwheat spiked with 10-60 ng/g of FTB was 78-88.7%.

Monosodium urate, hydroxyapatite, and calcium pyrophosphate crystals induce tumor necrosis factor-alpha expression in a mononuclear cell line.

OBJECTIVE: Tumor necrosis factor-alpha (TNF-alpha) is thought to be important in chronic inflammation of joints characteristic of crystal induced arthritis. Monocytes are instrumental in maintaining that inflammation. We investigated production of mRNA and protein for TNF-alpha in vitro in a murine mononuclear cell line, after exposure to relevant crystal types. METHODS: Using the cell line designated RAW 264.7, cells were grown in standard medium and exposed to varying amounts of monosodium urate (MSU), hydroxyapatite (HA), and calcium pyrophosphate dihydrate (CPPD) crystals for differing time periods. Analysis of TNF-alpha mRNA induced by such exposure was by Northern hybridization; analysis of TNF-alpha protein was by ELISA: RESULTS: RNA analyses of cells treated with various levels of MSU, HA, and CPPD crystals showed strong induction of TNF-alpha transcripts. ELISA on culture supernatants confirmed high level TNF-alpha peptide secretion resulting from that transcriptional upregulation. Time course studies showed peak accumulation of TNF-alpha mRNA 1-6 h post-treatment. Study of the signalling pathway involved in TNF-alpha transcriptional upregulation indicated that increased phospholipase A2 activity was required. CONCLUSION: These observations suggest that crystals in joints can directly stimulate production of TNF-alpha, and that the source of that cytokine may be the monocytes known to be present and playing an important role in chronic joint disease.

The striatal adenosinergic modulation of ethanol-induced motor incoordination in rats: possible role of chloride flux.

Previous studies from our laboratory have provided strong evidence that brain adenosine modulates acute ethanol (i.p.)-induced motor incoordination (MI) through receptor mediated mechanism(s). Recently, we have reported the involvement of the striatum in ethanol-induced MI as well as the striatal adenosinergic modulation of the ethanol-induced motor deficit. The present study was thus designed to further characterize the modulatory effect of striatal adenosine on ethanol-induced MI and to look for its functional correlation with chloride flux within the rat striatum. Intrastriatal microinfusion of adenosine A1 receptor agonist N6-cyclohexyladenosine (CHA) and antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), significantly accentuated and attenuated, respectively, the motor incoordinating effect of ethanol while having no effect on the normal motor coordination in saline-treated control animals. These data confirmed the role of striatal adenosine in ethanol-induced MI. The selectivity of interactions between adenosine A1 agonist and antagonist and ethanol was further confirmed by the study in which neither intrastriatal CHA nor DPCPX significantly altered the MI induced by sodium pentobarbital. Previously, we have shown that intrastriatal Ro15-4513 not only significantly attenuated ethanol-induced MI but also blocked its accentuation by intrastriatal CHA. It is well known that Ro15-4513 antagonizes many, but not all, CNS effects of ethanol by blocking the ethanol potentiation of GABA-stimulated uptake of chloride. Therefore, experiments using striatal microsac preparations were carried out to investigate the possible modulation of chloride conductance by CHA and its relationship to ethanol. High concentrations of CHA (10 and 100 nM) increased the total chloride uptake by the striatal microsacs. Corresponding to the ethanol-adenosine interaction observed behaviorally, a much lower concentration (1 nM) of CHA, being ineffective itself, significantly enhanced the stimulatory action of ethanol on chloride uptake. This effect was blocked by either Ro15-4513 (100 nM) or DPCPX (10 nM). The modulatory effect of GABA and/or ethanol on chloride influx was also evaluated, and the results supported the appropriateness to use striatal microsac preparations in the present study. Overall, the data suggested a functional interaction between ethanol and striatal adenosine and further supported the hypothesis that striatal adenosine might, in part, modulate ethanol-induced MI through its effect on chloride conductance through chloride channels coupled to GABA-benzodiazepine receptor complex.

Dispersion characteristics of [3H]-labeled adenosine agonist/antagonist following their intrastriatal microinfusion.

Following their intrastriatal microinfusion, the dispersion patterns of an adenosine receptor agonist (N6-cyclohexyladenosine) and an antagonist (8-cyclopentyl-1,3-dipropylxanthine) within the striatal tissue were investigated in Sprague-Dawley rats. The [3H]-labeled ligands were microinfused into the striatum of conscious rats through preimplanted guide cannulae in the volume of either 200 (0.1 microCi) or 1000 nl (0.5 microCi) and the animals were killed 15 or 30 min later. The diffusion of the radioactive ligands was evaluated by measuring the radioactivity in the striatal tissue samples using a tissue punching technique. When the volume of microinfusion was 200 nl, the diffusion within the striatum was limited as the radioactivity remained confined to the immediate vicinity of microinfusion site regardless of the postmicroinfusion time (15 or 30 min). The pattern of tissue diffusion was similar at 15 min after the intrastriatal microinfusion of 1000 nl of [3H]-ligands. At 30 min after the intrastriatal microinfusion of 1000 nl volume, a relatively larger area of striatal tissue was covered by the drug solution. In addition, the 1000 nl intrastriatal microinfusion probably resulted in the diffusion of some of the drug solution into the extrastriatal area since small but significant radioactivity was detected at sites outside the striatum. The intrastriatal diffusion of the [3H]-ligand solution was not uniform in all directions from the site of microinfusion. The relationship between the amount of radioactivity remaining at the site of microinfusion and the postmicroinfusion time was inverse. Additionally, at the same postmicroinfusion time (15 or 30 min), a lower percent of the total microinfused radioactivity was found remaining at the microinfusion site with the 1000 nl microinfusion volume than that with the 200 nl volume. Overall, the diffusion patterns of intrastriatal adenosine agonist and antagonist were similar. The results of the present investigation suggest that both the microinfusion volume and the postmicroinfusion time may be important factors in determining the diffusion pattern and tissue content of intrastriatally microinfused adenosine drugs. This information could be important for the correct understanding and interpretation of the data from studies involving drug microinfusions.