Differential gene expression associated with inflammation in peripheral blood cells of patients with pneumoconiosis.

OBJECTIVES: To study expression changes in inflammation-related genes in peripheral blood of patients with pneumoconiosis and to explore the possibility of these genes as pneumoconiosis biomarkers. METHODS: Peripheral blood samples of patients with pneumoconiosis patients and controls were collected, and total RNA of the blood cells were extracted and reverse transcribed to cDNA. Screenings of deferentially expressed genes associated with inflammation between patients with pneumoconiosis and controls were performed using real-time quantitative PCR array and the expressions of the three most upregulated genes were confirmed by real-time PCR. RESULTS: The expression of 11 genes was significantly altered in patients with pneumoconiosis compared with those of the control. Among these 11 genes, 8 genes were upregulated and 3 were downregulated. Preliminary results indicated that interleukin 6 (IL-6) mRNA expression in patients with pneumoconiosis was higher than that in controls (P=0.019). The level of IL6 mRNA expression in the patients was higher than that in non-smoking controls, but it was neither affected by type and stage of pneumoconiosis nor by time of contact with dust. CONCLUSIONS: IL6 was possibly involved in the development of pneumoconiosis.

Cytogenetic analysis of peripheral blood lymphocytes, many years after exposure of workers to low-dose ionizing radiation.

The present study aims to measure chromosomal aberrations and micronuclei in peripheral blood lymphocytes from 25 subjects exposed to 0.10-0.33 Gy external or internal irradiation 32-41 years ago using conventional cytogenetic analysis methods. The frequencies of total chromosome-type aberrations and micronucleus significantly increased in the exposed group compared with that in age-matched control group (p<0.001); chromatid-type aberrations showed no difference between the two groups (p>0.05). When exposed subjects were divided into two groups based on exposure dose, higher levels of dicentric plus translocation frequencies were observed in the ≥0.15 Gy dose group compared with those in the <0.15 Gy dose group, though the difference was not significant. Borderline association between exposure dose and dicentric frequency was detected in the exposed group (r=0.358; p=0.079). These results suggest that the genotoxic effects of ionizing radiation remain in subjects exposed to low-dose radiation even decades after exposure.

Assessment of retrospective dose estimation, with fluorescence in situ hybridization (FISH), of six victims previously exposed to accidental ionizing radiation.

The present study aims to evaluate the use of the fluorescence in situ hybridization (FISH) translocation assay for retrospective dose estimation of acute accidental exposure to radiation in the past. Reciprocal translocation analysis by FISH with three whole-chromosome probes was performed on normal peripheral blood samples. Samples were irradiated with 0-5Gy (60)Co γ-rays in vitro, and dose-effect curves were established. FISH-based translocation analyses for six accident victims were then performed, and biological doses were estimated retrospectively by comparison with the dose-effect curves. Reconstructed doses by FISH were compared with estimated doses obtained by analysis of di-centrics performed soon after exposure, or with dose estimates from tooth-enamel electron paramagnetic resonance (EPR) data obtained at the same time as the FISH analysis. Follow-up FISH analyses for an adolescent victim were performed. Results showed that dose-effect curves established in the present study follow a linear-quadratic model, regardless of the background translocation frequency. Estimated doses according to two dose-effect curves for all six victims were similar. FISH dose estimations of three adult victims exposed to accidental radiation less than a decade prior to analysis (3, 6, or 7 years ago) were consistent with those estimated with tooth-enamel EPR measurements or analyses of di-centrics. Estimated doses of two other adult victims exposed to radiation over a decade prior to analysis (16 or 33 years ago) were underestimated and two to three times lower than the values obtained from analysis of di-centrics or tooth-enamel EPR. Follow-up analyses of the adolescent victim showed that doses estimated by FISH analysis decrease rapidly over time. Therefore, the accuracy of dose estimates by FISH is acceptable only when analysis is performed less than 7 years after exposure. Measurements carried out more than a decade after exposure through FISH analysis resulted in underestimation of the biological doses compared with values obtained through analysis of di-centrics and tooth-enamel EPR.

[Serum levels of CC-Chemokine ligand 18 in the patients with pneumoconiosis].

OBJECTIVE: To measure the peripheral serum levels of CC-chemokine ligand-18 (CCL-18) in patients with pneumoconiosis, and to investigate the feasibility of the index asa potential biomarker for pneumoconiosis. METHODS: Seventy-seven male patients with pneumoconiosis (stage 1:40 cases, stage 2:22 cases, stage 3:15 cases), including 42 cases of silicosis and 35 cases of coal worker pneumoconiosis, were enrolled as subjects, and 162 healthy male physical examinees in our hospital were used as controls. A fasting blood sample (3 ml) was collected from the peripheral venous blood of each patient or control. The CCL-18 concentration in serum was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The serum CCL-18 concentrations of the patients with pneumoconiosis were significantly higher than those of the controls [(116.70 ± 82.85) ng/ml vs. (83.34 ± 64.83) ng/ml]; (Z = -2.389, P < 0.05). The serum CCL-18 concentrations of the patients with silicosis were significantly higher than those of the patients with coal worker pneumoconiosis (147.02 ± 93.32 ng/ml vs. 96.43 ± 47.19 ng/ml; Z = -3.030, P < 0.05). There were no significant differences in serum CCL-18 concentration among different stages of pneumoconiosis (P > 0.05). The degree of respiratory impairment was positively correlated with the serum CCL-18 concentration in patients with pneumoconiosis (r = 0.611, P < 0.01). CONCLUSION: Serum CCL-18 level can be used as a potential biomarker for pneumoconiosis.

[The endoscopic and clinical features of Indigo Naturalis-associated ischemic lesions of colonic mucosa].

OBJECTIVE: By analysing the clinical features of Indigo Naturalis-associated ischemic lesion of colon mucosa to improve the precautionary and therapeutic level of the disease. METHODS: Thirteen patients diagnosed as Indigo Naturalis-associated ischemic lesion of colon mucosa in Peking University Third Hospital from 2005 to 2010 were reviewed. The endoscopic and clinical features were analysed. RESULTS: The 13 patients with an average age of (60.6 ± 14.1) years old were prescribed Chinese traditional medicine containing Indigo Naturalis for psoriasis or idiopathic thrombocytopenic purpura (ITP). The ratio of males to females was 1:1.6. The typical manifestations were abdominal pain and bloody stool with watering diarrhea before bloody stool in 61.5% patients. Endoscopic and pathological characteristics were coincident with ischemic lesion and more like a chronic index. Vasodilatic medicine was effective and the average hemostatic time was (1.7 ± 0.8) days. The prognosis was well and no recurrence was found during 3 months follow-up. CONCLUSIONS: Patients having psoriasis or ITP treated with Chinese traditional medicine containing Indigo Naturalis have an inclination to colon mucosa lesions, even ischemic lesion. Careful assessment and observation before prescribing are necessary in these patients.

Follow-up study by chromosome aberration analysis and micronucleus assays in victims accidentally exposed to 60Co radiation.

The goal of this study was to assess the persistence of chromosomal aberrations and micronuclei of three victims 2 y after accidental radiation exposure to Co gamma rays. Traditional chromosome aberration analysis was performed by scoring the dicentric chromosomes (dic) and rings (r) in peripheral blood lymphocytes. Micronuclei were detected using the cytokinesis block micronucleus assay. G-banding and semi-automatic karyotype analysis was used to record translocations (t), inversions (inv) and deletions (del). The frequency of unstable chromosomal aberrations (dicentrics and rings) remained at high levels 6 mo after the accident. Two years after exposure, the frequency was reduced to 4-11% in the three victims. However, stable chromosome aberrations, which were detected by G-banding and included t, inv, and del, remained at a high level and have an obvious dose-dependent relationship even 2 y post-exposure. The frequency of micronuclei decreased faster than that of chromosome aberrations, reaching almost a normal level two years after the accident, especially for the child victim. Unstable chromosome aberrations reduced gradually, but the stable aberration remained at a high level along with the time-lapse. The micronucleus assay was less valuable for assessing long-term effects after high dose irradiation.

[Fecal calprotectin in differential diagnosis of irritable bowel syndrome].

OBJECTIVE: To assess the value of fecal calprotectin as a non-invasive screening biomarker in differential diagnosis of irritable bowel syndrome compared with fecal occult blood test (FOBT), erythrocyte sedimentation (ESR) or C reactive protein (CRP). METHODS: Subjects were a total of 240 persons, including 60 patients with irritable bowel syndrome, 60 patients with colorectal cancer, 60 patients with chronic inflammation, and 60 healthy controls. 5 g fecal samples were collected within one week of endoscopy or before surgical operation. Fecal calprotectin was measured by an enzyme-linked immunosorbent assay (ELISA) kit in spot stool samples. At the same time, FOBT was measured; the results of ESR and CRP in hospital lab were collected. RESULTS: The median of fecal calprotectin concentrations were 12.21 mg/kg and 15.36 mg/kg in IBS and healthy controls, respectively. There was no statistical significance of calprotectin concentration between patients with IBS and healthy controls (P>0.05). The median of fecal calprotectin concentrations were 159.00 mg/kg and 466.00 mg/kg in colorectal cancer and chronic inflammation respectively. There were statistical significance between patients with chronic inflammation, colorectal cancer, and others (P<0.01). The maximal calprotctin concentration was with chronic inflammation; the medium with colorectal cancer; the minimum with IBS and healthy controls. When the cut-off limit was set as 50 mg/kg of fecal calprotectin, the positive rates of colorectal cancer, chronic inflammation, IBS and healthy controls were 85.0%, 91.7%, 10%, and 5%,respectively. Fecal calprotectin was much superior to FOBT, ESR and CRP. CONCLUSION: Fecal calprotectin as a non-invasive screening biomarker in the differential diagnosis of IBS and symptomatic chronic large intestinal organic disease was better than FOBT, ESR and CRP. It was simple, inexpensive, repeatable and no-invasive. It can be used as a biomarker in exclusion from related organic diseases before the diagnosis of irritable bowel syndrome.

The actin gene promoter-driven bar as a dominant selectable marker for nuclear transformation of Dunaliella salina.

A 5'-flanking region of an actin gene from the green unicellular alga Dunaliella salina (D. salina) was cloned using a genome-walking method by PCR and its structural features were characterized. Two repetitive sequences found, over 75 bp in length each, were located at position -573 and -424 bp,respectively, relative to the AUG codon. The actin gene promoter region of D. salina displayed a consensus sequence of GCTC (G/C) AAGGC, a CCAAT motif and two TATA-like motifs that did not have a canonical sequence of a TATA box. The 5' flanking region of the actin gene was exploited to direct expression of the bialaphos resistance gene (bar) from Streptomyces hygroscopicus as a dominant marker in the nuclear transformation of D. salina. Direct selection of bar resistant transformants was achieved by allowing a 24 h period of recovery of cells transformed by biolistic procedure, followed by growth of the cells for one week under standard condition prior to harvesting and plating on the solid medium containing 0.5 microg/mL of phosphinothricin (PPT). Five colonies picked from the plate were analyzed, of which the integration of the bar gene was demonstrated in the nuclear genome. Southern blotting revealed that only one of five transformants contained a single copy of the bar gene whereas others contained multiple copies,suggesting that nuclear transformation of D. salina mainly occurred through illegitimate recombination events,resulting in ectopic integration of the introduced DNA. The integration patterns of the foreign DNA in this experiment appeared not to influence the bar gene expression in the transformants containing single or multiple inserts. The bar gene expression in the five transformants was verified by RT-PCR, confirming transcription of the chimeric DNA. These transformants were maintained on agar plates in the absence of PPT for more than seven months and retained resistance to the herbicide at 1 microg/mL. This work demonstrates that the actin gene promoter-driven expression of the bar gene may be used as a dominant selectable marker for nuclear transformation of D. salina.

Characteristics and therapeutic efficacy of sulfasalazine in patients with mildly and moderately active ulcerative colitis.

AIM: To investigate the characteristics and short-term efficacy of sulfasalazine (SASP) in patients with mildly and moderately active ulcerative colitis (UC). METHODS: Two hundred and twenty-eight patients with mildly and moderately active UC were recruited, 106 patients in 1993-1995, and 122 patients in 2000-2002, they were assigned as the 1990s group (n = 106) and the 2000s group (n = 122), prospectively. The general characteristics, clinical manifestations, colonoscopic and histological data were compared between the two groups. The short-term efficacy and safety of SASP 3 g per d were evaluated. RESULTS: Between 2000s and 1990s groups, the gender ratio of men to women was 1:1.18 and 1:1.04, 57.4% and 50.9% of the patients were between 30 and 49 years old. The gender ratio and age of UC patients were not significantly different. The total course of 50.0% and 37.1% of UC patients was less than 1 year (P<0.05), 10.6% and 31.2% of the cases had a duration of more than 5 years (P<0.05) in 2000s and 1990s groups, respectively. The most common clinical type was first episode in 2000s group and chronic relapse in 1990s group. The patients showed a higher frequency of abdominal pain and tenderness in 1990s group than in 2000s group. Erosions were found in 84.4% and 67.9% of patients in 2000s and 1990s groups (P<0.05). Rough and granular mucosa (67.9% vs 43.4%, P<0.05) and polyps (47.2% vs 32.8%, P<0.05) were identified in 1990s group more than in 2000s group. There were no significant differences in clinical, colonoscopic and histological classifications. After SASP (1 g thrice per d) treatment for 6 wk, the clinical, colonoscopic and histological remission rates were 71.8%, 21.8% and 16.4%, respectively. In 79 patients with clinical remission, 58.2% and 67.1% remained grade 1 in colonoscopic and histological findings, respectively. The overall effects in first episode type (complete remission in 10, 18.9%, partial remission in 28, 52.8%, and improvement in 9, 17.0%) were better than in chronic relapse type (complete remission in 3, 7.5%; partial remission in 16, 40.0%; and improvement in 15, 37.5%) and chronic persistent type (complete remission in 1, 5.9%; partial remission in 6, 35.3%; and improvement in 6, 35.3%) respectively (P<0.05). In 110 patients treated with SASP, 18 patients (16.4%) had adverse reactions. Except for two cases of urticaria and one case of WBC decrease, none of the patients had to stop the treatment because of severe adverse reactions. CONCLUSION: Patients with mildly and moderately active UC in 2000s group had a shorter disease course, milder clinical manifestations, more first episode type and higher frequency of acute mucosal lesions in colonoscopy than in 1990s group. The patients in 1990s group had higher proportion of chronic relapse type and chronic mucosal change in colonoscopy than in 2000s group. The short-term efficacy of SASP could be mainly remission of clinical manifestations. But more than half of the patients still had light inflammation in colonoscopy and histology. The overall effects of SASP in first episode type were better than those in other types. SASP was a safe and effective drug to treat mildly and moderately active UC.

[Expression of calprotectin in colon mucosa and fecal of patients with ulcerative colitis].

OBJECTIVE: To investigate the correlation between the disease activity of ulcerative colitis (UC) and the immunohistochemical distribution of calprotectin in colon mucosa,as well as the levels of calprotectin in fecal. METHODS: Monoclonal antibody against calprotectin was used to investigate the distribution of these proteins in colon mucosa from 42 patients with ulcerative colitis and 20 healthy controls. ELISA assay was used to measure the concentrations of calprotectin in fecal. The disease activity of UC was determined by Truelove-Witts histological criteria. RESULTS: Calprotectin was demonstrated in the majority of granulocytes and macrophages in colon mucosa of patients with active UC, while negative in patients with unactive UC and healthy controls. A strong calprotectin immunoreactivity was presented in ulcerative and erosion lesions in the colon. The concentration of calprotectin was significantly higher in patients with active UC than that with unactive UC and healthy controls (P<0.01). Both the distribution of calprotectin in colon mucosa and the levels of calprotectin in fecal in UC were significantly correlated with the histological grades respectively (r=0.89, P=0.000 1;r=0.849, P<0.01), and the two parameters were correlated well (r=0.90, P=0.000 7). CONCLUSION: We suggest that calprotecin in fecal is mostly derived from the colon mucosa in active UC. The level of in fecal calprotectin fecal accurately reflects the degree of disease activity of UC. The morphological RESULTS confirm the findings of enhanced fecal calprotectin levels in patients with active UC.

Effects and mechanism of the selective COX-2 inhibitor, celecoxib, on rat colitis induced by trinitrobenzene sulfonic acid.

OBJECTIVE: To investigate the action of celecoxib (a selective COX-2 inhibitor) in a rat model of colitis induced by trinitrobenzene sulfonic acid (TNBS). METHODS: Rats were randomized into four groups. Colitis was induced in groups 1 and 2 by intracolonic administration of TNBS (25 mg/mL) in 50% ethanol (0.25 mL). The rats in group 1 received oral celecoxib (1.25 mg/kg) and those in group 2 received distilled water (1 mL/0.3 kg), beginning 3 h before induction of colitis and continuing twice daily thereafter for up to 7 days. The rats in group 4 received oral celecoxib (1.25 mg/kg) twice daily for 7 days and those in group 3 were healthy controls. All rats that survived 7 days were killed and both the severity of colonic mucosal damage and the prostaglandin E2 (PGE2) concentrations of the colonic mucosa were assessed. RESULTS: The colonic mucosal damage scores for groups 1 and 2 were 11.15 +/- 3.30 and 8.50 +/- 2.82, respectively, both of which were significantly higher than the score for the healthy controls (0.62 +/- 0.09; P < 0.01, P < 0.01). The score of group 1 was significantly higher than that of group 2 (P < 0.05). No difference was found between the scores of groups 3 and 4. The mucosal concentrations of PGE2 in groups 1 and 2 were 12.00 +/- 4.33 pg/microg and 17.20 +/- 9.62 pg/microg, respectively, both of which were significantly higher than the concentration in the healthy controls (6.02 +/- 3.39 pg/microg; P < 0.05, both). The PGE2 concentration of group 1 was decreased significantly compared with that of group 2 (P < 0.05). No difference was found between groups 3 and 4. CONCLUSION: The results suggest that treatment with celecoxib exacerbates inflammation-associated colonic injury in experimental colitis induced by TNBS. This preliminary study shows that the mechanism is related to suppression by the COX-2 inhibitor of the PG derived from COX-2, but further study is needed to identify if there are other related mechanisms.

[Cloning and functional analyses of promoters of two carbonic anhydrase genes from Dunaliella salina].

The cloning vectors pMD-DCA1 and pMD-CA containing the promoters of duplicated carbonic anhydrase 1 (DCA1) and carbonic anhydrase (CA) genes, respectively, from Dunaliella salina, and expression vector pDM307 containing bar-NOS polyA fragment were digested with EcoR I. The bar-NOS polyA fragment was fused, respectively, to the fragments of the vectors pMD-DCA1 and pMD-CA to form transgenic D. salina expression vectors pMDDC-B and pMDC-B. The micro-shots were prepared by coating two constructs (pMDDC-B and pMDC-B) with gold particle. Each sample was bombarded once, twice, and thrice, respectively, with micro-projectile gun at a rupture pressure of 690 kPa in helium gas. The screening culture of the bombarded alga cells was performed in PKS liquid and solid medium containing 3 mg/L phosphinothricin (PPT) to develop the transformed cells of D. salina. Analyses of the transformed cells were carried out through PCR, Southern blotting, and Northern blotting. The results of screening culture showed that the expression of the external bar gene of vectors pMDDC-B and pMDC-B was stable and transient, respectively, in the transformed D. salina cells. In the meantime, the transformed efficiency of particle bombardment twice was higher than that of once or thrice particle bombardment at a rupture pressure of 690 kPa in helium gas. PCR and Southern blotting analyses indicated that the external bar gene was integrated into the genome of the cells. Northern analysis indicated that expression efficiency of the bar gene driven by DCA1 promoter was regulated by the gradient concentration of sodium chloride, and the positive blotting signal intensity of the bar mRNA was highest in the medium containing 2 mol/L of sodium chloride. The findings of the present study suggest that promoter of the DCA1 gene may be an inducible promoter following a hyperosmotic shock with high activity and safety in the research of transgenic D. salina. The tandem GT sequences of the promoter region of DCA1 and CA genes may be related to the molecular mechanisms of the extreme halo-toleration of the unicellular green alga, D. salina.

[Clinical characteristics of non-steroidal anti-inflammatory drugs associated gastroduodenal ulcer bleeding].

OBJECTIVE: To study the clinical characteristics of non-steroidal anti-inflammatory drugs (NSAIDs) associated gastroduodenal ulcer bleeding. METHODS: Patients who were hospitalized in our ward because of gastroduodenal bleeding from Jan, 1997 to Dec, 2001 were divided into two groups according to consumption of NSAIDs or not in the week previous to the onset of bleeding. RESULTS: 446 patients were investigated with 86 in NSAIDs group and 360 in non-NSAIDs group. There was no significant difference in sex, way of bleeding, history of peptic ulcer, bleeding site in stomach or duodenum, presence of erosion and the need of endoscopic injection therapy or not between the two groups. However, the patients in NSAIDs group were older than those in non-NSAIDs group. Hemoglobin level was lower in NSAIDs group (P = 0.004). There was more gastric ulcer and complex ulcer in the NSAIDs group than in non-NSAIDs group (P < 0.001). NSAIDs users had more ulcers (P < 0.001). Helicobacter pylori (Hp) was present in 72.5% of the non-NSAIDs group and 53.4% of the NSAIDs group (P = 0.001). In an another study, we found that age of the patients or Hp state didn't affect the clinical characteristics of NSAIDs ulcer bleeding. CONCLUSIONS: The clinical characteristics of NSAIDs associated gastroduodenal bleeding should be better understood so as to decrease the occurrence of NSAIDs ulcer and its complication.

[Cloning and characterization of Hsp70a cDNA fragment of Dunaliella salina].

The present study is to obtain heat shock protein 70a cDNA fragment from Dunaliella salina. Two pairs of degenerate primers were designed according to conserved motifs of DIDLGTT,DQGNRTTP,PAYFNDS and ATKDAG of the homologous amino acid sequences and used to amplify hsp70a cDNA fragment from heat-shock-treated Dunaliella salina by nest PCR technique. The resulting PCR products were inserted into T-vector then transformed into JM109. Ten colonies were selected to determine their sequences. Homologous analysis of the deduced amino acid sequences were performed by BLAST and subsequently compared with GenBank data. Three of ten nucleotide sequences were obtained,of which there was 372 bp coding 126 amino acids. The sequences shared high homology with hsp70a,with identity 96% to Chlamydomonas reinhardtii, 94% to Petunia, 93% to Pisumsativum, 92% to tomato, 92% to human,90% to Drosophila and 89% to yeast respectively. It can be concluded that the cloned sequence is putatively hsp70a cDNA fragment from Dunaliella salina.

[Biological dose assessment by the analyses of chromosomal aberrations and CB micronuclei in two victims accidentally exposed to 60Co gamma-rays].

Biological doses were estimated by using the yields of dicentrics plus rings(dic+r) and cytokinesis-block micronuclei (CBMN) for two victims of the accident radiation occurred on Mar 6,2000 in the City of Xuchang(victim A), and Jun 26,2000 in the City of Kaifeng(victim B), Henan Province,respectively. The results indicated that the equvalent whole body doses based on the standard dose-response curves established by the analyses of dic+r and CBMN were estimated to be 1.44Gy and 1.43Gy(victim A), and 0.15Gy and 0.22Gy(victim B). These doses were very consistent with the mean doses calculated from physical measurement and conformable to the clinical dianosis. The dic+r aberrations of victim A did not accord with Poisson distribution. The analyses of chromosomal aberrations and CBMN are an extremely reliable method in biological dosimetry. The radiation which victim A was exposed to was heterogeneous.