Flow Cytometric Analysis of the Toxicity of Nitrofen in Cultured Keratinocytes

Lactate dehydrogenase (LDH) release test, 3 H-thymidine (3 H-TdR) and 3 H-leucine (3 H-Leu) incoopration tests and flow cytometric analysis (FCM) of cell cycle were empoyed to elucidate cellular and molecular mechanism of nitrofen-induced toxicity in cultured keratinocytes.The results showed that cell morphologic damages were observed after exposure to 1.0 mmol/L and 10.0 mmol/L nitrofen. LDH release increased in a dose- and time-dependent manner. Depressions in 3H -TdR and 3 H-Leu incorpration were found even at 0.01 mmol/L, and increased with the exposure dose. Cell cycle was analyzed from the DNA- histogram with propidium iodde stain. The results showed that there was no pronounced alteration in cell cycle after cells exposed to 0.01 and 0.1 mmol/L nitrofen. At dose of 1.0 mmol/L, S phase cells increased 2 times of that of control. With the increase of dose, G2/M phase cells became to increase about 5 times of that of the control. At 1 .0 mmol/L, time course of cell cycle after exposure was observed. At the beginning of exposure, cells in S phase and G2/M phase were about 8 .7 % and 11 %. Following 24 h incubation with nitrofen, cells in S phase increased to 18.0% with almost no change in G2/M. 72 h after exposure, G2/M phase cells increased to 63 .3%. The forve results demonstrated that S phase and G2/M phase blockage in cultured keratinocytes after exposed to nitrofen seems of importance in the mechanism of nitrofen-induced toxicity.

THE CLINICAL SIGNIFICANCE OF FLOW CYTOMETRIC DEOXYRIBONUCLEIC ACID MEASUREMENT OF DEPARA-FFINIZED SPECIMEN IN BLADDER TUMOR

A retrospective study of flow cytometric measurements on paraffin-embedded tumor specimens from 188 patients with bladder tumor was conducted. The results were analyzed in combination with the morphological variation of bladder tumors. It was found that the DNA ploid pottern, degree of infiltration and the multiplicity of bladder tumor were closely related with tumor recurrence, among which the DNA ploid pattern was most significant. In aneuploid bladder tumors the recurrent rate and mean annual recurrence frequency were 76.7% and 1.46, and those in the diploid bladder tumors were 18.7% and 0.33 respectively. Aneuploid was the most indicative parameter of the recurrence in bladder tumors. In addition, according to the DNA ploid pattern and DNA index (DI), the aneuploid tumors in our group were divided into 4 types, namely, tetraploid tumors, npn-euploid with DI(?)1.5, non-euploid tumors with DI>1.5 and two-aneuploid tumors. The results showed that the recurrent rate of tetraploid tumors was relatively lower and it became higher and higher in the following order: non-euploid tumors with DI(?)1.5, non-euploid tumors with DI>1.5, and two-aneuploid tumors. This indicates that there are different biological behaviors in tumors with different ploid pattern. Finally, the correlation between DNA ploid pattern and tumor metastasis was also discussed.

Clinical and biological relevance of flow cytometric determination of P-glycoprotein expression in acute non-lymphocytic leukemia

This study was supported by grants from Science and Technology Committee of Jiangsu and Health Bureau of Jiangsu. Objective To evaluate the characteristics of P glycoprotein(P gp) expression of acute non lymphocytic leukemia (ANLL) at different status and the prognostic and biological features in ANLL at diagnosis. Methods Monoclonal antibody UIC2 and indirect immunofluorescence assay by flow cytometry were used to determine P gp expression of 169 patients with ANLL, including 152 previously untreated, 7 refractory and 10 at remission. Results P gp was expressed in 28.9% of the previously untreated ANLL cases and P gp was lower than that in 71.4% of the refractory cases (P<0.05). No P gp expression was found in the patients at remission. For previously untreated ANLL, P gp was highly expressed in hybrid acute leukemia (66.7%) and acute monoblastic leukemia (47.4%). P gp expression was highly associated with surface markers [cluster of differentiation (CD) 34, CD7, CD14, CD42b and CD61] and unfavorable cytogenetic abnormalities. About 23% of P gp +ANLL obtained complete remission, which was significantly lower than that (76%) in P gp - cases. Conclusions P gp expression is higher in refractory ANLL cases than that in cases at diagnosis or at remission. P gp is an index of poor prognosis in adults with ANLL. P gp +ANLL cases have unique clinical and biological characteristics.

Inducible nitric oxide synthase expression is related to angiogenesis,bcl—2 and cell proliferation in hepatocellular carcinoma

In this study, we examined the expression of inducible nitric oxide s ynthase (iNOS) and vascular endothelial growth factor (VEGF) by immunohistoc hemi cal staining in 76 tissue sections collected from hepatocellular carcinoma (HCC) patients undergoing hepatectomy. Microvascular density (MVD) was determined by counting endothelial cells immunostained using anti-CD34 antibody. We performe d DNA-flow cytometric analyses to elucidate the impact of iNOS and VEGF expressi o n on the cell cycle of HCC. Most of the HCC cells that invaded stroma were mark edly immunostained by iNOS antibody. The iNOS stain intensity of the liver tissu e close to the tumor edge was stronger than that of HCC tissue, and the stronges t was the hepatocytes closer to the tumor tissue. However, iNOS expression in 10 normal hepatic samples was undetectable. VEGF positive expression ratio was 84. 8% in iNOS positive expression cases, and the ratio was 35.3% in negative cases. There was significant correlation (P=0.000) between iNOS and VEGF expressi on. Moreover, iNOS expression was significantly associated with bcl-2 and MVD, but w ithout p53 expression. DNA-flow cytometric analyses showed that combined expres s ion of iNOS and VEGF had significant impact on the cell cycle in HCC. PI (Proli ferating Index) and SPF (S-phase fraction) in the combined positive expression o f iNOS and VEGF group was significantly higher than that in the combined negativ e group. The present findings suggested that iNOS expression was significantly a ssociated with angiogenesis, bcl-2 and cell proliferation of HCC.

Clinical features and treatment outcomes of intraocular lymphoma:a single-center experience in China

AIM:To investigate the clinical manifestations,diagnostic approaches,treatments,and outcomes of intraocular lymphoma.METHODS:In this retrospective study,16 patients(28 eyes)with intraocular lymphoma were recruited in the Department of Ophthalmology,Peking Union Medical College Hospital,from 2004 to 2019.All patients underwent comprehensive ophthalmic examinations.Vitreous specimens of 13 patients were sent for cytopathology examination and other adjunctive diagnostic procedures.Three patients were diagnosed with intraocular lymphoma according to analysis of the histopathological results of systemic lymphoma by one clinician.Twenty-three eyes were treated with intravitreal administration of methotrexate,4 eyes could not receive ocular treatment due to life-threatening lymphoma,and 1 eye did not require ocular treatment because the fundus lesions regressed after systematic chemotherapy.RESULTS:In 28 eyes,25 eyes were diagnosed with vitreoretinal lymphoma,and 3 eyes were diagnosed with ciliary body lymphoma,all of which were non-Hodgkin diffuse large B cell lymphomas.The final visual acuity improved in 15 eyes(54%),remained unchanged in 5 eyes(18%),and decreased in 8 eyes(29%).Anterior segment inflammation disappeared or reduced in 8 and 5 eyes,respectively;and 15 eyes had no anterior segment reaction.Twenty eyes had mild vitreous opacity,1 eye had mild vitritis,and 7 eyes had pars plana vitrectomy combinedwith silicone oil tamponade.Fundus lesions disappeared in 9 eyes and were relieved in 5 eyes;4 eyes showed no changes,and the remaining 10 eyes’fundus were normal.CONCLUSION:The clinical manifestations of intraocular lymphoma are diverse,and the misdiagnosis rate is high.Cytopathological analysis of vitreous is one of the gold standards for the diagnosis.Immunohistochemistry,gene rearrangement and flow cytometric immunophenotypic analysis can improve the diagnostic rate.Ocular chemotherapy or radiotherapy regimens may preserve visual acuity,and a multidisciplinary team can provide individualized treatment for the patients.

Effect of Exogenous p16ink4a and hRb1 Genes on Cell Cycle Regulation of Osteosarcoma Cell

To study the effect on regulation of cell cycle of osteosarcoma cell line MG63 tranceduced with exogenous p16ink4a and hRbl genes, pIRES-p16ink4a-hRb1, pIRES-p16ink4a and pIREShRbl plasmids were constructed by gene recombination technology. The recombinant plasmid was transferred into osteosarcoma cell line MG63 by metafectene, and the resistant clones were selected by G418 selective medium, mRNA and protein expression of osteosarcoma cell line were assayed by RT-PCR and Western-Blot respectively. Cell cycle and apoptosis were analyzed by subG1 flow cytometric. Cell proliferation was tested by MTT. In the genome of these transfected target cells, the expression of p16ink4a and hRb1 mRNA and protein were detected respectively in vitro. It was demonstrated with subG1 flow cytometric analysis and MTT method that p16ink4a and hRbl genes cooperation more significantly inhibited cell growth and induced a more marked G1 arrest and apoptosis than p16ink4a/hRb1 alone （P〈0.01）. Coexpression of exogenous p16ink4a with hRbl broke the regulatory feedback loop of p16ink4a-cyclinD1/CDK-hRbl and played a more significant role in inhibiting cell growth as well as inducing cell apoptosis than p16ink4a or hRbl did alone in vitro.

Immunomodulatory effects of Caulerpa racemosa var peltata polysaccharide and its selenizing product on T lymphocytes and NK cells in mice

A polysaccharide, CrvpPS, was isolated from Caulerpa racemosa var peltata. It was reacted with nano-selenium in distilled water containing ascorbic acid (Vit C) to form a stable CrvpPS-nano-Se complex. The immunomodulatory effects of CrvpPS and CrvpPS-nano-Se on T lymphocytes subgroups and NK cells in mice were investigated. After intragastric administration for 10 days separately, both CrvpPS and CrvpPS-nano-Se showed significant stimulatory functions to thymus gland of mice. Moreover, the CrvpPS-nano-Se induced the percentage of CD3+, CD3+CD4+, NK cells and the CD4+/CD8+ value to increase significantly (P<0.05) when analyzed by flow cytometry, which is better than the CrvpPS, sucrose-nano-Se, and even the positive drug levamisole.

Study of the Effects of the Chinese Herbal Prescription Combinedwith Copper and Ferum on the Malignancy ot Cancer Cells

As compared with normal cells, cancer cells or malignant cells were morphologically abnor-mal : their contact inhibition and normal growth order were lost, the DNA content and ploid increased and suchkind of cells were transplantable. It the malignancy should be decreased or the malignant cells reversed, theabove abnormal changes could be reduced or disappear. BALB/c mice bearing ascites liver cancer wereused, Chinese herbal prescription combined with copper and ferrum (CHPCCF) was given by gavage for 10days, and then some cell-biological parameters were measured; further , the ascites cancer cells (controland treatment) were removed and retransptanted to another mice and observed. The results showed that inCHPCCF treatment group, DNA content of the cancer cells was decreased, and the proliferation index wasreduced (control : 83 . 4 ± 2 . 6, CHPCCF group : 78. 8 ± 1 . 5 ; or control : 67. 2 ± 1 . 3 , CHPCCF group : 64. 2 ±l . 6, P < 0. 02) , the number of the cancer cells in Gl phase increased obviously, but, those of S + G2Mphases decreased ( P < 0. 05 ￣ 0. 01 ) ; on the DNA histogram, the diploid peak became higher and bigger,but aneuploid or multiploid peaks became smaller. Furthermore, retransplanted experiments showed that in2/10 animals, the tumors did not grow, and in other 8/10 animals , the tumors grew, but the tumors' sizewere smaller than that of the control ; the growth inhibition rate was 71 . 7% ￣ 88. 3% ; and tumors ' grewslowly ; the growth curve of the tumors in CHPCCF group was considerably lower than that of the control ; thesurvival period of retransplanted animals was prolonged significantly (from 26. 1 ± 11 . 8 to 38. 1 ± 9. 6, or to39 . 6 ± 7 . 2 days, P<0 . 01 ); the increase in life span was 46% and 52% respectively. The results suggestedthat CHPCCF could reduce the malignancy of mice liver cancer cells.