[Clinical value of p16INK4a immunocytochemistry in cervical cancer screening].

Objective: To evaluate the value of p16INK4a detected by p16INK4a immunostaining as a new generation of cervical cytology for primary screening and secondary screening in population-based cervical cancer screening, and in improving cytological diagnosis. Methods: Between 2016 and 2018, 5 747 non-pregnant women aged 25-65 years with sexual history were recruited and underwent cervical cancer screening via high-risk (HR)-HPV/liquid-based cytological test (LCT) test in Shenzhen and surrounding areas. All slides were immuno-stained using p16INK4a technology, among them, 902 cases were offered p16INK4a detection during primary screening, and the remaining 4 845 cases were called-back by the virtue of abnormal HR-HPV and LCT results for p16INK4a staining. Participants with complete LCT examination, HR-HPV test, p16INK4a staining and histopathological examination results were included in this study. The performance of p16INK4a in primary and secondary screening, and in assisting cytology to detect high grade squamous intraepithelial lesion [HSIL, including cervical intraepithelial neoplasia (CIN) Ⅱ or Ⅲ] or worse [HSIL (CIN Ⅱ)+ or HSIL (CIN Ⅲ)+] were analyzed. Results: (1) One-thousand and ninety-seven cases with complete data of p16INK4a and histology were included. Pathological diagnosis: 995 cases of normal cervix, 37 cases of low grade squamous intraepithelial lesion (LSIL), 64 cases of HSIL and one case of cervical cancer were found. Among them, 65 cases of HSIL (CIN Ⅱ)+ and 34 cases of HSIL (CIN Ⅲ)+ were detected. The positive rate of p16INK4a in HSIL (CIN Ⅱ)+ was higher than that in CINⅠ or normal pathology (89.2% vs 10.2%; P<0.01). (2) p16INK4a as primary screening for HSIL (CIN Ⅱ)+ or HSIL (CIN Ⅲ)+ was equally sensitive to primary HR-HPV screening (89.2% vs 95.4%, 94.1% vs 94.1%; P>0.05), but more specific than HR-HPV screening (89.8% vs 82.5%, 87.7% vs 80.2%; P<0.05). p16INK4a was equally sensitive and similarly specific to cytology (≥LSIL; P>0.05). (3) The specificity of LCT adjunctive p16INK4a for detecting HSIL (CIN Ⅱ)+ or HSIL (CIN Ⅲ)+ were higher than that of LCT alone or adjunctive HR-HPV (P<0.01), while the sensitivity were similar (P>0.05). (4) p16INK4a staining as secondary screening: p16INK4a was significantly more specific (94.1% vs 89.7%, 91.9% vs 87.4%; P<0.01) and comparably sensitive (84.6% vs 90.8%, 88.2% vs 91.2%; P>0.05) to cytology for triaging primary HR-HPV screening. HPV 16/18 to colposcopy and triage other HR-HPV with p16INK4a was equally sensitive (88.2% vs 94.1%; P=0.500) and more specific (88.3% vs 83.0%; P<0.01) than HPV 16/18 to colposcopy and triage other HR-HPV with LCT≥ atypical squamous cells of undetermined significance (ASCUS), and the referral rate decreased (14.0% vs 19.4%; P=0.005). Conclusions: For primary screening, p16INK4a is equally specific to cytology and equally sensitive to HR-HPV screening. p16INK4a alone could be an efficient triage after primary HR-HPV screening. In addition, p16INK4a immunostaining could be used as an ancillary tool to cervical cytological diagnosis, and improves its accuracy in cervical cancer screening.

[The value of p16(INK4a) cytology for early diagnosis of cervical cancer].

Objective: To investigate the use of p16(INK4a) immuno-stained cytology as the primary screening for cervical cancer prevention. Methods: From March to August 2018, 902 women from Shenzhen and surrounding area were recruited for cervical cancer screening with ThinPrep Cytologic Test (TCT), cobas4800 HPV test, and p16(INK4a) co-test. Colpo/biopsies were performed using the point of interest biopsy protocol of directed and random cervical biopsies plus endocervical curettage for all women, any of whose tests was positive. Two senior cytopathologists interpreted TCT and p16(INK4a) test. The performance of p16(INK4a) for early detection of CIN2+ and inter-observer reproducibility of the interpretation of p16(INK4a) were evaluated. Results: The positive rates of HPV test, p16(INK4a) co-test and TCT diagnosed as LSIL/AGC or higher grade were 8.1% (73/902), 6.8% (61/902) and 4.7% (42/902), respectively. Colposcopy referring rate was 79.6% (109/137), among which 10 cases were diagnosed as CIN2+ (5 cases of CIN2 and 5 cases of CIN3). The sensitivity and specificity for CIN2+ of p16(INK4a) test, TCT (LSIL/AGC or higher grade) and HPV test were 90.0%, 80.0%, 100.0% and 90.9%, 91.9%, 82.5%, respectively. Compared to TCT and HPV test, there was no significant difference in sensitivity and specificity between p16(INK4a) and TCT/HPV test (P>0.05). The Kappa value of the 2 cytopathologists in interpreting p16(INK4a) and TCT was 0.944 and 0.425, respectively (P<0.05). Conclusions: p16(INK4a) for cervical cancer screening is equally sensitive to HPV test and specific to TCT while subjective difference of cytopathologists' interpretation of p16(INK4a) is small. Therefore, p16(INK4a) can be used as a new cervical cancer screen method for its better diagnostic performance.

The effect of maternal obesity on fatty acid transporter expression and lipid metabolism in the full-term placenta of lean breed swine.

This study was conducted to evaluate the influence of back-fat thickness (BF), at mating of sows, on the maternal and newborn circulating lipids, expression of placental fatty acids (FA) transporters and lipid accumulation in placenta. Full-term placentas were obtained by vaginal delivery from BFI (9-14 mm; n = 37), BFII (15-19 mm; n = 43) and BFIII (20-27 mm; n = 38) sows according to BF at mating, and frozen placental sections were analysed for fat accumulation. Blood samples were collected from the sows of day 105 pregnancy and from cord blood at delivery. mRNA and protein expression levels were evaluated with real-time RT-PCR and Western blotting. Our results demonstrated that BFII females had significantly increased litter weight and placental efficiency, decreased maternal triglyceride (TG) and non-esterified fatty acids (NEFA) levels, decreased maternal IL-6, TNFα and leptin levels compared to BFIII females (p < .05). BFIII sows were associated with significantly decreased newborn TG levels, increased newborn glucose, IL-6 and TNFα levels compared to BFI or BFII sows (p < .05). BFI and BFII females had significantly decreased placental TG, NEFA and cholesterol (CHOL) contents compared to BFIII females (p < .05). Moreover, decreased CD36, FATP1, FABP4, and FABP1 mRNA and protein and FATP4 protein expression, and increased LPL activity were also observed in BFIII group compared with BFII group (p < .05). PPARγ mRNA and protein and lipogenic genes such as SREBP-1c, ACSL1, ACCα, FAS and SCD mRNA expression were downregulated or upregulated, respectively, in the placentas of BFIII sows compared to BFI or BFII sows (p < .05). Overall, this study demonstrated that there is no advantage, in terms of litter live size, litter weight and placental FA transport and metabolism, in performing the mating of sows with BF>19 mm.

[Comparison of contrast sensitivity and wavefront aberrations with myopic].

Objective: Correlation analysis of visual acuity, wavefront aberrations and contrast sensitivity in myopia. Methods: Retrospective study. One hundred and twelve patients with myopia(209 eyes) from April 2013 to August 2015 were enrolled in our study. All subjects were divided into various groups to investigate the relationship between wavefront aberrations and contrast sensitivity in myopic eyes.The correlations between ocular aberrations and contrast sensitivity(4 spatial frequency) in myopia eyes were analyzed using multivariate stepwise regression. Results: The AULCSF in the BCVA 1.2 were 1.32±0.10,1.30±0.12 respectively in the light and dark conditions,, which were higher than those in the BCVA 1.0 (t=-3.58 and-2.48, P<0.05). There was no significant difference between AULCSF in dark glare condition.At 4mm and 6mm pupil diameters,The difference in Z(-)(33) of ocular higher-order aberrations between the BCVA 1.0 and the BCVA 1.2 was statistically significant (t=2.09, P=0.04; t=-2.05, P=0.04). Differences between the other ocular higher-order aberrations and corneal aberrations were not statistically significant.The spherical aberrations of the low contrast sensitivity group were (0.019±0.010), (0.136±0.117) and(0.006±0.003)µm separately under the condition of bright light, dark light and dark glare light, which were higher than other groups (0.013±0.006) , (0.083±0.054) , (0.004±0.002) µm (t=1.10, 2.65, 2.44, P<0.05). The values of AULCSF for the larger spherical aberrations under dark light and dark glare light conditions were 1.281±0.126 and 1.216±0.154 respectively which were lower than the AULCSF 1.281±0.126, 1.216±0.154 of the another spherical aberrations group (t=2.14, 1.98, P<0.05). It was found that the S(All) RMS and spherical aberrations under different frequencies and illuminating conditions were negatively correlated with CS.Vertical coma was positively correlated with CS. Conclusions: Better BCVA may achieve better visual quality.In the case of the same BCVA, there are differences in visual quality.Higher order aberrations are the main factor affecting this result, especially the spherical aberrations.Total aberrations and spherical aberration had a negative correlation with visual quality.Vertical coma had a positive affects with visual quality. (Chin J Ophthalmol, 2018, 54: 748-755).

Correlation between Cystatin C and retinopathy of type-two diabetes mellitus patients.

Diabetic retinopathy is one of most common diabetic microvascular complications. In recent years the incidence of the disease has increased, hence early diagnosis and treatment are of great importance. In order to find reliable biological indexes to diagnose and treat type-two diabetes mellitus promptly, this study focused on the correlation between Cystatin C (Cys C) and retinopathy of type-two diabetes mellitus patients. One hundred and eighty type-two diabetes mellitus patients and one hundred healthy controls (the control group) were chosen in this study. Of the patients ninety-eight patients had typetwo diabetes mellitus without retinopathy (non-diabetic retinopathy group) and eighty-two had typetwo diabetes mellitus with retinopathy (diabetic retinopathy group). Correlation of Cys C and typetwo diabetic retinopathy was analyzed by examining the waist-hip ratio, fasting blood glucose (FBG), total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), glycosylated hemoglobin (HbA1c), and Cys C of both groups. The results showed that FBG, TC, TG, LDL-C, HbA1c, Cys C in the type-two diabetes mellitus patients group were higher than those of the control group (P less than 0.05). Age, course of diabetes, FBG, HbA1c, and Cys C levels were statistically significant in both the DR group and NDR group (P less than 0.05). The result of logistic regression analysis indicates that there was a positive correlation between type-two diabetic retinopathy development and age, course of diabetes, and Cys C level (P less than 0.05). Thus, it can be seen that changes of Cys C levels can assist early diagnosis and treatment of diabetic retinopathy to some extent. The patients with high Cys C level, long course of diabetes, and old age are more likely to have diabetic retinopathy.

Clinical effect of intravitreous injection of triamcinolone acetonide in treating cystoid macular edema.

Cystoid macular edema (CME), a commonly seen sign for multiple fundus diseases, is able to induce visual deterioration. The incidence rate of CME is constantly increasing; however, the existing clinical treatments cannot achieve satisfactory curative effects. To explore the curative effect of intravitreous injection of triamcinolone acetonide (TA) in treating CME, this study carried out a clinical test on 39 patients (42 eyes) from The First Affiliated Hospital of Zhengzhou University who developed CME induced by central retinal vein occlusion (CRVO). All 42 eyes received intravitreous injection of 40 mg/ml TA (0.1 ml) and then were followed up for 11-23.5 months. Eyes were examined by slit-lamp microscope, fundus fluorescein angiography (FFA) and optical coherence tomography (OCT) and best corrected visual acuity (BCVA), and intraocular pressure (IOP) of those eyes were detected before and after treatment. Average vision of eyes was 0.1 before treatment, and the vision improved in one month (vision ≥ 0.2: 100%; vision ≥ 0.5: 42.9%) and three months (vision ≥ 0.2: 64.3%; vision ≥ 0.5: 21.4%) after treatment; but as time went on, the vision of some patients declined; at the last follow-up, patients with vision ≥ 0.2 accounted for 28.6% and those with vision ≥0.5 accounted for 7.1%; compared to before treatment, 71.4% patients had improved vision and the remaining 28.6% had declined vision. Some patients were observed with high IOP during treatment, and 7 eyes were found with secondary cataract in posterior capsule of lens at the last follow-up. Intravitreous injection of triamcinolone acetonide proved to have significant short-term curative effect on CEM which is non-sensitive to conventional therapies, but it is likely to induce high IPO and posterior capsular opacification.

Fatty acid composition evaluation of edible parts of Eriocheir sinensis in intensive ponds of Gucheng waters.

In Gucheng Lake, Jiangsu Province, China, randomly selected crabs were fed 2 diet types. Crab crude oil methyl ester analysis was performed using gas chromatography-mass spectrometry. Fatty acid compositions in the 3 edible parts of a crab - the hepatopancreas, gonad, and muscle - were analyzed. C16:1 and C18:2 were significantly higher in most commercial pellet feed-feeding crabs than in small trash fish-feeding crabs, while the opposite was observed for eicosapentaenoic and docosahexaenoic acids. Phytanic acid reached 1.93% in the hepatopancreas of small trash fish-feeding crabs. Furan fatty-acid-DiMe (11,5) contents in the testes of small trash fish-feeding crabs was 1.49%. These values were higher in male crabs than in female crabs. According to a standard ratio of 1:1:1 which meaning the saturated fathy acid (SFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA) were 33.33 each, fatty acid structure analysis of crab edible parts showed that SFA:MUFA:PUFA of crab edible parts was 2.3-4.1:2.9-5.0:1.3-4.8. The highest muscle score was 29.53 in male trash fish-feeding crabs, and the lowest hepatopancreas score was -40.81 in female commercial pellet feed-feeding crabs. The n-6/n-3 ratio was 0.36-2.48. Muscle ratio was the lowest in female commercial pellet feed-feeding crabs. Thus, small trash fish-feeding and commercial pellet feed-feeding crabs are healthy foods. Overall, for consumption, the males of small trash fish-feeding crabs were better than the females, the muscle was better than the gonads and hepatopancreas, and the testis was better than the ovary.

Molecular cloning, expression, and regulation of the ovalbumin gene in pigeon oviduct epithelial cells.

The full-length pigeon ovalbumin (OVA) gene cDNA was cloned and sequenced by reverse transcription-polymerase chain reaction (RT-PCR) and rapid-amplification of cDNA ends. A 386-amino acid protein was predicted for the obtained sequence, which had 67% identity with the chicken protein. Similar to chicken OVA, the pigeon OVA gene is a non-inhibitory serine protease inhibitor. Quantitative PCR analysis revealed that pigeon OVA mRNA was highly expressed in the oviduct, and trace amounts were detected in other tissues. During the reproductive cycle, pigeon oviduct OVA mRNA expression reached its peak during the egg-laying stage, decreased with brooding, and then increased again during the squab-feeding period. Moreover, the relative OVA expression level in pigeon oviduct epithelial cells could be upregulated by a constant concentration of steroid hormones.

Effects of heat treatment of soy protein isolate on the growth performance and immune function of broiler chickens.

This study was conducted to evaluate the effects of oxidative modification of soy protein isolate (SPI) after exposure to heat on the growth performance and immune function of broilers. The SPI was heated in an oven at 100°C for 1, 4, and 8 h, respectively, and resultant oxidative status was evaluated. A total of 320 one-day-old Arbor Acres chickens were randomly divided into 4 treatment groups with 8 replicates of 10 birds, and fed diets supplemented with the native SPI or 1 of the 3 heat-treated SPI for 21 d. The results showed that heat exposure of SPI for 4 and 8 h caused an increase in protein carbonyl (P < 0.05), and a simultaneous decrease in sulfhydryl and free amine groups (P < 0.05) compared with native SPI. The BW of broilers fed diets supplemented with SPI heated for 8 h were significantly lower than that of broilers fed diets supplemented with native SPI (P < 0.05). Compared with native SPI, heat-treated SPI (heated for 8 h) diminished liver weight at 14 d (P = 0.01), spleen (P < 0.01) and bursa (P < 0.05) weights at 21 d; and the content of IgG in serum and duodenal mucosa of broilers (at 14 d) was decreased when diets supplemented with heat-treated SPI (heated for 8 h; P < 0.01). No significant differences were observed in the mucosa secretory IgA contents of broilers among the treatment groups (P > 0.05). Compared with native SPI, a significant increases were observed in the content of adrenocorticotropic hormone and cortisol in serum of broilers fed the heat-treated SPI (heated for 8 h) at 21 d (P < 0.05); and the myeloperoxidase activities in serum (at 14 d) and mucosa of broilers were increased when diets supplemented with heat-treated SPI (heated for 8 h; P < 0.05). The present study suggests that protein oxidation of SPI is induced by heating, and oxidized protein may negatively affect the immune function of broilers.

Human bronchus and intestine express the same mucin gene.

The amino acid and sugar composition of mucins from various organs is similar but not identical. This could arise by one or more of the following: organ-specific processing of a single core protein, organ-specific splicing of a single mucin mRNA, or organ-specific expression of various mucin genes. To begin to investigate the source of this variability, we examined (a) immunological cross-reactivity and (b) cDNA cross-hybridization, among several mucin-secreting organs of the human body. Peptide-directed antibodies raised against both nondeglycosylated (LS) and deglycosylated (HFB) intestinal mucin strongly stained mucous cells in the bronchial epithelium and submucosal glands, indicating homology between mucins of the bronchus and intestine at the peptide level. By screening a bronchus cDNA library with an intestinal mucin cDNA, SMUC-41, we isolated a bronchus mucin cDNA, HAM-1. This cDNA is 96% homologous to the first repeat of SMUC-41. HAM-1 hybridized to restriction fragments of human genomic DNA identical to those hybridizing to SMUC-41 on Southern blots. SMUC-41 also hybridized to polydisperse transcripts in the bronchus, cervix, gall bladder, and mammary gland, indicating mucin homology among all these organs at the RNA level. We conclude that the bronchus and intestine express a common mucin gene, which is likely co-expressed by at least several other mucin-secreting organs.

Characterization of new pancreatic cancer-reactive monoclonal antibodies directed against purified mucin.

Although mucins have been found to be useful in the diagnosis of pancreatic cancer, the carbohydrate and peptide structures of pancreatic mucins are still not well characterized. Monoclonal antibodies were produced using mucins purified from xenografts of a human pancreatic cancer cell line as the immunogen. One of these, Ia3, reacted with almost all pancreatic, gastric, and colorectal carcinomas examined by immunoperoxidase staining, but with few normal tissues. Ia3 antigen was elevated in sera of 50.4% of individuals with gastrointestinal tumors, but its levels did not correlate with those of CA15-3, CA19-9, or DU-PAN-2. Serum Ia3 antigens migrated more slowly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the polymorphic epithelial mucins recognized by DF3 or 115D8. Ia3 reacted only with native, and not with partially deglycosylated, pancreatic cancer xenograft mucins. Periodate or neuraminidase treatment destroyed this reactivity, but protease had little effect. The antigen recognized by another antibody, Nd2, was not detected in normal pancreatic, colonic, or gastric tissues but was present in approximately 60% of the pancreatic and gastric carcinomas examined. Nd2 reactivity with native and partially deglycosylated mucin was lost after pretreatment with protease and beta-mercaptoethanol. We conclude that, while Ia3 reacts with carbohydrates, Nd2 reactivity appears to be dependent on the integrity of the mucin protein core. The antigenic determinants of Ia3 and Nd2 are different from those of B72.3, CA19-9, DU-PAN-2, SPan-1, and several breast cancer mucin-directed antibodies. These results suggest that the malignancy-associated structures identified by Ia3 and Nd2 may provide new information on the carbohydrate and peptide structure of pancreatic cancer mucins.

Heterogeneity of mucin gene expression in normal and neoplastic tissues.

To determine the relative expression of distinct mucin genes in normal and neoplastic tissue, antibodies and cDNA probes that recognize the core tandem repeat sequences of membrane-bound (MUC1) and secreted (MUC2 and MUC3) mucins were used for immunohistochemical and RNA Northern and slot-blot analysis. MUC1 mRNA was detected in all epithelial tissues tested. MUC1 core peptide, recognized by monoclonal antibodies 139H2 and DF3, was highly expressed on apical membranes of bronchus, breast, salivary gland, pancreas, prostate, and uterus, and was sparsely expressed in gastric surface cells, gallbladder, small intestine, and colonic epithelium. In contrast, MUC2 and MUC3 gene expression was primarily restricted to the intestinal tract. MUC2 mRNA was highly expressed in normal jejunum, ileum, and colon, compared with very low levels in normal bronchus and gallbladder. MUC3 mRNA was highly expressed in normal jejunum, ileum, colon, and gallbladder. Immunohistochemical studies using antibodies against synthetic MUC2 (anti-MRP) and MUC3 (anti-M3P) peptides indicate that MUC2- and MUC3-producing cells in the gastrointestinal tract are distinct. Goblet cells of the small intestine and colon reacted strongly with anti-MRP, whereas M3P reactivity was restricted to columnar cells of small intestinal villi, surface colonic epithelium, and gallbladder. Mucin protein epitopes and mRNA levels were frequently altered in adenocarcinomas compared to corresponding normal tissues. Alterations included increased expression, aberrant expression, and, less frequently, loss of expression. Increased MUC1 immunoreactivity was observed in most adenocarcinomas of the breast, lung, stomach, pancreas, prostate, and ovary. In addition, with the exception of prostate cancer, focal aberrant expression of MUC2 and MUC3 epitopes was frequently observed. Increased MUC1, MUC2, and MUC3 epitopes were present in colon adenocarcinomas of all histological subtypes, with the greatest increase of MUC2 epitopes observed in colloid (mucinous) colon cancers. MUC2 or MUC3 mRNA levels were increased in colloid colon cancer compared with normal colon, however in well- and moderately well-differentiated colon cancers MUC1, 2 and 3 mRNA levels were decreased. Compared with corresponding normal tissue, MUC1 mRNA levels were increased in breast cancer and well-differentiated lung cancers, and MUC3 mRNA was increased in gastric adenocarcinomas. Normal stomach lacked both MUC2 and MUC3 immunoreactivity and mRNA, however, MUC2 and MUC3 proteins and mRNA were highly expressed in gastric intestinal metaplasia. In conclusion, mucin genes are independently regulated and their expression is organ- and cell type-specific. Furthermore, neoplastic transformation is associated with dys-regulated expression of both membrane-bound and secreted mucin core protein epitopes and may be due to altered mucin mRNA levels and/or altered mucin glycosylation.

Relationship of pancreatic cancer apomucin to mammary and intestinal apomucins.

Pancreatic cancer mucins have several carbohydrate antigens that are potentially useful in the detection of pancreatic cancers, but little is known about the core polypeptides of pancreatic cancer mucins. In this study, purified mucin from SW1990 pancreatic cancer xenografts was deglycosylated by treatment with hydrogen fluoride to give pancreatic cancer apomucin. Consistent with near-complete removal of carbohydrate, the apomucin had 10- to 70-fold decreased binding of lectins and, unlike the native mucin, served as an acceptor for polypeptidyl N-acetylgalactosaminyl transferase. Antibodies prepared against the apomucin did not bind to native mucin, and antibodies that bound to native mucin did not bind to apomucin. On the basis of cross-reaction with deglycosylated colon cancer mucin and intestinal mucin repeat peptide, apomucins from SW1990 pancreatic cancer xenografts contain the intestinal mucin repeat peptide. On the basis of binding of breast cancer-reactive monoclonal antibodies 139H2, DF3, and HMFG-2, apomucins from SW1990 pancreatic cancer xenografts also have the mammary mucin repeat peptide. Using complementary DNA probes specific for intestinal mucin and breast mucin sequences, both types of apomucin mRNA were detected in nude mouse xenografts of SW1990 cells. In immunohistochemical staining, antibody against deglycosylated SW1990 mucin stained normal breast and pancreas but not normal colon. Some pancreatic and mammary cancers and most colonic cancers, however, were stained by antibodies against both intestinal apomucin and mammary apomucin. We conclude that pancreatic cancers can produce mucins with the intestinal repeat peptide as well as those with mammary repeat peptide sequences.

Cathepsin B expression and localization in glioma progression and invasion.

The poor prognosis of human malignant gliomas is due to their invasion and recurrence, the molecular mechanisms of which remain poorly characterized. We have accumulated substantial evidence implicating the cysteine protease cathepsin B in human glioma malignancy. Increases in cathepsin B expression were observed throughout progression. In primary brain tumor tissue, transcript abundance (Northern blot analysis) increased in low-grade astrocytoma to high-grade glioblastoma from 3- to 6-fold, respectively, above normal brain levels. This increase correlated with increases in protein abundance (from + to ) as measured by immunohistochemistry. Furthermore, in glioblastoma cell lines increases in transcript abundance (ranging from 3- to 12-fold) were accompanied by increases in enzyme activity (44-133 nmol/min x mg protein). Altered subcellular localization was observed both immunohistochemically and by indirect immunofluorescence confocal microscopy and was found to correlate with increased grade. In addition, this increase in cathepsin B expression and altered subcellular localization correlated with histomorphological invasion and clinical evidence of invasion as detected by magnetic resonance imaging. These data support the hypothesis that cathepsin B plays a role in human glioma progression and invasion.

Dissecting complex epigenetic alterations in breast cancer using CpG island microarrays.

It is now clear that aberrant DNA methylation observed in cancer cells is not restricted to a few CpG islands, but affects multiple loci. When this epigenetic event occurs at the 5'-end of the regulatory region of genes, it is frequently associated with transcriptional silencing. To investigate further this widespread event in the tumor genome, we developed a novel microarray containing 7776 short GC-rich tags tethered to glass slide surfaces. This DNA chip was used to study 17 paired tissues of breast tumors and normal controls. Amplicons, representing differential pools of methylated DNA fragments between tumors and normal controls, were cohybridized to the microarray panel. Hypermethylation of multiple CpG island loci was then detected in a two-color fluorescence system. Approximately 1% (on average, 83 loci) of these CpG islands examined were hypermethylated in this patient group. Hierarchical clustering segregated these tumors based on their methylation profiles and identified a group of CpG island loci that corresponds to the hormone-receptor status of breast cancer. This observation was independently confirmed by examining a single locus, the promoter of the human glypican 3 gene, which was predominately hypermethylated in the hormone receptor-negative tumors. Our findings support the notion that hypermethylation of critical CpG island loci influences cancer development and produces distinct epigenetic signatures for particular tumor subtypes.

CpG island arrays: an application toward deciphering epigenetic signatures of breast cancer.

CpG island hypermethylation is a frequent epigenetic event in cancer. We have recently developed an array-based method, called differential methylation hybridization (DMH), allowing for a genome-wide screening of CpG island hypermethylation in breast cancer cell lines (T. H-M. Huang et al., Hum. Mol. Genet., 8: 459-470, 1999). In the present study, DMH was applied to screen 28 paired primary breast tumor and normal samples and to determine whether patterns of specific epigenetic alterations correlate with pathological parameters in the patients analyzed. Amplicons, representing a pool of methylated CpG DNA derived from these samples, were used as hybridization probes in an array panel containing 1104 CpG island tags. Close to 9% of these tags exhibited extensive hypermethylation in the majority of breast tumors relative to their normal controls, whereas others had little or no detectable changes. Pattern analysis in a subset of CpG island tags revealed that CpG island hypermethylation is associated with histological grades of breast tumors. Poorly differentiated tumors appeared to exhibit more hypermethylated CpG islands than their moderately or well-differentiated counterparts (P = 0.041). This early finding lays the groundwork for a population-based DMH study and demonstrates the need to develop a database for examining large-scale methylation data and for associating specific epigenetic signatures with clinical parameters in breast cancer.

Preliminary study on the microbiology of Campylobacter pyloridis and gastric histopathology.

Biopsy samples were taken endoscopically from the antral-mucosa of 693 patients with peptic ulcer and chronic gastritis presenting dyspepsia symptoms. Campylobacter pyloridis cultures were positive in 59 of 98 (60.2%) cases and histopathologically the organisms were found in 411 of 693 cases (59.3%). Pathologically, Campylobacter pyloridis was positive in 273 out of 300 patients with chronic superficial gastritis (91.0%), in 102 of 249 patients with chronic atrophic gastritis (40.9%), in 36 out of 144 patients with chronic atrophic gastritis with intestinalization or dysplasia (25.0%). We found that there was a significant association between the presence of Campylobacter pyloridis and chronic superficial gastritis, also the degree of lymphocyte infiltration showed a strong inverse association with the presence of Campylobacter pyloridis, suggesting that a local immune response might exert an important action in the eradication of this organism. These findings support the view that Campylobacter pyloridis, may be etiologically related to chronic gastritis and peptic ulceration, even though its role still remains to be determined.

Immunolocalization of cathepsin B in human glioma: implications for tumor invasion and angiogenesis.

The poor prognosis of patients with malignant gliomas is at least partially due to the invasive nature of these tumors. In this study, the authors investigated the possibility that the cysteine protease cathepsin B (CB) is a participant in the process of glial tumor cell invasion. To accomplish this, an immunohistochemical analysis was made of the localization of antibodies to CB in biopsies of five specimens of normal brain, 16 astrocytomas, 33 anaplastic astrocytomas, and 33 glioblastomas multiforme. Staining was scored according to the percentage of positive cells and the intensity of the stain, graded from 0 to 3+. Staining for CB was not seen in any of five samples of normal brain except for occasional neuronal cell bodies and microglia. Only five (31%) of 16 astrocytomas showed a small percentage of positive cells (0.01%-3%) that were stained in a light, diffuse cytoplasmic pattern (1+). Twenty-nine (87.8%) of 33 anaplastic astrocytomas showed positive light, granular staining in 2% to 40% of cells. In anaplastic astrocytoma, the staining within a tumor was heterogeneous with intensities of 1+ (17%), 1+ to 2+ (29%), or 2+ (55%). In contrast, all 33 (100%) glioblastomas were positive in 10% to 90% of cells. The staining was present in a coarse, granular pattern with an intensity of 2+ (12%) or 3+ (88%). Tumor cells infiltrating into brain adjacent to malignant gliomas stained positively in 26 cases that could be evaluated for glioblastoma multiforme; these invading cells frequently followed penetrating blood vessels as typical "secondary structures of Scherer." Moderate to intense CB staining associated with endothelial proliferation in high-grade tumors was also observed, especially in regions of tumor infiltration into adjacent normal brain. These results provide evidence consistent with the hypothesis that CB is functionally significant in the process of tumor invasion and angiogenesis in the clinical progression of the malignant phenotype in astrocytes.

Cholesterol oxidation products. Their occurrence and detection in our foodstuffs.

The structural similarity of cholesterol oxidation products (COP) to native cholesterol and their xenobiotic effects prompt researchers to study the long-term effects of the assimilation of these compounds into our tissues. COP are present in our food system. The level of exposure changes as our food products and our food choices alter. Therefore, the presence of COP in our food system has to be carefully monitored and their presence in processed foods minimized by optimizing processing and storage conditions. This review will briefly discuss the chemistry of some commonly-occurring COP and their biological significance. A more in-depth survey of the literature on the pitfalls of COP determination is included. It is the intention of the author to impress the readers that 'exogenous' COP can easily form during sample preparation. These artifacts will hinder our understanding of factors that promote COP formation in foods. The effects of heating, dehydrating, packaging and the presence of highly unsaturated lipids on the levels of COP in cholesterol-containing foods are evaluated to gauge the levels of exposure to different consumer groups.

Primary and secondary changes in the brains of suckling Balb/c mice with experimental hemorrhagic fever.

To testify whether primary changes caused by the virus with its related factors and secondary changes caused by hypotension in the brains of patient with epidemic hemorrhagic fever (EHF) could be repeated in the animal model, suckling Balb/c mice were inoculated IP with 100 LD50/0.05 ml of Chen strain of hemorrhagic fever virus. After the onset of the disease, paraformaldehyde and glutaraldehyde were used for fixation by perfusion through left ventricle. Sections stained with HE and MAb against EHF virus by immunocytochemical method (4-step PAP) showed diffuse viral antigen deposition. All brains were diffusely scattered with single cell acidophilic necrosis which are believed to be caused primarily by the virus. 33.3% of the brains also showed symmetrical distribution of cerebral infarct-like necrosis which are believed to be caused secondarily by hypotension. This result supports our previous study on the autopsy of brains of EHF patients.