[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.

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.

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.

Isolation of microorganisms and substances inhibitory to aflatoxin production.

Aflatoxins are toxic and carcinogenic secondary metabolites produced by Aspergillus flavus and A. parasiticus. The contamination of crops, feeds, and foods with aflatoxins can have serious effects on the health of humans and animals. Although many studies have been done to develop aflatoxin-control strategies, most are limited in their effectiveness. As part of an effort to develop control procedures, we have devised simple and safe methods that are useful for identifying microorganisms that effectively inhibit aflatoxin production by fungi. These include the microtitre agar plate assay using norsolorinic acid-accumulating mutant fungi, the ultraviolet light photography method using an instant film, the tip culture method, a convenient RNA extraction method for reverse transcription-polymerase chain reaction (RT-PCR) analysis, and other methods. Results of a recent trial have shown that Achromobacter xylosoxidans significantly inhibited aflatoxin production by A. parsiticus, and that the main inhibitory substance produced by the bacterium was cyclo(L-leucyl-L-prolyl). This result confirms that the methods described herein are useful for identifying microorganisms that inhibit aflatoxin production by fungi and could contribute to the development of methods to reduce aflatoxin contamination in commodities.

DNA methylation and allelic losses on chromosome arm 14q in oligodendroglial tumours.

Cytogenetic and molecular genetic studies have shown frequent losses on the long arm of chromosome 14 in different types of human gliomas. Using differential methylation hybridization as a genome-wide screening approach to determine DNA methylation patterns in gliomas, we recently identified two DNA fragments in 14q23.1 (CGI-clone musical sharp396) and 14q32.12 (CGI-clone musical sharp519) that were differentially methylated between astrocytic gliomas and mixed oligoastrocytomas. To validate this observation, we examined these 14q32.12 locus for methylation in an extended series of 43 astrocytic and oligodendroglial gliomas. All tumours were additionally investigated for loss of heterozygosity (LOH). Microsatellite analysis showed LOH in seven of 28 (25%) oligodendroglial tumours and three of 15 (20%) astrocytic tumours. Seven tumours demonstrated LOH at all informative 14q loci whereas three tumours carried partial deletions defining a commonly deleted region at 14q22.3-q32.1 between the microsatellite markers D14S282 and D14S995. Methylation-specific PCR analysis of the 14q32.12 locus revealed hypermethylation in 12 of 43 gliomas (28%). Hypermethylation was restricted to tumours with oligodendroglial differentiation (12 of 28 tumours, 43%). However, none of the hypermethylated tumours demonstrated LOH on 14q and vice versa. In total, 19 of 28 oligodendroglial tumours (68%) showed either hypermethylation at the 14q32.12 locus or LOH at 14q22.3-q32.2. Taken together, our data lend further support for the location of one or more yet to be identified glioma-associated tumour suppressor gene(s) on 14q. In addition, the restriction of 14q32.12 methylation to oligodendroglial tumours suggests a role for epigenetic DNA modifications in these particular gliomas.

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.

Hypermethylation of ribosomal DNA in human breast carcinoma.

We examined the methylation status of the transcribed domain of ribosomal DNA (rDNA) in 58 patients with breast cancer. The mean percent of methylation was significantly higher in breast tumours than that of normal control samples (P < 0.0001). This increased rDNA methylation was associated with oestrogen receptor non-expression (P < 0.0273) and with moderately or poorly differentiated tumours as compared to well differentiated tumours (P < 0.0475). Our results suggest that rDNA can be a useful marker for monitoring aberrant methylation during breast tumour progression.

Expression of the endogenous galactose-binding protein galectin-3 correlates with the malignant potential of tumors in the central nervous system.

BACKGROUND: The 31-kilodalton beta-galactoside-binding protein galectin-3 has been associated with cellular transformation and metastasis. Because neural tissues contain large amounts of glycoconjugates, and endogenous carbohydrate-binding proteins have been described in the human brain, the authors examined the expression of galectin-3 in human brain tumors and metastases to the central nervous system. METHODS: Brain tumors were categorized by the World Health Organization system and galectin-3 expression by immunoperoxidase staining using a quantitative staining score. RESULTS: Glioblastomas (Grade 4 astrocytomas) all stained strongly for galectin-3, whereas low grade astrocytomas (Grade 2) did not express the endogenous lectin. Anaplastic astrocytomas (Grade 3) exhibited intermediate expression. The staining score was significantly associated with tumor grade (P < 0.001). Normal brain tissue and benign tumors did not express galectin-3, whereas metastases to the brain were all positive for galectin-3 expression. Metastases expressed significantly more galectin-3 than the primary tumors from which they were derived (P = 0.003). CONCLUSIONS: Galectin-3 expression correlates with the malignant potential of tumors in the central nervous system.

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.

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.

Stable differentiation of a human colon adenocarcinoma cell line by sodium butyrate is associated with multidrug resistance.

Colorectal cancers are often composed of cell types representing various differentiated cell lineages, however little is known concerning the relationship of differentiation and drug resistance in these cancers. The present study was performed to develop and characterize a stable, differentiated clone of the human colon cancer cell line LS174T and to characterize the drug resistance of this cell line in relation to its undifferentiated parental cell line. LS174T cell line was treated with the differentiating agent sodium butyrate (0.5 mM) for 30 days, then recultured in standard medium. Foci of flat-appearing cells appeared and were isolated using cloning rings, and subcloned. One subclone was designated LS174T-D. The LS174T-D clone maintains a stable, differentiated phenotype in standard culture conditions in the absence of sodium butyrate. It is characterized by the formation of a polarized monolayer with dome formation and the presence of prominent apical microvilli and tight junctions. This cell line demonstrated reduced growth in soft agar and nude mice compared with the parental cell line. LS174T-D cells expressed immunoreactive intestinal mucin antigens and brush border enzymes dipeptidyl aminopeptidase (DAP)-IV and aminopeptidase. The activities of DAP-IV and aminopeptidase were increased 5.6-fold and 3.4-fold, respectively, in LS174T-D compared with parental cells. Proliferation assays demonstrated that, compared with the parental cell line, LS174T-D cells were more resistant to doxorubicin (93-fold), cisplatin (23-fold), 5-fluorouracil (12-fold), 5-fluorodeoxyuridine (31-fold), and methotrexate (12.5-fold). Intracellular uptake of (3H)-5-fluorodeoxyuridine did not differ significantly in the differentiated and undifferentiated cell lines. Levels of mdr-1 p-glycoprotein measured by Western blot and RNA Northern blot assays were also similarly low in both cell lines. However, total glutathione content and glutathione-S-transferase activities were increased in LS174T-D cells by sixfold and threefold, respectively, compared with parental cells. Depletion of glutathione by pretreatment with DL-buthionine sulfoximine reversed LS174T-D resistance to cisplatin. Long-term treatment with sodium butyrate induces or selects for colon cancer cells with features of enterocytic differentiation. This stably differentiated cell line is associated with glutathione-mediated multidrug resistance, and provides a model for further studies of differentiation in normal and cancerous colon.

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.

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.

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.

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.

Expression of native and deglycosylated colon cancer mucin antigens in normal and malignant epithelial tissues.

Immunohistochemical techniques were used to determine the distribution and cellular location of the mature and precursor forms of a colonic-type mucin in normal and malignant epithelial tissues. The antisera used in this study were prepared against native human colon cancer mucin (LS), partially deglycosylated mucin (HFA or GalNAc-apomucin), and fully deglycosylated mucin (HFB or apomucin). These antisera reacted with most mucin-producing cells of the normal gastrointestinal tract, salivary ductular cells, bronchial epithelial cells, some bronchial mucous glands, and squamous epithelial cells of the esophagus. Breast, endometrium, ovary, prostate, liver, and thyroid were nonreactive. In most normal organs, HFB reactivity was present in the supranuclear and perinuclear cytoplasm and LS and HFA were located primarily in goblet cell vacuoles, apical cytoplasm, and luminal secretions. These findings are consistent with the expected subcellular locations of apomucin and more "mature" mucins. LS, HFA, and HFB were frequently expressed in adenocarcinomas of the colon, stomach, pancreas, and lung. Lymphoma, sarcoma, and melanoma specimens were nonreactive. Alterations in the expression of these mucin antigens in malignant tissues included loss of subcellular compartmentalization, increased intensity of staining, and disappearance of staining. In addition, de novo expression of HFB was observed in one of five breast carcinomas and three of five ovarian mucinous cystadenocarcinomas. These data demonstrate that LS, HFA, and HFB are useful for studying the organ specificities and biosynthetic pathways of one type of mucin in normal and malignant tissues.

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.