The Immuno-fluorescence Quantity Analysis of α-Tubulin and γ-Tubulin Protein in Precancerous Lesion and Carcinoma of the Breast and Its Significance

OBJECTIVE To investigate the changes and values of the expression of α-tubulin and γ-tubulin in atypical ductal hyperplasia （ADH）, ductal carcinoma in situ （DCIS） and invasive ductal carcinoma （IDC） of the breast. The relationship between centrosome abnormalities and breast tumor development was further discussed. METHODS There were three groups including ADH, DCIS and IDC with 30 cases in each group. They were analyzed by immuno-fiuorescence quantity analysis. The expression levels of α-tubulin and γ-tubulin protein in these tissues were detected by flow cytometry immuno-fiuorescence analysis and compared with the results from normal tissues. Immunohistochemistry was also performed in this research. RESULTS The results showed significant differences of the average of the positive （FITC labeled） cells （P=0.000） among the four groups. The level of the IDC group was the highest, while normal breast tissue showed the lowest level. The results suggested that the expression levels of α-tubulin and γ-tubulin both increased as the grade of cellular proliferation and differentiation increased. The expressions showed significant differences among all the groups, except between the ADH and DCIS. There were no significant differences between α-tubulin and γ-tubulin expression in each group （P〈0.05）, as there was agreement in the immuno-fluorescence and immunohistochemical analysis for protein expression. CONCLUSION There is abnormal expression of centrosome tubulin as an early event in the development of breast tumor. Furthermore these aberrations may play a key role during oncogenesis and promote cellular transformation to malignancy. The immuno-fiuorescence quantitive analysis and immunohistochemistry can complement each other.

Designing DNA cage-based immuno-fluorescence strategy for rapid diagnosis of clinical cervical cancer tissues

Exploiting a tissue diagnosis method to abstain the involuted operating and consume valuable reagents while realizing high-speed and inexpensive pathological grading technology to supply a better scheme for cancer therapy is a significant method of cancers detection. A promising immuno-fluorescence strategy was rationally designed and synthesized by loading ruthenium complex into cervical cancer-targeted DNA-cage, which was well used to realize high-speed and inexpensive diagnosis of clinical cervical cancer tumor tissues avoiding the traditional multi-stage process, thus demonstrating high application potential in clinical pathological grading and surgical judgment. Moreover, it has been finding that Apts-DNA@Ru can enrichment in the tumor region, interestingly, no enrichment in normal cervical cancer tissue. It has the potential to realize the integration of in vivo diagnose and further synchronous treatment in the near future. Thence, this study demonstrates a strategy for integration of cancer-targeted DNA-cage and fluorescent Ru POP as alternative IHC reagents for next-generation more rapid convenient cancer detection.

Significance of antineutrophil cytoplasmic antibody in adult patients with Henoch-Schnlein purpura presenting mainly with gastrointestinal symptoms

AIM： To test the clinical significance of antineutrophil cytoplasmic antibody （ANCA） in evaluation of adult Henoch-Schonlein purpura （HSP） patients presenting mainly with abdominal symptoms. METHODS： Twenty-eight consecutive HSP patients who presented predominantly with abdominal symptoms were enrolled in this study. Control subjects included 27 ageand sex-matched patients with peptic ulcer disease, colon cancer, acute gastroenteritis, irritable bowel syndrome and colonic polyps. ANCA was measured by indirect immunofluorescence （IIF） in all patients, and follow-up ELISA was performed in patients with positive IIF tests. RESULTS： ANCA was detected in 9 HSP patients by IIF （2 were positive for c-ANCA and 7 were positive for p-ANCA）. No ANCA was found in the control group. The sensitivity and specificity of a positive ANCA test （either c- or p-ANCA） were 32.1% and 100% respectively. Only one out of the 9 patients with positive ANCA by IIF had positive ANCA by ELISA and the antigen was myeloperoxidase （MPO）. The patients positive for ANCA had higher HSP clinical scores, and were more likely to have renal function impairment. Patients with late purpura development were also associated with more severe clinical manifestations. CONCLUSION： A positive ANCA test is associated with more severe symptoms in HSP. After inflammatory bowel disease is excluded, a positive ANCA test provides a clue to the diagnosis of HSP presenting predominantly with abdominal symptoms.

Antibody to El peptide of hepatitis C virus genotype 4 inhibits virus binding and entry to HepG2 cells in vitro

AIM： To analyze the neutralizing activity of antibodies against E1 region of hepatitis C virus （HCV）. Specific polydonal antibody was raised via immunization of New Zealand rabbits with a synthetic peptide that had been derived from the E1 region of HCV and was shown to be highly conserved among HCV published genotypes. METHODS： Hyper-immune HCV E1 antibodies were incubated over night at 4 ℃ with serum samples positive for HCV RNA, with viral loads ranging from 615 to 3.2 million IU/mL. Treated sera were incubated with HepG2 cells for 90 min. Blocking of viral binding and entry into cells by anti E1 antibody were tested by means of RTPCR and flow cytometry. RESULTS： Direct immunostaining using FITC conjugated E1 antibody followed by Flow cytometric analysis showed reduced mean fluorescence intensity in samples pre-incubated with E1 antibody compared with untreated samples. Furthermore, 13 out of 18 positive sera （72%） showed complete inhibition of infectivity as detected by RT-PCR. CONCLUSION： In house produced E1 antibody, blocks binding and entry of HCV virion infection to target cells suggesting the involvement of this epitope in virus binding and entry. Isolation of these antibodies that block virus attachment to human cells are useful as therapeutic reagents.

Fluorescence imaging analysis of the glioma microenvironment

Glioma is the most malignant brain cancer.The neurons,macrophages,T cells and other immune ellls constitute the glioma immunosuppressive microenvironment.The accurate spatial distri-bution of these cells in the glioma microenvironment and its relationship with glioma metastasis is unknown.We constructed a mouse gliomna cell line stably expressing the large Stokes shifted yellow fluorescent protein and applied it to the multicolor immunofluorescence imaging.The inaging data revealed that the neurons were sparsely distributed in the glioma core and the mumber of neurons decreased by 90%compared with normal brain site.The spatial distribution of monocyte-macrophages and microglia is heterogeneous.The monocyte macrophages and T cells were heavily recrnuited into the glioma core and metastasis.There was no significant difference in the distribution of microglia amnong glioma core,margin,and normal brain site.Our results provided new perspectives for targeting immune regulation cells and developing new immuno-therapy strategies for glioma.

Expression of Auto-antiboby in Patients wih Acute Pancreatitis and its Clinical Significance

The expression of an auto-antibody in patients with edematous acute pancreatitis and its possible clinical significance was investiaged. Eighteen cases of acute pancreatitis were chosen as experimental group and 25 subjects served as control group. Venous blood samples were taken in both groups at 4 time points: at the day of admission, the 2nd, 4th and 7th day of hospitalization. By using indirect immuno-fluorescence tests the expression of auto-antibody in the samples was semiquantitatively detected. Other biochemical indexes, such as serum amylase, urine amylase, were determined simultaneously. As well, the clinical signs or symptoms were mornitored. It was found that the expression of the auto-antibody was gradually enhanced with the development of acute pancreatitis. The inceased positive expression of auto-antibody showed a correlationship with the improvement of biochemical indexes (r=0.951) and clinical manifestations (r=0.996). There was significant difference between experimental and control groups (P<0.05). During the recovery period of acute pancreatitis, gradually increased auto-antibody expression was detectable. This antibody is against the interstitial structure of the pancreas.

Application of an indirect immunofluorescent staining method for detection of Salmonella enteritidis in paraffin slices and antigen location in infected duck tissues

AIM： To detect Salmonella enteritidis （S. enteritidis） in paraffin slices and antigen location in infected duck tissues. METHODS： The rabbits were immunized with purified bacillus to obtain S. enteritidis-specific antibody, which were then extracted by the caprylic-ammonium sulphate method, purified through High-Q columns. An indirect immuno-fluorescent staining method （IFA） was established to detect the S. enteritidis antigen in paraffin slices. Detected S. enteritidis in each organ tissue of ducklings experimentally infected with S. enteritidis. RESULTS： The gland of Garder, heart, kidney, spleen, liver, brain, ileum, jejunum, bursa of Fabricius from S. enteritidis experimentally infected ducklings were positive or strongly positive, and the S. enteritidis antigen mainly distributed in the infected cell cytoplasm.CONCLUSION： IFA is an intuitionist, sensitive and specific method in detecting S. enteritidis antigen in paraffin wax slices, and it is a good method in diagnosis and antigen location of S. enteritidis. We also conclude that the gland of Garder, heart, kidney, spleen, liver, ileum, jejunum are target organs in S. enteritidis infections of duck, and S. enteritidis is an intracellular parasitic bacterium.