Ongoing angiogenesis in blood vessels of the abdominal aortic aneurysm

Pathogenesis of the abdominal aortic aneurysm has been attributed to neovascularization of the aortic wall. However, it is not clear whether angiogenesis persists in the aneurysm. In sections of aneurysms, we determined the immunohistochemical distributions of the alpha v beta 3 integrin, tenascin and endothelial nitric oxide synthase (eNOS), which are markers respectively, of angiogenesis, matrix remodeling and vasoregulatory function. In addition, we used reverse transcription followed by in situ PCR, to determine the distribution of alpha v mRNA. All aneurysm specimens exhibited extensive increases of wall vascularization as compared with the control aortic wall, and showed the presence of perivascular inflammatory exudates containing macrophages and lymphocytes. The neovascularization consisted of thick-walled vessels in the media and adventitia, and capillaries in the subintima. The majority of vessels stained positively for the alpha v beta 3 antigen and eNOS. Tenascin was deposited as bands that circumscribed thick-walled vessels. The distribution of av mRNA was extensive and was positive even in those vessels that failed to stain for the alpha v beta 3 protein. No staining was evident in control aortas for the alpha v beta 3 antigen, tenascin or alpha v mRNA. The upregulation of av mRNA and the alpha v beta 3 integrin in blood vessels surrounded by a matrix expressing tenascin, indicates that angiogenesis is an ongoing process in the mature aortic aneurysm.

Less DeltamtDNA4977 than normal in various types of tumors suggests that cancer cells are essentially free of this mutation

Levels of mtDNA(4977) deletions (DeltamtDNA(4977)) have been found to be lower in tumors than in adjacent non-tumoral tissues. In 87 cancer patients, DeltamtDNA(4977) was detected by multiplex polymerase chain reaction (PCR) amplification in 43 (49%) of the tumors and in 74 (85%) of the samples of non-tumoral tissues that were adjacent to the tumors. DeltamtDNA(4977) deletions were detected in 24% of the breast tumors, 52% of the colorectal tumors, 79% of the gastric tumors, and 40% of the head and neck tumors as compared with 77, 83, 100, and 90% of the adjacent respective non-tumoral tissues at the same DNA template dilution. Based on limiting dilution PCR of 16 tumors and their adjacent non-tumoral tissues, it was found that the amount of DeltamtDNA(4977) was 10- to 100-fold lower in the tumor than in the respective control non-tumoral tissues. Real-time PCR experiments were performed to quantify the number of DeltamtDNA(4977) deletions per cell, by determining the mitochondrial-to-nuclear DNA ratio. In all of the cases of breast, colorectal, gastric, and head and neck cancer the proportion of DeltamtDNA(4977) in tumors was lower than that of the respective non-tumoral tissue. Traces of DeltamtDNA(4977) in tumors were apparently due to contamination of tumor tissue with surrounding non-tumoral tissue, as evidenced by tumor microdissection and in situ PCR techniques, suggesting that tumors are essentially free of this mutation. Although the metabolic effect of DeltamtDNA(4977) may be minimal in normal (non-tumor) tissue, in tissue under stress, such as in tumors, even low levels of DeltamtDNA(4977) deletions may be intolerable.

Absence of Human Papillomavirus DNA in Nongenital Seborrheic Keratosis

Seborrheic keratosis (SK) is a benign epidermal tumor of unknown etiology. Because of its wart-like morphology, human papillomavirus (HPV) has been suggested as a possible causative agent. Viral involvement, however, has not been confirmed yet despite extensive research. The aim of this study was to evaluate the presence of HPV 6/11, 31, 33 DNA in nongenital SK. We analyzed 40 biopsy specimens taken from patients with nongenital SK using in situ polymerase chain reaction (PCR) and PCR with tissue extracts. The SK specimens (n=40), analyzed by in situ PCR, were negative for all HPV probes tested (types 6/11, 31, 33). Control slides (condyloma acuminatum, n=3) were positive for type 6/11, 31, and 33 HPV probes tested. Melasma samples (n=4), the negative controls, were consistently negative. No HPV DNA band was detected by PCR with the tissue extracts from paraffin-embedded SK samples, while condyloma acuminatum, the positive controls, showed DNA bands of the correct molecular weights. Our results show that HPV type 6/11, 31, and 33 cannot be recognized as causative agents for nongenital SK, which is in contrast to the previous studies. Further studies are required to reveal the presence of other types (more than 90) of HPV DNA.

The Prevalence of Epstein-Barr Virus in Uterine Cervical Cancer: Detection by PCR and In Situ PCR Methods / 대한산부인과학회잡지

OBJECTIVE: Uterine cervical cancer is the most common malignant tumor in Korean women. Human papillomaviruses are associated in 85-90% of the cases. However, other cofactors are considered to be important in carcinogenesis. There is an evidence that the uterine cervix is the site of shedding of the Epstein-Barr viruses(EBV). Furthermore the virus has been detected in cervical intraepithelial neoplasia and invasive carcinoma of the uterine cervix. We studied to evaluate the role of EBV in cervical carcinogenesis. METHODS: Non-neoplastic cervices(12), carcinoma in situ(32), microinvasive squamous cell carcinomas(9), invasive squamous cell carcinomas(37) and adenocarcinomas and adenosquamous carcinomas(14) were studied for EBV infection. PCR and in situ PCR for EBNA-1 were done and subtyping was done using PCR for EBNA 3C. RESULTS: In non-neoplastic cervix, EBV was detected in 16.7% by PCR and found in normal epithelial cells and lymphocytes in in situ PCR. By PCR technique, EBV was detected in 65.6% of CIS, 66.3% and 51.4% of microinvasive and invasive squamous cell carcinomas, 57.1% of adenocarcinomas and adenosquamous carcinomas. EBV subtyping was done in EBV positive cases by PCR and all showed type 1. CONCLUSION: EBV was detected in higher frequency in cervical cancer than in non-neoplastic cervix. However the frequency was not correlated to the invasion depth and histologic types of cervical carcinomas. EBV was detected in tumor cells as well as normal epithelial cells and lymphocytes also. It was suggested that EBV may play a role in cervical cancers but the mechanism in carcinogenesis remains to be solved.

Localization of HBsAg and Hepatitis B Virus DNA in Renal Tissues from HBsAg Positive Patients with the Membranoproliferative Glomerulonephritis / 대한신장학회잡지

Hepatitis B virus(HBV) infection has been suggested as the etiologic agent in membranoproliferative glomerulonephritis(MPGN), but the mechanism by which HBV infection leads to MPGN in human has not been established. To localize the HBV antigen and HBV-DNA in the kidney tissue, we examined paraffin sections of kidney biopsies which were positive for HBsAg by immunohistochemical study from 13 HBV carriers with MPGN (HBV-MPGN). Polymerase chain reaction(PCR) and in situ PCR(ISP) were used for the HBV DNA amplification and localization in kidney tissues. Primers used in PCR and ISP were from the S, C, and X HBV-DNA regions. Immunohistochemical study showed HBsAg deposits on the mesangium and glomerular capillaries. Arteriolar deposits were also occasionally observed. PCR for the S, C, and X regions were positive in 11 patients(85%), 11 patients(85%), and 9 patients (69%), respectively. The PCR findings were further confirmed by direct sequencing of PCR products and the amplification of HSP70 gene as a control. ISP showed the amplified HBV-DNA at the glomeruli and renal tubules. For S region, ISP was positive in 7 patients. For C and X regions, ISP was positive in 8 patients, respectively. 5 patients showed the positive signals for both the glomeruli and tubules, while 4 patients were positive at the tubules only. These 4 patients seemed to have the longer disease durations when compared to the other 5 patients (52.8 months vs. 11.8 months), but it was not statistically significant. In conclusion, the detection and the localization of HBV antigen and DNA in renal tissues indicate the presence of the complete virion in the kidney. These results suggest that HBV may infect the kidneys of HBV carriers with MPGN.

Study on the Detection of Chromosome X, Y Using in situ PCR / 대한산부인과학회잡지

Untill now, classical karyotyping remains the most reliable and conclusive method, but sometimes very rapid and sensitive method for the detection of chromosomal abnormality is needed. The primed in situ labelling(PRINS) is very rapid and sensitive method for the detection of chromosomal aneuploidy and X-linked disorder instead of fluorescen in situ hybridization. The purpose of this study was to prepent the application of PRINS protocol to the rapid detection of chromosomes X and Y in both metaphases and interphase nuclei. The PRINS reaction was done in 10 amniocytes. We compared the PRINS result with conventional cytogenetic karyotyping. The result was the same with cytogenetic karyotyping. In the future, PRINS will become very useful and sensitive method for the chromosomal aneuploidy detection and X-linked disorder diagnosis.

A Study of Standardization of the In situ PCR ( Polymerase Chain Reaction ) on the Tissues of Borderline Leprosy Patients / 대한피부과학회지

BACKGROUND: PCR from paraffin-embedded tissues has been recently applied on the diagnosis of leprosy, PCR is a highly sensitive technique and the amplified product is detected by gel electrophoresis and Southern blot hybridization. But conventional PCR does not give an information about histopathologic features. On the other hand in situ PCR informs the histopathological location of DNA amplified inside the cells and detected by in sita hybridization or immunohistoche mical method. OBJECTIVE: In situ PCR was applied on the paraffin-embedded tissues of borderline leprosy patients. The optimal condition of in situ PCR in leprosy was searched with the parameters of pretreatment of the tissues, fixation time of paraformaldehyde and total volume of PCR. METHODS: The paraffin-embedded tissues of ten borderline leprosy patients were subjected to in situ PCR. Amplified DNA product within the cells was incorporated with Digoxigenin and was directly detected by immunohistochemical method using anti-Dig-alkaline phosphatase conjugated antibody. The tissues were pretreated with 0.2N HCl or various concentration of proteinase K such as 10, 15 and 100ug/ml and various incubation time of proteinase K from 3.5 minutes to 15 minutes. Fixation was performed before pretreatment. and after PCR for 15 minutes, after pretreatment and after PCR for 15 minutes, only after pretreatment for 15 minutes or after pretretmant for 60 minutes. The total volume of PCR was 40, 50 or 60ul. RESULTS: 1. The amplified DNA was detected using the HCl-pretreated tissues in four of seven BL patients and one of three BT pat ients. 2. The signals were detected in the cytoplasm of most histiocytes and proliferated Schwann cells, some secretory cells of sweat glands and a few endothelial cells in the tissues of the BL patients and the cells composing of the granuloma in those of the BT patients. 3. Proteinase K-treated tissues showed positive reaction in only one tissue used in 10ug/ml of proteinase K for 10 minutes. 4. The proper time for fixation of paraformaldehyde was before treatment of proteinase K or HCl and after PCR reaction. 5. The 40ul of total volume for PCR reaction was enough and minimized the loss of tissues during PCR reaction. CONCLUSION: When in situ PCR was applied on the paraffin-ernbedded tissues of borderline leprosy patients, pretreatment such as concentration and incubation time of proteinase K or HCL was the most critical parameter.

Detection of Herpes Simplex Virus Type-1 DNA by In Situ Polymerase Chain Reaction

BACKGROUND: Standard solution-phase PCR cannot localize the amplified DNA products in cells or tissue sections. Recently, in situ PCR technique which combines PCR with in situ hybridization was developed and applied to detect target DNA or gene expression in the tissue sections. OBJECTIVE: The purpose of this study was to detect the presence of HSV type-1 DNA in herpes simplex lesions by using hot start PCR in situ hybridization and hot start in situ PCR and to compare the sensitivity and specificity of the two methods. The sensitivity and specificity of multiple overlapping primers and a single primer pair in hot start in situ PCR were also compared. METHODS: We performed hot start PCR in situ hybridization and in situ PCR with multiple overlapping primers, and hot start in situ PCR with a single primer pair in paraffin-embedded, formalin-fixed tissues. RESULTS: HSV type-1 DNA was detected in 4 (80%) of.5 cases of herpes simplex and negative in all cases of herpes zoster, verruca vulgaris, and normal skins. One negative case of herpes simplex could not be detected by HSV type-1 specific primers because it might be caused by HSV type-2. There was no difference in the sensitivity, specificity, and intensity of signals between the three methods. CONCLUSION: Hot start in situ PCR with a single primer pair is a simpler, easier, and more rapid technique for detecting the HSV type-1 DNA in lesional tissue sections with similar sensitivity and specificity than hot start PCR in situ hybridization and hot start in situ PCR using multiple overlapping primers.