Laser Dissection Microscopy Research Articles

Highly Tissue Substructure-Specific Effects of Human Papilloma Virus in Mucosa of HIV-Infected Patients Revealed by Laser-Dissection Microscopy-Assisted Gene Expression Profiling

Human papilloma virus (HPV) causes focal infections of epithelial layers in skin and mucosa. HIV-infected patients on highly active antiretroviral therapy (HAART) appear to be at increased risk of developing HPV-induced oral warts. To identify the mechanisms that allow long-term infection of oral epithelial cells in these patients, we used a combination of laser-dissection microscopy (LDM) and highly sensitive and quantitative, non-biased, two-step multiplex real-time RT-PCR to study pathogen-induced alterations of specific tissue subcompartments. Expression of 166 genes was compared in three distinct epithelial and subepithelial compartments isolated from biopsies of normal mucosa from HIV-infected and non-infected patients and of HPV32-induced oral warts from HIV-infected patients. In contrast to the underlying HIV infection and/or HAART, which did not significantly elaborate tissue substructure-specific effects, changes in oral warts were strongly tissue substructure-specific. HPV 32 seems to establish infection by selectively enhancing epithelial cell growth and differentiation in the stratum spinosum and to evade the immune system by actively suppressing inflammatory responses in adjacent underlying tissues. With this highly sensitive and quantitative method tissue-specific expression of hundreds of genes can be studied simultaneously in a few cells. Because of its large dynamic measurement range it could also become a method of choice to confirm and better quantify results obtained by microarray analysis.

Systemic distribution of steroid sulfatase and estrogen sulfotransferase in human adult and fetal tissues.

Estrogens play a key role in various target tissues. Enzymes involved in the biosynthesis and metabolism of these sex steroids also regulate estrogenic actions in these tissues. Estrone sulfate (E1S) is a major circulating plasma estrogen that is converted into the biologically active estrogen, estrone (E1), by steroid sulfatase (STS). E1 is also sulfated and reverted into E1S by estrogen sulfotransferase (EST). These two enzymes have recently been shown to play important roles in the in situ estrogen actions of various sex steroid-dependent human tumors. However, the distribution of STS and EST in normal adult and fetal human tissues remains largely unknown. Therefore, in this study, in addition to examining the tissue distribution of both STS and EST mRNA in human adult and fetal tissues using RT followed by quantitative PCR, we studied the activity of these enzymes using (3)H-labeled E1/E1S as substrates in the homogenates of various human adult tissues. We also examined the localization of STS and EST protein in human adult and fetal tissues using immunohistochemistry, and that of EST mRNA in the adult kidney using laser dissection microscopy and PCR. STS mRNA, enzyme activity, and immunoreactivity were either absent or detected at very low levels in all adult and fetal tissues examined in this study. EST mRNA expression, however, was detected in all of the tissues examined, except for adult spleen and pancreas. EST enzyme activities were consistent with those of mRNA expression in the great majority of the tissues examined. Marked EST immunoreactivity was detected in hepatocytes, adrenal gland (adult, zona fasciculate to the reticularis; fetus, fetal zone), and epithelial cells of the gastrointestinal tract, smooth muscle cells of the tunica media in aorta, Leydig cells of the testis, and syncytiotrophoblast of the placenta. Patterns of EST immunolocalization were similar between adult and fetal human tissues, but EST immunoreactivity was detected in the urinary tubules of adult kidney, whereas in the fetal kidney, it was localized in the interstitial cells surrounding the urinary tubules. In the adult kidney, the presence of EST mRNA was also confirmed in the cells of urinary tubules using laser dissection microscopy and RT-PCR. Although the number of human tissues available for examination in this study was limited, our results suggest that between the enzymes involved in estrogen activation or inactivation, EST and not STS is the more widely expressed enzyme in various peripheral tissues in humans. We speculate that EST may play an important role in protecting peripheral tissues from possible excessive estrogenic effects.

Analysis of in vivo endothelial cell activation applying RT-PCR following endothelial cell isolation by laser dissection microscopy.

Laser dissection microscopy was applied to isolate endothelial cells from tumors obtained from mice treated with TNF-alpha. RNA integrity was demonstrated from whole sections and dissected cells after acetone fixation and hematoxylin staining. RT-PCR for GAPDH, CD31, VCAM-1, ICAM-1, and E-selectin was successfully performed on these samples. These data demonstrate the feasibility of analyzing local endothelial cell responses in diseased tissues at the level of gene expression.