Pulmonary adenofibroma in a sika deer

A solitary firm nodule was found in the lung of a sika deer (Cervus nippon yesoensis). Histologically, it was a biphasic lesion composed of epithelial and stromal cell elements and exhibited a leaf-like growth pattern. The epithelial cells were immunohistochemically positive for pancytokeratin, cytokeratin 7, napsin A, and thyroid transcription factor-1, and the stromal cells were positive for vimentin and partially positive for desmin and α-smooth muscle actin. These observations were consistent with pulmonary adenofibroma, which is an extremely rare lesion in humans. To the best of our knowledge, this is the first reported case of pulmonary adenofibroma in an animal

Quantification of Chitinase mRNA Levels in Human and Mouse Tissues by Real-Time PCR: Species-Specific Expression of Acidic Mammalian Chitinase in Stomach Tissues.

Chitinase hydrolyzes chitin, which is an N-acetyl-D-glucosamine polymer that is present in a wide range of organisms, including insects, parasites and fungi. Although mammals do not contain any endogenous chitin, humans and mice express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Because the level of expression of these chitinases is increased in many inflammatory conditions, including Gaucher disease and mouse models of asthma, both chitinases may play important roles in the pathophysiologies of these and other diseases. We recently established a quantitative PCR system using a single standard DNA and showed that AMCase mRNA is synthesized at extraordinarily high levels in mouse stomach tissues. In this study, we applied this methodology to the quantification of chitinase mRNAs in human tissues and found that both chitinase mRNAs were widely expressed in normal human tissues. Chit1 mRNA was highly expressed in the human lung, whereas AMCase mRNA was not overexpressed in normal human stomach tissues. The levels of these mRNAs in human tissues were significantly lower than the levels of housekeeping genes. Because the AMCase expression levels were quite different between the human and mouse stomach tissues, we developed a quantitative PCR system to compare the mRNA levels between human and mouse tissues using a human-mouse hybrid standard DNA. Our analysis showed that Chit1 mRNA is expressed at similar levels in normal human and mouse lung. In contrast, the AMCase expression level in human stomach was significantly lower than that expression level observed in mouse stomach. These mRNA differences between human and mouse stomach tissues were reflecting differences in the chitinolytic activities and levels of protein expression. Thus, the expression level of the AMCase in the stomach is species-specific

Expression of Chit1 and AMCase mRNAs in normal human tissues.

<p>Quantification of Chit1 (A) and AMCase (B) mRNAs in human tissues. Both chitinases were quantified by real-time PCR using the human standard DNA. All of the values are expressed as number of molecules per 10 ng of total RNA in y axis.</p

Analysis of Chit1 and AMCase mRNAs and reference gene mRNAs in lung and stomach tissues.

<p>The expression levels of the five genes, which were determined using cDNAs prepared from normal human lung (A) and stomach (B) tissues, were quantified by real-time PCR. The upper panel indicates the actual value, and the lower panel shows the logarithm of each value. The expression level of the human Chit1 gene was set to 1.0; the values on the bars indicate the relative expression levels compared to the expression level of the human Chit1 gene.</p

Expression of Chit1 and AMCase mRNAs in human and mouse tissues.

<p>The expression levels of Chit1 (A) and AMCase (B) mRNAs in eight human and mouse tissues were quantified on the same scale by real-time PCR using the hybrid standard DNA. Filled bars, human tissues; hatched bars, mouse tissues. All of the values are expressed as number of molecules per 10 ng of total RNA in y axis. The upper panel indicates the actual value, and the lower panel shows the logarithm of each value. Part of mouse data has been reported in our previous paper <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067399#pone.0067399-Ohno1" target="_blank">[17]</a>.</p

Strategy for comparing Chit1 and AMCase mRNA levels between human and mouse tissues.

<p>(A) The expression levels of the Chit1 and AMCase genes in human and mouse tissues were compared. (B) Schematic representation of the human-mouse hybrid standard DNA used for the quantification. The human and mouse standard DNAs were ligated using the <i>Eco</i>RI site at a one-to-one ratio into a DNA fragment and used as the human-mouse hybrid standard DNA.</p

Strategy for the comparison of the gene expression levels of five human genes.

<p>(A) We compared the expression levels of the Chit1 and AMCase genes. To evaluate the chitinase levels, we used two housekeeping genes (GAPDH and β-actin) and pepsinogen C (progastricsin), which served as a reference gene for the stomach. Using these three reference genes, we evaluated the gene expression levels of Chit1 and AMCase in human tissues. (B) Schematic representation of the standard DNA used for real-time PCR. The target fragments for AMCase, pepsinogen C, Chit1, GAPDH and β-actin cDNAs, in addition to their flanking sequences and restriction sites, were ligated at a one-to-one ratio into a DNA fragment and used as the standard DNA for the analysis of the human genes.</p

The levels of chitinases activity and protein expression in mouse and human stomach tissues.

<p>(A) The chitinolytic activity in the stomach extracts from mouse and human stomach tissues. Filled bars, human tissues; hatched bars, mouse tissues. (left) AMCase activity was determined using the synthetic chitin substrate of 4MU-chitobioside. Chitinolytic activity at pH 2.0 and pH 5.0 were expressed in y axis. (right) Chit1 activity was measured using 4MU-chitotriose at pH 5.2, as previously described in Materials and Methods. AMCase activity was also measured at pH 2.0. All graph points are the mean of triplicate measurements and representative of multiple experiments. (B) Representative patterns of Western blotting of mouse and human AMCase in mouse or human stomach proteins. Proteins were run on SDS-polyacrylamide gels and analyzed by Western blotting using anti-human or anti-mouse AMCase antibodies. Stomach soluble proteins (6.3 µg) were separated on SDS-polyacrylamide gels and transferred onto Immobilon membranes, and probed with AMCase specific antibodies.</p

Analysis of the expression levels in human and mouse lung and stomach tissues.

<p>The expression levels of the five genes, which were determined using the cDNAs prepared from normal human and mouse lung (A) and stomach (B) tissues, were quantified by real-time PCR. Filled bars, human tissues; hatched bars, mouse tissues. The upper panel indicates the actual value, and the lower panel shows the logarithm of each value. The expression level of the human Chit1 gene was set to 1.0; the values on the bars indicate the relative expression levels compared to the expression level of the human Chit1 gene. Part of mouse data has been reported in our previous paper <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067399#pone.0067399-Ohno1" target="_blank">[17]</a>.</p