Bill A. Rampy

Novel Expression Patterns of PI3K/Akt/mTOR Signaling Pathway Components in Colorectal Cancer

BACKGROUND The phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway plays a critical role in the growth and progression of colorectal cancer (CRC). The purpose of our study was 2-fold: (1) to determine the expression levels of several key components of this pathway, including p85alpha, Akt1, Akt2, p-mTOR(Ser2448), and p-p70S6K(Thr389) in CRCs; and (2) to correlate the expression of these proteins with cancer stage and location (left versus right side). STUDY DESIGN Immunohistochemistry for p85alpha, Akt1, Akt2, p-mTOR(Ser2448), and p-p70S6K(Thr389) was performed on normal colon and CRCs from 154 patients. RESULTS All proteins investigated were significantly overexpressed in CRCs compared with matched normal colonic tissue from the same patient (p < 0.0001). PI3K pathway component proteins were moderately correlated across normal and malignant colon tissues; correlations tended to be stronger in normal tissues as compared with the same correlations in cancers. Expression levels of p85alpha were significantly higher in stage IV cancers than in stage I to III cancers (p = 0.0005). p85alpha expression was also significantly increased in the adjacent normal colonic mucosa of patients with stage IV CRC compared with earlier stages (p = 0.003). Finally, expression of Akt1, Akt2, and p-p70S6K(Thr389) was higher in left-sided CRCs compared with CRCs in the right colon (p = 0.007, p = 0.0008, and p = 0.04, respectively). CONCLUSIONS The PI3K/Akt/mTOR pathway components, p85alpha, Akt1, Akt2, p-mTOR(Ser2448), and p-p70S6K(Thr389) are highly overexpressed in CRCs, providing the rationale for targeting this pathway therapeutically in CRC patients. The increased expression of p85alpha in the adjacent normal mucosa of stage IV patients suggests an important field defect, which may contribute to the growth and progression of these cancers.

Distribution of Histochemically Reactive Zinc in the Forebrain of the Rat

The major cytoarchitectonic regions of the rat brain that stain with the Timm-Danscher metal stain were tested with the fluorescent probe for zinc, 6-methoxy 8-para toluene sulfonamide quinoline (TSQ). Throughout most of the striatum, cerebral cortex and limbic system, the diffuse, even neuropil staining produced by the Timm-Danscher method was mirrored by comparable fluorescence in TSQ-stained sections. Blockade of the TSQ fluorescence by prior treatment with sulphide indicated that the Timm-Danscher and the TSQ procedures both labeled the same pool of endogenous metal, which is inferred to be the zinc that is in axonal boutons. It is concluded that the Timm-Danscher staining generally indicates zinc-containing axonal boutons. The distribution of the zinc-containing axonal boutons throughout the forebrain is described.

A Rapid and Noninvasive Method for Detecting Tissue-Limited Mosaicism: Detection of i(12)(p10) in Buccal Smear from a Child with Pallister-Killian Syndrome

Pallister-Killian syndrome (PKS), a rare disorder, is characterized by tissue-limited or tissue-specific mosaicism. The characteristic chromosome abnormality associated with PKS is i(12p), which is seen predominantly in skin fibroblast cultures. Diagnosis of i(12p) has been carried out on buccal smears before and was shown to be an easy and feasible method. All previously published studies used alpha-satellite probes for the diagnosis and as such have several pitfalls. Our approach, using dual-color, locus-specific probes, has high specificity and sensitivity for the diagnosis of i(12p). Using statistical analysis, we have also confirmed that the signal pattern in interphase nuclei is consistent with isochromosome 12p.

Serous cystadenoma of the pancreas with papillary features: A diagnostic pitfall on fine-needle aspiration biopsy

Serous cystadenoma of the pancreas is an uncommon neoplasm that occasionally exhibits papillary differentiation. The cytomorphologic structure of pancreatic serous cystadenoma has been rarely described, and, to our knowledge, such papillary morphologic structure has never been reported on fine-needle aspiration cytologic examination. We present a case of serous cystadenoma of the pancreas in a 77-year-old woman. Endoscopic ultrasonography showed a well-demarcated solid/cystic mass in the midbody of the pancreas, suggestive of solid pseudopapillary tumor. Aspiration cytologic examination, performed under endoscopic ultrasound guidance, showed a predominantly papillary epithelial neoplasm consistent with the radiologic impression. Gross and histologic examination of the excised specimen revealed a pancreatic serous cystadenoma with multifocal papillae. This case illustrates the cytomorphologic structure of serous cystadenoma that presents with prominent papillary differentiation on aspiration cytologic examination. The unusual cytologic appearance of this tumor introduces significant diagnostic challenges to the pathologist. Serous cystadenoma must be included in the differential diagnosis of pancreatic neoplasms with papillary morphologic structure as evaluated by fine-needle aspiration cytologic examination.

Fine needle aspiration cytology of hepatobiliary cystadenoma with mesenchymal stroma

Hepatobiliary cystadenomas (HBCs) with mesenchymal stroma (MS) are rare cystic neoplasms occurring exclusively in women. Hepatobiliary cystadenoma consists of a mucin‐producing cyst lining epithelium underlined by a dense MS cell layer. In the current study, the authors review the fine needle aspiration cytology of HBC with MS and identify characteristic cytologic features that suggest such an uncommon neoplasm on aspirates.

Helicobacter pylori cag Pathogenicity Island's Role in B7-H1 Induction and Immune Evasion

During Helicobacter pylori (H. pylori) infection CD4+ T cells in the gastric lamina propria are hyporesponsive and polarized by Th1/Th17 cell responses controlled by Treg cells. We have previously shown that H. pylori upregulates B7-H1 expression on GEC, which, in turn, suppress T cell proliferation, effector function, and induce Treg cells in vitro. In this study, we investigated the underlying mechanisms and the functional relevance of B7-H1 induction by H. pylori infection to chronic infection. Using H. pylori wild type (WT), cag pathogenicity island (cag PAI-) and cagA - isogenic mutant strains we demonstrated that H. pylori requires its type 4 secretion system (T4SS) as well as its effector protein CagA and peptidoglycan (PG) fragments for B7-H1 upregulation on GEC. Our study also showed that H. pylori uses the p38 MAPK pathway to upregulate B7-H1 expression in GEC. In vivo confirmation was obtained when infection of C57BL/6 mice with H. pylori PMSS1 strain, which has a functional T4SS delivery system, but not with H. pylori SS1 strain lacking a functional T4SS, led to a strong upregulation of B7-H1 expression in the gastric mucosa, increased bacterial load, induction of Treg cells in the stomach, increased IL-10 in the serum. Interestingly, B7-H1-/- mice showed less Treg cells and reduced bacterial loads after infection. These studies demonstrate how H. pylori T4SS components activate the p38 MAPK pathway, upregulate B7-H1 expression by GEC, and cause Treg cell induction; thus, contribute to establishing a persistent infection characteristic of H. pylori.

Reduced CD1d expression in colonic epithelium in microscopic colitis.

CD1d is a member of a major histocompatibility complex class I-like molecule family. Its function may involve presenting microbial nonpeptide or lipidic antigens to T lymphocytes, therefore to serve as an important factor in normal mucosal immunity of the gastrointestinal tract. In this study, the expression level of CD1d in microscopic colitis (ie, collagenous and lymphocytic colitis) was examined, and compared with that in normal colonic mucosa. Formalin-fixed, paraffin-embedded colon biopsies with diagnosis of lymphocytic colitis (19 cases), collagenous colitis (6 cases), and no pathologic change (20 cases) were studied immunohistochemically using monoclonal antibodies against human CD1d, CD3, CD4, and CD8. CD1d staining in the epithelium and lamina propria was graded along a scale of 0 to 4. Intraepithelial CD3-positive lymphocytes were counted in an area of 300 epithelial cells for each specimen. The results show that CD1d was expressed in normal colonic epithelial cells, primarily on the basolateral membranes with a concentrated intracellular pool in the subnuclear region. The expression level was markedly reduced in both lymphocytic colitis (P<0.001) and collagenous colitis (P<0.001), along with a significant increase in the number of intraepithelial CD3/CD8 lymphocytes (P<0.001). These findings suggest that microscopic colitis is associated with decreased epithelial expression of CD1d, an important immunoregulatory molecule in the gastrointestinal tract.

Hepatic alcohol dehydrogenase deficiency induces pancreatic injury in chronic ethanol feeding model of deer mice

The single most common cause of chronic pancreatitis (CP, a serious inflammatory disease) is chronic alcohol abuse, which impairs hepatic alcohol dehydrogenase (ADH, a major ethanol oxidizing enzyme). Previously, we found ~5 fold greater fatty acid ethyl esters (FAEEs), and injury in the pancreas of hepatic ADH deficient (ADH-) vs. hepatic normal ADH (ADH+) deer mice fed 3.5g% ethanol via liquid diet daily for two months. Therefore, progression of ethanol-induced pancreatic injury was determined in ADH- deer mice fed ethanol for four months to delineate the mechanism and metabolic basis of alcoholic chronic pancreatitis (ACP). In addition to a substantially increased blood alcohol concentration and plasma FAEEs, significant degenerative changes, including atrophy and loss of acinar cells in some areas, ultrastructural changes evident by such features as swelling and disintegration of endoplasmic reticulum (ER) cisternae and ER stress were observed in the pancreas of ethanol-fed ADH- deer mice vs. ADH+ deer mice. These changes are consistent with noted increases in pancreatic injury markers (plasma lipase, pancreatic trypsinogen activation peptide, FAEE synthase and cathepsin B) in ethanol-fed ADH- deer mice. Most importantly, an increased levels of pancreatic glucose regulated protein (GRP) 78 (a prominent ER stress marker) were found to be closely associated with increased phosphorylated eukaryotic initiation factor (eIF) 2α signaling molecule in PKR-like ER kinase branch of unfolded protein response (UPR) as compared to X box binding protein 1S and activating transcription factor (ATF)6 - 50kDa protein of inositol requiring enzyme 1α and ATF6 branches of UPR, respectively, in ethanol-fed ADH- vs. ADH+ deer mice. These results along with findings on plasma FAEEs, and pancreatic histology and injury markers suggest a metabolic basis of ethanol-induced pancreatic injury, and provide new avenues to understand metabolic basis and molecular mechanism of ACP.