James A. Radosevich

Immunohistochemical localization of modified C-reactive protein antigen in normal vascular tissue.

BACKGROUND The prototypic acute phase reactant, C-reactive protein (CRP), is a serum soluble, cyclic pentameric protein, the concentration of which increases markedly within hours of any tissue-damaging, inflammatory event. However, upon dissociation of its pentameric quaternary structure, CRP subunits undergo a spontaneous and irreversible conformational change. The resulting molecule, termed modified CRP or mCRP, has reduced aqueous solubility and a propensity to aggregate into a matrix-like lattice structure. METHODS Using monoclonal antibodies, normal human tissues were immunohistochemically screened for the presence of CRP as well as mCRP antigens. RESULTS Significant levels of mCRP were detected in the walls of blood vessels associated with normal human tissues. These data indicate that mCRP is a naturally occurring form of CRP and that it is a tissue-based rather than serum-based molecule. SIGNIFICANCE This report describes the localization of a stable form of CRP, mCRP, in blood vessels associated with normal human tissues.

The Yin and Yang of nitric oxide: Reflections on the physiology and pathophysiology of NO·

Nitric oxide (NO·) is an arginine‐derived nitrogen‐based radical that is rapidly becoming one of the most important molecular species to be discovered. Over the past decade, an explosion of evidence has revealed the extreme complexity of function of this seemingly simple inorganic molecule. It is now evident that NO· demonstrates a functional dualism, playing a pivotal role in numerous physiologic and pathophysiologic processes. Whether this molecule is beneficial or detrimental is dependent upon the tissue of generation, the level of production, the oxidative/reductive (redox) environment in which this radical is generated, and the presence or absence of NO· transduction elements. Nitric oxide is generated by three independent isoenzymes that resemble the p‐450 enzyme superfamily in both form and function. It ultimately alters enzymatic function through covalent modification, redox interactions, and interactions with metallic functional centers. This radical is a key figure in a number of pathophysiologic processes by means of similar yet uncoordinated interactions. In consideration of the already broad spectrum of roles attributed to NO·, it seems highly likely that this molecule will be implicated in an ever widening variety of functions relative to the practice of otolaryngology–head and neck surgery. This article reviews the enzymology, signal transduction mechanisms, physiology, and pathophysiology of NO· as it pertains to head and neck cancer.

Nitric Oxide Synthase Type 3 is Increased in Squamous Hyperplasia, Dysplasia, and Squamous Cell Carcinoma of the Head and Neck

The implication of nitric oxide (NO) in the multistep process of carcinogenesis prompted us to examine the expression of endothelial constitutive nitric oxide synthase (NOS3) in head and neck squamous cell carcinoma (HNSCCa). Eleven paraffin-embedded samples of normal oral mucosa, 3 reactive oral lesions, 13 samples of squamous dysplasia, and 120 specimens of HNSCCa were immunostained with an anti-NOS3 monoclonal antibody and graded on a 0 to 4+ scale of intensity. Normal squamous mucosa demonstrated very little NOS3 expression. Areas of normal mucosa, reactive mucosa, and dysplastic lesions associated with inflammation tended to demonstrate regional expression of NOS3. Reactive mucosal lesions, squamous dysplasia, and HNSCCa demonstrated a significant (p < .0001) increase in global expression of NOS3. Therefore, NOS3 is expressed very little in histologically normal squamous mucosa, while squamous hyperplasia, dysplasia, and HNSCCa express significantly more NOS3. Regional variation in NOS3 expression appears to be associated with perilesional inflammation.

Expression of the double-stranded RNA-dependent protein kinase (p68) in human breast tissues

P68 is a potent inhibitor of protein synthesis in virally infected cells and has been suggested to function in noninfected cells as a tumor suppressor gene. We have previously demonstrated that p68 expression correlates directly with cellular differentiation and inversely with proliferative activity in normal epithelium and in several human tumor systems. In order to determine the role of p68 in human breast cancer, we utilized immunohistochemistry and mapped the expression of p68 in tissue from 200 breast biopsy specimens. A total of 434 foci, ranging from normal breast tissue to infiltrating carcinoma were examined. We found that p68 was present at basal levels in normal lobular and luminal ductal epithelial cells, with higher levels present in myoepithelial cells. Nonproliferative fibrocystic lesions showed variable expression of p68, with high levels seen within foci of apocrine metaplasia and low levels in cystically dilated terminal duct units. Low levels of p68 were seen in typical ductal proliferations, lobular neoplasia (atypical lobular hyperplasia and lobular carcinoma in situ), and in fibroadenomas. Foci of atypical ductal hyperplasia in situ and invasive ductal carcinoma generally showed higher levels of p68 expression. Among the infiltrating carcinomas, p68 expression correlated with nuclear grade. This suggests that the ability of p68 to inhibit cellular proliferation may be impaired in breast cancer and that its expression, although modestly paralleling cellular differentiation, is not a predictive indicator of improved survival.

Nitric oxide: Perspectives and emerging studies of a well known cytotoxin

The free radical nitric oxide (NO•) is known to play a dual role in human physiology and pathophysiology. At low levels, NO• can protect cells; however, at higher levels, NO• is a known cytotoxin, having been implicated in tumor angiogenesis and progression. While the majority of research devoted to understanding the role of NO• in cancer has to date been tissue-specific, we herein review underlying commonalities of NO• which may well exist among tumors arising from a variety of different sites. We also discuss the role of NO• in human physiology and pathophysiology, including the very important relationship between NO• and the glutathione-transferases, a class of protective enzymes involved in cellular protection. The emerging role of NO• in three main areas of epigenetics—DNA methylation, microRNAs, and histone modifications—is then discussed. Finally, we describe the recent development of a model cell line system in which human tumor cell lines were adapted to high NO• (HNO) levels. We anticipate that these HNO cell lines will serve as a useful tool in the ongoing efforts to better understand the role of NO• in cancer.

The H+/K+-ATPase (proton) pump is expressed in human laryngeal submucosal glands.

Objectives/Hypothesis: Diagnosis and treatment of gastroesophageal and laryngopharyngeal reflux disease has significantly increased over recent years. The larynx is highly sensitive to the effects of LPRD and is similarly responsive to proton pump inhibitor pharmacotherapy. The hypothesis of the study was that proton pump activity exists in the human larynx and plays a functional role in normal and/or pathological laryngeal tissue. Study Design: Pathological investigation. Methods: Two fresh human cadaveric larynges (one male and one female larynx) were obtained as part of an exempt protocol from the Human Subjects Committee and were formalin fixed and paraffin embedded. Banked human stomach tissue was also obtained for use as comparative positive and negative control specimens. Sections were immunostained with monoclonal antibodies reactive with both alpha and beta subunits of the H+/K+‐ATPase (proton) pump. Specimens were reviewed for staining pattern and intensity. Results: Stomach parietal cells (known to produce gastric acid) exhibited strongly positive staining for both the alpha and beta subunits of the proton pump. There was no staining in stomach cells that were not morphologically consistent with the parietal cell. In the human larynx there were strong focal and identical staining patterns in the serous cells and ducts of the minor seromucinous glands by both alpha and beta monoclonals to the proton pump. There was variable staining in the laryngeal epithelium that was thought to be consistent with artifact staining resulting from tissue processing. Conclusion: The H+/K+‐ATPase (proton) pump is present in serous cells and ducts of submucosal glands in the human larynx. Proton pump inhibitor pharmacotherapy may have a site of action in seromucinous glands of the human larynx, with possible relevance for patients treated for chronic laryngitis with or without laryngopharyngeal reflux disease.

Endothelial constitutive nitric oxide synthase (ecNOS) localization in normal and neoplastic salivary tissue

Nitric oxide (NO·) has been implicated in the process of carcinogenesis in various organs. This study was designed to investigate the expression of endothelial constitutive nitric oxide synthase (ecNOS) in normal and neoplastic salivary tissues.

Expression of p68 in human colon cancer.

p68 is an interferon-inducible protein kinase which is a key factor in the regulation of both viral and cellular protein synthesis. Since p68 plays a central role in cellular protein synthesis, we hypothesized that it would parallel translational activity, and thereby correlate with cellular differentiation in both normal and neoplastic cell types. Using the anti-p68 monoclonal antibody, TJ4C4, we have previously noted a correlation of p68 expression with the degree of cellular differentiation in the human upper aerodigestive tract and lung. During normal human fetal development, p68 is abundantly expressed in the upper aerodigestive tract and lungs, but considerably less so in the colon. In order to determine if this fetal expression pattern correlated with the pattern seen in adult colon and colonic adenocarcinomas, we analyzed the expression pattern of p68 in 80 patients with adenocarcinoma of the colon. Using light microscopic evaluation of immunoperoxidase-stained tissue sections, a spectrum of p68 expression was noted among the tissue samples. Increased p68 levels were noted in the majority of tumors with increased cellular differentiation, and those tissues with decreased p68 were, in general, less differentiated. These data are consistent with the concept that the expression of p68 parallels the degree of cellular differentiation, and are consistent with previously reported studies using this antibody. The limited fetal expression pattern of p68 in the colon and the variable correlation of p68 with differentiation suggests that p68, as well as other translational regulators, can be important in assessing the biological potential of tumors arising in the colon.

Distribution of CD45RA and CD45RO T-lymphocyte subsets in rheumatoid arthritis synovial tissue

We have characterized the lymphocytes in the synovium of patients with rheumatoid arthritis (RA) by immunohistochemistry using monoclonal antibodies directed against B lymphocytes, T lymphocytes, and antibodies directed against CD45RA and CD45RO, which define T-cell subsets. Both CD45RA+ and CD45RO+ T lymphocytes were detected in the perivascular regions. CD45RA+ lymphocytes were present primarily in perivascular areas of moderate to large lymphocytic infiltration. Some synovial perivascular lymphocytic aggregates were organized into focal areas of CD45RA+ B lymphocytes surrounded by CD45RO+ T lymphocytes. In areas of diffuse lymphocytic infiltration, the T lymphocytes were CD45RO+. These data suggest that both CD45RO+ and CD45RA+ T lymphocytes enter the RA synovial tissue via the synovial vasculature and that, once in the tissue, the CD45RA+ T lymphocytes may undergo activation/maturation and acquire the CD45RO phenotype.

Immunohistochemical evaluation of ras oncogene expression in pulmonary and pleural neoplasms.

SummaryWe undertook an immunohistochemical analysis of human bronchopulmonary epithelial neoplasms and pleural mesotheliomas using a monoclonal antibody which recognizes ras oncogene products (p21ras). The monoclonal antibody, RAP-5, recognizes both unaltered and certain mutated p21ras. Formalin fixed and paraffin embedded tissue samples of 187 lung epithelial tumors and 27 pleural mesotheliomas were investigated; normal and bronchiectatic lungs were similarly studied. Normal lung and pleural tissue did not immunostain except for occasional type II pneumocytes. Reactive type II pneumocytes adjacent to carcinomas and bronchiectasis immunostained consistently. Twenty four/34 (71%) squamous carcinomas immunostained. Only 8/50 (16%) adenocarcinomas immunostained focally and weakly whereas 19/24 (79%) bronchioloalveolar carcinomas immunostained. Eleven/18 (61%) large cell carcinomas immunostained with variable intensity. Eleven/13 (85%) carcinoids, 6/7 (85%) well differentiated neuroendocrine carcinomas, and 18/21 (86%) intermediate cell neuroendocrine carcinomas immunostained while none of 20 small cell neuroendocrine carcinomas immunostained. Only a few mesotheliomas were immunostained focally. Two/14 (14%) epithelial type and 1/9 (11%) biphasic type mesotheliomas immunostained weakly; none of 4 spindle cell mesotheliomas immuno stained.We conclude that while at least occasional cases of most types of pulmonary epithelial neoplasms express p21ras, the frequency and intensity of the expression are distinctly greater in certain tumor types such as squamous, bronchioloalveolar, and neuroendocrine neoplasm except for the small cell type. Contrary to these lung epithelial neoplasms, most mesotheliomas did not immunostain for p21ras. Whether the enhanced p21ras expression may point to a different mechanism of transformation or may merely reflect differentiation features remains undetermined.