Immunohistochemical detection of EGFR, p185(erbB-2), Bcl-2 and p53 in breast carcinomas in pre-menopausal and post-menopausal women.

Young pre-menopausal women with breast carcinomas have an overall worse prognosis than older, post-menopausal women. Because histologic grade is a major predictor of tumor behavior and correlates with biomarker expression, we assessed the immunohistochemical expression of epidermal growth factor receptor (EGFR), p185(erbB-2) and Bcl-2, and nuclear accumulation of p53 in breast carcinomas in pre- and post-menopausal women with equivalent histologic grades. This allowed identification of differences in biomarker expression and prognostic significance in pre- and post-menopausal women independent of histologic grade. We investigated 100 infiltrating ductal carcinomas (IDC) in pre-menopausal women, ages 45 years and younger, and 100 IDC in post-menopausal women, ages 65 years and older. The IDC were selected so that the proportions of high and low/moderate grade carcinomas were equal in the pre- and post-menopausal groups. Prognostic utility of biomarker expression in pre- and post-menopausal groups was determined by product limit and multivariate analysis of survival. There were statistically significant differences in cytoplasmic expression of EGFR and Bcl-2 and nuclear accumulation of p53, but not in the expression of p185(erbB-2), in carcinomas of high vs. low histologic grade. There was no difference in the expression of EGFR, p185(erbB-2) or Bcl-2, or in nuclear accumulation of p53 in these IDC from pre- vs. post-menopausal women. Bcl-2 and the nuclear accumulation of p53 were of prognostic significance in our overall study population; however, when assessing pre- and post-menopausal women separately, Bcl-2 and p53 were of prognostic significance only in pre-menopausal, but not post-menopausal women. In summary expression of EGFR and Bcl-2 and nuclear accumulation of p53 were significantly associated with histologic grade, but not with menopausal status. In addition, there were differences in the prognostic effectiveness of these biomarkers in pre- vs. post-menopausal women.

The efficient operation of the surgical pathology gross room.

The gross room is the area where pathology specimens from operating rooms are transferred for pathology review and analysis, serving as the bridge between the treating physician and diagnostic surgical pathologist. Reaching the correct diagnosis for a specimen depends on the proper handling and processing of tissue transferred to this very busy area. We review here the basic function and management of the gross room including a brief discussion of common specimen types, biohazard exposure and safety, and collection of tissue for research.

Acute granulomatous lupus pneumonitis: the first case report.

Acute lupus pneumonitis is a rare form of pulmonary involvement in systemic lupus erythematosus (SLE). We present herein a patient with acute lupus pneumonitis who presented with acute onset of fever, cough, dyspnea and a miliary pattern on chest radiographs and computer tomography. Lung histopathology revealed bronchocentric granulomatosis. To our knowledge, this is the first documented case of granulomas in lung parenchyma believed to be caused by SLE. The differential diagnoses of acute lupus pneumonitis and the pertinent literature are discussed.

Molecular pathology of chondroid neoplasms: part 1, benign lesions.

This two-part review presents an overview of the molecular findings associated with both benign and malignant chondroid neoplasms. This first part presents a brief review of methods in molecular pathology along with a review of the cytogenetic and molecular genetic findings in benign chondroid neoplasms. Clinical aspects of the various lesions are briefly discussed, and each tumor is illustrated with representative radiographic and pathologic images. Malignant chondroid neoplasms will be considered in the second part of this review.

Molecular pathology of chondroid neoplasms: part 2, malignant lesions.

This is the second part of a two-part review presenting an overview of the molecular findings associated with both benign and malignant chondroid neoplasms. The first part presented a brief review of modern methods in molecular pathology, along with a review of the cytogenetic and molecular genetic findings in benign chondroid neoplasms. This second part reviews the cytogenetic and molecular genetic findings in malignant chondroid neoplasms. Clinical aspects of the various lesions are briefly discussed, and each tumor is illustrated with representative radiographic and pathologic images.

Race- and age-dependent alterations in global methylation of DNA in squamous cell carcinoma of the lung (United States).

OBJECTIVE: The current study investigated the race- and age-dependent alterations in global DNA methylation on the development and progression of squamous cell carcinomas (SCCs) of the lung. METHODS: Methylation status was evaluated in SCC and in the associated uninvolved bronchial mucosa (UBM) and epithelial hyperplasia (EH) of 53 Whites and 23 African Americans by using an antibody specific for 5-methylcytosine (5-mc). A low 5-mc score indicates global hypomethylation of DNA. RESULTS: 5-mc scores of SCC were significantly lower compared to 5-mc scores of UBM and EH in Whites (p < 0.05). In African Americans, 5-mc scores of SCCs were not significantly different from 5-mc scores of UBM and EH, suggesting an involvement of methylation in the development of SCCs in Whites, but not in African Americans. 5-mc scores were lower in younger subjects compared to older subjects in Whites. Since cancers in younger subjects tend to be more aggressive than cancers in older subjects, these observations may suggest that hypomethylation may have contributed to aggressiveness cancers of younger Whites. Hypomethylation of SCCs in White men was associated with shorter survival from the disease. CONCLUSIONS: These preliminary results suggest that the methylation status of DNA may affect the development, aggressiveness, and prognosis of SCCs in Whites.

The response of extracellular signal-regulated kinase (ERK) to androgen-induced proliferation in the androgen-sensitive prostate cancer cell line, LNCaP.

The mechanisms by which androgens stimulate proliferation of prostate cancer cells are poorly understood. It has been proposed that androgen stimulation may induce the mitogen-activated protein (MAP) kinase system in prostate cancer cells and lead to cellular proliferation. We attempted to evaluate the role of the extracellular signal-regulated kinase (ERK) pathway in the stimulation by androgens of prostate cancer cell proliferation. Androgen-sensitive prostate cancer cell line (LNCaP) cells plated on sterile glass coverslips were treated with 10(-8) M dihydrotestosterone (DHT) or epidermal growth factor (EGF) (10 ng/ml) for periods ranging from 1 min to 96 h. The proliferative index of the cells, evaluated by immunoperoxidase staining of cells with an antibody to Ki-67, was increased at least two-fold at all time points from 5 min to 48 h following exposure to either DHT or EGF. Immunohistochemical evaluation of ERK1/2 and pERK (activated ERK) demonstrated high levels of ERK1/2 in untreated LNCaP cells, while pERK was expressed at much lower levels. Following treatment with DHT, no change in staining intensity for either ERK1/2 or pERK was observed, while treatment with EGF resulted in no change in ERK1/2, but significantly increased cytoplasmic staining for pERK at all time points beyond 2 min. These results were confirmed by Western blot analysis of ERK1/2 and pERK expression in these cell lines following treatment with DHT or EGF. Our findings suggest that the proliferative response of prostate cancer cells to androgens, unlike the proliferative response to EGF, is not mediated by the activation of ERK1/2, and that currently undefined pathways other than those involving ERK1/2 are involved.

Altered global methylation of DNA: an epigenetic difference in susceptibility for lung cancer is associated with its progression.

Alterations in global DNA methylation have been observed in many cancers, but whether such alterations represent an epigenetic difference in susceptibility for the disease is unknown. The status of global DNA methylation also has not been reported in intact or specific types of cells involved in the carcinogenic process. To address these issues in lung carcinogenesis, we evaluated the status of global DNA methylation by using a monoclonal antibody specific for 5-methylcytosine (5-mc) in randomly selected lung specimens of 60 cigarette smokers who developed squamous cell carcinoma (SCC) and 30 cigarette smokers who did not. 5-mc immunostaining scores of DNA of SCC (0.61 +/- 0.42) and associated hyperplastic lesions (0.82 +/- 0.27) was significantly lower than those of DNA of histologically normal bronchial epithelial cells (0.99 +/- 0.52) and hyperplastic lesions (1.2 +/- 0.22) of noncancer specimens. The ratio of 5-mc scores between SCC and matched uninvolved bronchial epithelial cells was significantly associated with advanced stage and size of the tumor. The results suggest that alteration in global DNA methylation is an important epigenetic difference in susceptibility for the development of lung cancer. The reduced global DNA methylation in SCC compared with epithelial hyperplasia and its association with tumor size and disease stage is suggestive of its involvement in the progression of SCC. The results also indicate that normal methylation of DNA in epithelial hyperplastic lesions may prevent the transformation of these lesions to invasive cancer. If these results are confirmed, the status of DNA methylation in early lesions such as epithelial hyperplasia could be used to identify smokers who are at risk for the development of SCC.

The expression of fatty acid synthase (FASE) is an early event in the development and progression of squamous cell carcinoma of the lung.

Correlation of elevated levels of the lipogenic enzyme, fatty acid synthase (FASE), with advanced stages of some cancers has drawn attention to this enzyme as a possible marker of poor prognosis. Because recent studies have shown that cancer cells are dependent on fatty acid synthetic activity and pharmacologic inhibitors of this enzyme are selectively cytotoxic to cancer cells, expression of FASE also may provide a potential target for intervention in the neoplastic process. To determine the potential usefulness of expression of FASE in the neoplastic process of the lung, we evaluated its pattern of expression immunohistochemically in archival specimens from 60 human lung specimens with squamous cell cancer (SCC) and associated "preneoplastic" lesions compared with its expression in the normal bronchial epithelium of 60 noncancer specimens. The expression of FASE was significantly higher in SCC associated uninvolved bronchial epithelium (mean = 0.40+/-0.03, median = 0.38) compared with its expression in the bronchial epithelium of noncancer specimens (mean = 0.18+/-0.02, median = 0.16) indicating its early expression. We also observed a statistically significant step-wise increase in FASE expression from SCC associated uninvolved bronchial epithelium (mean = 0.40+/-0.03, median = 0.38) to epithelial hyperplasia (0.58+/-0.04, median = 0.57) to SCC (1.53+/-0.06, median = 1.50). The results suggested that expression of FASE is an early event in the development and progression of SCC of the lung. The inhibition of fatty acid synthesis by inhibiting enzymatic function with metabolic analogues may be a useful strategy in the treatment of SCCs. The expression of FASE in early lesions such as SCC associated uninvolved bronchial epithelium and epithelial hyperplasia might also provide a potential means for intervention early in the neoplastic process in the lung or even preventing their malignant transformation to invasive carcinomas.

The accumulation of ascorbic acid by squamous cell carcinomas of the lung and larynx is associated with global methylation of DNA.

BACKGROUND: Cigarette smokers are known to have lower concentrations of circulating ascorbic acid than nonsmokers. In contrast, there is evidence that the extracellular fluid lining of the alveolus, which comes in close contact with cigarette smoke, and the alveolar macrophages of smokers are enriched with ascorbic acid. The clinical significance of these observations is unknown. METHODS: The authors measured the ascorbic acid concentrations and radiolabeled methyl incorporation (which is inversely related to the degree of DNA methylation in vivo) of paired samples of squamous cell carcinoma (SCC) and adjacent uninvolved mucosa of the lung and larynx (n = 22). RESULTS: Cancerous tissues had significantly higher ascorbic acid concentrations (mean +/- standard deviation [SD, 485 +/- 77; median, 483 ng/mg protein) compared with their matched uninvolved tissues (mean +/- SD, 151 +/- 52; median, 72 ng/mg protein; P < 0.0001). The radiolabeled methyl incorporation was significantly higher in cancerous tissues (mean +/- SD, 31,419 +/- 2629; median, 31,416 counts per minute [CPM]/microg DNA) compared with their matched uninvolved tissues (mean +/- SD, 11,883 +/- 1567; median, 11,444 CPM/microg DNA; P < 0.0001). The Spearman correlation between ascorbic acid concentrations and radiolabeled methyl incorporation by DNA in SCCs was inverse and statistically significant (r = -0.58, P = 0.008), indicating a beneficial effect of accumulated ascorbic acid in global methylation of DNA. In the uninvolved tissues, this correlation was inverse but statistically not significant (r = -0.20, P =0.35). CONCLUSIONS: Cancerous tissues of the lung and larynx demonstrated their ability to accumulate ascorbic acid. The accumulation of ascorbic acid by these tissues seemed to facilitate global methylation of DNA.

American Academy of Pediatrics. Committee on Child Abuse and Neglect and Committee on Community Health Services. Investigation and review of unexpected infant and child deaths.

Although there is a continuing need for timely review of child deaths, no uniform system exists for investigation in the United States. Investigation of a death that is traumatic, unexpected, obscure, suspicious, or otherwise unexplained in a child younger than 18 years requires a scene investigation and an autopsy. Review of these deaths requires the participation of pediatricians and other professionals, usually as a child death review team. An appropriately constituted team should evaluate the death investigation process, review difficult cases, and compile child death statistics.

Extraordinary conservation of cysteines among homologous Chironomus silk proteins sp185 and sp220.

Aquatic larvae of the midge, Chironomus tentans, synthesize a 185-kDa silk protein (sp185) with the cysteine-containing motif Cys-X-Cys-X-Cys (where X is any residue) every 20-28 residues. We report here the cloning and full-length sequence of cDNAs encoding homologous silk proteins from Chironomus pallidivittatus (sp185) and Chironomus thummi (sp220). Deduced amino acid sequences reveal proteins of nearly identical mass composed of 72 blocks of 20-28 residues, 61% of which can be described by the motif X5-8-Cys-X5-(Trp/Phe/Tyr)-X4-Cys-X-Cys-X-Cys. Spatial arrangement of these residues is preserved more than surrounding sequences. cDNA clones enabled us to map the genes on polytene chromosomes and identify for the first time the homolog of the Camptochironomus Balbiani ring 3 locus in Chironomus thummi. The apparent molecular weight difference between these proteins (185 vs 220 kDa) is not attributable to primary structure and may be due to differential N-linked glycosylation. DNA distances and codon substitutions indicate that the C. tentans and C. pallidivittatus genes are more related to each other than either is to C. thummi; however, substitution rates for the 5'- and 3'-halves of these genes are different. Blockwise sequence comparisons suggest intragenic variation in that some regions evolved slower or faster than the mean and may have been subjected to different selective pressures.

Florida Department of Corrections' substance abuse programs.

The Florida Department of Corrections has worked diligently to plan and implement a system of comprehensive institutional and community-based programs. These programs strive to establish a functional, cost-effective continuum of care for incarcerated individuals while providing necessary linkages essential to transferring inmates back into society with the knowledge and social skills necessary to lead a drug-free life. It is believed that a viable working model has been developed that will offer inmate services and, ultimately, afford them the opportunity and appropriate linkages to continue treatment as needed after incarceration.

2-acetamido-2-deoxy-alpha-D-galactosidases of Clostridium perfringens. Separation and characterization of an exoglycosidase and an oligosaccharidase.

Two distinct 2-acetamido-2-deoxy-alpha-D-galactosidases have been separated from filtrates of cultured Clostridium perfringens by electrophoresis in 6.5% poly(acrylamide) gels. One of the enzymes had a mobility of 0.32--0.36 (relative to Bromophenol Blue) and was identified as the exoglycosidase, 2-acetamido-2-deoxy-alpha-D-galactosidase. It appears to be the same enzyme as that reported in 1972 by McGuire et al. The second of the two enzymes, having a relative mobility of 0.42--0.46, corresponds to the oligosaccharidase reported in 1972 by Huang and Aminoff. The A-specificities of human type-A erythrocytes and of water-soluble glycoproteins having A-activity are both destroyed by incubation with the 2-acetamido-2-deoxy-alpha-D-galactosidase, but not on incubation with the oligosaccharidase. A concomitant rise in blood-group O(H) activity, as indicated by the use of a lectin from Ulex europeus, occurred in the A-erythrocytes treated with the exoglycosidase 2-acetamido-2-deoxy-alpha-D-galactosidase.

Cell surface carbohydrate recognition and the viability of erythrocytes in circulation.

The presence of sialic acid on the cell surface is crucial for the survival of mammalian erythrocytes in circulation. In contrast, sialidase-treated chicken erythrocytes retain their viability in circulation. Galactose oxidase treatment of chicken red blood cells has no effect on their viability. However, after sialidase treatment, galactose oxidase treatment results in the rapid elimination of the chicken erythrocytes from circulation. This is compatible with the interpretation that consecutive treatment with the 2 enzymes abolishes the ability of the chicken erythrocytes to regenerate the sialic acid on the cell surface. Mammalian asialo-erythrocytes are sequestered in the liver and spleen. We have shown that at the cellular level there is a preferential recognition of sialidase-treated as compared to normal erythrocytes by mononuclear spleen cells and Kupffer cells of the liver. This recognition manifests itself in both autologous and homologous systems by rosette-like adhesions. These adhesions may represent the normal physiological mechanism for the removal of senescent erythrocytes from circulation by liver and spleen since it has been previously reported that older erythrocytes contain decreased amounts of sialic acid. The mechanisms in mammals for the elimination from circulation of asialo-erythrocytes and asialo-glycoproteins, while analogous in many respects, are definitely not identical.

Effect of galactose oxidase, with and without prior sialidase treatment, on the viability of erythrocytes in circulation.

Previous studies have shown that sialidase-treated mammalian erythrocytes were rapidly eliminated from circulation. In contrast, chicken asialoerythrocytes remained fully viable. This investigation was undertaken to ascertain the reason for this difference in behavior as well as to determine the extent of the similarity of the physiological mechanism for the elimination from circulation of asialoglycoproteins and mammalian asialoerythrocytes. To that end, erythrocytes from dogs, rabbits, and chickens were each subjected to the action of galactose oxidase (D-galactose:oxygen 6-oxidoreductase; EC 1.1.3.9) both before and after sialidase (acylneuraminyl hydrolase; EC 3.2.1.18) treatment. The viability of the autologously transfused erythrocytes in circulation was monitored by Na2-51CrO4 labeling. Galactose oxidase had no deleterious effect on the viability of dog or chicken erythrocytes, nor did it restore the viability of dog or rabbit asialoerythrocytes. On the other hand, desialated chicken erythrocytes, which were fully viable, were rendered nonviable upon treatment with galactose oxidase. It may be concluded therefore that (a) the physiological mechanism of elimination of mammalian asialoerythrocytes from circulation is not the same as that for plasma asialoglycoproteins and (b) the treatment of chicken asialoerythrocytes with galactose oxidase results in the oxidation at carbons 6 of the galactosyl- or N-acetylgalactosaminyl residues, thereby rendering the erythrocytes nonviable.

Role of sialic acid in survival of erythrocytes in the circulation: interaction of neuraminidase-treated and untreated erythrocytes with spleen and liver at the cellular level.

Sialidase (neuraminidase; acylneuraminyl hydrolase; EC 3.2.1.18)-treated erythrocytes obtained from different species are susceptible to rapid elimination from the circulation and are sequestered in the liver and spleen. The present studies were concerned with the mechanism of this clearance and how it may relate to the normal physiological process of removing senescent erythrocytes from the circulation. The results obtained indicate a preferential recognition of sialidase-treated as compared to normal erythrocytes by mono-nuclear spleen cells and Kupffer cells of the liver. This recognition manifests itself in both autologous and homologous systems by adhesion of the complementary cells in the form of rosettes, and as such could explain the removal of enzyme-treated erythrocytes from the circulation with their accumulation in liver and spleen. This phenomenon may represent a normal physiological mechanism for removal of senescent erythrocytes containing decreased sialic acid.

Effect of sialidase on the viability of erythrocytes in circulation.

Sialic acid has been detected on the erythrocyte surface of a number of different species of animals. The objective of this investigation was to determine the physiological significance of these sialyl residues to the viability of erythrocytes in circulation. Methods have been described for the determination of total sialic acid on red blood cells and the conditions under which it may be released with sialidase. Chicken, dog, goat, and rabbit were chosen for these studies because of the differences in the amount (3 X 10(6) - 72 X 10(6) resides per erythrocyte), and type (N-acetyl-or N-glycolyl-neuraminic acids) of sialic acid found on the surface of their erythrocytes. Radioactive tagging with Na251CrO4 was used to monitor the effect of sialidase on the viability of erythrocytes upon autologous transfusion. By the two criteria used to assess the viability of erythrocytes-the percentage of erythrocytes surviving 24 hr after the autologous transfusion, and the half-life of those red blood cells in circulation that survive the first 24 h after the autologous transfusion, and the half-life of those red blood cells in circulation that survive the first 24 hr-it is apparent that the presence of sialic acid on the cell surface is crucial for the survival of nonnucleated mammalian erythrocytes. The loss of viability of dog erythrocytes can be elicited by the removal of approximately 10% of the total sialic acid. In marked contrast to the behavior of mammalian erythrocytes, sialidase-treated chicken erythrocytes appear to retain their viability in circulation.