Raymond T. Jones

Cellular change in human disease: A new method of pathological analysis*

Abstract The concept of evaluation of cellular alterations as a method for studying the pathogenesis and the manifestations of human disease is presented. Such study is based on evaluation by chemical and morphologic methods performed with human tissues obtained at immediate autopsy and is discussed in the context of cellular reaction to injury. The techniques discussed are readily available and comparable in resources required and difficulty of execution to many other specialized laboratory procedures presently in use. Now is an appropriate time to apply established cell biological principles to the study of human disease.

Bleomycin-induced chromosome breaks as a risk marker for lung cancer: a case-control study with population and hospital controls

Environmental exposure to carcinogens and individual susceptibility play significant roles in cancer risk. Suboptimal DNA repair capability, measured by quantifying mutagen-induced chromosome breaks, might explain variable host susceptibility to environmental carcinogens. In an ongoing lung cancer case-control study, we compared individual sensitivity to bleomycin-induced chromosome breaks in 152 non-small cell lung cancer patients with 94 population controls and 85 hospital controls with no history of cancer. Mutagen sensitivity was measured by mean number of chromatid breaks per cell in cultured peripheral blood lymphocytes treated with bleomycin. Non-parametric tests and chi(2) tests were used to determine the statistical significance of the crude case-control comparisons, followed by logistic regression to adjust for important covariates. The mean number of bleomycin-induced breaks per cell was 1.01 for the cases compared with 0.86 for hospital controls (P < 0.01) and 0.89 for population controls (P < 0.01). The mean number of breaks per cell was 1.01 for those >65 years old and 0.81 for those < or = 65 years old (P < 0.01) among population controls. Defining bleomycin sensitive as >0.84 break/cell (the median level in population controls), 67% of the cases were bleomycin sensitive compared with 49% of the hospital controls [adjusted odds ratio (OR) = 2.69, 95% confidence interval (CI) = 1.44, 5.04], and 51% of the population controls (adjusted OR = 2.18, 95% CI = 1.13, 4.21). Our data indicate that the increased number of bleomycin-induced chromosome breaks was significantly associated with an increased risk of lung cancer in the first 331 subjects.

Aging changes in the human aortic valve in relation to dystrophic calcification

To elucidate the pathogenesis of aging changes and their relation to age associated calcification, a morphological study of 27 human aortic valves was carried out. Nine valves were obtained from immediate autopsies and 18 valves from routine autopsies done within four hours after death. Calcium deposition was present deep in the zona fibrosa along a zone of lipid accumulation. Fibrocytes in the zona fibrosa showed predominant age associated changes, i.e., a massive accumulation of residual bodies in the cytoplasm probably derived from autophagic vacuoles. Light microscopic lipid accumulation corresponded with both intracellular accumulation of electron dense spherules and membranous vesicles derived from degenerate fibrocytes. Calcium deposition in various stages, including needle shaped hydroxyapatite crystals, was seen in close association with these cellular degradation products rather than collagen or elastic fibers. Dystrophic calcification in the aortic valve appears to result from cellular aging and death followed by petrification of cellular degradation products, which may progress to calcific aortic stenosis.

The application of electron microscopy and cellular biochemistry to the autopsy: Observations on cellular changes in human shock*

A method based on the utilization of electron microscopy, morphometric analysis, tissue culture, and biochemical analysis in the study of human autopsies is described. In this method rapid sampling immediately following somatic death is conducted in order to make meaningful the application of such techniques. In addition to describing the procedure, we present some new findings relating to cellular changes associated with shock. As in cellular pathobiology, it is of utmost importance that ultrastructural changes be correlated with alterations in chemistry and function.

Less Efficient G2-M Checkpoint Is Associated with an Increased Risk of Lung Cancer in African Americans

Cell cycle checkpoints play critical roles in the maintenance of genomic integrity. The inactivation of checkpoint genes by genetic and epigenetic mechanisms is frequent in all cancer types, as a less-efficient cell cycle control can lead to genetic instability and tumorigenesis. In an on-going case-control study consisting of 216 patients with non-small cell lung cancer, 226 population-based controls, and 114 hospital-based controls, we investigated the relationship of gamma-radiation-induced G2-M arrest and lung cancer risk. Peripheral blood lymphocytes were cultured for 90 hours, exposed to 1.0 Gy gamma-radiation, and harvested at 3 hours after gamma-radiation treatment. gamma-Radiation-induced G2-M arrest was measured as the percentage of mitotic cells in untreated cultures minus the percentage of mitotic cells in gamma-radiation-treated cultures from the same subject. The mean percentage of gamma-radiation-induced G2-M arrest was significantly lower in cases than in population controls (1.18 versus 1.44, P < 0.01) and hospital controls (1.18 versus 1.40, P = 0.01). When dichotomized at the 50th percentile value in combined controls (population and hospital controls), a lower level of gamma-radiation-induced G2-M arrest was associated with an increased risk of lung cancer among African Americans after adjusting for baseline mitotic index, age, gender, and pack-years of smoking [adjusted odd ratio (OR), 2.25; 95% confidence interval (95% CI), 0.97-5.20]. A significant trend of an increased risk of lung cancer with a decreased level of G2-M arrest was observed (P(trend) = 0.02) among African Americans, with a lowest-versus-highest quartile adjusted OR of 3.74 (95% CI, 0.98-14.3). This trend was most apparent among African American females (P(trend) < 0.01), with a lowest-versus-highest quartile adjusted OR of 11.75 (95% CI, 1.47-94.04). The results suggest that a less-efficient DNA damage-induced G2-M checkpoint is associated with an increased risk of lung cancer among African Americans. Interestingly, we observed a stronger association of DNA damage-induced G2-M arrest and lung cancer among African Americans when compared with Caucasians. If replicated, these results may provide clues to the exceedingly high lung cancer incidence experienced by African Americans.

P53 gene protein overexpression predicts results of trimodality therapy in esophageal cancer patients.

BACKGROUND P53 protein overexpression in esophageal cancer and its correlation with response and survival after chemoradiation was retrospectively investigated. METHODS Pretreatment and resection specimens were stained by automatic p53 immunohistochemical staining technique. RESULTS P53 was expressed in 84.0% of esophagoscopy (EGD) biopsies; 71.4% of patients with metastasis of thoracoscopy/laparoscopy lymph nodes (TS/LS LN) identified by hematoxylin/eosin (H/E) were p53 (+); 14.2% of patients with negative TS/LS LN by H/E were p53 (+). Eleven out of 18 patients with p53 (+) in pretreatment EGD remained p53 (+) after chemoradiation; 38.8% of these patients had a pathological complete response (pCR). The median survival of this group was 15 months. Of 4 patients with p53 (-) pretreatment EGD, all of those were still p53 (-) after chemoradiation; 75% of these patients had pCR. The median survival was 30 months. In patients with p53 (+) TS/LS LN, 23% had a pCR after chemoradiation with a median survival of 16 months. In patients with p53 (-) TS/LS LN, 50.0% had a pCR with a median survival of 31.5 months. CONCLUSIONS P53 protein overexpression in pretreatment EGD and TS/LS LN may predict response to chemoradiation and survival in esophageal cancer patients.

Isolation and characterization of epithelial cells from bovine pancreatic duct

SummaryEpithelial cells derived from bovine pancreatic duct have been grown continuously in culture for 30 weeks (approximately 90 doublings of the cell population). The cells were grown in Eagles minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 0.1 mM nonessential amino acids, and antibiotics. In confluent cultures, the cells are multilayered and form circular structures. When tested at various passages, the cells neither formed colonies in soft agar nor produced tumors after inoculation into athymic, nude mice. Hydrocortisone (1 and 5 μg per ml) and insulin (1,5 and 10 μg per ml) had no effect on the growth of the cells. β-Retinyl acetate inhibited growth rate and cell yield at a concentration of 5 μg per ml but was not growth-inhibitory at lower concentrations. By electron microscopy the cells have numerous mitochondria, Golgi and microvilli. Mucous droplets were observed in a small proportion of the cells. Desmosome-like structures and occluding junctions were observed more frequently between cells that had been transferred as aggregates than between cells transferred as single cells. Cytochemical studies indicated that some cells produce PAS positive granules that were not removed after treatment of the cultures with diastase. Eleven cell clones were isolated from the mass culture. The growth rates of the clones are different as well as the period of time in which the clones can be propagated in vitro.

Reproductive and hormonal factors and the risk of nonsmall cell lung cancer.

Although exposure to estrogen may directly influence or modify the association between cigarette smoking and lung cancer risk, results from epidemiologic studies examining the association between reproductive and hormonal factors and risk of lung cancer among women have been inconsistent. Between 1998 and 2008, 430 women diagnosed with nonsmall cell lung cancer, 316 hospital controls and 295 population controls were recruited into the multi‐center Maryland Lung Cancer Study. Conditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) according to reproductive and hormonal exposures adjusting for age, smoking, passive smoking, education and household income. Results were similar for hospital and population based controls, so the control groups were combined. Reduced risks of lung cancer were observed among women with greater parity (≥5 vs. 1–2 births: OR = 0.50, 95% CI = 0.32, 0.78, p‐trend = 0.002) and later ages at last birth (≥30 vs. <25 years old: OR = 0.68, 95% CI = 0.48, 0.98, p‐trend = 0.04). After mutual adjustment parity, but not age at last birth, remained significantly inversely associated with risk (p‐trend = 0.01). No associations were found for nonsmall cell lung cancer risk with age at menarche, age at first birth, menopausal status, oral contraceptive use or menopausal hormone use, including use of oral estrogens. Compatible with findings from recent epidemiologic studies, we observed a reduction in the risk of nonsmall cell lung cancer with increasing number of births. Other reproductive and hormonal exposures, including menopausal hormone therapy use, were not associated with risk.

Organ explant culture of adult Syrian golden hamster pancreas

SummaryAn organ explant culture system has been developed for long term maintenance of adult pancreatic tissue from the Syrian golden hamster. Gastric and duodenal lobe explants of up to 0.5 cm2 size were placed in tissue culture dishes (60 mm2) on Gelfoam sponge rafts to which was added 5 ml of CMRL medium 1066 supplemented with heat inactivated newborn bovine serum,l-glutamine hydrocortisone, insulin, and antibiotics. Dishes were placed in a controlled atmosphere chamber, which was gassed with 45% O2 50% N2, and 5% CO2 and incubated at 36.5°C. Viability of the tissues was determined by light and electron microscopy as well as by [3]thymidine incorporation. Explants were viable for up to 70 d. Zymogen granule-containing cells characteristic of acinar cells and mucuscontaining cells characteristic of ductal cells were present throughout this period. However, endocrine cells were only present for the 1st wk in culture.

A review of in vitro and in vivo culture techniques for the study of pancreatic carcinogenesis.

Techniques have been developed for long‐term organ explant culture of bovine, hamster, and human pancreatic ducts in enriched medium. The explants were then exposed to chemical carcinogens and studied by morphologic and biochemical techniques. Untreated explants have also been successfully xenotransplanted into athymic “nude” mice. Techniques have also been developed to isolate human and bovine pancreatic ductal cells for cell culture. These as well as techniques developed by other authors are discussed.