Extrapulmonary tissue responses in cynomolgus macaques (Macaca fascicularis) infected with highly pathogenic avian influenza A (H5N1) virus.

The mechanisms responsible for virulence of influenza viruses in humans remain poorly understood. A prevailing hypothesis is that the highly pathogenic virus isolates cause a severe cytokinemia precipitating acute respiratory distress syndrome and multiple organ dysfunction syndrome. Cynomolgus macaques (Macaca fascicularis) infected with a human highly pathogenic avian influenza (HPAI) H5N1 virus isolate (A/Vietnam/1203/2004) or reassortants of human influenza virus A/Texas/36/91 (H1N1) containing genes from the 1918 pandemic influenza A (H1N1) virus developed severe pneumonia within 24 h postinfection. However, virus spread beyond the lungs was only detected in the H5N1 group, and signs of extrapulmonary tissue reactions, including microglia activation and sustained up-regulation of inflammatory markers, most notably hypoxia inducible factor-1alpha (HIF-1alpha), were largely limited to this group. Extrapulmonary pathology may thus contribute to the morbidities induced by H5N1 viruses.

Formaldehyde gas inactivation of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials.

AIMS: To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using formaldehyde gas. METHODS AND RESULTS: B. anthracis, B. subtilis, and G. stearothermophilus spores were dried on seven types of indoor surfaces and exposed to approx. 1100 ppm formaldehyde gas for 10 h. Formaldehyde exposure significantly decreased viable B. anthracis, B. subtilis, and G. stearothermophilus spores on all test materials. Significant differences were observed when comparing the reduction in viable spores of B. anthracis with B. subtilis (galvanized metal and painted wallboard paper) and G. stearothermophilus (industrial carpet and painted wallboard paper). Formaldehyde gas inactivated>or=50% of the biological indicators and spore strips (approx. 1x10(6) CFU) when analyzed after 1 and 7 days. CONCLUSIONS: Formaldehyde gas significantly reduced the number of viable spores on both porous and nonporous materials in which the two surrogates exhibited similar log reductions to that of B. anthracis on most test materials. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide new comparative information for the decontamination of B. anthracis spores with surrogates on indoor surfaces using formaldehyde gas.

Decontamination assessment of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surfaces using a hydrogen peroxide gas generator.

AIMS: To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using hydrogen peroxide gas. METHODS AND RESULTS: Bacillus anthracis, B. subtilis, and G. stearothermophilus spores were dried on seven types of indoor surfaces and exposed to > or =1000 ppm hydrogen peroxide gas for 20 min. Hydrogen peroxide exposure significantly decreased viable B. anthracis, B. subtilis, and G. stearothermophilus spores on all test materials except G. stearothermophilus on industrial carpet. Significant differences were observed when comparing the reduction in viable spores of B. anthracis with both surrogates. The effectiveness of gaseous hydrogen peroxide on the growth of biological indicators and spore strips was evaluated in parallel as a qualitative assessment of decontamination. At 1 and 7 days postexposure, decontaminated biological indicators and spore strips exhibited no growth, while the nondecontaminated samples displayed growth. CONCLUSIONS: Significant differences in decontamination efficacy of hydrogen peroxide gas on porous and nonporous surfaces were observed when comparing the mean log reduction in B. anthracis spores with B. subtilis and G. stearothermophilus spores. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide comparative information for the decontamination of B. anthracis spores with surrogates on indoor surfaces using hydrogen peroxide gas.

Chromosome 3p tumor-suppressor gene alterations in cervical carcinomas.

Loss of heterozygosity (LOH) on chromosome 3p is a common event in cervical cancer and typically occurs in a dispersed pattern involving several loci. This implies that more than one resident tumor-suppressor gene is involved in the genesis of these tumors; however, specific targets remain to be identified. The region of 3p14.2-pter encompasses a region of frequent loss and contains at least three tumor-suppressor genes: fragile histidine triad (FHIT), transforming growth factor-beta receptor II (T beta R-II), and Von Hippel-Lindau. To identify those loci within 3p14.2-pter that are important in cervical cancer, invasive tumors were first subjected to high-density LOH analysis. With 25 microsatellite markers, LOH was detected in seven of 15 cervical carcinomas (47%). Losses always included markers mapping to 3p22, and markers at this location were exclusively lost in two tumors, implicating this as a site of a cervical tumor-suppressor gene. Because it is a known tumor-suppressor gene located at 3p22 and thus a potential target for inactivation in these tumors, the T beta R-II gene was subsequently screened for mutation and altered expression levels. Whereas no tumor-derived mutations were detected in any of the tumors, six of ten tumors showed T beta R-II transcript levels reduced by > or = 50% when compared with normal cervical epithelium. Nine of 15 (60%) tumors exhibited LOH at 3p22 or reduced expression of T beta R-II, suggesting that reduced T beta R-II levels contribute to cervical tumorigenesis. Two cases exhibited silent germline polymorphisms of T beta R-II: one corresponding to a C1167T transversion and the other to an A1266G transition. The FHIT gene, which is located at 3p14.2, also frequently incurred LOH and abnormal transcription in these tumors. LOH of FHIT was observed in five of the 15 tumors analyzed. Neither mutations nor homozygous deletions of FHIT were detected in the tumors. However, aberrantly short transcripts of the FHIT gene were evident in six of nine (67%) tumors. Only one of these also displayed LOH, indicating that this gene was altered in at least 10 of 15 (67%) tumors. These results provide evidence that the inactivation of two known tumor-suppressor genes, TbetaR-II and FHIT, on chromosome 3p is involved in cervical carcinogenesis. Mol. Carcinog. 30:159--168, 2001.

Alterations in inflammatory cytokine gene expression in sulfur mustard-exposed mouse skin.

Cutaneous exposure to sulfur mustard (bis(2-chloroethyl) sulfide, HD), a chemical warfare agent, produces a delayed inflammatory skin response and severe tissue injury. Despite defined roles of inflammatory cytokines produced or released in response to skin-damaging chemicals, in vivo cytokine responses associated with HD-induced skin pathogenesis are not well understood. Additionally, there is little information on the in vivo temporal sequence of gene expression of cytokines postexposure to HD. The goal of these studies was to identify in vivo molecular biomarkers of HD skin injury within 24 hours after HD challenge. Gene expression of interleukin 1beta (IL-1beta), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 6 (IL-6), and interleukin 1alpha (IL-1alpha) in the mouse ear vesicant model was examined by quantitative reverse transcription-polymerase chain reaction (RT-PCR). An increase in IL-1beta mRNA levels was first observed at 3 hours. IL-1beta, GM-CSF, and IL-6 mRNA levels were dramatically increased at 6-24 hours postexposure. IL-1alpha mRNA levels were not increased following HD exposure. Immunohistochemical studies demonstrated that IL-1beta and IL-6 protein was produced at multiple sites within the ear, including epithelial cells, inflammatory cells, hair follicles, sebaceous glands, the dermal microvasculature, smooth muscle, and the dermal connective tissue. An increase in the intensity of staining for IL-1beta, and IL-6 was observed in localized areas at 6 hours and was evident in multiple areas at 24 hours. Positive staining for GM-CSF immunoreactive protein was localized to the inflammatory cells within the dermis. The number of immunostaining cells was increased as early as 1 hour following HD exposure. These studies document an early increase in the in vivo expression of inflammatory cytokines following cutaneous HD exposure. An understanding of the in vivo cytokine patterns following HD skin exposure may lead to defining the pathogenic mechanisms of HD injury and the development of pharmacological countermeasures.

Peroxisome proliferator-activated receptor gamma activation in human breast cancer.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor family of ligand-activated transcription factors. This study was designed to evaluate ligand activation of PPARgamma in human breast cancer cells. DNA binding by endogenous PPARgamma in gel shift assays and activation of PPARgamma by prostanoid and thiazolidinedione ligands in reporter gene assays differed between the cell lines. The PPARgamma ligands elicited an anti-proliferative effect in MTT proliferation assays. Our data point to a variable, cell-specific response to different gamma-ligands, which holds significance for further studies on the role of PPARgamma in mediating breast cancer growth and progression.

Expression of cytochrome P450 2A3 in rat esophagus: relevance to N-nitrosobenzylmethylamine.

N-nitrosobenzylmethylamine (NBzMA) must be metabolically activated to exert its carcinogenic potential and is a potent inducer of tumors in the rat esophagus. The activation is believed to occur in the esophagus. Although the pathways of NBzMA metabolism are well studied, the principal cytochrome P450 enzyme(s) (P450) responsible for catalyzing its activation is unknown. Several preliminary studies have suggested that this enzyme may belong to the P450 2A family. We report here that P450 2A3 expressed in a baculovirus system metabolizes NBzMA, predominantly by methylene hydroxylation. To determine whether or not P450 2A3 is present in the rat esophagus, the relative level of P450 2A3 mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The mRNA levels of P450 2A3 were compared with the levels of P450 2A1 and 2A2 mRNA in the esophagus, liver, lung and nasal mucosa. P450 2A3 mRNA was detected in rat nasal mucosa, lung and esophagus, but not in liver, whereas P450 2A1 and 2A2 mRNAs were detected only in the liver. To determine the relative expression of P450 2A3 in each tissue, quantitative RT-PCR with PCR-MIMICS used as internal standards was performed. The expression level in the nasal mucosa was by far the greatest. The expression in the lung and esophagus was 60- and 1600-fold less, respectively. Using antibodies to P450 2A4/5 and P450 2A10/11 a 50 kDa immunoreactive protein was detected in all three tissues by western blot analysis. This is consistent with the expression of P450 2A3 in these tissues. However, the amount of protein detected in the nasal mucosa was much greater than that in the esophagus or lung. The expression of P450 2A protein was similar in the lung and esophagus. The rate of coumarin 7-hydroxylation in cultured rat esophagus was very low. This is a reaction efficiently catalyzed by P450 2A5, 2A6 and 2A10. In summary, our results clearly demonstrate the presence of P450 2A3 protein and mRNA in the esophagus, but the amounts are low and may not be sufficient to account for NBzMA activation in this tissue.

Altered localization of E-cadherin and alpha-catenin in rat esophageal tumors.

An alteration in the localization of E-cadherin and its associated proteins has been observed in many epithelial neoplasms. No data exist, however, for the expression of these proteins in an animal model system for esophageal cancer or in cultured rat esophageal epithelial cell lines. The present study investigated the localization of E-cadherin and its associated protein, alpha-catenin, in rat esophageal epithelial cell lines of differing tumorigenic potential; in tumors induced after transplantation of these cell lines into syngeneic hosts; and, in esophageal tumors induced in rats by the carcinogen, N-nitrosomethylbenzylamine (NMBA). Immunofluorescent staining of the cultured cell lines revealed staining for both E-cadherin and alpha-catenin at cell-cell boundaries. Western blot analysis confirmed the membrane-bound localization of E-cadherin and alpha-catenin in the cells. However, tumors induced by these cell lines in syngeneic rats showed reduction in the expression of both E-cadherin and á-catenin in the plasma membrane of invasive epithelial cells. Immunohistochemical analysis of NMBA-induced esophageal neoplasms in rats revealed E-cadherin and alpha-catenin to be abnormally expressed in poorly differentiated tumors when compared to well differentiated tumors. These results suggest that the microenvironment may have an important role in regulating the expression of these adhesion molecules in rat esophageal epithelial cells, and that alteration in the cellular localization of E-cadherin and alpha-catenin may be indicative of tumor progression in NMBA-induced rat esophageal cancer.

Altered expression of cyclin D1 and cyclin-dependent kinase 4 in azoxymethane-induced mouse colon tumorigenesis.

Alterations in the expression of the cell cycle regulators, cyclin D1 and cyclin-dependent kinase 4 (Cdk4), have been implicated in malignancies of both humans and experimental animal models. We hypothesize that altered expression of cyclin D1 and Cdk4 may also be involved in mouse colon tumorigenesis induced by the chemical carcinogen, azoxymethane (AOM). In the present study, SWR/J mice were given AOM by i.p. injection at a dose of 10 mg/kg once a week for 8 weeks, and colonic tissue and tumors were isolated 18 weeks later. The expression and localization of cyclin D1 and Cdk4 were examined by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical analyses. Cyclin D1 and Cdk4 mRNA levels in tumor samples were increased 1.3-fold (P < 0.01) and 1.2-fold (P < 0.01), respectively, when compared with control mouse colon tissue. Control colon epithelium was uniformly negative for cyclin D1 immunoreactivity, whereas minimal Cdk4 nuclear staining was confined to the lower portion of the crypts within the control tissue. Both cyclin D1 and Cdk4 immunoreactive cells were markedly increased in preneoplastic lesions and in adenomas isolated from AOM-treated mice. Furthermore, some morphologically normal colon crypts from AOM-treated mice showed positive cyclin D1 immunoreactivity. These findings suggest that overexpression of cyclin D1 and Cdk4 occurs early in the AOM-induced mouse colon tumorigenesis and may contribute to tumor progression in this model.

Expression of cell cycle proteins in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced mouse lung tumors.

Cyclin D1 dysregulation and differential inactivation of p16INK4a and Rb have been observed in human lung cancer. In chemically induced mouse lung tumors, the p16INK4a gene is a target of inactivation, and Rb is reduced at the mRNA level (Northern blot) although similar at the protein level (Western blot) when compared to normal lung tissues. The expression of cyclin D1, cdk4, p16INK4a, and Rb protein was examined by immunohistochemistry in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mouse lung tumors. Immunohistochemical staining revealed exclusive nuclear staining of both cyclin D1 and cdk4 that was light to moderate in normal mouse lung tissues, but intense in lung adenomas and adenocarcinomas. Western blot analysis confirmed the increased expression of cyclin D1 and cdk4 in lung tumors compared to normal lung. Immunohistochemical analyses of lung tumors showed focal areas which lacked p16INK4a staining. Expression of p16INK4a, as determined by RT-PCR, was variable in lung tumors. Mutations in p16INK4a were not found by SSCP analysis. Immunohistochemical analyses of normal lung tissues showed intense staining for Rb protein in alveolar epithelial cells and in other lung cell types; however, in the lung tumors the staining intensity was reduced and the distribution was altered. Expression of Rb was detected in normal lung tissues but was barely detectable by Northern blot hybridization in lung tumors. Western blot analysis indicated the presence of both hypophosphorylated and hyperphosphorylated Rb protein in lung tumors and in normal lung tissues. These results suggest that alterations in the cell cycle proteins, cyclin D1, cdk4, p16INK4a, and Rb, may play a role in the acquisition of autonomous growth by adenomas. Furthermore, they demonstrate the importance of immunohistochemical studies to examine expression in tissues that contain multiple cell types, such as the lung, and in tumors that by nature are heterogeneous.

Alterations in the expression of alpha6beta4 integrin and p21/WAF1/Cip1 in N-nitrosomethylbenzylamine-induced rat esophageal tumorigenesis.

Integrin alpha6beta4 is altered in many neoplastic cells, but no data exist to show this happens in esophageal neoplasms. To examine the expression of this integrin in rat esophageal tumorigenesis induced by N-nitrosomethylbenzylamine (NMBA), (alpha6 and beta4 expression was evaluated in normal esophageal epithelium, in NMBA-induced preneoplastic lesions, and in papillomas by quantitative reverse transcription (RT)-polymerase chain reaction (PCR) and immunohistochemical analysis. Because the 34 subunit of this integrin has been found to cause cell-cycle arrest by the induction of p21/WAF1/Cip1, the expression of p21/WAF1/Cip1 was also analyzed by RT-PCR. Compared with the levels in normal epithelium, the alpha6A, alpha6B, and beta4 integrin levels in esophageal papillomas were 1.9-, 2.2-, and 2.1-fold lower, respectively. RT-PCR analysis showed no significant differences in integrin levels between preneoplastic and normal samples, and northern blot analysis of the beta4 integrin produced results in agreement with the RT-PCR results. The p21/WAF1/Cip1 level was decreased 1.6-fold in preneoplastic tissues and 3.1-fold in papilloma samples when compared with the mRNA levels in normal epithelium. Immunostaining showed that alpha6beta4 integrin was localized at the basolateral surface of the basal cells in normal esophageal epithelium. In preneoplastic lesions, however, the expression of this integrin was not polarized and was expressed in basal cells as well as in suprabasal cells. Beta4 expression was significantly reduced and alpha6A expression was decreased and delocalized in papillomas. These findings suggest that alteration in alpha6beta4 integrin and p21/WAF1/Cip1 expression may be an important biomarker for tumor progression in NMBA-induced rat esophageal tumorigenesis.

Mutated and wild-type p53 expression and HPV integration in proliferative verrucous leukoplakia and oral squamous cell carcinoma.

The frequencies of overexpression and mutation in the p53 tumor suppressor gene were examined in proliferative verrucous leukoplakia and oral squamous cell carcinoma with immunohistochemistry and single-strand conformation polymorphism analysis of DNA fragments amplified by polymerase chain reaction. Ten samples each of normal oral mucosa, proliferative verrucous leukoplakia, and squamous cell carcinoma were immunostained with antibodies against p53 protein; 8 of 10 cases of proliferative verrucous leukoplakia cases and 7 of 10 cases of oral squamous cell carcinoma were positive for p53 protein. Minimal staining was observed in normal oral tissues. The quantified labeling indexes demonstrated a range that corresponded to lesion progression. Single-strand conformation polymorphism analysis revealed p53 gene mutations within exons 5 to 8 in 40% (4 of 10) of the squamous cell carcinoma samples. Two of the 4 mutated squamous cell carcinoma samples lacked p53 expression. No p53 mutations were detected in proliferative verrucous leukoplakia tissues. Human papillomavirus 16 was identified in 2 of 7 p53 positive oral squamous cell carcinoma samples. Human papillomavirus 16 and 18 were identified in two of eight p53 positive proliferative verrucous leukoplakia samples. One p53 negative squamous cell carcinoma sample was positive for human papillomavirus 16 and had a mutation in exon 6 of the p53 gene. Human papillomavirus infection along with p53 expression plays a yet to be defined role in the pathogenesis of a limited number of cases of proliferative verrucous leukoplakia and squamous cell carcinoma. p53 immunohistochemistry, p53 gene mutations, and human papillomavirus infection prevalence do not provide a means to differentiate between leukoplakia and carcinoma and do not provide a predictive test for progression of leukoplakia to carcinoma.

Biodegradation of dimethylsilanediol in soils.

The biodegradation potential of [14C]dimethylsilanediol, the monomer unit of polydimethylsiloxane, in soils was investigated. Dimethylsilanediol was found to be biodegraded in all of the tested soils, as monitored by the production of 14CO2. When 2-propanol was added to the soil as a carbon source in addition to [14C]dimethylsilanediol, the production of 14CO2 increased. A method for the selection of primary substrates that support cometabolic degradation of a target compound was developed. By this method, the activity observed in the soils was successfully transferred to liquid culture. A fungus, Fusarium oxysporum Schlechtendahl, and a bacterium, an Arthrobacter species, were isolated from two different soils, and both microorganisms were able to cometabolize [14C]dimethylsilanediol to 14CO2 in liquid culture. In addition, the Arthrobacter sp. that was isolated grew on dimethylsulfone, and we believe that this is the first reported instance of a microorganism using dimethylsulfone as its primary carbon source. Previous evidence has shown that polydimethylsiloxane is hydrolyzed in soil to the monomer, dimethylsilanediol. Now, biodegradation of dimethylsilanediol in soil has been demonstrated.

Absence of p16/MTS1 gene mutations in human prostate cancer.

The tumor suppressor gene p16/MTS1, located on chromosome 9p21, is a cell cycle regulatory gene which is frequently altered in human cancers. The role of this gene in prostate cancer is unknown. To determine the frequency of deletions and point mutations of p16/MTS1 in human prostate cancer, we examined 18 cancer and matched benign and hyperplastic tissue specimens. Deletions of p16/MTS1 were detected by semi-quantitative multiplex polymerase chain reaction in which a portion of exon 2 of the p16/MTS1 gene and a control marker, the glyceraldehyde 3-phosphate dehydrogenase gene, were amplified simultaneously. 'Cold' single-stranded conformational polymorphism (SSCP) analysis was performed to examine exons 1 and 2 of the p16/MTS1 gene for point mutations. Our data indicate no evidence for intragenic homozygous deletion in the prostate tumors. One prostate tumor and matched benign tissue showed mobility shifts. Direct DNA sequencing of the SSCP positive samples showed a G --> A transition in codon 140 which would result in an amino acid change from alanine to threonine. Our results indicate that deletions and point mutations in the p16/MTS1 gene are rare and do not play a major role in human prostate carcinogenesis.

Overexpression of cyclin D1 and cyclin E in N-nitrosomethylbenzylamine-induced rat esophageal tumorigenesis.

Dysregulation of G1 cyclins has been implicated in several human malignancies. To further investigate the role of G1 cyclins in chemical carcinogenesis, the expression of cyclin D1 and cyclin E was analyzed by RT-PCR and immunohistochemical studies in N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis. Cyclin D1 mRNA levels were increased 2.8-fold in 25 week (P < 0.05) and 6.8-fold in 45 week (P < 0.01) papillomas induced by NMBA, when compared with normal rat esophageal epithelium. Cyclin E mRNA levels were increased 6.2-fold in 25 week (P < 0.01) and 6.9-fold in 45 week (P < 0.01) papillomas. Immunohistochemical staining revealed exclusive nuclear staining of both cyclin D1 and cyclin E. Furthermore, there was a sequential increase in cyclin D1- and cyclin E-positive cells from normal epithelium, to preneoplastic lesions, to papillomas. These findings suggest that overexpression of cyclin D1 and cyclin E occur relatively early in rat esophageal tumorigenesis and participate in tumor progression in this model.

Expression of the somatostatin receptor subtype-2 gene predicts response of human pancreatic cancer to octreotide.

BACKGROUND: Somatostatin inhibits proliferation of many solid tumors. The current study examines whether inhibition of the growth of pancreatic cancer by the somatostatin analog, octreotide, requires tumor expression of somatostatin receptors. METHODS: We studied five human pancreatic cancer cell lines, Capan-1, Capan-2, CAV, MIA PaCa-2, and Panc-1. Solid tumors were established in nude mice (n = 20/cell line) by flank injection of tumor cells. Subcutaneous octreotide (500 micrograms/kg/day) was administered by osmotic pumps to 10 of the animals in each group, and the other 10 received control infusions of saline solution. On day 36, the tumors were excised and weighed. Plasma levels of the putative trophic peptides cholecystokinin, epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin were assessed by radioimmunoassay. Each of the five cell lines was assayed for the presence of cell surface somatostatin receptors by using whole cell competitive binding assays with 125I-somatostatin. Expression of the somatostatin receptor subtype-2 (SSR2) gene was determined with reverse transcriptase-polymerase chain reactions. Southern blot hybridization was used to assess the presence of the SSR2 gene. RESULTS: Octreotide inhibited tumor growth in the MIA PaCa-2 group (512 +/- 75 mg control versus 285 +/- 71 mg treated; p < 0.05) but had no significant effect on tumor weight in the other four cell lines. Plasma levels of cholecystokinin, epidermal growth factor, insulin-like growth factor-1, and insulin were not altered by chronic octreotide infusion. Cell surface somatostatin receptors and SSR2 gene expression were detected only in the MIA PaCa-2 tumors. The gene for the SSR2 receptor was found in all five tumor lines. CONCLUSIONS: Octreotide-mediated inhibition of pancreatic cancer growth is dependent on expression of somatostatin receptors. The expression of somatostatin receptors should be considered in the design and interpretation of clinical trials with somatostatin analogs for treatment of pancreatic cancer.

Transforming growth factor-alpha overexpression in proliferative verrucous leukoplakia and oral squamous cell carcinoma: an immunohistochemical study.

Proliferative verrucous leukoplakia is a unique type of oral leukoplakia that has a high risk of malignant transformation. The aim of this study was to examine the expression of transforming growth factor-alpha in proliferative verrucous leukoplakia, oral squamous cell carcinoma, and normal mucosa. Transforming growth factor-alpha, a potent mitogen, is known to play an important role in various neoplasms including oral squamous cell carcinoma. Immunohistochemical localization of transforming growth factor-alpha in archival paraffin-embedded sections was performed with commercially available monoclonal antibodies. Ten cases each of normal mucosa, proliferative verrucous leukoplakia, and oral squamous cell carcinoma were stained. Quantification of the staining intensity, expressed as the cytoplasmic optical density, was done with the Roche Image Analysis System. The data were statistically analyzed with the one-way analysis of variance and Tukey tests. Notably, the mean cytoplasmic optical density of proliferative verrucous leukoplakia was significantly higher than the mean cytoplasmic optical density of normal mucosa (p < 0.01). The mean cytoplasmic optical density of proliferative verrucous leukoplakia was slightly higher than that of oral squamous cell carcinoma, however, this difference was not significant (p > 0.05). The mean cytoplasmic optical density values demonstrate that increased transforming growth factor-alpha immunoreactivity occurs in proliferative verrucous leukoplakia and oral squamous cell carcinoma relative to normal mucosa.

Characterization of a murine model of acute lung injury (ALI): a prominent role for interleukin-1.

This report describes a model developed to study local and systemic events that occur as a result of acute lung injury (ALI). C57BL/6J mice were injected with a single intravenous dose (2, 4, and 6 micrograms) of 12-O-tetradecanoylphorbol-13-acetate (TPA). At 1, 2, 4, 12, 24, and 48 h, after injection, plasma was collected by sinus orbital puncture, bronchoalveolar lavage (BAL) was performed and cells and fluid were collected, lungs were perfused, and pulmonary tissue was isolated and processed for histological, immunochemical, and gene expression studies. The results indicate a dose-dependent increase in animal distress and a decrease in survival. TPA induced an early systemic response, reflected as an initial decrease in numbers of peripheral blood neutrophils at 1 h, followed at 2 h by a sustained increase. There was dose- and time-dependent increase in IL-1 beta mRNA synthesis, detected using RT-PCR, and in immunoreactive IL-1 alpha produced by both tissue-fixed pulmonary cells and cells within alveolar spaces. Infiltration of neutrophils into pulmonary tissue and increased protein content in BAL fluid was detected 2 h after injection of TPA. Disruptions in pulmonary architecture accompanied by the presence of highly vacuolated macrophages within the alveolar spaces and interstitial tissue were evident after IV injection of TPA. The study shows that injection of TPA induces reproducible dose- and time-dependent alterations in cell types, numbers, state of activation, and production of soluble mediators in the peripheral circulation within BAL and pulmonary tissue. Thus, this model offers a means to examine the cellular basis for the local and systemic alterations observed during ALI.

Alterations in transforming growth factor-alpha and epidermal growth factor receptor expression during rat esophageal tumorigenesis.

Transforming growth factor-alpha (TGF-alpha) stimulates cell proliferation through interaction with its receptor, the epidermal growth factor receptor (EGFR), by activating its tyrosine kinase activities. The simultaneous overexpression of TGF-alpha and EGFR by tumor cells is thought to trigger the autocrine growth pathway, leading to uncontrolled proliferation. To examine their roles in rat esophageal tumorigenesis induced by the chemical carcinogen N-nitrosomethylbenzylamine (NMBA), TGF-alpha, and EGFR expression was evaluated in normal rat esophageal epithelium, in NMBA-induced preneoplastic lesions, and in papillomas by quantitative reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemical analyses. Compared with the levels in normal epithelium, the TGF-alpha and EGFR mRNA levels in esophageal papillomas were 3.6 and 1.9 times higher, respectively. In the preneoplastic epithelium, although a trend of increased TGF-alpha and EGFR mRNA levels was observed, collectively there were no significant differences between preneoplastic and normal samples by RT-PCR analysis. In situ hybridization and immunohistochemical staining showed increased levels of TGF-alpha and EGFR mRNA and protein products in papillomas and in pronounced hyperplastic and dysplastic lesions. TGF-alpha and EGFR expression correlated with each other and with the expression of proliferating cell nuclear antigen, a marker for cell proliferation. These results suggest that disregulation of TGF-alpha and EGFR expression may contribute to autonomous cell growth and may play an important role in rat esophageal tumorigenesis induced by NMBA.

Detection of genomic alterations in human cervical cancer by two-dimensional gel electrophoresis.

Two-dimensional gel electrophoresis was used to comprehensively scan the whole genome of 6 cervical intraepithelial neoplasia (CIN) lesions, 7 cervical squamous cell carcinomas, 1 cervical adenosquamous cell carcinoma, and 2 cervical adenocarcinomas for multiple genetic alterations, such as DNA amplification, chromosome deletion, loss of heterozygosity, and chromosome translocation, as compared with the paired normal tissues. DNA spot analysis of the genomic 2-dimensional gels was performed by a computer color overlay system and by spot recognition software allowing for objective spot comparison and quantitation. Nine spots were found to be amplified in the cervical carcinomas while two amplified spots were detected in the CIN III lesions. Fourteen DNA spots were either reduced in their intensity or absent in cervical carcinomas as compared to their normal paired tissues. Reduction of intensity in 6 spots was observed in the 5 CIN III lesions. These genetic alterations may represent changes in cancer genes that are associated with human cervical carcinogenesis. Further characterization of these alterations may be significant to the understanding of cervical tumorigenesis and to the development of biomarkers for clinical trials in cancer chemoprevention.