A new mouse model to explore the initiation, progression, and therapy of BRAFV600E-induced lung tumors

Mutationally activated BRAF(V600E) (BRAF(VE)) is detected in approximately 6% of human malignancies and promotes sustained MEK1/2-ERK1/2 pathway activation. We have designed BRaf(CA) mice to express normal BRaf prior to Cre-mediated recombination after which BRaf(VE) is expressed at physiological levels. BRaf(CA) mice infected with an Adenovirus expressing Cre recombinase developed benign lung tumors that only rarely progressed to adenocarcinoma. Moreover, BRaf(VE)-induced lung tumors were prevented by pharmacological inhibition of MEK1/2. BRaf(VE) expression initially induced proliferation that was followed by growth arrest bearing certain hallmarks of senescence. Consistent with Ink4a/Arf and TP53 tumor suppressor function, BRaf(VE) expression combined with mutation of either locus led to cancer progression.     

Expression of mos in astrocytic tumors and its potential role in neoplastic progression

The c-mos gene and its protein product mos, components of the mitogen-activated protein kinase transduction pathway, are known to be involved in the control of meiosis and mitosis. Apart from a study on lung carcinomas, there is little information about its role in human neoplasia. The aim of this study was to investigate expression of mos in astrocytic tumors and to correlate it with accumulation of p53. We studied expression of mos in 62 cases of supratentorial astrocytic tumor. Intracytoplasmic immunostaining for mos was found in 28 (45%) cases: 3 of 20 (15%) grade 2 astrocytomas, 9 of 20 (45%) grade 3 anaplastic astrocytomas, and 16 of 22 (73%) glioblastomas. Immunopositivity for mos correlated significantly (P < 0.01) with tumor grade but not with p53 expression. In contrast to the findings in relation to lung tumors, immunopositivity for mos in astrocytic tumors did not predict recurrence-free or overall survival time. Cytoplasmic immunostaining was observed in scattered large cortical neurons adjacent to tumors, possibly due to stress-induced abortive entry into the cell cycle. The correlation of mos immunopositivity with tumor grade may reflect the expansion of more malignant mos-positive clones. This study provides evidence that mos may be involved in the neoplastic progression of a proportion of astrocytic tumors.     

Light scattering from cervical cells throughout neoplastic progression: influence of nuclear morphology,DNA content,and chromatin texture

A number of noninvasive fiber optic optical technologies are under development for real-time diagnosis of neoplasia. We investigate how the light scattering properties of cervical cells are affected by changes in nuclear morphology, DNA content, and chromatin texture, which occur during neoplastic progression. We used a Cyto-Savant computer-assisted image analysis system to acquire quantitative nuclear features measurements from 122 Feulgen-thionin-stained histopathologic sections of cervical tissue. A subset of the measured nuclear features was incorporated into a finite-difference time-domain (FDTD) model of cellular light scattering. The magnitude and angular distribution of scattered light was calculated for cervical cells as a function of pathologic grade. The nuclear atypia strongly affected light scattering properties. The increased size and elevated DNA content of nuclei in high-grade lesions caused the most significant changes in scattering intensity. The spatial dimensions of chromatin texture features and the amplitude of refractive index fluctuations within the nucleus impacted both the angular distribution of scattering angles and the total amount of scattered light. Cellular scattering is sensitive to changes in nuclear morphology that accompany neoplastic progression. Understanding the quantitative relationships between nuclear features and scattering properties will aid in the development of noninvasive optical technologies for detection of precancerous conditions.     

Tolerance induction to self-MHC antigens in fetal and neonatal mouse B cells

We have studied the mechanisms of tolerance induction to self-MHC antigens in mouse B cells during fetal development and the post-natal period. To monitor the fate of autoreactive B cell clones, we used the 3-83 micro delta B cell receptor (BCR)-transgenic (Tg) and -knock-in (KI) mouse models. These BCR-Tg and -KI B cells recognize the MHC class I molecules H-2K(k) and H-2K(b), with a high or moderate affinity, respectively. We compared the fate of BCR-Tg and -KI B cells in H-2K(b)-bearing animals and H-2K(b)-negative controls at various stages of their fetal development and post-natal life. Our data show that, in contrast to what occurs in adult B cells, anergy is the main component of tolerance induction in 3-83 micro delta BCR-Tg K(b+) autoreactive fetuses, while 3-83 BCR-KI fetuses primarily use receptor editing. Interestingly, autoreactive B cell deletion is absent or merely marginal before birth. Our results indicate that tolerance induction is effective as early as embryonic day 16.5 and that in the fetus and neonate, like in the adult, the main mechanism of B cell tolerance functioning in the 3-83 KI system is receptor editing. In contrast, in the 3-83 micro delta mice where receptor editing is hindered, adult and fetal B cells differ in their preferential use of mechanisms leading to self-tolerance (i.e. deletion versus anergy).     

Expression of cytotoxic proteins by neoplastic T cells in mycosis fungoides increases with progression from plaque stage to tumor stage disease

Granzyme B (GrB) and T-cell-restricted intracellular antigen (TIA-1) are cytotoxic proteins that are specifically expressed by cytotoxic CD4 or CD8 positive T cells and natural killer cells. Recent studies demonstrated frequent expression of GrB and TIA-1 by neoplastic cells in primary cutaneous CD30(+) large T-cell lymphomas and lymphomatoid papulosis but not in CD30(-) large T-cell lymphomas. In the present study, 74 biopsies from 54 patients with mycosis fungoides (MF) were investigated for the expression of GrB and TIA-1 using immunohistochemistry on paraffin sections. Staining of more than 10% of the neoplastic T cells for GrB or TIA-1 was considered positive. All but two follow-up biopsies had been obtained from patients without extracutaneous disease at the time of biopsy. Expression of TIA-1 and GrB was found in 33 (45%) and 14 (19%) of 74 MF biopsies, respectively. Comparison of biopsies from T3NoMo-stage MF (n = 27) and T2NoMo-stage MF (n = 45) showed increased expression of TIA-1 (55 versus 37%) and GrB (33 versus 9%) in T3NoMo-stage MF. Evaluation of multiple sequential biopsies from successive stages of MF also revealed an increase in the GrB/TIA-1 expression with tumor progression in five of eight cases. A clearcut relation between the expression of TIA-1 and/or GrB and the type of skin lesion biopsied was found. Considering all 74 biopsies, expression of TIA-1 and GrB was found in 18 of 50 (35%) and 5 of 50 (10%) patches or plaques, 9 of 16 (55%) and 3 of 16 (20%) tumors without blastic transformation, and 6 of 8 (75%) and 6 of 8 (75%) tumors with blastic transformation (defined as >50% blast cells). Correlation between GrB/TIA-1 expression in first diagnostic biopsies from patches or plaques from 40 patients with T2NoMo-stage MF and clinical follow-up data did not reveal differences in clinical behavior and survival between patients with (n = 14) or without (n = 26) expression of cytotoxic proteins, indicating that MF expressing cytotoxic proteins should not be considered as a separate group.     

Spatial and temporal distribution of nerves, ganglia, and smooth muscle during the early pseudoglandular stage of fetal mouse lung development.

Neural tissue and smooth muscle appear early in the developing fetal lung, but little is known of their origin and subsequent distribution. To investigate the spatial and temporal distribution of nerves, ganglia, and airway smooth muscle during the early pseudoglandular stage, fetal mouse lungs at embryonic days (E) 11 to 14 were immunostained as whole-mounts and imaged by confocal microscopy. At E11, the primordial lung consisted of the future trachea and two budding epithelial tubules that were covered in smooth muscle to the base of the growing buds. The vagus and processes entering the lung were positive for the neural markers PGP 9.5 (protein gene product 9.5) and synapsin but no neurons were stained at this stage. An antibody to p75NTR revealed neural crest cells on the future trachea as well as in the vagus and in processes extending from the vagus to the lung. This finding indicates that even though neuronal precursors are already present at this stage, they are still migrating into the lung. By E12, neural tissue was abundant in the proximal part of the lung and nerves followed the smooth muscle-covered tubules to the base of the growing buds. At E13 and E14, a neural network of interconnected ganglia, innervated by the vagus, covered the trachea. The postganglionic nerves mainly followed the smooth muscle-covered tubules, but some extended out into the mesenchyme beyond the epithelial buds. Furthermore, we show in a model of cultured lung explants that neural tissue and smooth muscle persist and continue to grow and differentiate in vitro. By using fluorescent markers and confocal microscopy, we present the developing lung as a dynamic structure with smooth muscle and neural tissue in a prime position to influence growth and development.     

Dehydroepiandrosterone sulfate (DHEAS) and testosterone: relation to HIV illness stage and progression over one year

This study explored associations between serum dehydroepiandrosterone sulfate (DHEAS), free and total testosterone levels, and HIV illness markers, including viral load, and the behavioral problems of fatigue and depressed mood. Subjects were 169 HIV-positive men evaluated at baseline, 6, and 12 months for levels of DHEAS, total and free testosterone, HIV RNA, CD4, HIV symptoms, opportunistic illnesses, fatigue, and depression. Men with AIDS (N = 105), compared with men with less advanced illness, had lower mean levels of DHEAS. Baseline DHEAS was positively correlated with CD4 count, HIV symptom severity, and was inversely correlated with HIV RNA. Baseline DHEAS below the laboratory reference range (96 microg/dl) was associated with history of opportunistic infections and malignancies (adjusted odds ratio [OR], 4.4; 95% confidence interval [CI], 1.9-10.4) and with incidence of these complications or death over 1 year (adjusted OR, 2.6; 95% CI, 1-7.2). Initiating protease inhibitor combination therapy was associated with an increase in DHEAS over 6 months. Free testosterone was inversely correlated with HIV RNA, but there were no other significant associations between testosterone and HIV illness markers. No hormone was related to fatigue or depression. This study confirms that low serum DHEAS is associated with HIV illness markers, including viral load, and carries negative prognostic value. Further, protease inhibitor therapy may result in increased circulating DHEAS.     

Quantitation and localization of ENaC subunit expression in fetal, newborn, and adult mouse lung

The newborn lung is cleared of fetal liquid by active Na+ transport. The heterotrimeric (alpha, beta, gamma) epithelial Na+ channel, ENaC, mediates this process. To understand the role of individual ENaC subunits in Na+ transport during development, we quantified murine ENaC (mENaC) subunit messenger RNA (mRNA) expression levels of fetal, neonatal, and adult mouse lung by Northern blot analysis and studied regional expression by in situ hybridization. alphamENaC and gammamENaC mRNA expression increased sharply in late fetal gestation and reached near-adult levels by Day 1 of postnatal life. betamENaC expression increased more gradually through late fetal and early postnatal life and increased progressively until adulthood. In situ hybridization studies showed similar localization patterns of alphamENaC and gammamENaC subunit expression in fetal and postnatal lung. gammamENaC and alphamENaC subunits were initially localized to fetal lung bud tubules and by late gestation both subunits were expressed in all regions (acinar and bronchiolar) of the distal lung epithelium. betamENaC was detected from 16 d gestation onward and was expressed most intensely in small airways. There was little expression of betamENaC in the alveolar region. In postnatal lung all three subunits were expressed intensely in small airways. In adult lung, alphamENaC and gammamENaC were expressed in a pattern consistent with an alveolar type II (ATII) cell distribution. The timing of quantitative changes in mENaC subunit expression is consistent with a role of Na+ transport in liquid clearance of the perinatal lung. Intense expression of mENaC subunits in medium and small airway epithelium and in ATII cells suggests that these regions are a primary location for liquid absorption in the perinatal and postnatal murine lung.     

Fallopian tube ciliary beat frequency in relation to the stage of menstrual cycle and anatomical site

BACKGROUND: The cyclical changes in ciliary structure and motion within the human Fallopian tube are well documented. Previous investigators have studied ciliary beat frequency (CBF) in relation to menstrual cycle and anatomical site, but with conflicting results. METHODS: Using a technique that records variations in light intensity, we have studied the changes in CBF in relation to the menstrual cycle and anatomical site. Fallopian tubes were collected from 26 women who underwent hysterectomy for benign conditions. Menstrual history, hormone profile and endometrial biopsy results were used to determine the stage of the cycle. Fourteen women were in the proliferative phase, and 12 women in the secretory phase. RESULTS: Mean CBF for all subjects was 5.3 plus minus 0.2 Hz. There was no significant difference in CBF in relation to anatomical site. In the fimbrial region the ciliary beat was faster in the secretory (5.8 plus minus 0.3 Hz) as compared with the proliferative phase (4.9 plus minus 0.2 Hz), P < 0.02. CONCLUSIONS: It is possible that this increase in fimbrial CBF may contribute to ovum retrieval and transport after ovulation. However, the reproductive significance of the changes in CBF in relation to the menstrual cycle needs further investigation.     

Stage-related induction of chromosomal aberrations and SCE in mouse embryos treated transplacentally during organogenesis with MMC and DMBA

During organogenesis, mouse embryos were treated transplacentally with MMC and DMBA. The clastogenic and SCE-inducing effects of MMC and the clastogenic effects of DMBA were analyzed in metaphases from whole embryo suspensions. Positive effects were observed on all the days of pregnancy on which the embryos were analyzed, i.e., on days 10, 11, 12, and 13. Whereas the MMC-induced SCE-frequencies did not change significantly during the tested period, the clastogenic effects of MMC and DMBA varied drastically. Extremely high aberration rates were observed in embryos on day 11; on the other days the aberration rates were much lower. Factors that might have given rise to these stage-related effects are discussed.     

Metallothionein induction attenuates the progression of lung injury in mice exposed to long-term intermittent hypoxia

Inflammation Research - Intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), is prevalent in older adults and associated with inflammation. We previously showed that IH induces...     

The innate immune architecture of lung tumors and its implication in disease progression

Lung malignancies are the leading cause of cancer-related mortality. By virtue of its unique physiological function, the lung microenvironment is highly dynamic and constantly subjected to mechanical, chemical and pathogenic stimuli. Thus, the airways rely on highly organized innate defense mechanisms to rapidly protect against pathogens and maintain pulmonary homeostasis. However, in the context of lung malignancy, these defenses often provide collateral inflammatory insults that can foster tumor progression. This review summarizes the interactions between cancer cells, recruited immune cells and tissue-resident cell subpopulations, such as airway epithelial cells and alveolar macrophages, during homeostasis and disease. Furthermore, we examine the role of the lung immune landscape in response to current therapeutic interventions for cancer. Given the prevalence of lung malignancies, we propose that consideration of lung physiology as a whole is necessary to understand and treat these lethal diseases. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Expression in normal adult, fetal, and neoplastic tissues of a carbohydrate differentiation antigen recognised by antigranulocyte mouse monoclonal antibodies.

The distribution in paraffin fixed human tissues of a carbohydrate antigen defined by two monoclonal antibodies raised against human granulocytes has been studied by means of an immunoperoxidase technique. In addition to granulocytes, the antigen has been detected in adult tissues on identifiable cell types of the stomach, kidney, adrenal medulla, and brain and on the mucins of the gastrointestinal tract and other secretions. In fetal tissue, epithelial cells of the alimentary tract, lung, brain, and kidney express the antigen. Adenocarcinoma of the colon, stomach, breast, and lung are stained strongly, as are other types of lung cancer. The monoclonal antibodies give a staining pattern similar but not identical to other monoclonal antibodies raised against granulocytes or neoplastic cell lines which recognise the antigen 3-fucosyl N-acetyllactosamine. The use of antibodies against this oncofetal antigen in the study of differentiation and as tumour markers is discussed.     

Oncocytic carcinoma of the breast: frequency, morphology and follow-up

Oncocytic breast carcinomas are tumors composed of no fewer than 70% of oncocytic cells (World Health Organization). The purpose of this study was to determine the frequency, morphologic, immunohistochemical, and clinical features of invasive oncocytic carcinoma in a large series. Twenty-eight cases of putative oncocytic breast carcinoma (selected cases group) and 76 consecutive cases of invasive breast carcinoma (consecutive cases group) were analyzed. Immunohistochemistry for mitochondria, gross cystic disease fluid protein 15, chromogranin, estrogen receptor, progesterone receptor, androgen receptor, HER2/Neu, cytokeratin 7, cytokeratin 14, epithelial membrane antigen, and differentiation cluster 68 was performed. Score for mitochondria was based on intensity and percentage of immunopositive cells. Classes were as follows: (1) oncocytic carcinoma: at least 70%, 3+; (2) mitochondrion-rich carcinoma: 50% to 70%, 3+, or more than 50%, 2+; and (3) all the other cases were referred to as invasive breast carcinoma. Ultrastructural examination was available for 6 cases of oncocytic carcinoma. Morphologic and immunohistochemical features of the 3 groups were compared using Fisher exact test (P < .05). For overall survival analysis, Kaplan-Maier curves were compared using log-rank and Wilcoxon tests (P < .05). Our results suggest that oncocytic breast carcinoma is a morphologic entity with distinctive histologic and ultrastructural features. Mitochondrion-rich carcinomas are histologically similar to oncocytic carcinomas and constitute 19.7% of all invasive carcinomas, indicating that cytoplasmic eosinophilia in breast cancer cells is often due to accumulation of mitochondria. Oncocytic carcinomas and mitochondrion-rich carcinomas are more often grade III tumors and show human epidermal growth factor receptor 2 overexpression. Clinical features and overall survival of oncocytic carcinomas are not distinctive because they are similar to those of the other cases when matched for grade and stage.     

Role of anthrax toxins in dissemination, disease progression, and induction of protective adaptive immunity in the mouse aerosol challenge model

Anthrax toxins significantly contribute to anthrax disease pathogenesis, and mechanisms by which the toxins affect host cellular responses have been identified with purified toxins. However, the contribution of anthrax toxin proteins to dissemination, disease progression, and subsequent immunity after aerosol infection with spores has not been clearly elucidated. To better understand the role of anthrax toxins in pathogenesis in vivo and to investigate the contribution of antibody to toxin proteins in protection, we completed a series of in vivo experiments using a murine aerosol challenge model and a collection of in-frame deletion mutants lacking toxin components. Our data show that after aerosol exposure to Bacillus anthracis spores, anthrax lethal toxin was required for outgrowth of bacilli in the draining lymph nodes and subsequent progression of infection beyond the lymph nodes to establish disseminated disease. After pulmonary exposure to anthrax spores, toxin expression was required for the development of protective immunity to a subsequent lethal challenge. However, immunoglobulin (immunoglobulin G) titers to toxin proteins, prior to secondary challenge, did not correlate with the protection observed upon secondary challenge with wild-type spores. A correlation was observed between survival after secondary challenge and rapid anamnestic responses directed against toxin proteins. Taken together, these studies indicate that anthrax toxins are required for dissemination of bacteria beyond the draining lymphoid tissue, leading to full virulence in the mouse aerosol challenge model, and that primary and anamnestic immune responses to toxin proteins provide protection against subsequent lethal challenge. These results provide support for the utility of the mouse aerosol challenge model for the study of inhalational anthrax.     

Herpes virus in Alzheimer's disease: relation to progression of the disease

Studies regarding different viruses of the herpes family, such as cytomegalovirus (CMV), Epstein–Barr virus (EBV), or human herpes virus 6 (HHV-6) in Alzheimer's disease (AD) are scarce. DNA from peripheral blood leukocytes (PBL) and brain samples were analyzed for the presence of CMV, EBV, or HHV-6. All samples were negative for CMV. EBV positivity was 6% in AD brains, whereas 45% of PBL samples from AD patients and 31% from controls were positive for EBV (p = 0.05). HHV-6 showed a 23% positivity in PBL samples from AD and 4% from controls (p = 0.002). 17% of AD brains were HHV-6 positive. Within a group of elderly individuals, followed up for 5 years, EBV-positive or HHV-6–positive PBL increased in those who developed clinical AD. Virus serological positivity was also investigated, and IgG levels for CMV and EBV antigens were also increased in those subjects who developed AD during the follow-up. Our findings suggest that EBV and HHV-6 may be environmental risk factors for cognitive deterioration and progression to AD in elderly persons.     

Versican in nonsmall cell lung cancer: relation to hyaluronan, clinicopathologic factors, and prognosis

The aim of the study was to investigate the expression and prognostic role of versican in 212 patients with resected nonsmall cell lung cancer. Tumor samples were stained immunohistochemically, and the versican staining was evaluated both in tumor stroma and cancer cells. The staining results were compared to the clinical data of the patients, the tumor cell proliferation, and the expression of hyaluronan. In the whole material, low and high area percentages of stromal versican staining were observed in 135 and 77 carcinomas, respectively. Tumor cell-associated staining signal for versican was observed in 33 cases. In the whole material, the significant relationship between high stromal staining of versican and that of hyaluronan was noticed (P = .001). The expression of stromal versican was related to tumor type (P = .008) and high stromal staining was inversely correlated with poor tumor differentiation (P = .045), but not with tumor cell proliferation. Among adenocarcinomas, the high stromal staining of versican was associated with tumor recurrence (P = .024), higher tumor stage (P = .022), and lymph node metastases (P = .042). Versican expression was not related to patient outcome in the whole material, but among adenocarcinomas, the high stromal staining was related to poor disease-free survival (P = .0056). However, in Cox multivariate analysis with tumor stage, versican expression did not retain its prognostic significance. The results indicate that increased stromal versican is related to higher tumor recurrence rate and more advanced disease. Despite the important role of versican in nonsmall cell lung cancer, traditional clinicopathologic factors remained most significant in the current study.