Passive Immunization with a Single Monoclonal Neutralizing Antibody Protects against Cutaneous and Mucosal Mouse Papillomavirus Infections.

We have established a mouse papillomavirus (MmuPV1) model that induces both cutaneous and mucosal infections and cancers. In the current study, we use this model to test our hypothesis that passive immunization using a single neutralizing monoclonal antibody can protect both cutaneous and mucosal sites at different time points after viral inoculation. We conducted a series of experiments involving the administration of either a neutralizing monoclonal antibody, MPV.A4, or control monoclonal antibodies to both outbred and inbred athymic mice. Three clinically relevant mucosal sites (lower genital tract for females and anus and tongue for both males and females) and two cutaneous sites (muzzle and tail) were tested. At the termination of the experiments, all tested tissues were harvested for virological analyses. Significantly lower levels of viral signals were detected in the MPV.A4-treated female mice up to 6 h post-viral inoculation compared to those in the isotype control. Interestingly, males displayed partial protection when they received MPV.A4 at the time of viral inoculation, even though they were completely protected when receiving MPV.A4 at 24 h before viral inoculation. We detected MPV.A4 in the blood starting at 1 h and up to 8 weeks postadministration in some mice. Parallel to these in vivo studies, we conducted in vitro neutralization using a mouse keratinocyte cell line and observed complete neutralization up to 8 h post-viral inoculation. Thus, passive immunization with a monoclonal neutralizing antibody can protect against papillomavirus infection at both cutaneous and mucosal sites and is time dependent. IMPORTANCE This is the first study testing a single monoclonal neutralizing antibody (MPV.A4) by passive immunization against papillomavirus infections at both cutaneous and mucosal sites in the same host in the mouse papillomavirus model. We demonstrated that MPV.A4 administered before viral inoculation can protect both male and female athymic mice against MmuPV1 infections at cutaneous and mucosal sites. MPV.A4 also offers partial protection at 6 h post-viral inoculation in female mice. MPV.A4 can be detected in the blood from 1 h to 8 weeks after intraperitoneal (i.p.) injection. Interestingly, males were only partially protected when they received MPV.A4 at the time of viral inoculation. The failed protection in males was due to the absence of neutralizing MPV.A4 at the infected sites. Our findings suggest passive immunization with a single monoclonal neutralizing antibody can protect against diverse papillomavirus infections in a time-dependent manner in mice.

Comparison of the Toponomes of Alveolar Macrophages From Wild Type and Surfactant Protein A Knockout Mice and Their Response to Infection.

Background: Surfactant protein-A (SP-A) plays a critical role in lung innate immunity by regulating alveolar macrophages (AM), expression of inflammatory mediators, and other host defense proteins. The toponome imaging system (TIS), a serial immunostainer, was used to study the AM toponome because it characterizes the localization of multiple markers and identifies marker combinations in each pixel as combinatorial molecular phenotypes (CMPs). We used TIS to study the AM toponome from wild type (WT) and SP-A knockout (KO) mice and changes following Klebsiella pneumoniae exposure. Methods: WT or KO mice received intratracheal K. pneumoniae or vehicle and AM were obtained by bronchoalveolar lavage after one hour. AM were attached to slides and underwent TIS analysis. Images were analyzed to characterize all pixels. AM CMPs from WT vehicle (n=3) and infected (n=3) mice were compared to each other and to AM from KO (n=3 vehicle; n=3 infected). Histograms provided us with a tool to summarize the representation of each marker in a set of CMPs. Results: Using the histograms and other tools we identified markers of interest and observed that: 1) Both comparisons had conserved (present in all group members) CMPs, only in vehicle AM and only in infected AM, or common to both vehicle and infected AM, (i.e., unaffected by the condition). 2) the CMP number decreased with infection in WT and KO versus vehicle controls. 3) More infection-specific CMPs in WT vs KO AM. 4) When AM from WT and KO vehicle or infected were compared, there were more unique CMPs exclusive to the KO AM. 5) All comparisons showed CMPs shared by both groups. Conclusions: The decrease of CMPs exclusive to infected AM in KO mice may underlie the observed susceptibility of KO mice to infection. However, both KO groups had more exclusive CMPs than the corresponding WT groups, perhaps indicating a vigorous effort by KO to overcome deficits in certain proteins and CMPs that are dysregulated by the absence of SP-A. Moreover, the presence of shared CMPs in the compared groups indicates that regulation of these CMPs is not dependent on either infection or the presence or absence of SP-A.

Depo Medroxyprogesterone (DMPA) Promotes Papillomavirus Infections but Does Not Accelerate Disease Progression in the Anogenital Tract of a Mouse Model.

Contraceptives such as Depo-medroxyprogesterone (DMPA) are used by an estimated 34 million women worldwide. DMPA has been associated with increased risk of several viral infections including Herpes simplex virus-2 (HSV-2) and Human immunodeficiency virus (HIV). In the current study, we used the mouse papillomavirus (MmuPV1) anogenital infection model to test two hypotheses: (1) contraceptives such as DMPA increase the susceptibility of the anogenital tract to viral infection and (2) long-term contraceptive administration induces more advanced disease at the anogenital tract. DMPA treatments of both athymic nude mice and heterozygous NU/J (Foxn1nu/+) but ovariectomized mice led to a significantly increased viral load at the anogenital tract, suggesting that endogenous sex hormones were involved in increased viral susceptibility by DMPA treatment. Consistent with previous reports, DMPA treatment suppressed host anti-viral activities at the lower genital tract. To test the impact of long-term contraceptive treatment on the MmuPV1-infected lower genital tract, we included two other treatments in addition to DMPA: 17ß-estradiol and a non-hormone based contraceptive Cilostazol (CLZ, Pletal). Viral infections were monitored monthly up to nine months post infection by qPCR. The infected vaginal and anal tissues were harvested and further examined by histological, virological, and immunological analyses. Surprisingly, we did not detect a significantly higher grade of histology in animals in the long-term DMPA and 17ß-estradiol treated groups when compared to the control groups in the athymic mice we tested. Therefore, although DMPA promotes initial papillomavirus infections in the lower genital tract, the chronic administration of DMPA does not promote cancer development in the infected tissues in our mouse model.

The alveolar macrophage toponome of female SP-A knockout mice differs from that of males before and after SP-A1 rescue.

Using the Toponome Imaging System (TIS), a serial immunostainer, we studied the patterns of expression of multiple markers in alveolar macrophages (AM) from female mice lacking surfactant protein A (SP-A knockouts; KO) after "rescue" with exogenous SP-A1. We also used a 7-marker subset to compare with AM from males. AM were harvested 18 h after intrapharyngeal SP-A1 or vehicle, attached to slides, and subjected to serial immunostaining for 12 markers. Expression of the markers in each pixel of the image was analyzed both in the whole image and in individual selected cells. The marker combination in each pixel is referred to as a combinatorial molecular phenotype (CMP). A subset of antibodies was used to compare AM from male mice to the females. We found: (a) extensive AM heterogeneity in females by CMP analysis and by clustering analysis of CMPs in single cells; (b) AM from female KO mice respond to exogenous SP-A1 by increasing CMP phenotypic diversity and perhaps enhancing their potential innate immune capabilities; and (c) comparison of male and female AM responses to SP-A1 revealed that males respond more vigorously than females and clustering analysis was more effective in distinguishing males from females rather than treated from control.

A Pilot Proteomic Study of Vestibular Fluid From Patients With Vulvodynia.

OBJECTIVE: Many women are affected by vulvodynia, but medical therapies to date have proven ineffective. We performed a pilot study using gel-based proteomics to develop a map of proteins present in vaginal/vestibular secretions and identify proteins that could be considered for future evaluation as potential therapeutic targets. MATERIALS AND METHODS: We collected vestibular fluid from 4 controls and 4 patients with vulvodynia by placing a cotton swab in the vestibule and extracting the absorbed proteins. The proteins underwent 2-dimensional difference gel electrophoresis and mass spectrometry to develop a protein map. Immunohistochemistry was used to validate proteomic findings. RESULTS: A map was constructed of 32 of the more abundant proteins in vestibular fluid and their levels compared in control subjects and vulvodynia patients. Among these were annexin A1, interleukin 1 receptor antagonist, protein S100 A9, and a number of antiproteases and proteases. Many of these proteins differed by at least 50% between groups, but only annexin A1, one of the protease inhibitors, and immunoglobulin G κ chain were significantly different. The results with annexin A1 were validated by similar findings with immunohistochemistry. CONCLUSIONS: The findings of this pilot study demonstrate a set of vestibule mucosa proteins that differ significantly-either increasing or decreasing-in vulvodynia patients compared with controls, and several others that exhibited greater than 1.5-fold change but did not reach statistical significance. This study constitutes a proof-of-principle that an open, unbiased proteomic approach can identify molecular participants in vulvodynia, some of which had not been identified to date by hypothesis-driven studies.

Mouse Papillomavirus L1 and L2 Are Dispensable for Viral Infection and Persistence at Both Cutaneous and Mucosal Tissues.

Papillomavirus L1 and L2, the major and minor capsid proteins, play significant roles in viral assembly, entry, and propagation. In the current study, we investigate the impact of L1 and L2 on viral life cycle and tumor growth with a newly established mouse papillomavirus (MmuPV1) infection model. MmuPV1 L1 knockout, L2 knockout, and L1 plus L2 knockout mutant genomes (designated as L1ATGko-4m, L2ATGko, and L1-L2ATGko respectively) were generated. The mutants were examined for their ability to generate lesions in athymic nude mice. Viral activities were examined by qPCR, immunohistochemistry (IHC), in situ hybridization (ISH), and transmission electron microscopy (TEM) analyses. We demonstrated that viral DNA replication and tumor growth occurred at both cutaneous and mucosal sites infected with each of the mutants. Infections involving L1ATGko-4m, L2ATGko, and L1-L2ATGko mutant genomes generally resulted in smaller tumor sizes compared to infection with the wild type. The L1 protein was absent in L1ATGko-4m and L1-L2ATGko mutant-treated tissues, even though viral transcripts and E4 protein expression were robust. Therefore, L1 is not essential for MmuPV1-induced tumor growth, and this finding parallels our previous observations in the rabbit papillomavirus model. Very few viral particles were detected in L2ATGko mutant-infected tissues. Interestingly, the localization of L1 in lesions induced by L2ATGko was primarily cytoplasmic rather than nuclear. The findings support the hypothesis that the L2 gene influences the expression, location, transport, and assembly of the L1 protein in vivo.

Humans with inherited T cell CD28 deficiency are susceptible to skin papillomaviruses but are otherwise healthy.

We study a patient with the human papilloma virus (HPV)-2-driven "tree-man" phenotype and two relatives with unusually severe HPV4-driven warts. The giant horns form an HPV-2-driven multifocal benign epithelial tumor overexpressing viral oncogenes in the epidermis basal layer. The patients are unexpectedly homozygous for a private CD28 variant. They have no detectable CD28 on their T cells, with the exception of a small contingent of revertant memory CD4+ T cells. T cell development is barely affected, and T cells respond to CD3 and CD2, but not CD28, costimulation. Although the patients do not display HPV-2- and HPV-4-reactive CD4+ T cells in vitro, they make antibodies specific for both viruses in vivo. CD28-deficient mice are susceptible to cutaneous infections with the mouse papillomavirus MmuPV1. The control of HPV-2 and HPV-4 in keratinocytes is dependent on the T cell CD28 co-activation pathway. Surprisingly, human CD28-dependent T cell responses are largely redundant for protective immunity.

The environmental pollutant and tobacco smoke constituent dibenzo[def,p]chrysene is a co-factor for malignant progression of mouse oral papillomavirus infections.

HPV infections in the oral cavity that progress to cancer are on the increase in the USA. Model systems to study co-factors for progression of these infections are lacking as HPVs are species-restricted and cannot grow in preclinical animal models. We have recently developed a mouse papillomavirus (MmuPV1) oral mucosal infection model that provides opportunities to test, for the first time, the hypothesis that tobacco carcinogens are co-factors that can impact the progression of oral papillomas to squamous cell carcinoma (SCC). Four cohorts of mice per sex were included: (1) infected with MmuPV1 and treated orally with DMSO-saline; (2) infected with MmuPV1 and treated orally with the tobacco carcinogen, dibenzo[def,p]chrysene (DBP); (3) uninfected and treated orally with DMSO-saline, and (4) uninfected and treated orally with DBP. Oral swabs were collected monthly for subsequent assessment of viral load. Oral tissues were collected for in situ viral DNA/RNA detection, viral protein staining, and pathological assessment for hyperplasia, papillomas and SCC at study termination. We observed increased rates of SCC in oral tissue infected with MmuPV1 and treated with DBP when compared to mice treated with DBP or virus individually, each of which showed minimal disease. Virally-infected epithelium showed strong levels of viral DNA/RNA and viral protein E4/L1 staining. In contrast, areas of SCC showed reduced viral DNA staining indicative of lower viral copy per nucleus but strong RNA signals. Several host markers (p120 ctn, p53, S100A9) were also examined in the mouse oral tissues; of particular significance, p120 ctn discriminated normal un-infected epithelium from SCC or papilloma epithelium. In summary, we have confirmed that our infection model is an excellent platform to assess the impact of co-factors including tobacco carcinogens for oral PV cancerous progression. Our findings can assist in the design of novel prevention/treatment strategies for HPV positive vs. HPV negative disease.

Differences in the alveolar macrophage toponome in humanized SP-A1 and SP-A2 transgenic mice.

Alveolar macrophages (AMs) are differentially regulated by human surfactant protein-A1 (SP-A1) or SP-A2. However, AMs are very heterogeneous and differences are difficult to characterize in intact cells. Using the Toponome Imaging System (TIS), an imaging technique that uses sequential immunostaining to identify patterns of biomarker expression or combinatorial molecular phenotypes (CMPs), we studied individual single cells and identified subgroups of AMs (n = 168) from SP-A-KO mice and mice expressing either SP-A1 or SP-A2. The effects, as shown by CMPs, of SP-A1 and SP-A2 on AMs were significant and differed. SP-A1 AMs were the most diverse and shared the fewest CMPs with KO and SP-A2. Clustering analysis of each group showed 3 clusters where the CMP-based phenotype was distinct in each cluster. Moreover, a clustering analysis of all 168 AMs revealed 10 clusters, many dominated by 1 group. Some CMP overlap among groups was observed with SP-A2 AMs sharing the most CMPs and SP-A1 AMs the fewest. The CMP-based patterns identified here provide a basis for understanding not only AMs' diversity, but also most importantly, the molecular basis for the diversity of functional differences in mouse models where the impact of genetics of innate immune molecules on AMs has been studied.

Antibody-Mediated Immune Subset Depletion Modulates the Immune Response in a Rabbit (Oryctolagus cuniculus) Model of Epstein-Barr Virus Infection.

Epstein-Barr Virus (EBV) is a γ-herpesvirus which infects over 90% of the adult human population. Most notably, this virus causes infectious mononucleosis but it is also associated with cancers such as Hodgkin and Burkitt lymphoma. EBV is a species-specific virus and has been studied in many animal models, including nonhuman primates, guinea pigs, humanized mice, and tree shrews. However, none of these animal models are considered the "gold standard" for EBV research. Recently, rabbits have emerged as a viable alternative model, as they are susceptible to EBV infection. In addition, the EBV infection progresses after immune suppression with cyclosporine A (CsA), modeling the reactivation of EBV after latency. We sought to refine this model for acute or active EBV infections by performing antibody-mediated depletion of certain immune subsets in rabbits. Fourteen 16 to 20-wk old, NZW rabbits were intravenously inoculated with EBV and concurrently treated with either anti-CD4 T-cell antibody, anti-pan-T-cell antibody (anti CD45), CSA, or, as a control, anti-HPV antibody. Rabbits that received the depleting antibodies were treated with CsA 3 times at a dose of 15 mg/kg SC once per day for 4 d starting at the time of EBV inoculation then the dose was increased to 20 mg/kg SC twice weekly for 2 wk. Weights, temperatures, and clinical signs were monitored, and rabbits were anesthetized once weekly for blood collection. When compared with the control group, anti-CD4-treated rabbits had fewer clinical signs and displayed higher levels of viral DNA via qPCR in splenocytes; however, flow cytometry results showed only a partial depletion of CD4 T-cells. Treatment with anti-pan-T-cell antibody did not result in noticeable T-cell depletion. These data suggest the EBV-infected rabbit is a promising model for testing antiviral medications and prophylactic vaccines for EBV.

Using toponomics to characterize phenotypic diversity in alveolar macrophages from male mice treated with exogenous SP-A1.

BACKGROUND: We used the Toponome Imaging System (TIS) to identify "patterns of marker expression", referred to here as combinatorial molecular phenotypes (CMPs) in alveolar macrophages (AM) in response to the innate immune molecule, SP-A1. METHODS: We compared 114 AM from male SP-A deficient mice. One group (n = 3) was treated with exogenous human surfactant protein A1 (hSP-A1) and the other with vehicle (n = 3). AM obtained by bronchoalveolar lavage were plated onto slides and analyzed using TIS to study the AM toponome, the spatial network of proteins within intact cells. With TIS, each slide is sequentially immunostained with multiple FITC-conjugated antibodies. Images are analyzed pixel-by-pixel identifying all of the proteins within each pixel, which are then designated as CMPs. CMPs represent organized protein clusters postulated to contribute to specific functions. RESULTS: 1) We compared identical CMPs in KO and SP-A1 cells and found them to differ significantly (p = 0.0007). Similarities between pairs of markers in the two populations also differed significantly (p < 0.0001). 2) Focusing on the 20 most abundant CMPs for each cell, we developed a method to generate CMP "signatures" that characterized various groups of cells. Phenotypes were defined as cells exhibiting similar signatures of CMPs. i) AM were extremely diverse and each group contained cells with multiple phenotypes. ii) Among the 114 AM analyzed, no two cells were identical. iii) However, CMP signatures could distinguish among cell subpopulations within and between groups. iv) Some cell populations were enriched with SP-A1 treatment, some were more common without SP-A1, and some seemed not to be influenced by the presence of SP-A1. v) We also found that AM were more diverse in mice treated with SP-A1 compared to those treated with vehicle. CONCLUSIONS: AM diversity is far more extensive than originally thought. The increased diversity of SP-A1-treated mice points to the possibility that SP-A1 enhances or activates several pathways in the AM to better prepare it for its innate immune functions and other functions shown previously to be affected by SP-A treatment. Future studies may identify key protein(s) responsible for CMP integrity and consequently for a given function, and target it for therapeutic purposes.

Papillomavirus can be transmitted through the blood and produce infections in blood recipients: Evidence from two animal models.

Human papillomaviruses (HPV) contribute to most cervical cancers and are considered to be sexually transmitted. However, papillomaviruses are often found in cancers of internal organs, including the stomach, raising the question as to how the viruses gain access to these sites. A possible connection between blood transfusion and HPV-associated disease has not received much attention. Here we show, in rabbit and mouse models, that blood infected with papillomavirus yields infections at permissive sites with detectable viral DNA, RNA transcripts, and protein products. The rabbit skin tumours induced via blood infection displayed decreased expression of SLN, TAC1, MYH8, PGAM2, and APOBEC2 and increased expression of SDRC7, KRT16, S100A9, IL36G, and FABP9, as seen in tumours induced by local infections. Furthermore, we demonstrate that blood from infected mice can transmit the infection to uninfected animals. Finally, we demonstrate the presence of papillomavirus infections and virus-induced hyperplasia in the stomach tissues of animals infected via the blood. These results indicate that blood transmission could be another route for papillomavirus infection, implying that the human blood supply, which is not screened for papillomaviruses, could be a potential source of HPV infection as well as subsequent cancers in tissues not normally associated with the viruses.

Infrared microspectroscopy identifies biomolecular changes associated with chronic oxidative stress in mammary epithelium and stroma of breast tissues from healthy young women: implications for latent stages of breast carcinogenesis.

Studies of the decades-long latent stages of breast carcinogenesis have been limited to when hyperplastic lesions are already present. Investigations of earlier stages of breast cancer (BC) latency have been stymied by the lack of fiducial biomarkers needed to identify where in histologically normal tissues progression toward a BC might be taking place. Recent evidence suggests that a marker of chronic oxidative stress (OxS), protein adducts of 4-hydroxy-2-nonenal (4HNE), can meet this need. Specifically: (1) 4HNE immunopositive (4HNE+) mammary epithelial (ME) cells were found to be prevalent in normal (reduction mammoplasty) tissues of most women (including many teenagers) studied, representative of those living in the United States' high risk-posing environment and: (2) marked (> 1.5-fold) differences were identified between tissues of healthy young women with many vs. few 4HNE+ ME cells in the relative levels of transcripts for 42 of the 84 OxS-associated genes represented in SABioscience Oxidative-Stress/Oxidative-Defense PCR array. Herein we used synchrotron radiation-based Fourier-transform infrared (SR-FTIR) microspectroscopy to identify molecular changes associated with 4HNE adducts in basal and luminal ME cells in terminal ductal units (TDLU), which are the cells of origin of BC, and associated intralobular and interlobular stroma, known contributors to carcinogenesis. Multivariate analysis-derived wavenumbers differentiated 4HNE+ and 4HNE- cells in each of the anatomical compartments. Specifically, principal component and linear discriminant analyses of mid-infrared spectra obtained from these cells revealed unambiguous, statistically highly significant differences in the "biochemical fingerprint" of 4HNE+ vs. 4HNE- luminal and basal ME cells, as well as between associated intralobular and interlobular stroma. These findings demonstrate further SR-FTIR microspectroscopy's ability to identify molecular changes associated with altered physiological and/or pathophysiological states, in this case with a state of chronic OxS that provides a pro-carcinogenic microenvironment.

Down-regulation of HtrA1 activates the epithelial-mesenchymal transition and ATM DNA damage response pathways.

Expression of the serine protease HtrA1 is decreased or abrogated in a variety of human primary cancers, and higher levels of HtrA1 expression are directly related to better response to chemotherapeutics. However, the precise mechanisms leading to HtrA1 down regulation during malignant transformation are unclear. To investigate HtrA1 gene regulation in breast cancer, we characterized expression in primary breast tissues and seven human breast epithelial cell lines, including two non-tumorigenic cell lines. In human breast tissues, HtrA1 expression was prominent in normal ductal glands. In DCIS and in invasive cancers, HtrA1 expression was greatly reduced or lost entirely. HtrA1 staining was also reduced in all of the human breast cancer cell lines, compared with the normal tissue and non-tumorigenic cell line controls. Loss of HtrA1 gene expression was attributable primarily to epigenetic silencing mechanisms, with different mechanisms operative in the various cell lines. To mechanistically examine the functional consequences of HtrA1 loss, we stably reduced and/or overexpressed HtrA1 in the non-tumorigenic MCF10A cell line. Reduction of HtrA1 levels resulted in the epithelial-to-mesenchymal transition with acquisition of mesenchymal phenotypic characteristics, including increased growth rate, migration, and invasion, as well as expression of mesenchymal biomarkers. A concomitant decrease in expression of epithelial biomarkers and all microRNA 200 family members was also observed. Moreover, reduction of HtrA1 expression resulted in activation of the ATM and DNA damage response, whereas overexpression of HtrA1 prevented this activation. Collectively, these results suggest that HtrA1 may function as a tumor suppressor by controlling the epithelial-to-mesenchymal transition, and may function in chemotherapeutic responsiveness by mediating DNA damage response pathways.

Identification of mammary epithelial cells subject to chronic oxidative stress in mammary epithelium of young women and teenagers living in USA: implication for breast carcinogenesis.

Current knowledge of changes in the mammary epithelium relevant to breast carcinogenesis is limited to when histological changes are already present because of a lack of biomarkers needed to identify where such molecular changes might be ongoing at earlier during the of decades-long latent stages of breast carcinogenesis. Breast reduction tissues from young women and teenagers, representative of USA's high breast cancer incidence population, were studies using immunocytochemistry and targeted PCR arrays in order to learn whether a marker of chronic oxidative-stress [protein adducts of 4-hydroxy-2-nonenal (4HNE)] can identify where molecular changes relevant to carcinogenesis might be taking place prior to any histological changes. 4HNE-immunopositive (4HNE+) mammary epithelial cell-clusters were identified in breast tissue sections from most women and from many teenagers (ages 14-30 y) and, in tissues from women ages 17-27 y with many vs. few 4HNE+ cells, the expression of 30 of 84 oxidative-stress associated genes was decreased and only one was increased > 2-fold. This is in contrast to increased expression of many of these genes known to be elicited by acute oxidative-stress. The findings validate using 4HNE-adducts to identify where molecular changes of potential relevance to carcinogenesis are taking place in histologically normal mammary epithelium and highlight differences between responses to acute vs. chronic oxidative-stress. We posit that the altered gene expression in 4HNE+ tissues reflect adaptive responses to chronic oxidative-stress that enable some cells to evade mechanisms that have evolved to prevent propagation of cells with oxidatively-damaged DNA and to accrue heritable changes needed to establish a cancer.

Embracing the convenient care concept.

The landscape of primary care medicine is rapidly changing. The decline in interest, both in primary care fields and students choosing these career paths, has left a vacuum in the health care system that must be filled. One of the recent developments has been the birth of "convenient care centers," also known as "retail clinics." This form of health care delivery has mostly been entrepreneurial and based in retail organizations, such as drug stores. These walk-in clinics provide basic medical care for minor common medical conditions, such as sore throat, urinary tract infection, the common cold, and ear infections. Much of this care is provided not by physicians, but by nurse practitioners or physician assistants. After seeing the success of the earliest of these clinics, MinuteClinic by CVS, many other businesses joined the venture, and retail clinics popped up in Wal-Mart, Target, and many local grocery stores. Gradually, hospital systems, physician groups, and managed care companies have also entered the market, sometimes partnering with retail outlets, such as the local grocery store or Wal-Mart, and less often, starting a stand-alone facility. Only 12% of retail clinics are owned by hospital systems or physician groups, while 73% are owned by CVS, Walgreens, or Target. There is even a national nonprofit organization called the Convenient Care Association, started in 2006, and based in Philadelphia, PA. This new trend in delivering health care has been mostly, if not totally, ignored by the medical school practice plans, with the exception of the Mayo Clinic in Minnesota, which has developed several "express care" clinics as stand-alone facilities. As a medical school practice plan and a division of general internal medicine, we could continue to keep a blind eye toward this new trend in primary care medicine or embrace the concept. We aim to develop a new convenient care model integrating our College of Medicine practice plan in partnership with our College of Nursing graduate nursing program to form a stand-alone clinic within a bustling business district in downtown Philadelphia.

Altered gene expression in breast cancer liver metastases.

We previously developed a highly aggressive cell line from heart metastases of 4T1 breast carcinoma (designated 4THM), which produced liver metastases (designated 4TLM). In this study, gene array analysis (GAEA) compared gene expression profiles in 4TLM with profiles in 4T1 and 4THM primary tumors. GAEA demonstrated that 4T1 and 4THM tumors differed in about 250 genes. Over 1,000 genes, however, were expressed differently in 4TLM compared with primary tumors. A cohort of 16 genes showed significantly decreased expression in 4THM tumors, which decreased even further in 4TLM. Many of these genes have been implicated in breast cancer, and many are involved in cell adhesion and junctional complexes. Expression of multiple tight and adherence junction proteins was either downregulated or disappeared in 4TLM; downregulation of claudin 4, claudin 7 and gamma-catenin was confirmed by quantitative polymerase chain reaction, immunoblot, and immunocytochemical (ICC) analyses. At the protein level, intact ZO-1 was also observed in 4T1 tumors, but was not expressed in 4THM or 4TLM tumors. ICC demonstrated expression of gamma-catenin at the plasma membrane with 4T1 tumors, whereas staining appeared to be nuclear/perinuclear in 4THM tumors. Claudin 7 staining was also seen in monocyte/pmacrophage-like cells in liver around metastatic lesions by ICC, and it appeared that larger 4TLM tumors apparently reexpressed claudin 7 RNA and protein. Our results demonstrate that decreased or abnormal expression of a number of cell adhesion/junctional proteins, including claudin 4, 7, ZO-1 and gamma-catenin, correlates with liver metastases, and that cell adhesion molecules in the microenvironment are also altered.

Differentiation of xenografted human fetal lung parenchyma.

The goal of this study was to characterize xenografted human fetal lung tissue with respect to developmental stage-specific cytodifferentiation. Human fetal lung tissue (pseudoglandular stage) was grafted either beneath the renal capsule or the skin of athymic mice (NCr-nu). Tissues were analyzed from 3 to 42 days post-engraftment for morphological alterations by light and electron microscopy (EM), and for surfactant protein mRNA and protein by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry (ICC), respectively. The changes observed resemble those seen in human lung development in utero in many respects, including the differentiation of epithelium to the saccular stage. Each stage occurred over approximately one week in the graft in contrast to the eight weeks of normal in utero development. At all time points examined, all four surfactant proteins (SP-A, SP-B, SP-C, and SP-D) were detected in the epithelium by ICC. Lamellar bodies were first identified by EM in 14 day xenografts. By day 21, a significant increase in lamellar body expression was observed. Cellular proliferation, as marked by PCNA ICC and elastic fiber deposition resembled those of canalicular and saccular in utero development. This model in which xenografted lung tissue in different stages of development is available may facilitate the study of human fetal lung development and the impact of various pharmacological agents on this process.

Surfactant protein A, an innate immune factor, is expressed in the vaginal mucosa and is present in vaginal lavage fluid.

Surfactant protein A (SP-A), first identified as a component of the lung surfactant system, is now recognized to be an important contributor to host defence mechanisms. SP-A can facilitate phagocytosis by opsonizing bacteria, fungi and viruses, stimulate the oxidative burst by phagocytes and modulate pro-inflammatory cytokine production by phagocytic cells. SP-A can also provide a link between innate and adaptive immune responses by promoting differentiation and chemotaxis of dendritic cells. Because of the obvious relevance of these mechanisms to the host defence and 'gate keeping' functions of the lower genital tract, we examined human vaginal mucosa for SP-A protein and transcripts and analysed vaginal lavage fluid for SP-A. By immunocytochemistry, SP-A was identified in two layers of the vaginal epithelium: the deep intermediate layer (the site of newly differentiated epithelial cells); and the superficial layer (comprising dead epithelial cells), where SP-A is probably extracellular and associated with a glycocalyx. Transcripts of SP-A were identified by Northern blot analysis in RNA isolated from vaginal wall and shown, by sequencing of reverse transcription-polymerase chain reaction products, to be derived from each of the two closely related SP-A genes, SP-A1 and SP-A2. SP-A was identified in vaginal lavage fluid by two-dimensional gel electrophoresis, and confirmed by mass spectrometry. This study provides evidence, for the first time, that SP-A is produced in a squamous epithelium, namely the vaginal mucosa, and has a localization that would allow it to contribute to both the innate and adaptive immune response. The findings support the hypothesis that in the vagina, as in lung, SP-A is an essential component of the host-defence system. A corollary hypothesis is that qualitative and quantitative alterations of normal SP-A may play a role in the pathogenesis of lower genital tract inflammatory conditions.

Expression of UGT2B7, a UDP-glucuronosyltransferase implicated in the metabolism of 4-hydroxyestrone and all-trans retinoic acid, in normal human breast parenchyma and in invasive and in situ breast cancers.

Glucuronidation, mediated by UDP-glucuronosyltransferases (UGTs), affects the actions and disposition of diverse endo- and xenobiotics. In the case of catecholestrogens (CEs), glucuronidation is likely to block their oxidation to quinone estrogens that are the putative mediators of CEs' actions as initiators of cancers. The goal of this study was to determine whether UGT2B7, the isoenzyme with a high affinity for 4-hydroxyestrone, is expressed in human breast parenchyma. Glucuronidation of 4-hydroxyestrone has relevance to breast carcinogenesis because quinone metabolites of 4-hydroxylated CEs can form potentially mutagenic depurinating DNA adducts, and because in breast tissue estrone is likely to be the predominant estrogen available for 4-hydroxylation. Using reverse transcriptase-polymerase chain reaction, immunocytochemistry, immunoblot analyses, and assays of glucuronidation of 4-hydroxyestrone, we show that UGT2B7 is expressed in human mammary epithelium, and that its expression is dramatically reduced in invasive breast cancers. In many in situ carcinomas, however, 4-hydroxyestrone immunostaining was not only preserved but even more intense than in normal mammary epithelium. The finding of reduced UGT2B7 protein and glucuronidation of 4-hydroxyestrone in invasive cancers suggests a tumor-suppressor function for the enzyme. Recent identification of all-trans retinoic acid as a substrate of UGT2B7 suggests that this function includes the generation of retinoyl-beta-glucuronide, a potent mediator of actions of retinoids important for maintaining epithelia in a differentiated state. Current knowledge does not provide any ready explanation for the apparent increase in UGT2B7 expression in carcinomas in situ. However, this finding, together with reduced immunostaining at loci showing breach of the basement membrane (microinvasion), suggests involvement of UGT2B7-catalyzed reaction(s) in protection against invasion of surrounding tissue by cancer cells.