Label-free analysis of breast tissue polarity by Raman imaging of lipid phase

The formation of the basoapical polarity axis in epithelia is critical for maintaining the homeostasis of differentiated tissues. Factors that influence cancer development notoriously affect tissue organization. Apical polarity appears as a specific tissue feature that, once disrupted, would facilitate the onset of mammary tumors. Thus, developing means to rapidly measure apical polarity alterations would greatly favor screening for factors that endanger the breast epithelium. A Raman scattering-based platform was used for label-free determination of apical polarity in live breast glandular structures (acini) produced in three-dimensional cell culture. The coherent anti-Stokes Raman scattering signal permitted the visualization of the apical and basal surfaces of an acinus. Raman microspectroscopy subsequently revealed that polarized acini lipids were more ordered at the apical membranes compared to basal membranes, and that an inverse situation occurred in acini that lost apical polarity upon treatment with Ca(2+)-chelator EGTA. This method overcame variation between different cultures by tracking the status of apical polarity longitudinally for the same acini. Therefore, the disruption of apical polarity by a dietary breast cancer risk factor, ω6 fatty acid, could be observed with this method, even when the effect was too moderate to permit a conclusive assessment by the traditional immunostaining method.     

Polar protein transport between apical and basal cells during tobacco early embryogenesis

Key message

We found that protein trafficking between apical and basal cell can be unidirectional, which reveals the different roles of the two cells in the cell-to-cell communication between them during early embryogenesis.

Abstract

In most angiosperm species, asymmetric zygote division results in an apical cell and a basal cell that have distinct cell fates. Much has been speculated about possible communication between these cell types in relation to their cell fate determination. Here, we report on the use of photoactivatable green fluorescent protein (PA-GFP) in tobacco to trace intercellular communication between apical and basal cells during early embryogenesis. We found that PA-GFP was transported between apical and basal cells of a two-celled proembryo, and that protein trafficking was unidirectional toward the apical cell, highlighting different cell communication roles. Further ultrastructural analysis showed numerous plasmodesmata in the walls connecting the apical and basal cells, which may provide channels for protein trafficking. Our data show a possible unique method of cell-to-cell communication between apical and basal cells during early embryogenesis.     

Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane

An essential feature of mammary gland differentiation during pregnancy is the formation of alveoli composed of polarized epithelial cells, which, under the influence of lactogenic hormones, secrete vectorially and sequester milk proteins. Previous culture studies have described either organization of cells polarized towards lumina containing little or no demonstrable tissue-specific protein, or establishment of functional secretory cells exhibiting little or no glandular architecture. In this paper, we report that tissue-specific vectorial secretion coincides with the formation of functional alveoli-like structures by primary mammary epithelial cells cultured on a reconstituted basement membrane matrix (derived from Engelbreth-Holm-Swarm murine tumour). Morphogenesis of these unique three-dimensional structures was initiated by cell-directed remodelling of the exogenous matrix leading to reorganization of cells into matrix-ensheathed aggregates by 24 h after plating. The aggregates subsequently cavitated, so that by day 6 the cells were organized into hollow spheres in which apical cell surfaces faced lumina sealed by tight junctions and basal surfaces were surrounded by a distinct basal lamina. The profiles of proteins secreted into the apical (luminal) and basal (medium) compartments indicated that these alveoli-like structures were capable of an appreciable amount of vectorial secretion. Immunoprecipitation with a broad spectrum milk antiserum showed that more than 80% of caseins were secreted into the lumina, whereas iron-binding proteins (both lactoferrin and transferrin) were present in comparable amounts in each compartment. Thus, these mammary cells established protein targeting pathways directing milk-specific proteins to the luminal compartment. A time course monitoring secretory activity demonstrated that establishment of tissue-specific vectorial secretion and increased total and milk protein secretion coincided with functional alveolar-like multicellular architecture. This culture system is unique among models of epithelial cell polarity in that it demonstrates several aspects of epithelial cell polarization: vectorial secretion, apical junctions, a sequestered compartment and formation of a basal lamina. These lumina-containing structures therefore reproduce the dual role of mammary epithelia to secrete vectorially and to sequester milk proteins. Thus, in addition to maintaining tissue-specific cytodifferentiation and function, a basement membrane promotes the expression of tissue-like morphogenesis.     

Tissue distribution of cloxacillin after intramammary administration in the isolated perfused bovine udder

Background

Various intramammary suspensions containing cloxacillin benzathine are registered for use in cattle as antibiotics for intramammary use at drying off. To ensure antibacterial efficacy, the glandular tissue concentration of an antimicrobial agent must be sufficient. Since the possibilities to measure concentrations in the different areas of the glandular tissue in vivo are very limited, it was the aim of the present study to examine the distribution of cloxacillin in vitro using the isolated perfused bovine udder.

Methods

Mammary glands taken at slaughter from healthy lactating cows were perfused in vitro with warmed and gassed Tyrode solution. 600 mg cloxacillin benzathine were administered as Orbenin Extra Dry Cow by the intramammary route to six front and rear quarters each. Samples of glandular tissue - at different distances from and vertical to the teat right up to the udder base - were gathered from the treated quarters after 6 h. Perfusate was also sampled before and hourly after treatment for 6 h. The cloxacillin content of the tissue samples and perfusate samples was analysed by high performance liquid chromatography.

Results

The concentration of cloxacillin in the glandular tissue of front quarters measured 6 h after administration tended to decrease with increasing vertical distance from the teat. The decrease pattern of the concentration was not quite clear in rear quarters. A considerable variation in the tissue concentrations of cloxacillin was obvious, which reflects in vivo conditions. The concentrations measured in the perfusate samples were below the limit of quantification at all time points, indicating limited absorption of the antibiotic from the glandular tissue.

Conclusion

After intramammary administration of the dry off product containing cloxacillin benzathine concentrations of more than 0.5 μg/g (MIC) were reached in all regions of the front and rear quarters.     

DEAR1 Is a Dominant Regulator of Acinar Morphogenesis and an Independent Predictor of Local Recurrence-Free Survival in Early-Onset Breast Cancer

Background

Breast cancer in young women tends to have a natural history of aggressive disease for which rates of recurrence are higher than in breast cancers detected later in life. Little is known about the genetic pathways that underlie early-onset breast cancer. Here we report the discovery of DEAR1 (ductal epithelium–associated RING Chromosome 1), a novel gene encoding a member of the TRIM (tripartite motif) subfamily of RING finger proteins, and provide evidence for its role as a dominant regulator of acinar morphogenesis in the mammary gland and as an independent predictor of local recurrence-free survival in early-onset breast cancer.

Methods and Findings

Suppression subtractive hybridization identified DEAR1 as a novel gene mapping to a region of high-frequency loss of heterozygosity (LOH) in a number of histologically diverse human cancers within Chromosome 1p35.1. In the breast epithelium, DEAR1 expression is limited to the ductal and glandular epithelium and is down-regulated in transition to ductal carcinoma in situ (DCIS), an early histologic stage in breast tumorigenesis. DEAR1 missense mutations and homozygous deletion (HD) were discovered in breast cancer cell lines and tumor samples. Introduction of the DEAR1 wild type and not the missense mutant alleles to complement a mutation in a breast cancer cell line, derived from a 36-year-old female with invasive breast cancer, initiated acinar morphogenesis in three-dimensional (3D) basement membrane culture and restored tissue architecture reminiscent of normal acinar structures in the mammary gland in vivo. Stable knockdown of DEAR1 in immortalized human mammary epithelial cells (HMECs) recapitulated the growth in 3D culture of breast cancer cell lines containing mutated DEAR1, in that shDEAR1 clones demonstrated disruption of tissue architecture, loss of apical basal polarity, diffuse apoptosis, and failure of lumen formation. Furthermore, immunohistochemical staining of a tissue microarray from a cohort of 123 young female breast cancer patients with a 20-year follow-up indicated that in early-onset breast cancer, DEAR1 expression serves as an independent predictor of local recurrence-free survival and correlates significantly with strong family history of breast cancer and the triple-negative phenotype (ER⁻, PR⁻, HER-2⁻) of breast cancers with poor prognosis.

Conclusions

Our data provide compelling evidence for the genetic alteration and loss of expression of DEAR1 in breast cancer, for the functional role of DEAR1 in the dominant regulation of acinar morphogenesis in 3D culture, and for the potential utility of an immunohistochemical assay for DEAR1 expression as an independent prognostic marker for stratification of early-onset disease.     

Morphogenesis and secretory activity of mouse mammary cultures on EHS biomatrix

Summary The nature of the substratum profoundly influences the growth and function of epithelia in tissue culture. Mammospheres, hollow spherical structures, develop when epithelial clusters are plated on a biomatrix derived from the Engelbreth-Holm-Swarm murine tumour (EHS matrix). Morphologic examination of mammosphere development demonstrates that morphogenesis is a two stage process. Over the first 48 h the cells aggregate into spheres, drawing the matrix up and over themselves to become buried within the material. Changes in matrix morphology emphasize the importance of the quasi-fluid nature of the substratum on which the cells are plated. Lumen formation ensues over the next 2 to 5 days as the cells, polarized by basal contact with the matrix, differentiate. They form tight junctions at their apical borders and synthesize milk proteins, secreting caseins into the enlarging interior cavity and transferrin from their basal surfaces into the medium. These experiments demonstrate that the physical properties of the EHS matrix allow epithelial cells to develop the cuboidal shape necessary for secretory activity.Abbreviations EHS Engelbreth-Holm-Swarm biomatrix     

Association of cytokines with endothelium dependent flow mediated vasodilation (FMD) of systemic arteries in patients with non-ischemic cardiomyopathy

Background

Takotsubo cardiomyopathy is an acute cardiac syndrome characterized by transient LV regional wall motion abnormalities (with peculiar apical ballooning appearance), chest pain or dyspnea, ST-segment elevation and minor elevations of cardiac enzyme levels

Case presentation

A 68-year-old woman was admitted to the Emergency Department because of sudden onset chest pain occurred while transferring her daughter, who had earlier suffered a major seizure, to the hospital. The EKG showed sinus tachycardia with ST-segment elevation in leads V2–V3 and ST-segment depression in leads V5–V6, she was, thus, referred for emergency coronary angiography. A pre-procedural transthoracic echocardiogram revealed regional systolic dysfunction of the LV walls with hypokinesis of the mid-apical segments and hyperkinesis of the basal segments. Coronary angiography showed patent epicardial coronary arteries; LV angiography demonstrated the characteristic morphology of apical ballooning with hyperkinesis of the basal segments and hypokinesis of the mid-apical segments. The post-procedural course was uneventful; on day 5 after admission the echocardiogram revealed full recovery of apical and mid-ventricular regional wall-motion abnormalities.

Conclusion

Takotsubo cardiomyopathy is a relatively rare, unique entity that has only recently been widely appreciated. Acute stress has been indicated as a common trigger for the transient LV apical ballooning syndrome, especially in postmenopausal women. The present report is a typical example of stress-induced takotsubo cardiomyopathy in a Caucasian Italian postmenopausal woman.     

Age dependent association of endometrial polyps with increased risk of cancer involvement

Background

Most of gastric adenocarcinoma can be simply diagnosed by microscopic examination of biopsy specimen. Rarely the structural and cellular atypia of tumor cells is too insignificant to discriminate from benign foveolar epithelium.

Case presentation

A 67-year-old male presented with a gastric mass incidentally found on the abdominal computed tomography (CT) for routine medical examination. Gastric endoscopic examination revealed a huge fungating mass at the cardia and mucosal biopsy was performed. Microscopically the biopsy specimen showed proliferation of bland looking foveolar epithelia in the inflammatory background and diagnosed as foveolar epithelial hyperplasia. Because the clinical and endoscopic features of this patient were strongly suggestive of malignancy, the patient underwent radical total gastrectomy. The resected stomach revealed a huge fungating tumor at the cardia. The cut surface of the tumor was whitish gelatinous. Microscopically the tumor was sharply demarcated from surrounding mucosa and composed of very well formed glandular structures without significant cellular atypia, which invaded into the whole layer of the gastric wall. Tumor glands were occasionally complicated or dilated, and glandular lumina were filled with abundant mucin. Immunohistochemically the tumor cells revealed no overexpression of p53 protein but high Ki-67 labeling index. The tumor cells and intraluminal mucin were diffusely expressed MUC1 and MUC5AC and only focally expressed MUC2. On abdominal CT taken after 12 months demonstrated peritoneal carcinomatosis and multiple metastatic foci in the lung.

Conclusion

The clinicopathologic profiles of gastric extremely well differentiated adenocarcinoma of gastric phenotype include cardiac location, fungating gross type, very similar histology to foveolar epithelial hyperplasia, foveolar mucin phenotype, lack of p53 overexpressoin and high proliferative index.     

Three dimensional culture of fresh and vitrified mouse pre-antral follicles in a hyaluronan-based hydrogel: a preliminary investigation of a novel biomaterial for in vitro follicle maturation

Background

Folliculogenesis within the ovary requires interaction between somatic cell components and the oocyte. Maintenance of 3-dimensional (3-D) architecture and granulosa-oocyte interaction may be critical for successful in vitro maturation of follicles. Testing of novel biomaterials for the 3-D culture of follicles may ultimately lead to a culture model that can support the longer in vitro culture intervals needed for in vitro maturation of human oocytes from ovarian tissue biopsies.

Methods

A novel tyramine-based hyaluronan (HA) hydrogel was tested for its biocompatibility with ovarian follicles. The HA was prepared at concentrations from 2 to 5?mg/ml. HA hydrogel was also formulated and tested with matrix proteins (ECM). Enzymatically isolated pre-antral follicles from the ovaries of 10?C12?day SJL pups were divided amongst control (CT) and HA treatments. The growth of both fresh and vitrified follicles was assessed after encapsulation in the hydrogel. The basal culture medium was MEM alpha supplemented with FSH, LH, ITS and 5% FBS. Maturation was triggered by addition of hCG and EGF after in vitro culture (IVC). Outcome parameters monitored were follicle morphology, survival after IVC, antrum formation, GVBD and MII formation. Differences between treatments were analyzed.

Results

HA and ECM-HA encapsulated follicles looked healthy and maintained their 3-D architecture during IVC. In control cultures, the follicles flattened and granulosa:oocyte connections appeared fragile. Estradiol secretion per follicle was significantly higher by Day 12 in ECM-HA compared to HA or CT (4119, 703 and 1080?pg/ml, respectively). HA and ECM-HA cultured follicles had similar survival rates (62% and 54%, respectively), percent GV breakdown (96?C97%), MII formation (47?C48%) and oocyte diameters at the end of IVC. Control cultures differed significantly in percent GVBD (85%) and MII formation (67%) . Vitrified-warmed follicles encapsulated in HA had an oocyte maturation rate to MII of 54% as compared to 57% in non-embedded follicles.

Conclusions

Initial testing of this new and unique HA-based hydrogel was quite promising. The ease of follicle encapsulation in HA, its optical transparency and ability to be molded combined with its support of follicle growth, estradiol secretion and resumption of meiosis make this HA-hydrogel particularly attractive as model for 3-D ovarian follicle culture.     

Drosophila aPKC regulates cell polarity and cell proliferation in neuroblasts and epithelia

Cell polarity is essential for generating cell diversity and for the proper function of most differentiated cell types. In many organisms, cell polarity is regulated by the atypical protein kinase C (aPKC), Bazooka (Baz/Par3), and Par6 proteins. Here, we show that Drosophila aPKC zygotic null mutants survive to mid-larval stages, where they exhibit defects in neuroblast and epithelial cell polarity. Mutant neuroblasts lack apical localization of Par6 and Lgl, and fail to exclude Miranda from the apical cortex; yet, they show normal apical crescents of Baz/Par3, Pins, Inscuteable, and Discs large and normal spindle orientation. Mutant imaginal disc epithelia have defects in apical/basal cell polarity and tissue morphology. In addition, we show that aPKC mutants show reduced cell proliferation in both neuroblasts and epithelia, the opposite of the lethal giant larvae (lgl) tumor suppressor phenotype, and that reduced aPKC levels strongly suppress most lgl cell polarity and overproliferation phenotypes.     

The non-invasive documentation of coronary microcirculation impairment: role of transthoracic echocardiography

Background

Multiples indices have been described using tissue Doppler imaging (DTI) capabilities. The aim of this study was to assess the capability of one or several regional DTI parameters in separating control from ischemic myocardium.

Methods

Twenty-eight patients with acute myocardial infarction were imaged within 24-hour following an emergent coronary angioplasty. Seventeen controls without any coronary artery or myocardial disease were also explored. Global and regional left ventricular functions were assessed. High frame rate color DTI cineloop recordings were made in apical 4 and 2-chamber for subsequent analysis. Peak velocity during isovolumic contraction time (IVC), ejection time, isovolumic relaxation (IVR) and filling time were measured at the mitral annulus and the basal, mid and apical segments of each of the walls studied as well as peak systolic displacement and peak of strain.

Results

DTI-analysis enabled us to discriminate between the 3 populations (controls, inferior and anterior AMI). Even in non-ischemic segments, velocities and displacements were reduced in the 2 AMI populations. Peak systolic displacement was the best parameter to discriminate controls from AMI groups (wall by wall, p was systematically < 0.01). The combination IVC + and IVR< 1 discriminated ischemic from non-ischemic segments with 82% sensitivity and 85% specificity.

Conclusion

DTI-analysis appears to be valuable in ischemic heart disease assessment. Its clinical impact remains to be established. However this simple index might really help in intensive care unit routine practice.     

Percutaneous septal ablation for left mid-ventricular obstructive hypertrophic cardiomyopathy: a case report

Background

Mid-ventricular obstructive hypertrophic cardiomyopathy (MVOHC) is a rare type of cardiomyopathy. The diagnosis is based on the hourglass appearance on the left ventriculogram and the presence of pressure gradient between apical and basal chamber of the ventriculum on the hemodynamic assessment.

Case presentation

The present case represents successful percutaneous treatment with septal ablation to patient with MVOHC associated with systolic anterior motion of the mitral valve and obstruction at both the mid-ventricular and outflow levels.

Conclusion

Alcohol septal ablation has been proposed as less invasive alternatives to surgery in patients with MVOHC.     

Mechanical compression attenuates normal human bronchial epithelial wound healing

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

Amputation du gland lors de la circoncision: A propos de 19 cas

Purpose

To assess the clinical features and describe treatment difficulties associated with glandular amputation during circumcision in our daily urological practice in Senegal and to warn about the risks of this procedure, which remain underestimated in this country.

Patients and Methods

The authors report a retrospective series of 19 patients who suffered glandular amputation during circumcision and who were admitted to their urology unit. The medical charts of patients hospitalised during this period were studied.

Results

The mean age of these patients was 7.6 years. 84.2% of patients presented with complete glandular amputation. The mean time to consultation was 17.6 months generally in a context of urinary tract complications. Surgical treatment consisted of meatoplasty in 68.4% of cases. Four cases were treated by glandular reimplantation which unfortunately failed.

Conclusion

The severity of this complication and the difficulties of management in our regions must encourage prevention.     

Overgrowth of Douglas fir (Pseudotsuga menziesii Franco) stumps with regenerative tissue as an example of cell ordering and tissue reorganization

Main conclusion

Stump overgrowth may serve as a unique model for studying cellular reorganization and mechanisms responsible for cell polarity changes during the process of vascular tissue differentiation from initially unorganized parenchymatous cells. Cellular ordering and tissue reorganization during the overgrowth process of the transverse surfaces of Douglas fir stumps in forest stand was studied. At the beginning of stump overgrowth, the produced parenchymatous cells form an unorganized tissue. Particular parenchyma cells start arranging into more ordered structures which resemble rays. Application of digital image analysis software based on structure tensor was used. The analysis showed that at this stage of tissue development, cellular elements display a wide range of angular orientation values and attain very low coherency coefficients. The progress of the tissue differentiation process is associated with the formation of local regions with tracheids oriented circularly around the rays. This coincides with an increase in the range of angular orientations and greater values of coherency coefficients. At the most advanced stage of tissue development, with tracheids arranged parallelly in longitudinal strands, the degree of cell ordering is the highest what is manifested by the greatest values attained by coherency coefficients, and the narrow range of angular orientations. It is suggested that the ray-like structures could act as organizing centers in the morphogenetic field responsible for differentiation of the overgrowth tissue. The circular pattern of tracheids around rays in the initial phase of tissue development can be interpreted in terms of local rotation of the morphogenetic field which afterward is transformed into irrotational field. This transformation is noted by the presence of tracheids arranged parallelly in longitudinal strands. The possible involvement of a mechanism controlling cell polarity with respect to auxin transport is discussed.     

Amputation du gland après circoncision

Introduction

Circumcision is still the most commonly performed surgery in our society. It??s often performed by an unlicensed paramedic or even by a traditional practitioner outside the hospital under the non sterile conditions. Although, not technically difficult, circumcision may have serious complications that can endanger the sexuality or even the life of male children. The aim of this study is to emphasize the important problem of glandular amputation after circumcision.

Patients and methods

We report 8 cases of glandular amputation after circumcision performed outside the hospital within traditional methods.

Results

The mean age of these patients was 6 years (3 to 8 years). Glandular amputation occurred during circumcision in 4 cases including that 2 cases with total glandular amputation. On the other cases it was about glandular necrosis caused by inadequate use of electric lancet. An urgent reparation of glans was performed on only 3 cases. On the forth the amputed glans was unrepairable. We noticed only one case on wich glans revascularization succeeded. In case of glandular necrosis, patients consulted after 3 days. The treatment consisted of meatoplasty.

Conclusion

Glandular amputation is a rare but very severe complication of circumcision because it endangers the future sexuality of the child. So, the operation should be performed by educated and experienced personnel.