Yazarlar, maddi destek için Arizona Üniversitesi Patoloji Bölümü&#39;nü kabul etmek istiyorlar. Proje kısmen WZ&#39;ye Mark ve Jane Gibson bağış fonu tarafından desteklenmektedir.

Bu çalışmanın yürütülmesinden dolayı University of Arizona tarafından Kurumsal Değerlendirme Kurulu onayı alındı. Bilgilendirilmiş hasta onay formları elde edildi. 1. Hasta Seçimi NOT: Bir Kurumsal Gözden Geçirme Kurulu onayı Arizona Üniversitesi&#39;nden alınmıştır. Toplam 38 hasta çalışmaya alındı. Hastaların yaşları 32-86 arasında değişmekte olup, ortalama yaş 55, bunlardan 26&#39;sı menopoz sonrası, 12&#39;si menopoz öncesi idi. <ol><li> Her bir hastanın bilgilendirilmiş onayı alın. </li></ol> 2. Fallop Tüp Hücreleri Toplama Prosedürü NOT: Mevcut çalışmaya dahil olan hastaların profilaktik risk azalması veya malignite için total histerektomi ve bilateral salpingo-oofrektomi yapılması planlandı. Ameliyatın ayrıntıları, sitoloji çalışmasını yapan patolog için bilinmemektedir. <ol><li> Surgi bittikten hemen sonraRahim, iki taraflı yumurtalıklar ve bilateral fallop tüplerini içeren taze doku örneğini inceleyerek ikili fallop tüpünü tanımlayın. </li><li> Kan kaynağını kelepçelendirerek 30 dakika içinde fallop tüp hücrelerini koruyucunun içine yerleştirin (bakınız Malzeme Tablosu). Önerilen koruyucu, metanol bazlı, tamponlanmış bir koruyucu çözeltidir. NOT: Tüp hücresi toplanması için hafif bir dokunmatik fırça kullanın (gelecek adımlar). Fallop tüpünün uterusdan ayrıldığı ender durumlarda hariç olmak üzere, tubal fimbriadan hücreleri toplamak için bir fırça kafası takmak, genellikle fallop tüpü uterusa bağlandığında gerçekleşir. <ol><li> Küçük bir forseps yardımı ile fırçayı fimbrianın ucundan fallop tüpünün lümenine sokun (bkz . Malzeme Tablosu ). Yavaşça saat yönünde döndürün ve fırçayı sonbaharın infundibular kısmı boyunca öne doğru hareket ettirinOpian tüpünü, ardından ampülden isthusa ulaşana kadar. </li><li> Fırçayı dışarı çekmek için yavaşça döndürün ve fırçayı saat yönünün tersine tersine çevirin. Fırçanın lümen içindeki maksimum hızının 1 cm / s&#39;den daha yavaş olduğundan emin olun. </li><li> Fırça, solüsyonda 10 kez döndürüp döndürerek koruyucu bir solüsyonda (bir flakondaki) durulayın. </li><li> Şişe kapağını sıkıştırın. </li><li> Hastanın bilgilerini ve numunenin yanallığını kaydedin. </li><li> Şişeyi 4 ° C&#39;lik bir buzdolabında (6 ay kadar) saklayın. </li></ol></li></ol> 3. Tubal Sitoloji Slayt Hazırlama <ol><li> Slayt hazırlığı için örnek şişeyi otomatik işlemciye yükleyin. NOT: Otomatik işlemciyi slayt hazırlığı için kullanırız. Aşağıdaki adımlar, numune şişesinin işlemciye yerleştirilmesinden sonra otomatik olarak gerçekleştirilir: (i) Hücreler ve enkaz, numunedeki test filtresinin dönüşü ile ayrılır ve dağılır.şişe. (Ii) Tüp hücreleri, hafif bir vakumla filtrenin dış yüzeyinden toplanır. (Iii) Filtrenin ters çevrilmesi ve hücrelerin slayt içindeki dairesel bölgeye yapışmasına izin vermek için slaydın üzerine basılması. (Iv) Slayt, bir hücre fiksatif banyosunda sabitlenir ve boyama ve sitoloji analizi için hazırdır. Ne yazık ki, benzer kalitede sonuçlar verebilecek alternatif bir manuel yöntem yok. </li></ol> 4. Değiştirilmiş Papanicolaou Boyama Protokolü <ol><li> Tablo 1&#39;de gösterildiği gibi, otomatize non-gyn boyama prosedürünü çalıştırarak adım 3.1&#39;de hazırlanan slaytları boyayın. NOT: Boyama yöntemi, jinekolojik olmayan örnekler için rutin olarak kullandığımız Papanicolaou boyama tekniğinin bir değişikliğini temsil eder. Bazı slaytları leke yapmak için otomatik bir doku işlemcisi kullanıyoruz. Bu çalışmada Gyn boyama prosedürü üzerinden Non-Gyn boyama prosedürünü seçtik. Kullanılan Eosin, EA-65 yerine EA-50 olarak kullanılır.Jinekolojik numuneler (Pap smear örneği). Ek olarak, esasen skuamöz hücre boyaması için olan Gyn boyama prosedüründe OG-6, boyama prosedürüne dahil değildir, çünkü bu çalışmada skuamoz hücrelerin görülmesi beklenmemektedir. EA65 (Eosin Azure), Eozin Y, Hızlı yeşil FCF ve Bismarck kahverengi Y olmak üzere 3 boya içeren bir sayaç boyama çözeltisidir. Prosedür, Tablo 1&#39;de gösterilmektedir. </li><li> Aşağıdaki gibi manuel hızlı bir Pap boyama prosedürü uygulayın. <ol><li> Adım 3.1&#39;de hazırlanan slaydı, her bir daldırma yaklaşık 1 s süren 10 kat% 95 etanol içine daldırın. </li><li> Yaklaşık 1 saniye alan her daldırma ile, H2O, 10 kat içine slayt batırın. </li><li> Slayt 10 - 60 s boyunca hematoksilen içinde görünür hale getirilir. </li><li> Yaklaşık 1 saniye alan her daldırma ile, H2O, 10 kat içine slayt batırın. </li><li> Her daldırma yaklaşık 1 s süren slayt% 95 etanol içine 10 kez batırın. </li><li> EA65&#39;deki slaydı 1 - 3 dakika süreyle ortaya çıkarın. </li><li> Dip slayt intH 2 O, 10 defa, her bir daldırma yaklaşık 1 s sürer. </li><li> Her daldırma yaklaşık 1 s süren slayt% 95 etanol içine 10 kez batırın. </li><li> Slayt kaybolana kadar slaydonu ksilen içine daldırın. NOT: Bu, değiştirilmiş bir Papanicolaou boyama prosedürüdür. Bu yöntem yerinde ince iğne aspirasyonu ve STAT numuneleri gibi sınırlı zaman veya mekan olan vakalar için daha hızlı bir leke yöntemi olarak kullanılır. Bu prosedür, otomatize Non-Gyn boyama prosedürü olarak karşılaştırılabilir kaliteli lekeler sağlar. İşlem Tablo 2&#39;de gösterilmektedir. </li></ol></li></ol> 5. Hücreleri Slaytlara Döndürün NOT: Her numune için üç sitospin slayt yapılır. Bir slayt, sitoloji slaydı ile morfolojik karşılaştırma için boyanmış H &amp; E&#39;dir. Gelecekteki IHC boyama çalışması için iki lekelenmemiş slayt yapılmıştır. Bu bölümün ayrıntılı adımları bu makalenin odak noktası değildir; Bu nedenle, onları genişletilmiş d&#39;de tartışmayacağızetails. <ol><li> İncelenecek her numune için etiketli bir slayt, numune hunisi ve filtre ile bir sitosentrifüj hazırlayın. </li><li> 200 x g&#39;de 5 dakika santrifüj ederek hücreleri konsantre hale getirin. </li><li> Süpernatanın yaklaşık yarısını çıkarın ve yavaşça pipetle hücreleri tekrar askıya alın. </li><li> Her bir hücre süspansiyonunun 200 μL&#39;ini örnek hunisine ekleyin. </li><li> 5 dakika 250 x g&#39;de döndürün. Slaytları sitozentrifüjden dikkatlice çıkarın ve tespit için% 95 etanol&#39;e aktarın. </li><li> Boyamadan önce ve uzun süre saklama için hava kurur. NOT: Son slaytlardaki hücre yoğunluğuna bağlı olarak, adım 5.2 ve adım 5.3 atlanabilir veya ayarlanabilir. Bu çalışmada, yeterli hücre yoğunluğu için yüksek güç görünümü başına en az 2 hücre kümesi mevcut olmasını gerektirir. </li></ol> 6. Fimbriasyon Sonu (SEE-FIM) Protokolünü Bölümlendirme ve Kapsamlı Olarak İnceleme <ol><li> Tüm örneği düzeltin (rahim, bilateral fallop tüpleri ve yumurtalıklar dahil)Eksfoliyasyonu en aza indirgemek için grossing öncesi% 10 formalin içinde. </li><li> Küçük forseps ve makas kullanarak fallop tüplerini izostan dokudaki uterusdan hafifçe ve dikkatle ayırın. Yapışma varsa, küçük forseps ve bir makas kullanarak yumurtalık yüzeyden tırtıklı kenarı dikkatlice parçalayın; Bu adımlar, yumurtalık ve fallop tüpleri arasındaki yakınmalarından ötürü tümör çapraz kontaminasyonunu en aza indirgemek için çok önemlidir. </li><li> Numunenin geri kalanından gelen infundibulum ve fimbriale segmenti (distal 1.0 - 2.0 cm fallop tüpü) parçalayın. </li><li> Infundibulum ve fimbriaları boyuna olarak 2 mm aralıklarla kes. Histolojik muayene için tüm bölümleri toto&#39;ya gönderin. </li><li> Ayırma ve ampül 2 - 3 mm aralıklarla kesilip tamamen gönderin. </li></ol>

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Yazarlar, rekabet eden mali çıkarlarının olmadığını beyan ettiler.

Profilaktik bilateral salpenjektomi veya salpingo-ooforektominin, BRCA gen mutasyonları ve güçlü ailesel kanser öyküsü olan kadınlar gibi yüksek riskli popülasyonlarda HGSC gelişme riskini azalttığı gösterilmiştir. Bununla birlikte ameliyatın neden olduğu kısırlık ve cerrahi menopoz ciddi sonuçlar doğurur ve özellikle doğurganlık çağındaki kadınlar için zor bir karar verir. Bir önceki çalışmada tüp sitoloji ve histoloji 17 arasında mükemmel bir korelasyon gö...

Yumurtalık yüksek dereceli seröz karsinom (HGSC), en yaygın ve öldürücü yumurtalık kanseri türüdür ve halk sağlığı için büyük bir tehdit olmaya devam etmektedir. Son on yılda, araştırmacılar HGSC vakalarının büyük çoğunluğunun yumurtalık yerine 1 , 2 , 3 , 4 , 5 , 6 fallop tüplerinden kaynaklandığını öne sürmüşlerdir. Serum tubal intraepitelyal karsinom (STIC), HGSC 1 , 7 , 8 , 9 , 10 , 11&#39;in prekürsörü olarak yaygın olarak kabul edilmektedir. Şimdiye kadar, yumurtalık kanseri erken teşhisi zor olmaya devam etmektedir. Kombine kanser antijen 125 (CA-125) ve transvajinal ultrason ile mevcut tarama protokolüHasta mortalitesi üzerine çok az etki 12 , 13 . Over kanseri saptamasında endometriyal örnekleme son derece düşük hassasiyet gösterdi 14 . 15 , 16 nolu iki öncü çalışma, iyi huylu fallop tüplerinin laparoskopik doğrudan örneklenmesinin kullanımını araştırmış ve benign tübal epitelin sitolojik özelliklerini karakterize etmiştir. Fallop tüpünden direkt örneklemenin, tümörün görüntüleme ile görülemediği erken bir aşamada STIC veya HGSC&#39;nin saptanması için pratik ve doğrudan bir yol olabileceğine inanıyoruz. Nihai amaç, yüksek risk faktörlü hastalarda minimal invaziv laparoskopik taramanın faydası olmasına rağmen, şu andaki çalışmanın asıl amacı şudur: 1) Benign tübal epitelin bazal sitolojik özelliklerini oluşturmak; Ve 2) yumurtalık kanseri tespitinde tüp sitolojisinin hassasiyetini ve özgüllüğünü test etmekErs ve kanser öncülleri. Bu nedenle, bu protokolün HGSC ve onun prekürsörü STIC 17&#39;nin saptanmasında etkinliğini test etmek için aşağıdaki temel tüp sitolojisi yöntemini geliştirdik. Bu çalışmada, düzenli olarak alınan cerrahi örneklerin büyük bir bölümünü kullandık ve düzenli grossing prosedürlerine ek olarak cerrahi olarak kesilen fallop tüpünün doğrudan örneklenmesini yaptık. Bizim son çalışmada 17 malign veya malignite şüphesi sitolojik tanı son derece HGSC (% 100) histolojik tanı ile ilişkili olduğunu göstermiştir. Buna ek olarak, tubal sitolojik değerlendirme, bu çalışmaya katılan iki histolojik olarak doğrulanmış STIC olgusunda intratubal neoplaziyi saptamıştır. Tüp sitolojisinin erken tespitte büyük potansiyele sahip olduğuna ve hasta yönetimi için değerli bilgiler sağlayacağına inanıyoruz.

<table>
	<tbody>
		<tr>
			<td>Cytobrush plus GT gentle touch of Medscand sample collection kit</td>
			<td>CooperSurgical</td>
			<td>C0112</td>
			<td></td>
		</tr>
		<tr>
			<td>ThinPrep preservCyt solution</td>
			<td>HOLOGIC</td>
			<td>234005</td>
			<td></td>
		</tr>
		<tr>
			<td>ThinPrep 2000 Processor</td>
			<td>HOLOGIC</td>
			<td>70031-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Papanicolaou Stain, EA 65</td>
			<td>sigma aldrich</td>
			<td>HT40432</td>
			<td></td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>sigma aldrich</td>
			<td>652261</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>sigma aldrich</td>
			<td>493538</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>sigma aldrich</td>
			<td>214736</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>sigma aldrich</td>
			<td>H9627</td>
			<td></td>
		</tr>
		<tr>
			<td>Sakura Tissue Tek 2000 tissue processor</td>
			<td>SAKURA</td>
			<td>TISSUE-TEK VIP 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayo Dissecting Scissors,5.5 straight</td>
			<td>Pilgrim medical equiment</td>
			<td>FA710-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemware Forceps</td>
			<td>United state plastic corp</td>
			<td>76122</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Accu-Edge Trimming blades,short</td>
			<td>SAKURA</td>
			<td>4785</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Accu-Edge Trimming blades handle,short</td>
			<td>SAKURA</td>
			<td>4786</td>
			<td></td>
		</tr>
	</tbody>
</table>

tubal cytology of the fallopian tube as a promising tool for ovarian cancer early detection

Günümüzde, over yüksek dereceli seröz karsinomanın (HGSC) büyük çoğunluğunun fallop tüpünden kaynaklandığı yaygın olarak kabul edilmektedir. Bununla birlikte, yumurtalık kanseri tanımlaması için belirteçlerin veya araçların olmamasından dolayı, HGSC&#39;nin erken teşhisi zorlayıcı olmaya devam etmektedir. Fallop tüpünün doğrudan örneklenmesi, tümör gözle görünmüyorsa neoplastik hücrelerin saptanması için duyarlılığı artırabilir. Yeni alınan cerrahi örneklerden fallop tüp hücrelerini doğrudan toplamak için bir prosedür geliştirdik ve bu histolojik bulgularla mükemmel korelasyon gösterdi. Bu yaklaşım, gelecekte yüksek riskli hasta popülasyonlarında minimal invaziv laparoskopik taramanın kullanılmasına zemin hazırlamaktadır.

Önceki çalışmamız, hafif bir dokunuş fırçası kullanılarak kesilmiş fallop tüpünden doğrudan numuneleşmenin daha fazla sitolojik değerlendirme için niceliksel ve niteliksel olarak yeterli numuneye neden olabileceğini gösterdi. Buna ek olarak, otomatik slayt hazırlama ve modifiye Papanicolaou boyama kombinasyonu, patologun doğru tanı koymak için sitolojik ayrıntılarını daha iyi görselleştirmesini sağlar. İyi huylu fallop tüplerinden alınan bir numunenin ell...

Watch this Scientific Journal Video about Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection at JoVE.com

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

Yumurtalık kanseri erken teşhisinin bir aracı olarak fallop tüpünü direkt olarak cerrahi sonrası eksizyonu örnekleyerek bir tubal sitolojik yöntem araştırdık. Burada, yeni alınan cerrahi örneklerden fallop tüp hücreleri toplamak için bir protokol sunuyoruz.

Yumurtalık Kanseri Erken Teşhisi İçin Umut Verici Bir Araç Olarak Fallop Tüpünün Tubal Sitolojisi

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to ...

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JoVE Journal

Medicine

12.2K Görüntüleme.  University of Arizona College of Medicine.  Yumurtalık kanseri erken teşhisinin bir aracı olarak fallop tüpünü direkt olarak cerrahi sonrası eksizyonu örnekleyerek bir tubal sitolojik yöntem araştırdık. Burada, yeni alınan cerrahi örneklerden fallop tüp hücreleri toplamak için bir protokol sunuyoruz. 

Video: Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

In vitro Mezotelyal Gümrükleme Assay o Yumurtalık Kanseri Metastaz Erken Adımlar modeller

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

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A Human Fallopian Tube Model for Investigation of C. trachomatis Infections

Genital tract infections with Chlamydia trachomatis (C. trachomatis) are the most frequent transmitted sexually disease in women worldwide. Inefficient clearance or persistence of the pathogens may lead to ascending infections of the upper genital tract and are supposed to cause chronic inflammatory damage to infected tissues 1,2. As a consequence, severe clinical sequelae like pelvic inflammatory disease (PID), tubal occlusion and infertility may occur 3,4. 
Most of the research with C. trachomatis has been conducted in epithelial cell lines (e.g. HEp-2 cells and HeLa-229) or in mice. However, as with cell- culture based models, they do neither reflect the physiology of native tissue nor the pathophysiology of C. trachomatis genital tract infections in vivo 5. Further limitations are given by the fact that central signaling cascades (e.g. IFN-&gamma; mediated JAK/STAT signaling pathway) that control intracellular chlamydial growth fundamentally differ between mice and humans 6,7. We and others therefore established a whole organ fallopian tube model to investigate direct interactions between C. trachomatis and human fallopian tube cells ex vivo 8,9.
For this purpose, human fallopian tubes from women undergoing hysterectomy were collected and infected with C. trachomatis serovar D. Within 24 h post infection, specimen where analyzed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to detect Chlamydia trachomatis mediated epithelial damage as well as C. trachomatis inclusion formation in the fallopian tissue.

A Human Fallopian Tube Model for Investigation of C. trachomatis Infections

We describe an ex vivo infection model for visualisation of direct interactions from bacterial pathogens with human fallopian tube cells. The whole organ tissue model was established to investigate C. trachomatis induced pathology to the female fallopian tube under "life-like" conditions.

Incelenmesi için İnsan Fallop Tüp Modeli<em&gt; C. trachomatis</em&gt; Enfeksiyonlar

Genital tract infections with Chlamydia trachomatis (C. trachomatis) are the most frequent transmitted sexually disease in women worldwide. Inefficient ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Üç boyutlu heterotipik<em&gt; In Vitro</emYumurtalık Kanseri Başlatma ve İlerleme sırasında Stromal-Epitel Etkileşimleri&gt; Modelleme

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

Kanser Tedavisi İzleme Cerenkov Lüminesans Görüntüleme (CLI)

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

<em&gt; Ovo</emSarkoma için bir Xenograftmodel olarak&gt; CAM-testi

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Osteopathic Manipulative Treatment as a Useful Adjunctive Tool for Pneumonia

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in the hospitalization of 1.1 million patients2. With an average length of in-patient hospital stay of five days2, pneumonia and influenza comprise significant financial burden costing the United States $40.2 billion in 20053. Under the current Infectious Disease Society of America/American Thoracic Society guidelines, standard-of-care recommendations include the rapid administration of an appropriate antibiotic regiment, fluid replacement, and ventilation (if necessary). Non-standard therapies include the use of corticosteroids and statins; however, these therapies lack conclusive supporting evidence4. (Figure 1)

Osteopathic Manipulative Treatment (OMT) is a cost-effective adjunctive treatment of pneumonia that has been shown to reduce patients&#8217; length of hospital stay, duration of intravenous antibiotics, and incidence of respiratory failure or death when compared to subjects who received conventional care alone5. The use of manual manipulation techniques for pneumonia was first recorded as early as the Spanish influenza pandemic of 1918, when patients treated with standard medical care had an estimated mortality rate of 33%, compared to a 10% mortality rate in patients treated by osteopathic physicians6. When applied to the management of pneumonia, manual manipulation techniques bolster lymphatic flow, respiratory function, and immunological defense by targeting anatomical structures involved in the these systems7,8, 9, 10.

The objective of this review video-article is three-fold: a) summarize the findings of randomized controlled studies on the efficacy of OMT in adult patients with diagnosed pneumonia, b) demonstrate established protocols utilized by osteopathic physicians treating pneumonia, c) elucidate the physiological mechanisms behind manual manipulation of the respiratory and lymphatic systems. Specifically, we will discuss and demonstrate four routine techniques that address autonomics, lymph drainage, and rib cage mobility: 1) Rib Raising, 2) Thoracic Pump, 3) Doming of the Thoracic Diaphragm, and 4) Muscle Energy for Rib 1.5,11

Pneumonia is one of the top ten leading causes of death in the U.S. Osteopathic manipulative techniques (OMT) may be utilized as an adjunctive therapy in the treatment of pneumonia to enhance biomechanical and immune function; and ultimately, to reduce hospital stay, duration of antibiotics, incidence of respiratory failure, and mortality. In this video-article, we will review randomized controlled studies on the use of OMT in pneumonia patients, as well as demonstrate specific hands-on techniques that are routinely practiced by osteopathic physicians when treating pulmonary infections.

Pnömoni için Faydalı adjuvan Aracı Olarak Kemik Hastalıkları Manipülatif Tedavi

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Katı Örneklerinin From İlköğretim Yumurtalık Kanseri Hücreleri alma için yöntem

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

Etkinliğinin Değerlendirilmesi<em&gt; H. pylori</emOrtotopik Fare Modelinde Mesane Kanseri Tedavisinde Terapötik Aracı Olarak&gt; Protein HP-NAP

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

İçi periton Yumurtalık Kanseri Metastaz kantitasyonu

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

İnsan periton boşluğuna bir Organotipik Kültür Yumurtalık Kanseri Yaygınlaştırılması Erken Adımlar Modelleme

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...