Search results for: stromal TIL

Authors: Aliya Sekenova, Vyacheslav Ogay

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The derivation of human induced pluripotent stem cells (iPSCs) from somatic cells by direct reprogramming opens wide perspectives in the regenerative medicine. It means the possibility to develop the personal and, consequently, any immunologically compatible cells for applications in cell-based therapy. The purpose of our study was to develop the technology for the production of NK cells from T cell-derived induced pluripotent stem cells (TiPSCs) for subsequent application in adoptive cancer immunotherapy. Methods: In this study iPSCs were derived from peripheral blood T cells using Sendai virus vectors expressing Oct4, Sox2, Klf4 and c-Myc. Pluripotent characteristics of TiPSCs were examined and confirmed with alkaline phosphatase staining, immunocytochemistry and RT-PCR analysis. For NK cell differentiation, embryoid bodies (EB) formed from (TiPSCs) were cultured in xenogeneic serum-free medium containing human serum, IL-3, IL-7, IL-15, SCF, FLT3L without using M210-B4 and AFT-024 stromal feeder cells. After differentiation, NK cells were characterized with immunofluorescence analysis, flow cytometry and cytotoxicity assay. Results: Here, we for the first time demonstrate that TiPSCs can effectively differentiate into functionally active NK cells without M210-B4 and AFT-024 xenogeneic stroma cells. Immunofluorescence and flow cytometry analysis showed that EB-derived cells can differentiate into a homogeneous population of NK cell expressing high levels of CD56, CD45 and CD16 specific markers. Moreover, these cells significantly express killing activation receptors such as NKp44 and NKp46. In the comparative analysis, we observed that NK cells derived using feeder-free culture system have more high killing activity against K-562 tumor cells, than NK cells derived by feeder-dependent method. Thus, we think that our obtained data will be useful for the development of large-scale production of NK cells for translation into cancer immunotherapy.

Keywords: induced pluripotent stem cells, NK cells, T cells, cell diffentiation, feeder cell-free culture system

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Authors: Diwei Lin, Amanda Tan, Rajinder Singh-Rai

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We present the first reported case of a concomitant Leydig cell tumor (LCT) and paratesticular leiomyoma in an adult male with a known history of bilateral cryptorchidism. An 80-year-old male presented with a 2-month history of a left testicular lump associated with mild discomfort and a gradual increase in size on a background of bilateral cryptorchidism requiring multiple orchidopexy procedures as a child. Ultrasound confirmed a lesion suspicious for malignancy and he proceeded to a left radical orchidectomy. Histopathological assessment of the left testis revealed a concomitant testicular LCT with malignant features and paratesticular leiomyoma. Leydig cell tumors (LCTs) are the most common pure testicular sex cord-stromal tumors, accounting for up to 3% of all testicular tumors. They can occur at almost any age, but are noted to have a bi-modal distribution, with a peak incidence at 6 to 10 and at 20 to 50 years of age. LCT’s are often hormonally active and can lead to feminizing or virilizing syndromes. LCT’s are usually regarded as benign but can rarely exhibit malignant traits. Paratesticular tumours are uncommon and their reported prevalence varies between 3% and 16%. They occur in a complex anatomical area which includes the contents of the spermatic cord, testicular tunics, epididymis and vestigial remnants. Up to 90% of paratesticular tumours are believed to originate from the spermatic cord, though it is often difficult to definitively ascertain the exact site of origin. Although any type of soft-tissue neoplasm can be found in the paratesticular region, the most common benign tumors reported are lipomas of the spermatic cord, adenomatoid tumours of the epididymis and leiomyomas of the testis. Genetic studies have identified potential mutations that could potentially cause LCTs, but there are no known associations between concomitant LCTs and paratesticular tumors. The presence of cryptorchidism in adults with both LCTs and paratesticular neoplasms individually has been previously reported and it appears intuitive that cryptorchidism is likely to be associated with the concomitant presentation in this case report. This report represents the first documented case in the literature of a unilateral concomitant LCT and paratesticular leiomyoma on a background of bilateral cryptorchidism.

Keywords: testicular cancer, leydig cell tumour, leiomyoma, paratesticular neoplasms

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Authors: Mohammad Nasb, Muhammad Rehan, Chen Hong

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Background Meniscus defects critically alter knee function and lead to degenerative changes. Regenerative medicine applications including stem cell transplantation have showed a promising efficacy in finding alternatives to overcome traditional treatment limitations. However, stem cell therapy remains limited due to the substantially reduced viability and inhibitory microenvironment. Since tissue growth and repair are under the control of biochemical and mechanical signals, several approaches have recently been investigated (e.g., low intensity pulsed ultrasound [LIPUS]) to promote the regeneration process. This study employed LIPUS to improve growth and osteogenic differentiation of mesenchymal stem cells derived from human embryonic stem cells to improve the regeneration of meniscus tissue. Methodology: The Mesenchymal stromal cells (MSCs) were transplanted into the epicenter of the injured meniscus in rabbits, which were randomized into two main groups: a treatment group (n=32 New Zealand rabbits) including 4 subgroups of 8 rabbits in each subgroup (LIPUS treatment, MSC treatment, LIPUS with MSC and control), and a second group (n=9) to track implanted cells and their progeny using green fluorescence protein (GFP). GFP consists of the MSC and LIPUS-MSC combination subgroups. Rabbits were then subjected to histological, immunohistochemistry, and MRI assessment. Results: The quantity of the newly regenerated tissue in the combination treatment group that had Ultrasound irradiation after mesenchymal stem cells were better at all end points. Likewise, Tissue quality scores were also greater in knees treated with both approaches compared with controls and single treatment at all end points, achieving significance at twelve and twenty-four weeks [p < 0.05], and [p = 0.008] at twelve weeks. Differentiation into type-I and II collagen-expressing cells were higher in the combination group at up to twenty-four weeks. Conclusions: the combination of mesenchymal stem cells and LIPUS showed greater adhering to the sites of meniscus injury, differentiate into cells resembling meniscal fibrochondrocytes, and improve both quality and quantity of meniscal regeneration.

Keywords: stem cells, regenerative medicine, osteoarthritis, knee

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Authors: Roberto Coppo, Francesca Orso, Daniela Dettori, Elena Quaglino, Lei Nie, Mehran M. Sadeghi, Daniela Taverna

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Malignant melanoma is currently the fifth most common cancer in the white population and it is fatal in its metastatic stage. Several research studies in recent years have provided evidence that cancer initiation and progression are driven by genetic alterations of the tumor and paracrine interactions between tumor and microenvironment. Scattered data show that the Endothelial and Smooth muscle cell-Derived Neuropilin-like molecule (ESDN) controls cell proliferation and movement of stroma and tumor cells. To investigate the role of ESDN in the tumor microenvironment during melanoma progression, murine melanoma cells (B16 or B16-F10) were injected in ESDN knockout mice in order to evaluate how the absence of ESDN in stromal cells could influence melanoma progression. While no effect was found on primary tumor growth, increased cell extravasation and lung metastasis formation was observed in ESDN knockout mice compared to wild type controls. In order to understand how cancer cells cross the endothelial barrier during metastatic dissemination in an ESDN-null microenvironment, structure, and permeability of lung blood vessels were analyzed. Interestingly, ESDN knockout mice showed structurally altered and more permeable vessels compared to wild type animals. Since cell surface molecules mediate the process of tumor cell extravasation, the expression of a panel of extravasation-related ligands and receptors was analyzed. Importantly, modulations of N-cadherin, E-selectin, ICAM-1 and VAP-1 were observed in ESDN knockout endothelial cells, suggesting the presence of a favorable tumor microenvironment which facilitates melanoma cell extravasation and metastasis formation in the absence of ESDN. Furthermore, a potential contribution of immune cells in tumor dissemination was investigated. An increased recruitment of macrophages in the lungs of ESDN knockout mice carrying subcutaneous B16-F10 tumors was found. In conclusion, our data suggest a functional role of ESDN in the tumor microenvironment during melanoma progression and the identification of the mechanisms that regulate tumor cell extravasation could lead to the development of new therapies to reduce metastasis formation.

Keywords: melanoma, tumor microenvironment, extravasation, cell surface molecules

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Authors: Maria Francesca Lavadia-Gumabao, Mary Antoinette Salvamante-Torallo

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Introduction: The fibrous intrauterine adhesions forming inside the uterus and/or cervix in Asherman syndrome can obstruct the internal cervical orifice and may present as a case of outflow tract obstruction. Asherman syndrome is often overlooked since it has no specific presentation and is undetectable by routine physical examinations or diagnostic procedures such as an ultrasound. This paper highlights the delay and elusive diagnosis of Asherman syndrome which negatively impacted the patient’s fertility and quality of life. Case presentation: A 33-year-old woman (gravida 3, para 3) who presented with secondary amenorrhea for thirteen years associated with cyclic pelvic pain and secondary infertility sought a consultation at our institution for evaluation and specialty management. The patient had no other well-established risk factors for Asherman syndrome aside from pregnancy. For more than a decade, she delayed seeking medical care. At presentation, history taking, physical examination, and ultrasound were not helpful in identifying the cause of outflow tract obstruction. Diagnostic hysteroscopy was then performed, during which extensive scarring and fibrosis completely obscured the internal cervical orifice were observed, consistent with the diagnosis of Asherman syndrome (Grade 5B). The patient then underwent ultrasound guided hysteroscopy outflow tract dilatation and responded well to the treatment as she had her menstrual period a month after the procedure and no longer had cyclic pelvic pain with a repeat ultrasound finding of an unremarkable uterus. The hispathology result of the tissues retrieved revealed myometrial fragments with associated old hemorrhage benign endometrial stromal tissues, which failed to show endometrial glands. Conclusion: The delay and elusive diagnosis of Asherman syndrome can be brought about by poor health seeking behavior of patients and difficulty in detecting this condition by routine physical examinations or diagnostic procedures such as an ultrasound. It is, therefore, necessary to include Asherman syndrome in the differential diagnosis of secondary amenorrhea and secondary infertility. With expertise in hysteroscopy, early diagnosis, proper classification in the advent of hysteroscopy, and optimal management can improve patient outcomes.

Keywords: Asherman syndrome, outflow tract obstruction, secondary amenorrhea, infertility, hysteroscopy

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Authors: Akterono Budiyati, Gita Kusumo, Teguh Putra, Fritzie Rexana, Antonius Kurniawan, Aru Sudoyo, Ahmad Utomo, Andi Utama

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The presence of the programmed death ligand-1 (PD-L1) has been used in multiple clinical trials and approved as biomarker for selecting patients more likely to respond to immune checkpoint inhibitors. However, the expression of PD-L1 is regulated in different ways, which leads to a different significance of its presence. Positive PD-L1 within tumors may result from two mechanisms, induced PD-L1 expression by T-cell presence or genetic mechanism that lead to constitutive PD-L1 expression. Amplification of PD-L1 genes was found as one of genetic mechanism which causes an increase in PD-L1 expression. In case of colorectal cancer (CRC), targeting immune checkpoint inhibitor has been recommended for patients with microsatellite instable (MSI). Although the correlation between PD-L1 expression and MSI status has been widely studied, so far the precise mechanism of PD-L1 gene activation in CRC patients, particularly in MSI population have yet to be clarified. In this present study we have profiled 61 archived formalin fixed paraffin embedded CRC specimens of patients from Medistra Hospital, Jakarta admitted in 2010 - 2016. Immunohistochemistry was performed to measure expression of PD-L1 in tumor cells as well as MSI status using antibodies against PD-L1 and MMR (MLH1, MSH2, PMS2 and MSH6), respectively. PD-L1 expression was measured on tumor cells with cut off of 1% whereas loss of nuclear MMR protein expressions in tumor cells but not in normal or stromal cells indicated presence of MSI. Subset of PD-L1 positive patients was then assessed for copy number variations (CNVs) using single Tube TaqMan Copy Number Assays Gene CD247PD-L1. We also observed KRAS mutation to profile possible genetic mechanism leading to the presence or absence of PD-L1 expression. Analysis of 61 CRC patients revealed 15 patients (24%) expressed PD-L1 on their tumor cell membranes. The prevalence of surface membrane PD-L1 was significantly higher in patients with MSI (87%; 7/8) compared to patients with microsatellite stable (MSS) (15%; 8/53) (P=0.001). Although amplification of PD-L1 gene was not found among PD-L1 positive patients, low-level amplification of PD-L1 gene was commonly observed in MSS patients (75%; 6/8) than in MSI patients (43%; 3/7). Additionally, we found 26% of CRC patients harbored KRAS mutations (16/61), so far the distribution of KRAS status did not correlate with PD-L1 expression. Our data suggest genetic mechanism through amplification of PD-L1 seems not to be the mechanism underlying upregulation of PD-L1 expression in CRC patients. However, further studies are warranted to confirm the results.

Keywords: colorectal cancer, gene amplification, microsatellite instable, programmed death ligand-1

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Authors: Mark Gavriel, Ariel J. Jaffa, Dan Grisaru, David Elad

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The human endometrium-myometrium interface (EMI) is the uterine inner barrier without a separatig layer. It is composed of endometrial epithelial cells (EEC) and endometrial stromal cells (ESC) in the endometrium and myometrial smooth muscle cells (MSMC) in the myometrium. The EMI undergoes structural remodeling during the menstruation cycle which are essential for human reproduction. Recently, we co-cultured a layer-by-layer in vitro model of EEC, ESC and MSMC on a synthetic membrane for mechanobiology experiments. We also treated the model with progesterone and β-estradiol in order to mimic the in vivo receptive uterus In the present study we analyzed the cytokines profile in a single layer of EEC the hormonal treated in vitro model of the EMI. The methodologies of this research include simple tissue-engineering . First, we cultured commercial EEC (RL95-2, ATCC® CRL-1671™) in 24-wellplate. Then, we applied an hormonal stimuli protocol with 17-β-estradiol and progesterone in time dependent concentration according to the human physiology that mimics the menstrual cycle. We collected cell supernatant samples of control, pre-ovulation, ovulation and post-ovulaton periods for analysis of the secreted proteins and cytokines. The cytokine profiling was performed using the Proteome Profiler Human XL Cytokine Array Kit (R&D Systems, Inc., USA) that can detect105 human soluble cytokines. The relative quantification of all the cytokines will be analyzed using xMAP – LUMINEX. We conducted a fishing expedition with the 4 membranes Proteome Profiler. We processed the images, quantified the spots intensity and normalized these values by the negative control and reference spots at the membrane. Analyses of the relative quantities that reflected change higher than 5% of the control points of the kit revealed the The results clearly showed that there are significant changes in the cytokine level for inflammation and angiogenesis pathways. Analysis of tissue-engineered models of the uterine wall will enable deeper investigation of molecular and biomechanical aspects of early reproductive stages (e.g. the window of implantation) or developments of pathologies.

Keywords: tissue-engineering, hormonal stimuli, reproduction, multi-layer uterine model, progesterone, β-estradiol, receptive uterine model, fertility

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Authors: Manali Jain, Neeta Singh, Raunaq Fatima, Soniya Nityanand, Mandakini Pradhan, Chandra Prakash Chaturvedi

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Isolated Congenital Heart Defect (ICHD) is the major cause of neonatal death worldwide among all forms of CHDs. A significant proportion of fetuses with ICHD die in the neonatal period if no treatment is provided. Recently, stem cell therapies have emerged as a potential approach to ameliorate ICHD in children. ICHD is characterized by cardiac structural abnormalities during embryogenesis due to alterations in the cardiomyogenic properties of a pool of cardiac progenitors/ stem cells associated with fetal heart development. The stem cells present in the amniotic fluid (AF) are of fetal origin and may reflect the physiological and pathological changes in the fetus during embryogenesis. Therefore, in the present study, the cardiomyogenic potential of AF-MSCs derived from fetuses with ICHD (ICHD AF-MSCs) has been evaluated and compared with that of AF-MSCs of structurally normal fetuses (normal AF-MSCs). Normal and ICHD AF-MSC were analyzed for the expression of cardiac progenitor markers viz., stage-specific embryonic antigen-1 (SSEA-1), vascular endothelial growth factor 2 (VEGFR-2) and platelet-derived growth factor receptor-alpha (PDGFR-α) by flow cytometry. The immunophenotypic characterization revealed that ICHD AF-MSCs have significantly lower expression of cardiac progenitor markers VEGFR-2 (0.14% ± 0.6 vs.48.80% ± 0.9; p <0.01), SSEA-1 (70.86% ± 2.4 vs. 88.36% ±2.7; p <0.01), and PDGFR-α (3.92% ± 1.8 vs. 47.59% ± 3.09; p <0.01) in comparison to normal AF-MSCs. Upon induction with 5’-azacytidine for 21 days, ICHD AF-MSCs showed a significantly down-regulated expression of cardiac transcription factors such as GATA-4 (0.4 ± 0.1 vs. 6.8 ± 1.2; p<0.01), ISL-1 (2.3± 0.6 vs. 14.3 ± 1.12; p<0.01), NK-x 2-5 (1.1 ± 0.3 vs. 14.1 ±2.8; p<0.01), TBX-5 (0.4 ± 0.07 vs. 4.4 ± 0.3; p<0.001), and TBX-18 (1.3 ± 0.2 vs. 4.19 ± 0.3; p<0.01) when compared with the normal AF-MSCs. Furthermore, immunocytochemical staining revealed that both types of AF-MSCs could differentiate into cardiovascular lineages and express cardiomyogenic, endothelial, and smooth muscle actin markers, viz., cardiac troponin (cTNT), CD31, and alpha-smooth muscle actin (α-SMA). However, normal AF-MSCs showed an enhanced expression of cTNT (p<0.001), CD31 (p<0.01), and α-SMA (p<0.05), compared to ICHD AF-MSCs. Overall, these results suggest that the ICHD-AF-MSCs have a defective cardiomyogenic differentiation potential and that the defects in these stem cells may have a role in the pathogenesis of ICHD.

Keywords: amniotic fluid, cardiomyogenic potential, isolated congenital heart defect, mesenchymal stem cells

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Authors: Masoud Sakhinia, Sajjad Ahmad

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Introduction: Though knowledge of the compositional makeup and structure of the limbal niche has progressed exponentially during the past decade, much is yet to be understood. Identifying the precise profile and role of the stromal makeup which spans the ocular surface may inform researchers of the most optimum conditions needed to effectively expand LESCs in vitro, whilst preserving their differentiation status and phenotype. Limbal fibroblasts, as opposed to corneal fibroblasts are thought to form an important component of the microenvironment where LESCs reside. Methods: The corneal stroma was tissue engineered in vitro using both limbal and corneal fibroblasts embedded within a tissue engineered 3D collagen matrix. The effect of these two different fibroblasts on LESCs and hTCEpi corneal epithelial cell line were then subsequently determined using phase contrast microscopy, histolological analysis and PCR for specific stem cell markers. The study aimed to develop an in vitro model which could be used to determine whether limbal, as opposed to corneal fibroblasts, maintained the stem cell phenotype of LESCs and hTCEpi cell line. Results: Tissue culture analysis was inconclusive and required further quantitative analysis for remarks on cell proliferation within the varying stroma. Histological analysis of the tissue-engineered cornea showed a comparable structure to that of the human cornea, though with limited epithelial stratification. PCR results for epithelial cell markers of cells cultured on limbal fibroblasts showed reduced expression of CK3, a negative marker for LESC’s, whilst also exhibiting a relatively low expression level of P63, a marker for undifferentiated LESCs. Conclusion: We have shown the potential for the construction of a tissue engineered human cornea using a 3D collagen matrix and described some preliminary results in the analysis of the effects of varying stroma consisting of limbal and corneal fibroblasts, respectively, on the proliferation of stem cell phenotype of primary LESCs and hTCEpi corneal epithelial cells. Although no definitive marker exists to conclusively illustrate the presence of LESCs, the combination of positive and negative stem cell markers in our study were inconclusive. Though it is less traslational to the human corneal model, the use of conditioned medium from that of limbal and corneal fibroblasts may provide a more simple avenue. Moreover, combinations of extracellular matrices could be used as a surrogate in these culture models.

Keywords: cornea, Limbal Stem Cells, tissue engineering, PCR

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Authors: Paola Millare, Nelinda Catherine Pangilinan

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Background and Aim: Cervical cancer is the most commonly diagnosed gynecological malignancy during pregnancy. Owing to the rarity of the disease, and the complexity of all factors that have to be taken into consideration, standardization of treatment is very difficult. Cervical cancer is the second most common malignancy among women. The treatment of cancer during pregnancy is most challenging in the case of cervical cancer, since the pregnant uterus itself is affected. This report aims to present a case of cervical cancer in a pregnant woman and how to manage this case and several issues accompanied with it. Methods: This is a case of a 28 year-old, Gravida 4 Para 2 (1111), who presented with watery to mucoid, whitish, non-foul smelling and increasing in amount. Internal examination revealed normal external genitalia, parous outlet, cervix was transformed into a fungating mass measuring 5x4 cm, with left parametrial involvement, body of uterus was enlarged to 24 weeks size, no adnexal mass or tenderness. She had cervical punch biopsy, which revealed, adenocarcinoma, well-differentiated cervical tissue. Standard management for cases with stage 2B cervical carcinoma was to start radiation or radical hysterectomy. In the case of patients diagnosed with cervical cancer and currently pregnant, these kind of management will result to fetal loss. The patient still declined the said management and opted to delay the treatment and wait for her baby to reach at least term and proceed to cesarean section as route of delivery. Results: The patient underwent an elective cesarean section at 37th weeks age of gestation, with an outcome of a term, live baby boy APGAR score 7,9 birthweight 2600 grams. One month postpartum, the patient followed up and completed radiotherapy, chemotherapy and brachytherapy. She was advised to go back after 6 months for monitoring. On her last check up, an internal examination was done which revealed normal external genitalia, vagina admits 2 fingers with ease, there is a palpable fungating mass at the cervix measuring 2x2 cm. A repeat gynecologic oncologic ultrasound was done revealing cervical mass, endophytic, grade 1 color score with stromal invasion 35% post radiation reactive lymph nodes with intact paracolpium, pericervical, and parametrial involvement. The patient was then advised to undergo pelvic boost and for close monitoring of the cervical mass. Conclusion: Cervical cancer in pregnancy is rare but is a dilemma for women and their physicians. Treatment should be multidisciplinary and individualized following careful counseling. In this case, the treatment was clearly on the side of preventing the progression of cervical cancer while she is pregnant, however due to ethical reasons, the management deviates on the right of the patient to decide for her own health and her unborn child. The collaborative collection of data relating to treatment and outcome is strongly encouraged.

Keywords: cancer, cervical, ethical, pregnancy

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Authors: U. E. Mochalova, A. V. Demeneva, Shilkin A. G., J. Yu. Artiushina

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Objective. In medical ophthalmology OCT has been actively used in the last decade. It is a modern non-invasive method of high-precision hardware examination, which gives a detailed cross-sectional image of eye tissues structure with a high level of resolution, which provides in vivo morphological information at the microscopic level about corneal tissue, structures of the anterior segment, retina and optic nerve. The purpose of this study was to explore the possibility of using the OCT technology in complex ophthalmological examination in dogs and cats, to characterize the revealed pathological structural changes in corneal tissue in cats and dogs with some of the most common corneal diseases. Procedures. Optical coherence tomography of the cornea was performed in 112 animals: 68 dogs and 44 cats. In total, 224 eyes were examined. Pathologies of the organ of vision included: dystrophy and degeneration of the cornea, endothelial corneal dystrophy, dry eye syndrome, chronic superficial vascular keratitis, pigmented keratitis, corneal erosion, ulcerative stromal keratitis, corneal sequestration, chronic glaucoma and also postoperative period after performed keratoplasty. When performing OCT, we used certified medical devices: "Huvitz HOCT-1/1F», «Optovue iVue 80» and "SOCT Copernicus Revo (60)". Results. The results of a clinical study on the use of optical coherence tomography (OCT)of the cornea in cats and dogs, performed by the authors of the article in the complex diagnosis of keratopathies of variousorigins: endothelial corneal dystrophy, pigmented keratitis, chronic keratoconjunctivitis, chronic herpetic keratitis, ulcerative keratitis, traumatic corneal damage, sequestration of the cornea of cats, chronic keratitis, complicating the course of glaucoma. The characteristics of the OCT scans are givencorneas of cats and dogs that do not have corneal pathologies. OCT scans of various corneal pathologies in dogs and cats with a description of the revealed pathological changes are presented. Of great clinical interest are the data obtained during OCT of the cornea of animals undergoing keratoplasty operations using various forms of grafts. Conclusions. OCT makes it possible to assess the thickness and pathological structural changes of the corneal surface epithelium, corneal stroma and descemet membrane. We can measure them, determine the exact localization, and record pathological changes. Clinical observation of the dynamics of the pathological process in the cornea using OCT makes it possible to evaluate the effectiveness of drug treatment. In case of negative dynamics of corneal disease, it is necessary to determine the indications for surgical treatment (to assess the thickness of the cornea, the localization of its thinning zones, to characterize the depth and area of pathological changes). According to the OCT of the cornea, it is possible to choose the optimal surgical treatment for the patient, the technique and depth of optically constructive surgery (penetrating or anterior lamellar keratoplasty).; determine the depth and diameter of the planned microsurgical trepanation of corneal tissue, which will ensure good adaptation of the edges of the donor material.

Keywords: optical coherence tomography, corneal sequestration, optical coherence tomography of the cornea, corneal transplantation, cat, dog

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Authors: Yujie Yuan, Yiyang Fan, Hong Fan

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Objective: The RNA methylation recognition protein Aly/REF export factor (ALYREF) is considered one type of “reader” protein acting as a recognition protein of m5C, has been reported involved in several biological progresses including cancer initiation and progression. 5-methylcytosine (m5C) is a conserved and prevalent RNA modification in all species, as accumulating evidence suggests its role in the promotion of tumorigenesis. It has been claimed that ALYREF mediates nuclear export of mRNA with m5C modification and regulates biological effects of cancer cells. However, the systematical regulatory pathways of ALYREF in cancer tissues have not been clarified, yet. Methods: The expression level of ALYREF in pan-cancer and their normal tissues was compared through the data acquired from The Cancer Genome Atlas (TCGA). The University of Alabama at Birmingham Cancer data analysis Portal UALCAN was used to analyze the relationship between ALYREF and clinical pathological features. The relationship between the expression level of ALYREF and prognosis of pan-cancer, and the correlation genes of ALYREF were figured out by using Gene Expression Correlation Analysis database GEPIA. Immune related genes were obtained from TISIDB (an integrated repository portal for tumor-immune system interactions). Immune-related research was conducted by using Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) and TIMER. Results: Based on the data acquired from TCGA, ALYREF has an obviously higher-level expression in various types of cancers compared with relevant normal tissues excluding thyroid carcinoma and kidney chromophobe. The immunohistochemical images on The Human Protein Atlas showed that ALYREF can be detected in cytoplasm, membrane, but mainly located in nuclear. In addition, a higher expression level of ALYREF in tumor tissue generates a poor prognosis in majority of cancers. According to the above results, cancers with a higher expression level of ALYREF compared with normal tissues and a significant correlation between ALYREF and prognosis were selected for further analysis. By using TISIDB, we found that portion of ALYREF co-expression genes (such as BIRC5, H2AFZ, CCDC137, TK1, and PPM1G) with high Pearson correlation coefficient (PCC) were involved in anti-tumor immunity or affect resistance or sensitivity to T cell-mediated killing. Furthermore, based on the results acquired from GEPIA, there was significant correlation between ALYREF and PD-L1. It was exposed that there is a negative correlation between the expression level of ALYREF and ESTIMATE score. Conclusion: The present study indicated that ALYREF plays a vital and universal role in cancer initiation and progression of pan-cancer through regulating mitotic progression, DNA synthesis and metabolic process, and RNA processing. The correlation between ALYREF and PD-L1 implied ALYREF may affect the therapeutic effect of immunotherapy of tumor. More evidence revealed that ALYREF may play an important role in tumor immunomodulation. The correlation between ALYREF and immune cell infiltration level indicated that ALYREF can be a potential therapeutic target. Exploring the regulatory mechanism of ALYREF in tumor tissues may expose the reason for poor efficacy of immunotherapy and offer more directions of tumor treatment.

Keywords: ALYREF, pan-cancer, immunotherapy, PD-L1

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Authors: M. P. S. Tomar, Neelam Bansal

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Histomorphological, histochemical and scanning electron microscopic observations were recorded in developing cornea of buffalo fetuses. The samples from fetal cornea were collected in appropriate fixative from slaughter house and Veterinary Clinics, GADVASU, Ludhiana. The microscopic slides were stained for detailed histomorphological and histochemical studies. The scanning electron microscopic studies were performed at Electron microscopy & Nanobiology Lab, PAU Ludhiana. In present study, it was observed that, in 36 days (d) fetus, the corneal epithelium was well marked single layered structure which was placed on stroma mesenchyme. Cornea appeared as the continuation of developing sclera. The thickness of cornea and its epithelium increased as well as the epithelium started becoming double layered in 47d fetus at corneo-scleral junction. The corneal thickness in this stage suddenly increased thus easily distinguished from developing sclera. The separation of corneal endothelium from stroma was evident as a single layered epithelium. The stroma possessed numerous fibroblasts in 49d stage eye. Descemet’s membrane was appeared at 52d stage. The limbus area was separated by a depression from the developing cornea in 61d stage. In 65d stage, the Bowman’s layer was more developed. Fibroblasts were arranged parallel to each other as well as parallel to the surface of developing cornea in superficial layers. These fibroblasts and fibers were arranged in wavy pattern in the center of stroma. Corneal epithelium started to be stratified as a double layered epithelium was present in this age of fetal eye. In group II (>120 Days), the corneal epithelium was stratified towards a well marked irido-corneal angle. The stromal fibroblasts followed a complete parallel arrangement in its entire thickness. In full term fetuses, a well developed cornea was observed. It was a fibrous layer which had five distinct layers. From outside to inwards were described as the outer most layer was the 7-8 layered corneal epithelial, subepithelial basement membrane (Bowman’s membrane), substantia propria or stroma, posterior limiting membrane (Descemet’s membrane) and the posterior epithelium (corneal endothelium). The corneal thickness and connective tissue elements were continued to be increased. It was 121.39 + 3.73µ at 36d stage which increased to 518.47 + 4.98 µ in group III fetuses. In fetal life, the basement membrane of corneal epithelium and endothelium depicted strong to intense periodic Acid Schiff’s (PAS) reaction. At the irido-corneal angle, the endothelium of blood vessels was also positive for PAS activity. However, cornea was found mild positive for alcian blue reaction. The developing cornea showed strong reaction for basic proteins in outer epithelium and the inner endothelium layers. Under low magnification scanning electron microscope, cornea showed two types of cells viz. light cells and dark cells. The light cells were smaller in size and had less number of microvilli in their surface than in the dark cells. Despite these surface differences between light and dark cells, the corneal surface showed the same general pattern of microvilli studding all exposed surfaces out to the cell margin. which were long (with variable height), slight tortuous slender and possessed a micro villus shaft with a very prominent knob.

Keywords: buffalo, cornea, eye, fetus, ontogeny, scanning electron microscopy

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Authors: Aberdam Edith, Sangari Linda, Petit Isabelle, Aberdam Daniel

Abstract:

Background and Rationale: Transparent avascularised cornea is essential for normal vision and depends on limbal stem cells (LSC) that reside between the cornea and the conjunctiva. Ocular burns or injuries may destroy the limbus, causing limbal stem cell deficiency (LSCD). The cornea becomes vascularised by invaded conjunctival cells, the stroma is scarring, resulting in corneal opacity and loss of vision. Grafted autologous limbus or cultivated autologous LCS can restore the vision, unless the two eyes are affected. Alternative cellular sources have been tested in the last decades, including oral mucosa or hair follicle epithelial cells. However, only partial success has been achieved by the use of these cells since they were not able to uniformly commit into corneal epithelial cells. Human pluripotent stem cells (iPSC) display both unlimited growth capacity and ability to differentiate into any cell type. Our goal was to design a standardized and reproducible protocol to produce transplantable autologous LSC from patients through cell reprogramming technology. Methodology: First, keratinocyte primary culture was established from a small number of plucked hair follicles of healthy donors. The resulting epithelial cells were reprogrammed into induced pluripotent stem cells (iPSCs) and further differentiate into corneal epithelial cells (CEC), according to a robust protocol that recapitulates the main step of corneal embryonic development. qRT-PCR analysis and immunofluorescent staining during the course of differentiation confirm the expression of stage specific markers of corneal embryonic lineage. First appear ectodermal progenitor-specific cytokeratins K8/K18, followed at day 7 by limbal-specific PAX6, TP63 and cytokeratins K5/K14. At day 15, K3/K12+-corneal cells are present. To amplify the iPSC-derived LSC (named COiPSC), intact small epithelial colonies were detached and cultivated in limbal cell-specific medium. In that culture conditions, the COiPSC can be frozen and thaw at any passage, while retaining their corneal characteristics for at least eight passages. To evaluate the potential of COiPSC as an alternative ocular toxicity model, COiPSC were treated at passage P0 to P4 with increasing amounts of SDS and Benzalkonium. Cell proliferation and apoptosis of treated cells was compared to LSC and the SV40-immortalized human corneal epithelial cell line (HCE) routinely used by cosmetological industrials. Of note, HCE are more resistant to toxicity than LSC. At P0, COiPSC were systematically more resistant to chemical toxicity than LSC and even to HCE. Remarkably, this behavior changed with passage since COiPSC at P2 became identical to LSC and thus closer to physiology than HCE. Comparative transcriptome analysis confirmed that COiPSC from P2 are similar to a mixture of LSC and CEC. Finally, by organotypic reconstitution assay, we demonstrated the ability of COiPSC to produce a 3D corneal epithelium on a stromal equivalent made of keratocytes. Conclusion: COiPSC could become valuable for two main applications: (1) an alternative robust tool to perform, in a reproducible and physiological manner, toxicity assays for cosmetic products and pharmacological tests of drugs. (2). COiPSC could become an alternative autologous source for cornea transplantation for LSCD.

Keywords: Limbal stem cell deficiency, iPSC, cornea, limbal stem cells

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