Tubular Differentiation Research Articles

Estimation of tubular differentiation in female libyan breast cancer

We tried to evaluate tubular differentiation in the paraffin sections of 130 Libyan breast cancers to distinguish the less aggressive variants of infiltrating breast cancer from other variants and to found the relationship with clinicopathological features. We also compared our results with corresponding results on Finnish, and Nigerian female breast cancer patients. Methods: Histological samples from 130 patients of Libyan breast cancer were retrospectively studied by estimated the fraction of fields with tubular differentiation (FTD). The samples were screened at x10 magnification and the presence or absence of malignant tubular structures in each microscopic field was registered. They were compared with different clinicopathological features, and patient's survival. Results: The mean (±SD) value of FTD in Libya 23.4±21.6%, was higher than in Nigeria. but lower than reported in European breast cancer. There was statistically significant correlation between the FTD and some clinicopathological features, with the strongest association observed for large tumor (p < 0.0001). There was also correlation between FTD and histological grade (p = 0.001) and lymph node (LN) status (p = 0.007). The correlation with tumor staging was almost significant (p = 0.07). Conclusions: The results indicated that FTD are reliable prognostic indicators in Libyan female breast carcinomas, as they were among Finnish and Nigerian females. The FTD is a good parameter for identifying the aggressive tumor phenotype and provide significant prognostic support. The cut-offs (30% and 50%) might be applied as quantitative criterium for Libyan breast cancer to separate the patients into good, moderate and bad prognosis groups. The survival analysis indicated that short survival time was associated with low FTD values.

Immunohistochemical Characterization of a Renal Nephroblastoma in a &lt;i&gt;Trp&lt;/i&gt;53-mutant and Prolyl Isomerase 1-deficient Mouse

A nephroblastoma is a tumor arising from metanephric blastema occurring in childhood. Among laboratory rodents, nephroblastoma has been frequently reported in rats, but it remains exceedingly rare in mice. The present work describes a nephroblastoma in a young mouse homozygous for the specific Trp53 R172H point mutation coupled with targeted deletion of the Pin1 gene. The affected kidney was effaced by a biphasic tumor with an epithelial component arranged in tubules surrounded by nests of blastemal cells. Immunohistochemically, the neoplasm was diffusely positive for Wilms’ tumor antigen. The epithelial component expressed markers of renal tubular differentiation including wide-spectrum cytokeratin, E-cadherin and folate-binding protein. Furthermore, the neoplasm exhibited a high proliferative index and diffuse nucleocytoplasmic β-catenin expression. Based on histological and immunohistochemical features, a diagnosis of nephroblastoma potentially associated with Trp53 loss and oncogenic β-catenin activation has been proposed.

Dynamic expression of the sodium-vitamin C co-transporters, SVCT1 and SVCT2, during perinatal kidney development

Isoform 1 of the sodium-vitamin C co-transporter (SVCT1) is expressed in the apical membrane of proximal tubule epithelial cells in adult human and mouse kidneys. This study is aimed at analyzing the expression and function of SVCTs during kidney development. RT-PCR and immunohistochemical analyses revealed that SVCT1 expression is increased progressively during postnatal kidney development. However, SVCT1 transcripts were barely detected, if not absent, in the embryonic kidney. Instead, the high-affinity transporter, isoform 2 (SVCT2), was strongly expressed in the developing kidney from E15; its expression decreased at postnatal stages. Immunohistochemical analyses showed a dynamic distribution of SVCT2 in epithelial cells during kidney development. In renal cortex tubular epithelial cells, intracellular distribution of SVCT2 was observed at E19 with distribution in the basolateral membrane at P1. In contrast, SVCT2 was localized to the apical and basolateral membranes between E17 and E19 in medullary kidney tubular cells but was distributed intracellularly at P1. In agreement with these findings, functional expression of SVCT2, but not SVCT1 was detected in human embryonic kidney-derived (HEK293) cells. In addition, kinetic analysis suggested that an ascorbate-dependent mechanism accounts for targeted SVCT2 expression in the developing kidney during medullary epithelial cell differentiation. However, during cortical tubular differentiation, SVCT1 was induced and localized to the apical membrane of tubular epithelial cells. SVCT2 showed a basolateral polarization only for the first days of postnatal life. These studies suggest that the uptake of vitamin C mediated by different SVCTs plays differential roles during the ontogeny of kidney tubular epithelial cells.

Nephrogenic Adenoma

Nephrogenic adenoma (NA) has been considered as a metaplastic process of the urothelium. It has been suggested that this lesion is of renal tubular cell origin or differentiation. Immunohistochemical studies of NA emphasize its staining with α-methylacyl-coenzyme A racemase (AMACR), and prostatic adenocarcinoma may be a possible differential diagnosis. This reactivity was recently discussed as an artifact due to endogenous biotin. Kidney-specific cadherin (Ksp-cad) is a marker of distal nephron. CD10 and KIT are also expressed in the kidney. We studied the immunohistochemical expression of AMACR, p63, Ksp-cad, CD10, and KIT in 9 cases of NA (forming a total of 12 lesions). Practically all of the lesions stained for AMACR with 2 different antibodies and 2 high-sensitivity (multimer or polymer based) biotin-free methods (83% and 100%). The staining was similar for both methods in 9 of these 12 lesions. All of the NAs were negative for p63 and KIT, except 1 case, with focal reactivity for KIT. CD10 was expressed very focally in 4 of the 12 lesions (33%). We observed weak staining for Ksp-cad in 6 lesions (50%) and 3 (25%) showed a moderate positivity in 15% to 50% of the cells. In conclusion, positivity of NA for AMACR is not an artifact, as we confirmed using 2 different methods. Besides, p63, a basal cell marker, is usually negative. Immunoreactivity for Ksp-cad seems to support the differentiation of NA to distal nephron cells, at least in some of the cases. Other markers expressed by the nephron, such as CD10 and KIT, are usually negative in NA.

Prognostic value of tumour‐associated macrophages in canine mammary tumours

Tumour-associated macrophages (TAMs) have already been associated in human breast cancer to a poor prognosis. As a part of a tumoural microenvironment, TAMs have an important contribution influencing neoplastic progression. Hitherto, in canine mammary tumours (CMT) the prognostic value of TAMs has not been reported. In this study, MAC387 immunohistochemical expression was evaluated in 59 CMTs (20 benign and 39 malignant). The TAM value was significantly higher in malignant than benign CMT (P = 0.011). In malignant CMT, TAMs were associated with skin ulceration (P = 0.022), histological type (P = 0.044), nuclear grade (P = 0.031) and tubular differentiation (P = 0.042). The survival analysis revealed a significant association between tumours with higher levels of TAMs and the decrease in overall survival (P = 0.030). TAMs have proven to have a prognostic value. These findings suggest the future possibility of using TAMs as a novel therapeutic target in CMT.

Mechanisms of p53 activation and physiological relevance in the developing kidney

The tumor suppressor protein p53 is a short-lived transcription factor due to Mdm2-mediated proteosomal degradation. In response to genotoxic stress, p53 is stabilized via posttranslational modifications which prevent Mdm2 binding. p53 activation results in cell cycle arrest and apoptosis. We previously reported that tight regulation of p53 activity is an absolute requirement for normal nephron differentiation (Hilliard S, Aboudehen K, Yao X, El-Dahr SS Dev Biol 353: 354-366, 2011). However, the mechanisms of p53 activation in the developing kidney are unknown. We show here that metanephric p53 is phosphorylated and acetylated on key serine and lysine residues, respectively, in a temporal profile which correlates with the maturational changes in total p53 levels and DNA-binding activity. Site-directed mutagenesis revealed a differential role for these posttranslational modifications in mediating p53 stability and transcriptional regulation of renal function genes (RFGs). Section immunofluorescence also revealed that p53 modifications confer the protein with specific spatiotemporal expression patterns. For example, phos-p53(S392) is enriched in maturing proximal tubular epithelial cells, whereas acetyl-p53(K373/K382/K386) are expressed in nephron progenitors. Functionally, p53 occupancy of RFG promoters is enhanced at the onset of tubular differentiation, and p53 loss or gain of function indicates that p53 is necessary but not sufficient for RFG expression. We conclude that posttranslational modifications are important determinants of p53 stability and physiological functions in the developing kidney. We speculate that the stress/hypoxia of the embryonic microenvironment may provide the stimulus for p53 activation in the developing kidney.

Exosomes released by K562 chronic myeloid leukemia cells promote angiogenesis in a src-dependent fashion

Exosomes, microvesicles of endocytic origin released by normal and tumor cells, play an important role in cell-to-cell communication. Angiogenesis has been shown to regulate progression of chronic myeloid leukemia (CML). The mechanism through which this happens has not been elucidated. We isolated and characterized exosomes from K562 CML cells and evaluated their effects on human umbilical endothelial cells (HUVECs). Fluorescent-labeled exosomes were internalized by HUVECs during tubular differentiation on Matrigel. Exosome localization was perinuclear early in differentiation, moving peripherally in cells undergoing elongation and connection. Exosomes move within and between nanotubular structures connecting the remodeling endothelial cells. They stimulated angiotube formation over a serum/growth factor-limited medium control, doubling total cumulative tube length (P=0.003). Treatment of K562 cells with two clinically active tyrosine kinase inhibitors, imatinib and dasatinib, reduced their total exosome release (P<0.009); equivalent concentrations of drug-treated exosomes induced a similar extent of tubular differentiation. However, dasatinib treatment of HUVECs markedly inhibited HUVEC response to drug control CML exosomes (P<0.002). In an in vivo mouse Matrigel plug model angiogenesis was induced by K562 exosomes and abrogated by oral dasatinib treatment (P<0.01). K562 exosomes induced dasatinib-sensitive Src phosphorylation and activation of downstream Src pathway proteins in HUVECs. Imatinib was minimally active against exosome stimulation of HUVEC cell differentiation and signaling. Thus, CML cell-derived exosomes induce angiogenic activity in HUVEC cells. The inhibitory effect of dasatinib on exosome production and vascular differentiation and signaling reveals a key role for Src in both the leukemia and its microenvironment.

Ca2+ Influx through Reverse Mode Na+/Ca2+ Exchange Is Critical for Vascular Endothelial Growth Factor-mediated Extracellular Signal-regulated Kinase (ERK) 1/2 Activation and Angiogenic Functions of Human Endothelial Cells

VEGF is a key angiogenic cytokine and a major target in anti-angiogenic therapeutic strategies. In endothelial cells (ECs), VEGF binds VEGF receptors and activates ERK1/2 through the phospholipase γ (PLCγ)-PKCα-B-Raf pathway. Our previous work suggested that influx of extracellular Ca(2+) is required for VEGF-induced ERK1/2 activation, and we hypothesized that this could occur through reverse mode (Ca(2+) in and Na(+) out) Na(+)-Ca(2+) exchange (NCX). However, the role of NCX activity in VEGF signaling and angiogenic functions of ECs had not previously been described. Here, using human umbilical vein ECs (HUVECs), we report that extracellular Ca(2+) is required for VEGF-induced ERK1/2 activation and that release of Ca(2+) from intracellular stores alone, in the absence of extracellular Ca(2+), is not sufficient to activate ERK1/2. Furthermore, inhibitors of reverse mode NCX suppressed the VEGF-induced activation of ERK1/2 in a time- and dose-dependent manner and attenuated VEGF-induced Ca(2+) transients. Knockdown of NCX1 (the main NCX isoform in HUVECs) by siRNA confirmed the pharmacological data. A panel of NCX inhibitors also significantly reduced VEGF-induced B-Raf activity and inhibited PKCα translocation to the plasma membrane and total PKC activity in situ. Finally, NCX inhibitors reduced VEGF-induced HUVEC proliferation, migration, and tubular differentiation in surrogate angiogenesis functional assays in vitro. We propose that Ca(2+) influx through reverse mode NCX is required for the activation and the targeting of PKCα to the plasma membrane, an essential step for VEGF-induced ERK1/2 phosphorylation and downstream EC functions in angiogenesis.

Diffuse Expression of PAX2 and PAX8 in the Cystic Epithelium of Mixed Epithelial Stromal Tumor, Angiomyolipoma With Epithelial Cysts, and Primary Renal Synovial Sarcoma

Over the past decade, 3 novel, typically cystic renal neoplasms have been described: angiomyolipoma with epithelial cysts (AMLEC), mixed epithelial stromal tumor (MEST), and primary renal synovial sarcoma (SS). In all 3 neoplasms, the nature of the cystic epithelium is not clear; some have postulated that the cysts represent cystically dilated, entrapped renal tubular epithelium, whereas an alternative interpretation is that the epithelium represents epithelial differentiation by the stromal component of the neoplasm. The latter is supported by the extrarenal location of the epithelium in some cases. PAX2 and PAX8 are tissue-specific transcription factors expressed primarily in the renal and Müllerian systems and also in Wolffian duct structures (such as seminal vesicle). Their expression has not been examined in these lesions. We performed PAX2 and PAX8 immunohistochemistry on representative sections of cases of AMLEC (8 cases), MEST (8 cases), and renal SS (3 cases). The relative percentage and intensity (none, weak, moderate, and strong) of nuclear labeling were evaluated in both the benign adjacent renal tubules and the lesion's epithelial cysts. In the benign kidney, distal convoluted tubules (DCTs) labeled strongly for PAX2 and PAX8, whereas proximal convoluted tubules labeled minimally. The cystic epithelium of all 8 cases of AMLEC, including 5 that protruded beyond the renal capsule into the perirenal fat, demonstrated strong diffuse labeling for both PAX2 and PAX8. We also identified a mimic of entirely extrarenal AMLEC, angiomyolipoma with endosalpingiosis. PAX2 and PAX8 diffusely and strongly labeled the epithelial component of all 8 cases of MEST, including all architectural (phyllodes-like, large cysts, small cysts, clustered microcysts) and virtually all cytologic (hobnail, flat, cuboidal, columnar, apocrine, and clear cell) epithelial variants present. The epithelial cysts of all 3 cases of primary renal SS labeled diffusely and strongly for PAX2 and PAX8. Cyst epithelial labeling intensity was similar to that of renal DCT in all cases. The diffuse labeling for PAX2/PAX8 in the epithelial cysts of AMLEC, taken together with their consistent negativity for estrogen receptor and HMB45, supports the hypothesis that this epithelium represents entrapped, cystically dilated renal tubules that commonly herniate beyond the renal capsule. The diffuse labeling of the cyst epithelium of renal SS supports the previously proposed hypothesis that this cyst epithelium represents entrapped dilated renal tubules in a monophasic spindle cell lesion and not neoplastic epithelial differentiation. The diffuse labeling for PAX2/PAX8 in MEST epithelium, coupled with its usual estrogen receptor negativity, is consistent with the hypothesis that the epithelium of MEST demonstrates renal tubular differentiation and undergoes architectural and cytologic changes as it grows along with the stromal component. Whether this complex epithelium represents entrapped or neoplastic renal tubular epithelium remains an open question.

Discovery of Candidate Genes and Pathways Regulating Oviduct Development in Chickens1

Estrogen is a key regulator in the development of the female reproductive system. It also stimulates oviduct development in immature chicks. We identified candidate genes and pathways associated with the development of chicken oviducts. A pellet containing the synthetic estrogen analog diethylstilbestrol (DES) was implanted subcutaneously in 1-wk-old female chicks for 10 days. The pellet was removed from half the group for 10 days, and an additional dose was given for a further 10 days. Total RNA was extracted from the oviducts of DES-treated and untreated chicks and subjected to an Affymetrix chicken GeneChip analysis. We found differential expression of 2290 and 1745 transcripts from the oviducts that were treated with DES once and twice, respectively. We also found a twofold or greater change in the expression of 77 and 390 transcripts between the two control and DES-treated time points, respectively, while we found a change in the expression of 10 transcripts that were common to all groups. Analyses of real-time PCR and in situ hybridization of selected genes confirmed the validity of the gene expression patterns observed in the microarray analysis. In particular, CCRN4L, FAM26F, HAS2, NELF, and NTM were up-regulated in the DES-treated chicken oviducts. High-throughput analysis revealed that the differentially expressed genes were related to tubular formation, epithelial differentiation, hormone interactions, nerve development, and tissue remodeling in the chicken oviduct. This study provides novel insights into candidate genes regulating oviduct development and differentiation via estrogen. The identified genes may serve as biomarkers of reproductive tract development in chicks.

Angiogenic Functions of Voltage-gated Na+ Channels in Human Endothelial Cells: MODULATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) SIGNALING

Voltage-gated sodium channel (VGSC) activity has previously been reported in endothelial cells (ECs). However, the exact isoforms of VGSCs present, their mode(s) of action, and potential role(s) in angiogenesis have not been investigated. The main aims of this study were to determine the role of VGSC activity in angiogenic functions and to elucidate the potentially associated signaling mechanisms using human umbilical vein endothelial cells (HUVECs) as a model system. Real-time PCR showed that the primary functional VGSC α- and β-subunit isoforms in HUVECs were Nav1.5, Nav1.7, VGSCβ1, and VGSCβ3. Western blots verified that VGSCα proteins were expressed in HUVECs, and immunohistochemistry revealed VGSCα expression in mouse aortic ECs in vivo. Electrophysiological recordings showed that the channels were functional and suppressed by tetrodotoxin (TTX). VGSC activity modulated the following angiogenic properties of HUVECs: VEGF-induced proliferation or chemotaxis, tubular differentiation, and substrate adhesion. Interestingly, different aspects of angiogenesis were controlled by the different VGSC isoforms based on TTX sensitivity and effects of siRNA-mediated gene silencing. Additionally, we show for the first time that TTX-resistant (TTX-R) VGSCs (Nav1.5) potentiate VEGF-induced ERK1/2 activation through the PKCα-B-RAF signaling axis. We postulate that this potentiation occurs through modulation of VEGF-induced HUVEC depolarization and [Ca(2+)](i). We conclude that VGSCs regulate multiple angiogenic functions and VEGF signaling in HUVECs. Our results imply that targeting VGSC expression/activity could be a novel strategy for controlling angiogenesis.

Abstract 5135: Exosomes released by K562 chronic myeloid leukemia cells promote endothelial cell tubular differentiation through uptake and cell-to-cell transfer

Abstract We hypothesized that exosomes were a venue through which to transfer pro-angiogenic stimuli into and between endothelial cells during endothelial cell tubular differentiation. Exosomes are microvesicles of endocytic origin released by most normal and tumor cells that play an important role in cell-to-cell communication. Angiogenesis is recognized to be a factor in progression of chronic myeloid leukemia (CML). However, the mechanism through which this happens has not been elucidated. We first optimized and characterized secretion of exosomes from CML K562 cells, showing expected selective enrichment of exosomal markers CD63, CD81 and Tsg101 in exosomes compared to the K562 whole cell lysate. Next, these exosomes were stained with PKH, a fluorescent plasma membrane dye, and incubated with human umbilical vein endothelial cells (HUVECs) on Matrigel for measurement of exosome uptake under semi-physiologic conditions. We found that K562 exosomes were internalized by HUVECs during tubular differentiation, and that exosome uptake increased and exosomes redistributed over the time. Moreover, we observed a two-fold increase in cumulative length of vascular tubes formed on Matrigel in HUVEC exposed to K562 exosomes under limiting growth factor medium, with a maximum effect obtained with an exosome concentration of 10ug/ml (p=0.007). Next, we incubated HUVECs on Matrigel with PKH-labeled exosomes to dissect intracellular exosome movement and exosome-cell interactions during tube formation, using multi-dimensional confocal microscopy. Exosomes were perinuclear early in tubulogenesis; whereas, in later stages, exosomes were more peripheral in the cytoplasm and sub-membranous areas. Over time, the peripheral exosomes moved towards and then into nanotubular structures that were part of the tubulogenesis process. Nanotubes met and merged, connecting neighboring cells. Actin and tubulin staining was used to show the localization of exosomes in the nanotubes. Three-dimensional reconstruction of confocal images demonstrated that the actin and tubulin were in a loose network without clear organization. This analysis also revealed exosome presence within and movement through the nanotubes. The use of dual color PKH stain allowed demonstration that exosomes were shared along the nanotubular network between connected HUVEC cells, confirming cell-cell exosomal exchange. Time-lapse confocal microscopy further documented nanotubular exosome transfer. These results show that CML exosomes traffic into and between endothelial cells and stimulate tube formation in a physiologic in vitro model, suggesting they may play an important role in leukemia stimulation of bone marrow angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5135. doi:10.1158/1538-7445.AM2011-5135

Loss of Primary Cilia Upregulates Renal Hypertrophic Signaling and Promotes Cystogenesis

Primary cilia dysfunction alters renal tubular cell proliferation and differentiation and associates with accelerated cyst formation in polycystic kidney disease. However, the mechanism leading from primary ciliary dysfunction to renal cyst formation is unknown. We hypothesize that primary cilia prevent renal cyst formation by suppressing pathologic tubular cell hypertrophy and proliferation. Unilateral nephrectomy initiates tubular cell hypertrophy and proliferation in the contralateral kidney and provides a tool to examine primary cilia regulation of renal hypertrophy. Conditional knockout of the primary cilia ift88 gene leads to delayed, adult-onset renal cystic disease, which provides a window of opportunity to conduct unilateral nephrectomy and examine downstream kinetics of renal hypertrophy and cyst formation. In wild-type animals, unilateral nephrectomy activated the mTOR pathway and produced appropriate structural and functional hypertrophy without renal cyst formation. However, in ift88 conditional knockout animals, unilateral nephrectomy triggered increased renal hypertrophy and accelerated renal cyst formation, leading to renal dysfunction. mTOR signaling also increased compared with wild-type animals, suggesting a mechanistic cascade starting with primary ciliary dysfunction, leading to excessive mTOR signaling and renal hypertrophic signaling and culminating in cyst formation. These data suggest that events initiating hypertrophic signaling, such as structural or functional loss of renal mass, may accelerate progression of adult polycystic kidney disease toward end-stage renal disease.

Deletions of 16q in Wilms Tumors Localize to Blastemal-Anaplastic Cells and Are Associated with Reduced Expression of the IRXB Renal Tubulogenesis Gene Cluster

Wilms tumor is the most common pediatric renal neoplasm, but few molecular prognostic markers have been identified for this tumor. Somatic deletion in the long arm of chromosome 16 (16q) is known to predict a less favorable outcome in Wilms tumor, but the underlying molecular mechanisms are not known. We show that 16q deletions are typically confined to immature anaplastic-blastic tumor elements, while deletions are absent in maturing tumor components. The smallest region of deletion overlap mapped to a 1.8-Mb segment containing the IRXB gene cluster including IRX3, IRX5, and IRX6, of which IRX3 is a recently identified regulator of tubular maturation during nephrogenesis. Tumors with 16q deletion showed a lower overall mRNA expression of IRXB genes, and 16q-deleted tumor cells failed to express IRX3 while it was expressed in differentiating tubular tumor elements with intact 16q. Consistent with a role for IRX3 in tubular differentiation, gene sets linked to Notch signaling, Rho signaling, and ion channel activity were enriched in tumors with high IRX3 expression, while WTs with low expression were enriched for gene sets linked to cell cycle progression. Low mRNA levels of IRXB genes were associated with diffuse anaplasia, high-stage disease, and death. A disturbed balance between tubular differentiation and self-renewal of anaplastic-blastic elements may thus be one mechanism linking 16q deletion to adverse outcome in Wilms tumor.

CK7 expression in primary cutaneous squamous cell carcinoma

To evaluate cytokeratin 7 (CK7) immunoreactivity in invasive primary cutaneous squamous cell carcinomas (SCCs). Twenty-seven primary cutaneous SCCs from 25 patients were evaluated for tumor grade using hematoxylin and eosin-stained slides and for percentage and intensity of immunoreactivity for CK7. All cases exhibited features of SCC with an in situ component. No glandular or tubular differentiation was present. Staining intensity was graded on a scale of 0-3, with 0 indicating no reaction. Of immunoreactive cases, percentage of tumor staining and distribution of immunoreactivity was documented. Six of 27 SCCs (22%) exhibited immunostaining for CK7. Of those cases, three were poorly differentiated, exhibiting 2 to 3+ intensity in 5-15% of cells. Two were poorly differentiated, with 2 to 3+ intensity in 30-60% of cells. The remaining immunoreactive tumor was moderately differentiated, with 1+ intensity and 5% staining in an area of microinvasion. A subset of cutaneous SCCs, in particular, poorly differentiated tumors, may show focal-to-partial immunoreactivity for CK7. This is important to bear in mind when immunohistochemistry is used to distinguish SCC from simulants, such as porocarcinoma, or other adnexal carcinomas with squamous metaplasia.

Protective effects of melatonin and Glycyrrhiza glabra extract on ochratoxin A—induced damages on testes in mature rats

The effect of Glycyrrhiza glabra extract (GgE) as a natural antioxidant and melatonin (MEL) on ochratoxin A (OTA)-induced histopathological damages on the testes and oxidative stress was evaluated in male rats. The animals were assigned into four groups (n = 8) including control and test groups. The rats in control group received saline and the animals in the test groups received (200 µg/kg) of OTA, (15 mg/kg) of MEL + (200 µg/kg) OTA and (100 mg/kg) of GgE + (200 µg/kg) OTA, respectively, during 28 consecutive days. The serum total antioxidant power (TAOP) and total thiol molecules (TTM) production were assessed. Moreover, histopathological and histochemical studies were also performed. The results showed that the TAOP and TTM were decreased in OTA-exposed rats, while the animals that received MEL + OTA or GgE + OTA showed an enhancement in the serum TAOP and TTM levels. Histopathological analyses demonstrated that in OTA-exposed rats, the testicular degeneration, seminiferous tubule atrophy, dissociation of germinative epithelium, vasodilatation with vascular thrombosis, perivascular immune cell infiltration, hypertrophied leydic cells, giant cell formation, and negative tubular differentiation index (TDI) were observed. Surprisingly, both the biochemical and histopathological examinations showed that MEL and GgE, albeit with some differences, exerted a protective effect on OTA-induced damages. In conclusion, this data suggest that OTA contamination in animal feeds and human foods could cause reproductive abnormalities. Our data also indicate that OTA, at least partly by interfering in oxidative stress system, exerts its toxic effects on testes whereas MEL and GgE with antioxidant properties could fairly protect rats against OTA toxic effects.

First Heal Thyself: Rescue of Dysfunctional Endothelial Progenitor Cells Restores Function to the Injured Kidney

First Heal Thyself: Rescue of Dysfunctional Endothelial Progenitor Cells Restores Function to the Injured Kidney

THE EFFECT OF CHRONIC EXPOSURE WITH IMIDACLOPRID INSECTICIDE ON FERTILITY IN MATURE MALE RATS

Background This study was conducted to evaluate the effect of chronic exposure to imidacloprid (IM) insecticide on male testicular tissue, sperm morphology and testosterone levels in the serum of mature male rats. Materials and methods Animals were divided into and control-sham groups. The test group was subdivided into two groups of rats which were administered doses of 225 and 112 mg/kg IM per group. Each test group received the designated oral dose of IM once daily, for 60 days while the control-sham group received corn oil (0.2 ml/day) for the same time period. Results Clinical observations demonstrated decreased movement, staggering gait, occasional trembling, diarrhea and spasms in the test groups. No clinical signs were seen in control-sham rats. Light microscopic analyses revealed increased thickness of tunica albuginea, obvious edema in the sub-capsular and interstitial connective tissue, atrophied seminiferous tubules, arrested spermatogenesis, negative tubular differentiation and repopulation indexes, decreased Leydig cells/ mm2 of interstitial tissue, hypertrophy and cytoplasmic granulation of the Leydig cells, vasodilation and thrombosis, elevated death, as well as immature and decreased immotile sperm velocity. Hormonal investigations showed significant (p<0.05) decrease in serum testosterone levels. No hormonal changes were seen in the testosterone levels of the control-sham group. Conclusion The current data provide inclusive histological feature of chronic IM exposure in two doses with an emphasis on reproductive disorders including a histological adverse effect on testicular tissue, spermatogenesis, sperm viability, velocity and abnormality which potentially can cause infertility.

ZO-1 and ZONAB Interact to Regulate Proximal Tubular Cell Differentiation

<h3>Abstract</h3> Collateral lethality occurs when loss of one paralog renders cancer cells dependent on the remaining paralog. Combining genome scale CRISPR/Cas9 screens coupled with RNA-sequencing in over 900 cancer cell lines, we found that cancers of nervous system lineage, including adult and pediatric gliomas and neuroblastomas, required the nuclear kinase Vaccinia-Related Kinase 1 (VRK1) for their survival. <i>VRK1</i> dependency was inversely correlated with expression of its paralog VRK2. <i>VRK2</i> knockout (KO) sensitized cells to <i>VRK1</i> suppression, and conversely, VRK2 overexpression increased cell fitness in the setting of VRK1 suppression. DNA methylation of the <i>VRK2</i> promoter was associated with low VRK2 expression in human neuroblastomas, and adult and pediatric gliomas. Mechanistically, depletion of <i>VRK1</i> reduced Barrier-to-Autointegration Factor (BAF) phosphorylation during mitosis, resulting in DNA damage and apoptosis. Together, these studies identify VRK1 as a synthetic lethal target in <i>VRK2</i> promoter-methylated adult and pediatric gliomas and neuroblastomas. <h3>Statement of Significance</h3> We credential VRK1 as a target in adult and pediatric gliomas, and neuroblastomas with <i>VRK2</i> promoter methylation. This demonstrates the utility of paralog-driven synthetic lethal interactions for biomarker-linked, targeted therapeutics.

Expression of Galectin-3 in Renal Neoplasms: A Diagnostic, Possible Prognostic Marker

Galectin-3, a member of the lectin family, was shown to be expressed in normal distal tubular cells and in renal cell carcinomas (RCC). However, its diagnostic and prognostic significance in RCC is as yet undefined. To describe the expression of Galectin-3 among different histologic subtypes of renal neoplasms and to determine their diagnostic and prognostic significances. The expression of Galectin-3 was evaluated in 217 renal neoplasms by tissue microarray and immunohistochemistry with semiquantitative analysis. Strong expression of Galectin-3 was observed in 92 of 217 of renal neoplasms (42.4%). Although 22 of 23 oncocytomas (95.7%) and 19 of 21 chromophobe RCCs (90.5%) express Galectin-3, only 4 of 32 papillary RCCs (12.5%) and 47 of 137 clear cell RCCs (34.3%) express Galectin-3, suggesting that it may be used as a potential diagnostic marker. Galectin-3 expression was seen in 55% of high-grade (Fuhrman nuclear grades 3 and 4) versus 21% low-grade (grades 1 and 2) clear cell RCCs (P < .001). This study confirms that Galactin-3 is strongly overexpressed in renal cell neoplasms of distal tubular differentiation, that is, oncocytoma and chromophobe RCCs, suggesting it might be used as a possible differential diagnostic tool for renal cell neoplasm with oncocytic or granular cells. Furthermore, we observed a strong association of overexpression of Galectin-3 and high nuclear grade in clear cell RCC. These results also suggest a possible pivotal role for Galectin-3 in the differentiation and prognosis of clear cell RCC.