Significant Differences In mRNA Expression Research Articles

Antenatal betamethasone has a sex-dependent effect on the in vivo response to endothelin in adult sheep

Antenatal steroid administration is associated with multiple cardiometabolic alterations, including hypertension; however, the mechanisms underlying this phenomenon are unclear. The aim of the present study was to ascertain, in vivo, the contribution of the endothelin system to the development of hypertension in the adult offspring and the signaling pathway involved. Pregnant sheep were treated with two doses of betamethasone (n = 23) or vehicle (n = 22) at 80 days (~0.55) gestation and allowed to deliver at term. Adult sheep were chronically instrumented under general anesthesia to place vascular catheters and a femoral artery flow probe. Blood pressure and flow were recorded continuously, and femoral artery vascular resistance was calculated before and during administration of endothelin 1 (ET-1). Selective blockers (dantrolene, BQ123, niacinamide) or saline were administered simultaneously. Betamethasone-exposed animals exhibited a significant elevation in mean blood pressure (female: 98 ± 1.8 vs. 92 ± 2.1; males: 97 ± 3.4 vs. 90 ± 2.3; mmHg; P < 0.05). ET-1 elicited a significant increase in blood pressure (F = 56.4; P < 0.001) and in vascular resistance (F = 44.3; P < 0.001) in all groups. A betamethasone effect in the vascular resistance response to ET-1 (F = 25.7; P < 0.001) was present in females only, and the effect was partially blunted by niacinamide (F = 6.6; P < 0.01). Combined administration of niacinamide and BQ123, as well as of dantrolene abolished the betamethasone effect on vascular resistance. No significant differences in mRNA expression of ET(A) or ET(B) in endothelial or smooth muscle cells of resistance-size arteries were observed. We conclude that the betamethasone effect on vascular resistance is mediated by an enhanced response to ET-1 through ET(A) receptor via the cyclic ADPR/ryanodine pathway.

Synonymous SNP influences Adam12 mRNA expression level in synovial tissue

Synonymous SNP influences Adam12 mRNA expression level in synovial tissue

Visceral and post-Kala-Azar dermal leishmaniasis isolates show significant difference in their in vitro drug susceptibility pattern

Visceral leishmaniasis (VL) remains a major health problem in old world, and India accounts for half of the world burden. The widespread emergence of resistance to standard drug in India poses a major obstacle in the control of leishmaniasis. Post-Kala-Azar dermal leishmaniasis (PKDL) is considered as main source of drug resistance. Experimental data indicate that resistance against newer drugs is also imminent. Therefore, in vitro studies were carried out to test minimum parasiticidal concentration of five conventional and newly introduced anti-leishmanial drugs against 20 field isolates of Leishmania donovani obtained from visceral and post-Kala-Azar dermal leishmaniasis patients of India. Study revealed wide range of variation in minimum inhibitory concentration of sodium antimony gluconate (SAG). PKDL isolates displayed significantly lower susceptibility to SAG and miltefosine than VL isolates with P value of 0.0006 and 0.0243, respectively. All clinical isolates had higher IC(50) value for paromomycin and miltefosine as compared to reference strain indicating their vulnerability to develop unresponsiveness. However, isolates were uniformly susceptible to pentamidine and amphotericin B. The results of gene expression analysis of AQP1 were largely in agreement with phenotypic drug sensitivity results. Interestingly, significant down-regulation of AQP1 was observed in PKDL isolates as compared to VL isolates indicating their increased propensity for drug unresponsiveness. However, no significant difference in mRNA expression of LdMT and LdRos3 gene was found for two groups. The present study unravels valuable baseline scientific data showing variation in the drug susceptibility pattern in the L. donovani isolates. The information might have impact on the management and control of Indian visceral leishmaniasis.

Talens-Mediated DLEU1 Gene Silencing in Burkitt Lymphoma (BL): Implication of Lncrna DLEU1 Gene As a Potential Tumor Suppressor Gene in BL.

AbstractAbstract 2403 Background:Pediatric Burkitt Lymphoma (BL) is the most common histological subtype of Non Hodgkin Lymphoma (NHL) in children and adolescents (Cairo et al., Blood, 2007; Miles/Cairo, BJHaem., 2012). We previously identified in a subset analysis that children with BL and a 13q deletion, particularly 13q14.3, had significantly poorer outcome and inferior overall survival (OS) despite aggressive short, intensive multiagent chemotherapy (Poirel/Cairo et al., Leukemia, 2009; Nelson/Cairo/Sanger et al., BJHaem., 2009). Deleted in Lymphocytic Leukemia 1 (DLEU1) is a long non codign RNA gene with the BL classifier genes as reported by Dave et al (NEJM. 2006) on chromosome 13q14.3 region and the expression levels of RASSF1, TUBB2C and UBR1 were significantly higher in BL than in diffuse large B-cell lymphoma (DLBCL) (Day/Cairo et al., AACR, 2008). Sequence-specific Zinc Finger Nucleases (ZFN) and Transcription Activator-Like Effector Nucleases (TALENs) technologies have been developed for precision targeted genome editing in in vitro experiments with high efficiency and TALEN technology has been applied in a variety of organisms and stem cells for new experimental and therapeutic tools (Carlson et al., Mol Ther., 2012). Objectives:We hypothesize that 1) TALEN technology is suitable for the modification of and silencing of DLEU1 locus and 2) DLEU1 may have function as a tumor suppressor gene and therefore examined whether the loss of DLEU1 by TALEN-mediated DLEU1 knockdown Raji BL cells result in change in proliferation and programmed cell death. Methods:TALENs were constructed based on REAL (restriction enzyme and ligation) assembly methods (Sander et al., Nat. Biotech, 2011) for DLEU1 gene modification. DLEU1 TALEN pairs or combinations were transfected into 293T and Raji cells using lipofectamin reagent (Invitrogen) and Amaxa nucleofection kit, respectively. Genomic DNA and total RNA were extracted using genomic DNA extraction kit (Promega) and Trizol reagent (Invitrogen). Surveyor mutation detection assay (Transgenomic) was applied for TALENs validities and confirmed by sequencing analysis. Quantitative RT-PCR was performed by CFX96 Real-time system (Bio-rad) using qScript™ cDNA Synthesis Kit (Quantas) and SsoFast™ EvaGreen® Supermix (Bio-rad). Transfected cells were re-plated (1×104) into 48 well plates at 24hours post transfection and then counted every 24hrs for cell proliferation assay. For spontaneous apoptosis assay, DLEU1 TALENs transfected Raji cells were re-plated into 96 well plates at 24hours post-transfection and measured caspase 3/7 activities (Promega) using Clarity Luminescence microplate reader (Biotek) and statistical significance was determined by a one or two-tailed Student t-test. Results:Three pairs of functional DLEU1 TALENs (T1, T2, and T3) targeting endogenous DLEU1 gene generated and confirmed their validities with gene modification caused by non-homologous end joining (NHEJ) in TALENs mediated 293T [Figure 1]. Forty eight hours after transiently transfection in Raji cells, combined DLEU1 TALENs, T1/T2 (T12), T1/T3 (T13) and T2/T3 (T23) mediated Raji cells were dramatically reduced expression of DLEU1 mRNA (about 82–93% reduction) compared to untransfected Raji cells [92% reduction (p<0.002), 93% (p<0.0006), and 92% (p<0.0003)], respectively, and compared to empty vector as a control [82% reduction (p<0.05), 87% (p<0.0001), and 82% (p<0.001, respectively)]. T12 and T13-mediated DLEU1 knockdown Raji cells showed significantly an increase of cell proliferation [1.2-fold (p<0.02) and 1.5-fold (p<0.04) at day 5, respectively]. In spontaneous apoptotic assays, T13 mediated Raji showed significantly 20% (p<0.05) of reduction of caspase 3/7 activities. In comparison of mRNA expression of DLEU1 network genes in T13 mediated Raji cells, there were no significant differences in mRNA expression of c-myc compared to control, however, there was significantly reduced expression of RASSF1 and UBR1 mRNA (<7.7-fold, p<0.005 and <11.1-fold, p<0.0005), respectively, and significantly increased expression of TUBB2C mRNA (>7.5-fold, p<0.005). Conclusions:We demonstrate that 1) DLEU1-TALENs are useful tools for the modification of endogenous DLEU1 gene expression, 2) DLEU1 may have as a tumor suppressor gene in BL, and 3) DLEU1 TALEN mediated gene knockdown resulted in inhibition of BL apoptosis and increase in cell proliferation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Overexpression of Deleted Lymphocytic Leukemia 1 (DLEU1) Inhibits Proliferation of Burkitt Lymphoma (BL).

AbstractAbstract 2654 Background:Burkitt Lymphoma (BL) represents approximately 40% of all childhood and adolescents Non Hodgkin Lymphoma (Cairo et al., Blood, 2007; Miles/Cairo, BJHaem., 2012), and pediatric BL patients with chromosome del13q14.3 region had significantly poorer outcome and inferior overall survival despite aggressive short, intensive multiagent chemotherapy in subset analysis (Poirel/Cairo et al., Leukemia, 2009; Nelson/Cairo/Sanger et al., BJHaem., 2009). Deleted in Lymphocytic Leukemia 1 (DLEU1) is often deleted in B-CLL patients (Liu et al., Oncogene, 1997; Wolf et al., Hum. Mol. Genet., 2001) with the BL classifier genes (Dave et al., NEJM. 2006) on chromosome 13q14.3 region, is recognized to interact with Tubulin beta 2C (TUBB2C), RASSF1A, and E3 ubiquitin-protein ligase (UBR1), and p53 and their mRNA expression levels were significantly higher in BL than in diffuse large B-cell lymphoma (DLBCL) (Day/Cairo et al., AACR, 2008). DLEU1 may have function as a tumor suppressor gene, however, functions and mechanisms underlying DLEU1 expression in the cells are poorly understood. OBJECTIVE:We propose that DLEU1 may act as a tumor suppressor gene and sought out to examine whether gains of overexpression of DLEU1 in embryonic kidney cells and Burkitt Lymphoma cells result in inhibition of tumor proliferation METHODS:In order to get a full length of DLEU1 cDNA, total RNA was prepared from embryonic kidney epithelial 293T and Raji Burkitt lymphoma cells using Trizol (Invitrogen), and cDNA was synthesized by qScript™ cDNA Synthesis Kit (Quantas). The full length of cDNA encoding DLEU1 was fused into pEGFP-N3 vector. pEGFP-DLEU1fusion construct was transfected into 293T and Raji cells using lipofectamin or Amaxa Nucleofector™ kit V, respectively, followed by the manufacturer’s instruction. Twenty-four (293T) or Forty-eight hours (Raji) post transiently transfection, total RNA was extracted and 1ug of total RNA was used for cDNA synthesized as above. For comparison of mRNA expression of network genes, quantitative RT-PCR was performed using SsoFast™ EvaGreen® Supermix reagent by CFX96 Real-time system (Bio-rad) and statistical significance was performed by one-tailed Student t-test. For observation of expression of DLEU1 fusion protein, GFP only as control and GFP-DLEU1 transiently overexpressed 293T and Raji cells were observed under fluorescent microscope, and total lysates were prepared from both cells at 24 hours (293T) and 48hours (Raji) post-transfection and were hybridized with Rabbit polyclonal DLEU1 anitbody (Proteintech) on immune blotting. For cell proliferation assay, GFP-DLEU1 transiently overexpressed 293T and Raji cells were re-plated at 24hours post-transfection, and then counted every 24hrs. RESULTS:DLEU1 full length cDNA (234nt, 78amino acid) successfully fused into pEGFP expression vector and observed GFP fluorescence under fluorescent microscope in both 293T and Raji cells, and the predicted size of the fusion protein was confirmed by western blot (approximately 36kDa) [Figure 1]. The level of DLEU1 mRNA expression in GFP-DLEU1 transiently transfected 293T and Raji cells was significantly higher than GFP only transfected Raji cell as control [50% increase (p<0.05) and 25% increase (p<0.005), respectively]. In comparison of mRNA expression of DLEU1 network genes in DLEU1 overexpressed 293T cells, there were no significant differences in mRNA expression of c-myc compared to control, however, there was significantly increased expression of RASSF1 and UBR1 mRNA (>1.5-fold, p<0.03 and >1.2-fold, p<0.02), respectively, and significantly reduced expression of TUBB2C mRNA (<1.2-fold, p<0.03) in DLEU1 overexpressed 293T cells. In addition, in DLEU1 overexpressed Raji cells, there was no significant differences in c-myc mRNA similar to 293T cells, however, there was significantly increased expression of UBR1 mRNA (>1.1-fold, p<0.05), and significantly reduced expression of RASSF1 and TUBB2C mRNA (<1.4-fold, p<0.02 and <1.5-fold, p<0.03) in DLEU1 overexpressed Raji cells. DLEU1 transiently overexpressed cells showed an inhibition of cell proliferation in both 293T and Raji cells [60% reduction (p<0.05) at day 4 and 20% reduction (p<0.01) at day 5, respectively]. CONCLUSION:These results suggest that DLEU1 may act as a tumor suppressor gene and that following overexpression of DLEU1 resulted in inhibition of cell proliferation in Burkitt Lymphoma. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Relationship between the expressions of glucocorticoid receptors α, β, γ, p and heat shock protein 90 mRNA in patients with primary immune thrombocytopenia resistant to the glucocorticoid

To investigate the expressions of GRα, GRβ, GRγ, GRp and heat shock protein 90 (HSP90) in primary immune thrombocytopenia (ITP) patients and their correlations with glucocorticoid resistance. The expressions of glucocorticoid receptors (GRα, GRβ, GRγ, GRp) mRNA and HSP90 in peripheral blood mononuclear cells of 40 newly diagnosed ITP patients and 29 healthy volunteers were examined by real time PCR. Of them, 28 patients received glucocorticoid (GC) therapy divided into GC sensitive group (GCS) and GC resistant group (GCR) according to GC response. GRα, GRβ, GRγ, GRp, HSP90 mRNA and HSP90/GRα were analyzed in paired groups. The expression of HSP90 mRNA was significantly decreased in ITP patients \[0.91(0.48 - 2.21)\] than in normal subjects \[1.41(0.83 - 2.61)\] (P < 0.05). There were no significant differences in mRNA expressions of GRα, GRβ, GRγ, GRp and HSP90/GRα between ITP patients and normal controls. The expression of GRα mRNA in GCS patients was significant higher than in GCR patients (P < 0.05). Moreover, no significant differences in mRNA expressions of GRβ, GRγ, GRp and HSP90 and the ratio of HSP90 to GRα were observed between GCS and GCR patients. The expression of HSP90 mRNA decreased in adult ITP patients. GC resistance in adult ITP patients was associated with reduced expression of GRα. The very low expression of GRβ mRNA may be not involved in GC resistance in adult ITP.

Effect of electroacupuncture preconditioning at “Nèiguān” ( PC 6) on gene expression of myocardial opioid receptors in rats with ischemia-reperfusion

ObjectiveTo explore protective effects of electroacupuncture at “Nèiguān” (▪ PC 6) for preconditioning on myocardial ischemia-reperfusion injury (MIRI) and the mechanisms involved. MethodsForty-eight male Wistar rats were randomly divided into a sham operation group (Group N), a MIRI group (Group M) and an electroacupuncture (EA) group (Group E). The MIRI model was established by ligating the left anterior descending artery (LAD) for 30 min followed by reperfusion for 120 min. Partition sutures were passed under LAD without ligation for rats in Group N. Rats in Group E were pretreated with electroacupuncture (EA) applied at bilateral “Nèiguān” (▪ PC 6) for 20 min once a day for 3 consecutive days before ischemia. The infarct size plus the area at risk was evaluated by 2,3,5-triphenyltetrazolium chloride staining, and serum isoenzyme of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) levels were measured by biochemical methods. Myocardium morphological changes were observed under light microscopy. The mRNA expressions of myocardial δ and κ opioid receptors (DOR and KOR) were tested by real-time RT-PCR measurements. ResultsThe myocardial infarct size in Group E was more significantly decreased than that in Group M (P<0.05). The levels of CK-MB [(980 ± 92) U/L] and LDH [(2743 ± 124) U/L] in Group M were significantly higher than those in Group N [(312±41) U/L] and [(530 ± 56) U/L], respectively (both P<0.01). The levels of CK-MB [(572±70) U/L] and LDH [(1819±97) U/L] in Group E were significantly lower than those in Group M (both P<0.01). There were no significant differences in mRNA expressions of DOR and KOR between Group M and Group N (both P>0.05), but DOR expression in Group E was significantly higher than that either in Group M or in Group N (both P<0.01). No significant differences were found in KOR expression among the three groups (all P>0.05). ConclusionUp-regulation of expression of δ opioid receptors may be involved in protective effects of EA at Nèiguān (▪ PC 6) for preconditioning on MIRI.

Basic fibroblast growth factor suppresses meiosis and promotes mitosis of ovarian germ cells in embryonic chickens

Basic fibroblast growth factor (bFGF or FGF2) plays diverse roles in regulating cell proliferation, migration and differentiation during embryo development. In this study, the effect of bFGF on ovarian germ cell development was investigated in the embryonic chicken by in vitro and in vivo experiments. Results showed that a remarkable decrease in bFGF expression in the ovarian cortex was manifested during meiosis progression. With ovary organ culture, we revealed that meiosis was initiated after retinoic acid (RA) treatment alone but was decreased after combined bFGF treatment that was detected by real time RT-PCR, fluorescence immunohistochemistry and Giemsa staining. Further, no significant difference in mRNA expression of either RA metabolism-related enzymes (Raldh2 and Cyp26b1) or RA receptors was displayed after bFGF challenge. This result suggests that the suppression of bFGF on meiosis was unlikely through inhibition of RA signaling. In addition, as a mitogen, bFGF administration increased germ cell proliferation (via BrdU incorporation) in cultured organ or cells in vitro and also in developing embryos in vivo. In contrast, blockade of bFGF action by SU5402 (an FGFR1 antagonist) or inhibition of protein kinase C signaling showed inhibited effect of bFGF on mitosis. In conclusion, bFGF suppresses RA-induced entry of germ cells into meiosis to ensure embryonic ovarian germ cells to maintain at undifferentiated status and accelerate germ cell proliferation by binding with FGFR1 involving PKC activation in the chicken.

Transcriptional responses to loss or gain of function of the leucine-rich repeat kinase 2 (LRRK2) gene uncover biological processes modulated by LRRK2 activity

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are the most common genetic cause of Parkinson's disease (PD) and cause both autosomal dominant familial and sporadic PD. Currently, the physiological and pathogenic activities of LRRK2 are poorly understood. To decipher the biological functions of LRRK2, including the genes and pathways modulated by LRRK2 kinase activity in vivo, we assayed genome-wide mRNA expression in the brain and peripheral tissues from LRRK2 knockout (KO) and kinase hyperactive G2019S (G2019S) transgenic mice. Subtle but significant differences in mRNA expression were observed relative to wild-type (WT) controls in the cortex, striatum and kidney of KO animals, but only in the striatum in the G2019S model. In contrast, robust, consistent and highly significant differences were identified by the direct comparison of KO and G2019S profiles in the cortex, striatum, kidney and muscle, indicating opposite effects on mRNA expression by the two models relative to WT. Ribosomal and glycolytic biological functions were consistently and significantly up-regulated in LRRK2 G2019S compared with LRRK2 KO tissues. Genes involved in membrane-bound organelles, oxidative phosphorylation, mRNA processing and the endoplasmic reticulum were down-regulated in LRRK2 G2019S mice compared with KO. We confirmed the expression patterns of 35 LRRK2-regulated genes using quantitative reverse transcription polymerase chain reaction. These findings provide the first description of the transcriptional responses to genetically modified LRRK2 activity and provide preclinical target engagement and/or pharmacodynamic biomarker strategies for LRRK2 and may inform future therapeutic strategies for LRRK2-associated PD.

The role of RhoA/Rho kinase signalling pathway in myocardial fibrosis in rats with type 2 diabetes

The role of RhoA/Rho kinase signalling pathway in myocardial fibrosis in rats with type 2 diabetes

Androgen receptor W741C and T877A mutations in AIDL cells, an androgen-independent subline of prostate cancer LNCaP cells

The androgen-independent LNCaP (AIDL) cell line was generated by maintaining prostate cancer LNCaP cells in a hormone-deprived medium. Notably, synthetic androgen R1881-related gene response is attenuated in AIDL cells as compared to the parental LNCaP cells. The aim of this study was to clarify the mechanisms underlying androgen sensitivity in AIDL cells. We first examined the expression of androgen receptor (AR) and its co-regulators. However, no significant difference in mRNA expression was found between LNCaP and AIDL cells. Remarkably, AR protein levels were induced by R1881 and DHT in LNCaP cells, but not in AIDL cells. We next performed the cDNA sequencing to detect mutations in the AR gene. The T877A mutation was detected both in LNCaP and AIDL cells. Furthermore, AIDL cells harbored a missense substitution (TGG → TGT) in the AR gene, which caused a point mutation at codon 741 (W741C). Double T877A and W741C AR mutants have been previously reported to exhibit reduced androgen sensitivity. Hence, the low-androgen-sensitive responses of AIDL cells may be explained, at least in part, by AR gene mutations.

Yin Yang-1(YY-1) expression in idiopathic pulmonary fibrosis

Context: Yin Yang-1 (YY-1) is implicated in the pathogenesis of lung cancer which can be complicated with idiopathic pulmonary fibrosis (IPF).Objective: The aim of the study was to investigate whether YY-1 is involved in the pathogenesis of IPF and whether represents a common pathogenetic pathway which could explain the coexistence of these disorders.Materials and methods: Lung tissue from 52 patients (37 with IPF and 15 controls) and bronchoalveolar lavage fluid (BALF) from 34 patients (25 with IPF and 9 controls) were studied and YY-1 mRNA expression was evaluated by real-time PCR.Results: YY-1 was expressed in 8% (3/37) of IPF patients and in 6% (1/15) of healthy controls in tissue samples. In addition, 12% (3/25) of IPF patients and 33% (3/9) of healthy controls have expressed YY-1 gene in BALF samples. However, no statistical significant difference in mRNA expression between patients and controls has been detected in both tissue and BAL fluid samples.Discussion and conclusion: Our results do not support the hypothesis of YY-1 involvement in IPF. However, similar expression of YY-1 gene in two biological samples cannot exclude a possible role of this polymorphic gene in the pathway of IPF. Further studies in a larger scale of patients are needed.

Runx3 Expression Inhibits Proliferation and Distinctly Alters mRNA Expression of Bax in AGS and A549 Cancer Cells.

Runx3, a member of Runt-related transcription factor (Runx) proteins with tumor suppressor effect, is a tissue-restricted and cancer related transcription factor that regulate cell proliferation and growth, as well as differentiation. In the present study, exogenous Run3 was transiently expressed in AGS (human gastric adenocarcinoma), with undetectable Runx3 protein and in A549 (human lung carcinoma) with low levels of endogenous Runx3 protein. The GFP tagged Runx3 was transfected into AGS and A549 cells using fugene6 and PolyFect and Runx3 expression was confirmed by fluorescent microscopy and RT-PCR. The effect of Runx3 transfection on cell proliferation was determined by MTT assay and the results were confirmed by the trypan blue dye exclusion method. The effect of Runx3 expression on mRNA expression of BCL2-associated X protein (Bax) was evaluated using RT-PCR. In AGS and A549 cells, Runx3 expression inhibited cell proliferation (p < 0.01). The growth inhibition was less in A549 cells. We show that Runx3 expression increases Bax mRNA expression in AGS cells when compared with control (p < 0.05), but no significant differences in mRNA expression was observed in both examined cells. Runx3 expression has antiproliferative effect in AGS cell perhaps via increase in expression of Bax. The effect of Runx3 on A549 cells' viability which has endogenous level of Runx3 is not related to Bax. These findings implicate a complex regulation by Runx3 in inhibition of cell proliferation utilizing Bax.

Cytotoxicity and cytokine production by bovine alveolar macrophages challenged with wild type and leukotoxin-deficient Mannheimia haemolytica

Leukotoxin (LKT) is a virulence factor for Mannheimia haemolytica. In this study, bovine alveolar macrophages (BAMs) were challenged with wild type (wt) and LKT deficient (lkt(-)) M. haemolytica at a concentration of 1 bacterium/BAM and the cytokine response was quantified by ELISA and real-time reverse transcriptase-PCR. Significant increases in protein concentrations of tumor necrosis factor (TNF)-α and interleukin (IL)-10 were observed in supernatants obtained from BAMs challenged with the lkt(-) strain of M. haemolytica compared with wt challenged BAMs. There were no significant differences in mRNA expression of TNFα, IL-1ß, IL-6, IL-8 or IL-10 between BAMs challenged with the lkt(-) strain of M. haemolytica compared with wt challenged BAMs. BAMs challenged with the wt strain exhibited, on average, 43% more cytotoxicity than lkt(-) challenged BAMs (P<0.01).

Metastases of squamous cell carcinoma of the head and neck show increased levels of nucleotide excision repair protein XPF in vivo that correlate with increased chemoresistance ex vivo

Nucleotide excision repair (NER) is a key node of resistance of tumour cells to the anticancer drug cisplatin. Testicular germ cell tumours (TGCT) show exquisite sensitivity towards cisplatin, and this has been connected to low levels of the NER proteins ERCC1 and XPF. Tumours of some patients with advanced head and neck squamous cell carcinoma (HNSCC) regress well under cisplatin chemotherapy but prediction of responsiveness is poor. Little is known about the levels of ERCC1-XPF in HNSCC tissues and cell lines. We investigated mRNA and protein levels of ERCC1 and XPF in 13 HNSCC cell lines and seven testis tumour cell lines and examined the correlation between levels of ERCC1 and XPF and cellular resistance towards cisplatin. No significant difference in mRNA expression of either ERCC1 or XPF in the HNSCC cell lines compared to the testis tumour cell lines was observed. Significantly higher XPF protein levels were found in HNSCC cell lines compared to testis tumour cell lines resulting in cellular cisplatin resistance. The data indicate a contribution of XPF protein for the cisplatin resistance observed in HNSCC cell lines. Subsequently, XPF and ERCC1 protein expression was investigated in cancer tissue of 34 patients. XPF levels were significantly higher in metastases of HNSCC patients than in primary cancer tissue. These findings indicate a contribution of XPF protein for the observed chemoresistance in some HNSCC tissue. XPF protein may be a predictive marker for cisplatin responsiveness of metastases in HNSCC patients.

Ovarian matrix metalloproteinases are differentially regulated during the estrous cycle but not during short photoperiod induced regression in Siberian hamsters (Phodopus sungorus)

BackgroundMatrix metalloproteinases (MMPs) are implicated as mediators for ovarian remodeling events, and are involved with ovarian recrudescence during seasonal breeding cycles in Siberian hamsters. However, involvement of these proteases as the photoinhibited ovary undergoes atrophy and regression had not been assessed. We hypothesized that 1) MMPs and their tissue inhibitors, the TIMPs would be present and differentially regulated during the normal estrous cycle in Siberian hamsters, and that 2) MMP/TIMP mRNA and protein levels would increase as inhibitory photoperiod induced ovarian degeneration.MethodsMMP-2, -9, -14 and TIMP-1 and -2 mRNA and protein were examined in the stages of estrous (proestrus [P], estrus [E], diestrus I [DI], and diestrus II [DII]) in Siberian hamsters, as well as after exposure to 3, 6, 9, and 12 weeks of inhibitory short photoperiod (SD).ResultsMMP-9 exhibited a 1.6-1.8 fold decrease in mRNA expression in DII (p < 0.05), while all other MMPs and TIMPs tested showed no significant difference in mRNA expression in the estrous cycle. Extent of immunostaining for MMP-2 and -9 peaked in P and E then significantly declined in DI and DII (p < 0.05). Extent of immunostaining for MMP-14, TIMP-1, and TIMP-2 was significantly more abundant in P, E, and DI than in DII (p < 0.05). Localization of the MMPs and TIMPs had subtle differences, but immunostaining was predominant in granulosa and theca cells, with significant differences noted in staining intensity between preantral follicles, antral follicles, corpora lutea, and stroma classifications. No significant changes were observed in MMP and TIMP mRNA or extent of protein immunostaining with exposure to 3, 6, 9, or 12 weeks of SD, however protein was present and was localized to follicular and luteal steroidogenic cells.ConclusionsAlthough MMPs appear to be involved in the normal ovarian estrus cycle at the protein level in hamsters, those examined in the present study are unlikely to be key players in the slow atrophy of tissue as seen in Siberian hamster ovarian regression.

A dose-controlled system for air-liquid interface cell exposure and application to zinc oxide nanoparticles

BackgroundEngineered nanoparticles are becoming increasingly ubiquitous and their toxicological effects on human health, as well as on the ecosystem, have become a concern. Since initial contact with nanoparticles occurs at the epithelium in the lungs (or skin, or eyes), in vitro cell studies with nanoparticles require dose-controlled systems for delivery of nanoparticles to epithelial cells cultured at the air-liquid interface.ResultsA novel air-liquid interface cell exposure system (ALICE) for nanoparticles in liquids is presented and validated. The ALICE generates a dense cloud of droplets with a vibrating membrane nebulizer and utilizes combined cloud settling and single particle sedimentation for fast (~10 min; entire exposure), repeatable (<12%), low-stress and efficient delivery of nanoparticles, or dissolved substances, to cells cultured at the air-liquid interface. Validation with various types of nanoparticles (Au, ZnO and carbon black nanoparticles) and solutes (such as NaCl) showed that the ALICE provided spatially uniform deposition (<1.6% variability) and had no adverse effect on the viability of a widely used alveolar human epithelial-like cell line (A549). The cell deposited dose can be controlled with a quartz crystal microbalance (QCM) over a dynamic range of at least 0.02-200 μg/cm2. The cell-specific deposition efficiency is currently limited to 0.072 (7.2% for two commercially available 6-er transwell plates), but a deposition efficiency of up to 0.57 (57%) is possible for better cell coverage of the exposure chamber.Dose-response measurements with ZnO nanoparticles (0.3-8.5 μg/cm2) showed significant differences in mRNA expression of pro-inflammatory (IL-8) and oxidative stress (HO-1) markers when comparing submerged and air-liquid interface exposures. Both exposure methods showed no cellular response below 1 μg/cm2 ZnO, which indicates that ZnO nanoparticles are not toxic at occupationally allowed exposure levels.ConclusionThe ALICE is a useful tool for dose-controlled nanoparticle (or solute) exposure of cells at the air-liquid interface. Significant differences between cellular response after ZnO nanoparticle exposure under submerged and air-liquid interface conditions suggest that pharmaceutical and toxicological studies with inhaled (nano-)particles should be performed under the more realistic air-liquid interface, rather than submerged cell conditions.

Significant upregulation of antimicrobial peptides and proteins in lichen sclerosus

Lichen sclerosus (LS) is a chronic inflammatory T cell-driven sclerotic skin condition in which skin barrier disruption frequently occurs. Inflamed and injured epithelia are a particularly rich source of antimicrobial peptides and proteins (AMPs). We aimed to investigate for the first time the expression pattern of AMPs in lesions of LS as compared with healthy skin. Twenty-four women with LS as well as 10 healthy women were included in the study. In order to assess the expression of human beta-defensin (hBD)-1, hBD-2, hBD-3, psoriasin (S100A7), the cathelicidin LL-37 and RNase 7, real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry were performed on skin specimens obtained from lesional and healthy skin of the genital region, respectively. Median hBD-2 mRNA levels observed in LS were significantly higher than in controls (0.15 vs. 0.008; P = 0.0037). Moreover, psoriasin (98.2 vs. 28.1; P = 0.0052) mRNA expression was significantly higher in LS lesions as compared with controls. Significant differences in mRNA expression of hBD-2 and psoriasin were also confirmed by immunohistochemistry. For hBD-1, hBD-3, LL-37 and RNase 7, levels did not differ significantly or were significant only at the gene level but not protein level. We have demonstrated that hBD-2 and psoriasin expression levels in lesional skin of patients with LS are significantly increased when compared with healthy controls. Whether this observation simply reflects an innate defence response caused by an increased risk of local infection, or whether our data indicate a pathogenetic role of AMPs in LS, will be addressed in future studies.

Local expression of interferon-alpha and interferon receptors in cervical intraepithelial neoplasia.

The present study evaluated mRNA expression of interferon-alpha (IFN-alpha), IFN-alpha receptor subunits (IFNAR-1 and IFNAR-2) and an IFN-stimulated gene encoding the enzyme 2',5'-oligoadenylate synthetase (2'5'OAS) in biopsies on patients with varying grades of cervical intraepithelial neoplasia (CIN I, II and III). Uterine cervix biopsies were collected from women with CIN I, II and III (n = 28) and controls without CIN lesions or human papilloma virus (HPV) infection (n = 17). The presence of high and low-risk HPV DNA was determined using hybrid capture. The mRNA levels of IFNAR-1, IFNAR-2, IFN-alpha and 2'5'OAS were determined by RT-PCR with specific primers. The control group exhibited a greater frequency of IFNAR-1 expression (10/17; 58.3%) than the CIN samples (4/28; 14.2%) (P = 0.0018), while, the expression of IFNAR-2 was also greater in the control samples (11/17; 64.7%) than in the patients with lesions (2/28; 7.1%) (P = 0.0018). Importantly, simultaneous expression of both receptors was observed only in the control group (8/17; 47.0%) (P = 0.0001). Among the CIN samples, there was one case of low expression of mRNA of IFNAR-1 and IFNAR-2. IFN-alpha was present in 14.2% (4/28) of the CIN samples but was not expressed in the control group. mRNA 2'5'OAS were expressed in 28.5% (8/28) of the CIN samples and 11.7% (2/17) of the control samples (not statistically significant). Fifty percent (14/28) of the CIN samples were positive for HPV DNA. Cervical biopsy samples from control women or those without neoplasia or HPV infection displayed higher IFN-alpha receptor expression than those with CIN, while simultaneous expression of both IFN-alpha receptor subunits was found only in the control group. There was no significant difference in mRNA expression of IFN-alpha and 2'5'OAS between the control and CIN groups. Then we concluded that the samples obtained from patients with CIN present low levels of the IFN-alpha receptor mRNA.

Association of Estrogen Receptor α Gene Polymorphisms with Cytokine Genes Expression in Systemic Lupus Erythematosus

To analyze the association of estrogen receptor alpha (OR alpha) gene polymorphisms with cytokine genes expression in patients with systemic lupus erythematosus (SLE) and controls. Genomic DNA was extracted and polymorphisms of XbaI, Ukrainian (XX, Xx, or xx genotype) and PvuII (PP, Pp, or pp) in intron 1 of OR alpha gene were detected by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method. The messenger RNA (mRNA) levels of interleukin (IL)-10, IL-4, interferon (IFN)-gamma, and IL-2 were assessed by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). In patients with SLE with PpXx genotype, IL-10 and IL-4 mRNA expression was higher (P < 0.001 and P = 0.013, respectively), while in patients with SLE with Ppxx genotype IFN-gamma and IL-2 mRNA expression was lower than in controls (P < 0.001). There was no significant difference in mRNA expression of 4 cytokines among controls with various genotypes. OR alpha gene polymorphism may be associated with the expression of IL-10, IL-4, IL-2, and IFN-gamma in patients with SLE.