Generation of inducible hepatitis C virus transgenic mouse lines

Hepatitis C virus (HCV) is the causative agent of most cases of chronic liver disease, cirrhosis, and hepatocellular carcinoma (HCC) affecting more than 170 million people world-wide. Progress in elucidating the nature of HCV and the development of new therapeutic strategies is hampered fundamentally by the absence of adequate small animal models simulating natural HCV infection. The creation of conditional mouse lines with the tetracycline-controlled gene expression system holds new perspectives for simulation of wild-type HCV infection in a small animal model. Transgenic mice were established with tetracycline-inducible coexpression of HCV core or HCV open reading frame (ORF) and luciferase. In long-term induction experiments, mice were examined for immunopathological changes after expression of HCV proteins. Inducible and liver-specific expression of transgenes was detected by Western blot, immunoprecipitation, luciferase assay and in vivo imaging of bioluminescence of luciferase in genetically modified mice. Ectopic expression levels were determined quantitatively in the liver, kidney, heart and spleen of mice in the induced and non-induced state. During long-term induction an elevation of aminotransaminases (ALT) was observed only in HCV core/ORF-expressing mice, but HCV-specific immune response was not confirmed by in vitro immunological assays. The histology of liver sections provided evidence of steatosis, which was correlated with an inflammatory response. The inducible HCV-transgenic mouse lines provide further evidence of liver pathogenesis in the presence of inflammation during liver-specific expression of HCV proteins and offer new insights into the effects of temporally and spatially controlled protein expression of HCV.     

Xenopus laevis insulin receptor substrate IRS‐1 is important for eye development

Extracellular signal transduction into cells through ligand‐activated receptor tyrosine kinases, such as insulin‐like growth factor‐1 (IGF‐1) receptor (IGF‐1R) and insulin receptor (IR) is required for normal embryonic growth and development. The major mediators of IR and IGF‐1R are adaptor proteins of the insulin receptor substrate family, the best characterized member of which is IRS‐1. Insulin receptor substrate IRS‐1 has been shown to influence cell and body size and to interfere with differentiation. We have isolated IRS‐1 from Xenopus laevis embryos and analyzed for the first time its spatial and temporal expression pattern during embryogenesis. We found that Xenopus IRS‐1 is expressed maternally and constantly during embryogenesis. It is predominantly found in neural tissue at different stages. Furthermore, knock down of IRS‐1 in neural tissue by specific antisense morpholino oligonucleotides (MO) resulted in abnormal eye formation accompanied by reduction of the eye‐specific marker genes Rx1 and Pax6 and a decreased cell proliferation. Developmental Dynamics 240:1705–1715, 2011. © 2011 Wiley‐Liss, Inc.     

A novel bioassay for anti‐thyrotrophin receptor autoantibodies detects both thyroid‐blocking and stimulating activity

Autoantibodies to the thyrotrophin (TSH) receptor (anti‐TSHR) are unique, in that they are involved directly in the pathophysiology of certain autoimmune thyroid diseases (AITD). Thyroid‐stimulating antibodies (TSAb) act as agonists that activate the thyroid gland and cause Graves' disease. Other anti‐TSHR antibodies block TSH and can cause hypothyroidism. Thyroid‐blocking antibodies (TBAb) have not been studied as extensively as TSAb. We developed a TBAb bioassay based on a cell line that expresses a chimeric TSHR. The 50% inhibitory concentration of the chimeric Chinese hamster ovary (CHO)‐Luc cells was more than five‐fold lower compared with the wild‐type CHO‐Luc cells. We tested the performance of this bioassay using a thyroid‐blocking monoclonal antibody K1‐70, established an assay cut‐off and detected TBAb in 15 of 50 (30%) patients with AITD. Interestingly, the assay detects both TSAb and TBAb and measures the net activity of a mixture of both types of antibodies. There was a high correlation (R² 0·9, P < 0·0001) between the results of the TSAb assay and the negative percentage inhibition of the TBAb assay. The TBAb bioassay was approximately 20‐fold more sensitive than a commercially available TSHR binding assay (TRAb). In contrast to TRAb, sera with high levels of TBAb activity were able to be diluted several hundred‐fold and still exhibit blocking activity above the cut‐off level. Thus, this TBAb bioassay provides a useful tool for measuring the activity of anti‐TSHR antibodies and may help clinicians to characterize the diverse clinical presentations of patients with AITD.     

Developmental Changes in Lectin‐Binding Patterns of Three Nasal Sensory Epithelia in Xenopus laevis

The nasal cavity of adult Xenopus laevis (X. laevis) is composed of a series of three compartments: principal, middle, and inferior chambers. The principal chamber is lined with olfactory epithelium (OE), middle chamber with middle chamber epithelium (MCE), and inferior chamber with vomeronasal epithelium (VNE). In the present study, we examined developmental changes of lectin‐binding patterns of the OE, MCE, and VNE by the use of four biotinylated lectins; DSL, DBA, PNA, and UEA‐I. From Stage 59, just after the beginning of metamorphosis, the stainings of the free border for DBA and UEA‐I were decreased in the OE and MCE, respectively, but the stainings of secretory granules (SGs) in the OE became intense. From Stage 63, sensory cells positive for DSL were increased in these three epithelia, and positive stainings for UEA‐I and DBA increased in the SGs and Jacobson's glands (JGs), respectively. In addition, from 3 months after the end of metamorphosis, the stainings of sensory cells for PNA, DBA, and DSL changed in the OE, MCE, and VNE, respectively, and those of the SGs, Bowman's glands, and JGs also changed for several lectins. The present results showed that glycoconjugates expressed in three epithelia and their associated glands changed during and after the end of metamorphosis. These findings may indicate that the functional maturation of each epithelium depends not only on the maturation of sensory cells, but also on the maturation of the SGs in supporting cells of the OE and their associated glands after the end of metamorphosis. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

Micro‐Computed Tomography for Visualizing Limb Skeletal Regeneration in Young Xenopus Frogs

For studies of vertebrate limb regeneration it is often desirable to visualize the regenerated skeleton, which is mostly cartilage, and also section the specimen for histological or immunohistochemical visualization of other tissues. However, the normal skeletal staining techniques are incompatible with immunohistochemistry. Here, we describe a contrast‐based micro‐computed tomography (microCT) method for direct and nondestructive observation of regenerated cartilage spikes in Xenopus frog limbs. In addition, we show that contrast based microCT imaging is compatible with immunohistochemistry protocols. This approach provides versatile and high contrast images of the cartilage allowing us to measure the regenerated skeletal structure in detail as well as carrying out the other types of analysis. It opens a wide range of potential microCT applications in research on vertebrate limb regeneration. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Reduced expression of oestrogen receptor‐β is associated with tumour invasion and metastasis in oestrogen receptor‐α‐negative human papillary thyroid carcinoma

Oestrogens play an important role in the development and progression of papillary thyroid carcinoma (PTC) through oestrogen receptor (ER)‐α and ‐β, which may exert different or even opposing actions in PTC. The roles of ERβ in ERα‐negative PTC are still not clear. This study investigated the expression dynamics of ERβ1 (wild‐type ERβ) and its clinical significance in female ERα‐negative PTC patients. ERβ1 expression was detected in thyroid tissues of 136 female patients diagnosed with PTC. The relationships between ERβ1 expression and clinicopathological/biological factors were also analysed in female ERα‐negative PTC patients. The total score for ERβ1 was significantly lower in female ERα‐negative PTC patients with LNM or ETE when compared to those without LNM or ETE (Z = ?2.923, P = 0.003 and Z = ?3.441, P = 0.001). Accordingly, the total score for ERβ1 was significantly higher in ERα‐negative PTC patients expressing E‐cadherin compared to patients negative for E‐cadherin expression (Z = ?2.636, P = 0.008). The total score was lower in ERα‐negative PTC patients positive for VEGF expression compared to those negative for VEGF expression (Z = ?1.914, P = 0.056). This preliminary study indicates that reduced expression of ERβ1 in female ERα‐negative PTC patients is associated with greater progression of the disease. This may provide insights into the underlying molecular mechanisms of ERβ1 and could help design targeted approaches for treating or even preventing this disease.     

The Role of Sdf‐1α signaling in Xenopus laevis somite morphogenesis

Background: Stromal derived factor‐1α (sdf‐1α), a chemoattractant chemokine, plays a major role in tumor growth, angiogenesis, metastasis, and in embryogenesis. The sdf‐1α signaling pathway has also been shown to be important for somite rotation in zebrafish (Hollway et al., 2007). Given the known similarities and differences between zebrafish and Xenopus laevis somitogenesis, we sought to determine whether the role of sdf‐1α is conserved in Xenopus laevis. Results: Using a morpholino approach, we demonstrate that knockdown of sdf‐1α or its receptor, cxcr4, leads to a significant disruption in somite rotation and myotome alignment. We further show that depletion of sdf‐1α or cxcr4 leads to the near absence of β‐dystroglycan and laminin expression at the intersomitic boundaries. Finally, knockdown of sdf‐1α decreases the level of activated RhoA, a small GTPase known to regulate cell shape and movement. Conclusion: Our results show that sdf‐1α signaling regulates somite cell migration, rotation, and myotome alignment by directly or indirectly regulating dystroglycan expression and RhoA activation. These findings support the conservation of sdf‐1α signaling in vertebrate somite morphogenesis; however, the precise mechanism by which this signaling pathway influences somite morphogenesis is different between the fish and the frog. Developmental Dynamics 243:509–526, 2014. © 2013 Wiley Periodicals, Inc.     

New assay to detect low‐affinity interactions and characterization of leukocyte receptors for collagen including leukocyte‐associated Ig‐like receptor‐1 (LAIR‐1)

Leukocyte activity is controlled by numerous interactions between membrane receptors and ligands on the cell surface. These interactions are of low affinity making detection difficult. We developed a sensitive assay that could readily detect extremely weak interactions such as that between CD200 and the activating receptor CD200RLa (K_d>500 μM) at the protein level. We used the new technology to screen for interactions of inhibitory receptors for collagens. We confirmed that both human and mouse leukocyte‐associated Ig‐like receptor‐1, and in addition the related inhibitory leukocyte Ig‐like receptor subfamily B member 4 (CD85K, Gp49B), bound collagen specifically, whereas other cell surface proteins gave no binding. The monomeric affinities of the interactions were then determined to allow comparison with other leukocyte interactions and indicate conditions when these interactions might lead to inhibitory signals.     

A flk-1 promoter/enhancer reporter transgenic Xenopus laevis generated using the Sleeping Beauty transposon system: an in vivo model for vascular studies.

We have used the Sleeping Beauty (SB) transposable element to generate transgenic Xenopus laevis with expression of green fluorescent protein (GFP) in vascular endothelial cells using the frog flk-1 promoter. This is the first characterization of a SB-generated transgenic Xenopus that has tissue-restricted expression. We demonstrate that the transgene integrated into single genomic loci in two independent founder lines and is transmitted through the germline at the expected Mendelian frequencies. Transgene integration occurred through a noncanonical transposition process possibly reflecting Xenopus-specific interactions with the SB system. The transgenic animals express GFP in the same spatial and temporal pattern as the endogenous flk-1 gene throughout development and into adulthood. Overexpression of xVEGF122 in the transgenic animals disrupts vascular development that is visualized by fluorescent microscopy. These studies demonstrate the convenience of the SB system for generating transgenic animals and the utility of the xflk-1:GFP transgenic line for in vivo studies of vascular development.     

17β‐Estradiol Exposure Accelerates Skeletal Development in Xenopus laevis Tadpoles

Although it is well established that estrogen regulates skeletal growth and ossification in mammals, the effects of estrogen on skeletal development in amphibians are relatively uncharacterized. This study was conducted to characterize the impact of 17β‐estradiol exposure on skeletal development in Xenopus laevis tadpoles. On day 48 postfertilization, tadpoles were placed in tanks containing 50% Holtfreter's Solution ±17β‐estradiol at one of four concentrations (10^?11, 10^?10, 10^?9, and 10^?8 M). At 7–11 day intervals until day 91, 7–10 tadpoles per group were killed, fixed, measured, and staged. Specimens were then cleared and double‐stained for cartilage and bone, and 34 skeletal elements were analyzed for ossification. Results from the study indicate that both low (10^?11 M) and high (10^?8 M) concentrations of 17β‐estradiol have a significant stimulatory effect on tadpole development. Both the larval stage and ossification index of tadpoles exposed to 10^?11 or 10^?8 M 17β‐estradiol were significantly greater than those observed in control animals by day 91 postfertilization. These results are consistent with the hypothesis that endogenous and exogenous estrogen could play a role in the regulation of bone ossification in amphibians. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Role of p21‐activated kinase in cell polarity and directional mesendoderm migration in the Xenopus gastrula

The p21 activated kinases (Paks) are prominently involved in the regulation of cell motility. Using a kinase‐dead mutant of xPak1, we show that during Xenopus gastrulation, the kinase activity of Pak1 is required upstream of Cdc42 for the establishment of cell polarity in the migrating mesendoderm. Overactivation of Pak1 function by the expression of constitutively active xPak1 compromises the maintenance of cell polarity, by indirectly inhibiting RhoA function. Inhibition of cell polarization does not affect the migration of single mesendoderm cells. However, Pak1 inhibition interferes with the guidance of mesendoderm migration by directional cues residing in the extracellular matrix of the blastocoel roof, and with mesendoderm translocation in the embryo. Developmental Dynamics 238:1709–1726, 2009. © 2009 Wiley‐Liss, Inc.     

Differential immunostaining of various types of breast carcinomas for growth hormone‐releasing hormone receptor – Apocrine epithelium and carcinomas emerging as uniformly positive

Different classes of breast cancers were explored for their positivity for growth hormone‐releasing hormone receptors (GHRH‐R) in this pilot study, as no systematic evaluation of such tumors has been performed to date. Seventy‐two small primary breast carcinomas were evaluated for GHRH‐R expression by immunohistochemistry using a polyclonal antibody and a cutoff value of 10% staining. GHRH‐R positivity was detected in 58% of all cases, 20/23 (87%) of invasive lobular carcinomas (ILC) and 22/46 (48%) of invasive ductal carcinomas (IDC). GHRH‐R positivity was more frequent in grade 2 tumors (86%), as compared to grade 1 (18%) or grade 3 (47%) cancers. GHRH‐R expression was not associated with mitotic scores, the Ki‐67 labeling indices or nodal status. IDCs with casting‐type calcifications on the mammogram showed positivity for GHRH‐R in 9/12 (75%) cases. Most importantly, apocrine epithelium, and all 10 apocrine carcinomas added later to the study were GHRH‐R‐positive. These preliminary results suggest a greater than average GHRH‐R expression in ILCs and IDCs associated with casting‐type calcifications on the mammogram. Apocrine carcinomas seem uniformly positive for GHRH‐R. Whether these findings could indicate a potential role of GHRH‐antagonists in targeted treatment of these types of breast cancer requires further studies.     

Alpha‐melanocyte‐stimulating hormone attenuates behavioral effects of corticotropin‐releasing factor in isolated guinea pig pups

During a 3‐hr period of social isolation in a novel environment, guinea pig pups exhibit an initial active phase of behavioral responsiveness, characterized primarily by vocalizing, which is then followed by a stage of passive responsiveness in which pups display a distinctive crouch, eye‐closing, and extensive piloerection. Prior treatment of pups with alpha‐melanocyte‐stimulating hormone (α‐MSH) reduces each of the passive behaviors. The onset of passive responding during separation can be accelerated with peripheral injection of corticotropin‐releasing factor (CRF). To examine whether CRF produces its effects through a mechanism similar to that of prolonged separation, we examined the effect of administering α‐MSH to pups injected with CRF. As expected, CRF markedly enhanced passive responding during a 60‐min period of separation. α‐MSH delivered by either intracerebroventricular infusion or intraperitoneal injection significantly reduced each of the passive behavioral responses without significantly affecting active behavior. These findings, together with previous results indicating that it is the anti‐inflammatory property of α‐MSH that is responsible for its behavioral effects during prolonged separation, suggest that peripheral CRF speeds the induction of passive responding through a mechanism involving enhanced proinflammatory activity. © 2009 Wiley Periodicals, Inc. Dev Psychobiol 51: 399–407, 2009.     

Diagnostic value of B‐RAFV600E in difficult‐to‐diagnose thyroid nodules using fine‐needle aspiration: Systematic review and meta‐analysis

Fine‐needle aspiration (FNA) is routinely used in the preoperative evaluation of thyroid nodules. However, approximately 5–20% of thyroid nodules are considered indeterminate or suspicious cases that do not meet clinical standards. The B‐RAF^V600E mutation has been reported in FNA specimens. We conducted a systematic review to evaluate the diagnostic value of testing for B‐RAF^V600E in thyroid nodules that are difficult to diagnose by FNA. A systematic literature search was performed from January 1, 2002 to June 30, 2012. Articles were obtained by searching two electronic databases (MEDLINE and EMBASE), hand searching selected journals, and contacting authors. Article quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS) tool. Sensitivity, specificity, and other measures of accuracy were pooled using random effects models. Summary receiver operating characteristic (SROC) curves were used to summarize overall diagnostic accuracy. A total of 16 studies incorporating 1131 patients were included in a meta‐analysis on diagnostic accuracy of B‐RAF^V600E tests. Pooled sensitivity was 0.60 (95% confidence interval [CI]: 0.556–0.634), pooled specificity was 0.99 (95% CI 0.976–0.997), and the area under the curve of the SROC curve was 0.8376. Q index value was 0.7696. Our data suggest a potentially useful adjunct to evaluating thyroid nodules that are difficult to diagnose. The B‐RAF^V600E test has a high positive predictive value and could help clinicians formulate a more individualized treatment schedule. When supplemented with other noninvasive test methods, the B‐RAF^V600E test could be a powerful adjunct with extensive clinical applications. Diagn. Cytopathol. 2014;42:94–101. © 2013 Wiley Periodicals, Inc.