Extreme sarcoplasmic reticulum volume loss and compensatory T-tubule remodeling after Serca2 knockout

Cardiomyocyte contraction and relaxation are controlled by Ca(2+) handling, which can be regulated to meet demand. Indeed, major reduction in sarcoplasmic reticulum (SR) function in mice with Serca2 knockout (KO) is compensated by enhanced plasmalemmal Ca(2+) fluxes. Here we investigate whether altered Ca(2+) fluxes are facilitated by reorganization of cardiomyocyte ultrastructure. Hearts were fixed for electron microscopy and enzymatically dissociated for confocal microscopy and electrophysiology. SR relative surface area and volume densities were reduced by 63% and 76%, indicating marked loss and collapse of the free SR in KO. Although overall cardiomyocyte dimensions were unaltered, total surface area was increased. This resulted from increased T-tubule density, as revealed by confocal images. Fourier analysis indicated a maintained organization of transverse T-tubules but an increased presence of longitudinal T-tubules. This demonstrates a remarkable plasticity of the tubular system in the adult myocardium. Immunocytochemical data showed that the newly grown longitudinal T-tubules contained Na(+)/Ca(2+)-exchanger proximal to ryanodine receptors in the SR but did not contain Ca(2+)-channels. Ca(2+) measurements demonstrated a switch from SR-driven to Ca(2+) influx-driven Ca(2+) transients in KO. Still, SR Ca(2+) release constituted 20% of the Ca(2+) transient in KO. Mathematical modeling suggested that Ca(2+) influx via Na(+)/Ca(2+)-exchange in longitudinal T-tubules triggers release from apposing ryanodine receptors in KO, partially compensating for reduced SERCA by allowing for local Ca(2+) release near the myofilaments. T-tubule proliferation occurs without loss of the original ordered transverse orientation and thus constitutes the basis for compensation of the declining SR function without structural disarrangement.     

Glucocorticoid receptor disruption delays structural maturation in the lungs of newborn mice

In order to better understand the regulation of lung maturation by glucocorticoid-glucocorticoid receptor signaling, we studied glucocorticoid receptor (GR) hypomorphic mice with a mixed C57Bl6/129 sv background, in which disruption of exon 2 of the GR gene produces an N-terminal truncated GR protein. Four groups of mice were compared: homozygous mice that die at birth (non-survivors), homozygous mice that survive the neonatal period (survivors), heterozygotes and wild-type mice. Newborn non-survivors had 50% thicker airspace walls and a 46% decrease in the formation of secondary crests (the beginning of alveolar secondary septation) compared to either survivor or wild-type littermates (n = 9 mice in each group). The lung tissue to airspace ratio in homozygous mice not expressing wild-type GR (non-survivor and survivor) was increased compared to heterozygotes and wild-type mice that do express wild-type GR (0.91 +/- 0.08 vs. 0.49 +/- 0.02, n = 4 in each of the four subgroups), suggesting that complete morphological maturation of the lung is dependent on effective glucocorticoid signaling through a fully functional GR. Moreover, the relatively mature lung morphology of survivor versus non-survivor newborns suggests that a partial reduction in mesenchymal thickness is compatible with capillary remodeling, alveolar septation, and viable respiratory function after birth. Our findings suggest that in mice homozygous for disrupted GR, the severity of newborn respiratory insufficiency correlates with the degree of lung structural immaturity.     

Expression of Interleukin 9 in the Lungs of ?Transgenic Mice Causes Airway Inflammation,Mast Cell Hyperplasia,and Bronchial Hyperresponsiveness

Interleukin (IL)-9, a pleiotropic cytokine produced by the Th2 subset of T lymphocytes has been proposed as product of a candidate gene responsible for asthma. Its wide range of biological functions on many cell types involved in the allergic immune response suggests a potentially important role in the complex pathogenesis of asthma. To investigate the contributions of IL-9 to airway inflammation and airway hyperresponsiveness in vivo, we created transgenic mice in which expression of the murine IL-9 cDNA was regulated by the rat Clara cell 10 protein promoter. Lung selective expression of IL-9 caused massive airway inflammation with eosinophils and lymphocytes as predominant infiltrating cell types. A striking finding was the presence of increased numbers of mast cells within the airway epithelium of IL-9–expressing mice. Other impressive pathologic changes in the airways were epithelial cell hypertrophy associated with accumulation of mucus-like material within nonciliated cells and increased subepithelial deposition of collagen. Physiologic evaluation of IL-9–expressing mice demonstrated normal baseline airway resistance and markedly increased airway hyperresponsiveness to inhaled methacholine. These findings strongly support an important role for IL-9 in the pathogenesis of asthma.     

The use of genetically modified mice in cancer risk assessment: Challenges and limitations

Abstract

The use of genetically modified (GM) mice to assess carcinogenicity is playing an increasingly important role in the safety evaluation of chemicals. While progress has been made in developing and evaluating mouse models such as the Trp53^+/?, Tg.AC and the rasH2, the suitability of these models as replacements for the conventional rodent cancer bioassay and for assessing human health risks remains uncertain. The objective of this research was to evaluate the use of accelerated cancer bioassays with GM mice for assessing the potential health risks associated with exposure to carcinogenic agents. We compared the published results from the GM bioassays to those obtained in the National Toxicology Program’s conventional chronic mouse bioassay for their potential use in risk assessment. Our analysis indicates that the GM models are less efficient in detecting carcinogenic agents but more consistent in identifying non-carcinogenic agents. We identified several issues of concern related to the design of the accelerated bioassays (e.g., sample size, study duration, genetic stability and reproducibility) as well as pathway-dependency of effects, and different carcinogenic mechanisms operable in GM and non-GM mice. The use of the GM models for dose-response assessment is particularly problematic as these models are, at times, much more or less sensitive than the conventional rodent cancer bioassays. Thus, the existing GM mouse models may be useful for hazard identification, but will be of limited use for dose-response assessment. Hence, caution should be exercised when using GM mouse models to assess the carcinogenic risks of chemicals.     

A rice chloroplast transit peptide sequence does not alter the cytoplasmic localization of sheep serotonin N‐acetyltransferase expressed in transgenic rice plants

Ectopic overexpression of melatonin biosynthetic genes of animal origin has been used to generate melatonin‐rich transgenic plants to examine the functional roles of melatonin in plants. However, the subcellular localization of these proteins expressed in the transgenic plants remains unknown. We studied the localization of sheep (Ovis aries) serotonin N‐acetyltransferase (OaSNAT) and a translational fusion of a rice SNAT transit peptide to OaSNAT (TS:OaSNAT) in plants. Laser confocal microscopy analysis revealed that both OaSNAT and TS:OaSNAT proteins were localized to the cytoplasm even with the addition of the transit sequence to OaSNAT. Transgenic rice plants overexpressing the TS:OaSNAT fusion transgene exhibited high SNAT enzyme activity relative to untransformed wild‐type plants, but lower activity than transgenic rice plants expressing the wild‐type OaSNAT gene. Melatonin levels in both types of transgenic rice plant corresponded well with SNAT enzyme activity levels. The TS:OaSNAT transgenic lines exhibited increased seminal root growth relative to wild‐type plants, but less than in the OaSNAT transgenic lines, confirming that melatonin promotes root growth. Seed‐specific OaSNAT expression under the control of a rice prolamin promoter did not confer high levels of melatonin production in transgenic rice seeds compared with seeds from transgenic plants expressing OaSNAT under the control of the constitutive maize ubiquitin promoter.     

Neuromedin U – a new target in obesity

With the increasing prevalence of obesity, effective therapies are urgently required. When neuromedin U was administered intracerebroventricularly to rats there was a marked decrease in weight, whereas injection of an antibody to neuromedin U increased food intake. Unlike wild type, Nmu^-/- mice become obese when freely fed ordinary mouse chow. The plasma levels of insulin, leptin, total cholesterol, triglycerides and free fatty acids were higher in the Nmu^-/- than wild-type mice. Energy expenditure was lower in Nmu^-/- mice. The anorexigenic effect of neuromedin U was independent of the leptin signalling pathway. Transgenic mice overexpressing neuromedin U have lower body weight, less somatic and liver fat, were hypophagic, and had improved insulin sensitivity. These data establish neuromedin U as a target in obesity.     

An inducible transgenic mouse breast cancer model for the analysis of tumor antigen specific CD8+ T-cell responses

In Simian virus 40 (SV40) transgenic BALB/c WAP-T mice tumor development and progression is driven by SV40 tumor antigens encoded by inducible transgenes. WAP-T mice constitute a well characterized mouse model for breast cancer with strong similarities to the corresponding human disease. BALB/c mice mount only a weak cellular immune response against SV40 T-antigen (T-Ag). For studying tumor antigen specific CD8⁺ T-cell responses against transgene expressing cells, we created WAP-T_NP mice, in which the transgene additionally codes for the NP_118–126-epitope contained within the nucleoprotein of lymphocytic choriomeningitis virus (LCMV), the immune-dominant T-cell epitope in BALB/c mice. We then investigated in WAP-T_NP mice the immune responses against SV40 tumor antigens and the NP-epitope within the chimeric T-Ag/NP protein (T-Ag_NP). Analysis of the immune-reactivity against T-Ag in WAP-T and of T-Ag_NP in WAP-T_NP mice revealed that, in contrast to wild type (wt) BALB/c mice, WAP-T and WAP-T_NP mice were non-reactive against T-Ag. However, like wtBALB/c mice, WAP-T as well as WAP-T_NP mice were highly reactive against the immune-dominant LCMV NP-epitope, thereby allowing the analysis of NP-epitope specific cellular immune responses in WAP-T_NP mice. LCMV infection of WAP-T_NP mice induced a strong, LCMV NP-epitope specific CD8⁺ T-cell response, which was able to specifically eliminate T-Ag_NP expressing mammary epithelial cells both prior to tumor formation (i.e. in cells of lactating mammary glands), as well as in invasive tumors. Elimination of tumor cells, however, was only transient, even after repeated LCMV infections. Further studies showed that already non-infected WAP-T_NP tumor mice contained LCMV NP-epitope specific CD8⁺ T-cells, albeit with strongly reduced, though measurable activity. Functional impairment of these ‘endogenous’ NP-epitope specific T-cells seems to be caused by expression of the programmed death-1 protein (PD1), as anti-PD1 treatment of splenocytes from WAP-T_NP tumor mice restored their activity. These characteristics are similar to those found in many tumor patients and render WAP-T_NP mice a suitable model for analyzing parameters to overcome the blockade of immune checkpoints in tumor patients.     

Effect of mutant β‐catenin on liver growth homeostasis and hepatocarcinogenesis in transgenic mice

Background: Mutations in the Wnt signalling pathway molecule β‐catenin are associated with liver cancer. Aims: Our aim was to confirm the effects of stabilized β‐catenin on liver growth, identify whether those effects were reversible and cell autonomous or non‐cell autonomous and to model β‐catenin‐induced liver cancer in mice. Methods: Using a liver‐specific inducible promoter, we generated transgenic mice in which the expression of mutant β‐catenin can be induced or repressed within hepatocytes in mice of different ages. Results: Similar to other models, the hepatic expression of mutant β‐catenin in our model beginning in utero or induced in quiescent adult liver resulted in a two‐fold liver enlargement and development of disease with a latency of 1–5 months, and mice displayed elevated blood ammonia and altered hepatic gene expression. Our model additionally allowed us to discover that molecular and phenotypic abnormalities were reversible following the inhibition of transgene expression. Hepatocyte transplant studies indicated that mutant β‐catenin could not increase the growth of transgene‐expressing foci in either growth‐permissive or ‐restrictive hepatic environments, but still directly altered hepatocyte gene expression. Mice with continuous but focal transgene expression developed hepatic neoplasms after the age of 1 year. Conclusions: Our findings indicate that hepatocyte gene expression is directly affected by mutant β‐catenin in a cell autonomous manner. However, hepatomegaly associated with diffuse hepatocyte‐specific expression of mutant β‐catenin is secondary to liver functional alteration or non‐cell autonomous. Both phenotypes are reversible. Nevertheless, some foci of transgene‐expressing cells progressed to carcinoma, confirming the association of mutant β‐catenin with liver cancer.     

T-cell receptor gene transfer by lentiviral vectors in adoptive cell therapy

Adoptive cell therapy can be envisioned as a promising strategy for tumour immunotherapy. However, existing protocols of adoptive cell therapy still require optimisation as many factors, such as specificity, avidity, level of differentiation and amount of transferred T lymphocytes, can influence their immunocompetence and in vivo functionality. In particular, the need to reduce the in vitro expansion phase and to obtain large numbers of tumour-reactive T cells, as a favourable condition for cancer regression, make TCR gene transfer a potentially ideal tool to overcome the limits of adoptive cell therapy strategies. Here, the authors review the state-of-the-art and recent advances in TCR transfer with particular emphasis on lentiviral vector systems. Initial data from preclinical models and recent clinical trials encourage optimisation of a safe, simplified and stable transfer system. In this regard, HIV-based vectors are emerging as good alternative candidates over the most widely used oncoretroviral vectors due to their peculiar molecular features that fit the ideal conditions for donor T cell in vitro manipulation.