Immunosuppressive effects of streptozotocin-induced diabetes result in absolute lymphopenia and a relative increase of T regulatory cells.

OBJECTIVE Streptozotocin (STZ) is the most widely used diabetogenic agent in animal models of islet transplantation. However, the immunomodifying effects of STZ and the ensuing hyperglycemia on lymphocyte subsets, particularly on T regulatory cells (Tregs), remain poorly understood. RESEARCH DESIGN AND METHODS This study evaluated how STZ-induced diabetes affects adaptive immunity and the consequences thereof on allograft rejection in murine models of islet and skin transplantation. The respective toxicity of STZ and hyperglycemia on lymphocyte subsets was tested in vitro. The effect of hyperglycemia was assessed independently of STZ in vivo by the removal of transplanted syngeneic islets, using an insulin pump, and with rat insulin promoter diphtheria toxin receptor transgenic mice. RESULTS Early lymphopenia in both blood and spleen was demonstrated after STZ administration. Direct toxicity of STZ on lymphocytes, particularly on CD8(+) cells and B cells, was shown in vitro. Hyperglycemia also correlated with blood and spleen lymphopenia in vivo but was not lymphotoxic in vitro. Independently of hyperglycemia, STZ led to a relative increase of Tregs in vivo, with the latter retaining their suppressive capacity in vitro. The higher frequency of Tregs was associated with Treg proliferation in the blood, but not in the spleen, and higher blood levels of transforming growth factor-β. Finally, STZ administration delayed islet and skin allograft rejection compared with naive mice. CONCLUSIONS These data highlight the direct and indirect immunosuppressive effects of STZ and acute hyperglycemia, respectively. Thus, these results have important implications for the future development of tolerance-based protocols and their translation from the laboratory to the clinic.

Augmented myocardial methionine-enkephalin in a murine model of cardiac angiotensin II-overexpression.

INTRODUCTION: The endogenous opioid system has been reported to interact with both the cardiac sympathetic and renin-angiotensin systems in exerting a local regulatory action on the heart. The goal of this investigation was to examine how cardiac levels of enkephalin production are altered in the development of normotensive primary hypertrophy due to elevated intra-cardiac angiotensin II (Ang II) production. METHODS: Atrial and ventricular methionine-enkephalin (ME) levels were measured by quantitative radioimmunoassay in 14 and 28-week-old male transgenic mice (TG1306/1R) and control mice. The TG1306/1R exhibit cardiac specific Ang II overexpression and cardiac hypertrophy, but not hypertension. RESULTS: TG1306/1R mice had significantly higher heart/body weight ratios (15-20%) than control littermates at both 14 (p=0.02) and 28 weeks (p=0.04). Relative to controls, ME content was significantly elevated (approximately two-fold) in atria and ventricles in the older 28-week TG1306/1R mice only. A significant inverse correlation between heart size and ME level was observed for 28-week TG1306/1R only. CONCLUSIONS: We have provided evidence that a marked elevation of myocardial enkephalin level is observed in the established (but not early) phase of cardiac hypertrophy associated with cardiac-specific Ang II-overexpression. This study identifies a potentially important relationship between two endogenous peptidergic signalling systems involved in the regulation of growth and function of the hypertrophic heart.

Pancreatic-specific expression of the glucose transporter type 2 gene: identification of cis-elements and islet-specific trans-acting factors.

A defect in glucose sensing of the pancreatic beta-cells has been observed in several animal models of type II diabetes and has been correlated with a reduced gene expression of the glucose transporter type 2 (Glut2). In a transgenic mouse model, expression of Glut2 antisense RNA in pancreatic beta-cells has recently been shown to be associated with an impaired glucose-induced insulin secretion and the development of diabetes. To identify factors that may be involved in the specific decrease of Glut2 in the beta-cells of the diabetic animal, an attempt was made to localize the cis-elements and trans-acting factors involved in the control of Glut2 expression in the endocrine pancreas. It was demonstrated by transient transfection studies that only 338 base pairs (bp) of the murine Glut2 proximal promoter are needed for reporter gene expression in pancreatic islet-derived cell lines, whereas no activity was detected in nonpancreatic cells. Three cis-elements, GTI, GTII, and GTIII, have been identified by DNAse I footprinting and gel retardation experiments within these 338 bp. GTI and GTIII bind distinct but ubiquitously expressed trans-acting factors. On the other hand, nuclear proteins specifically expressed in pancreatic cell lines interact with GTII, and their relative abundance correlates with endogenous Glut2 expression. These GTII-binding factors correspond to nuclear proteins of 180 and 90 kilodaltons as defined by Southwestern analysis. The 180-kilodalton factor is present in pancreatic beta-cell lines but not in an alpha-cell line. Mutation of the GTI or GTIII cis-elements decreases transcriptional activity directed by the 338-bp promoter, whereas mutation of GTII increases gene transcription. Thus negative and positive regulatory sequences are identified within the proximal 338 bp of the GLUT2 promoter and may participate in the islet-specific expression of the gene by binding beta-cell specific trans-acting factors.

The CNGCb and CNGCd genes from Physcomitrella patens moss encode for thermosensory calcium channels responding to fluidity changes in the plasma membrane.

Land plants need precise thermosensors to timely establish molecular defenses in anticipation of upcoming noxious heat waves. The plasma membrane-embedded cyclic nucleotide-gated Ca(2+) channels (CNGCs) can translate mild variations of membrane fluidity into an effective heat shock response, leading to the accumulation of heat shock proteins (HSP) that prevent heat damages in labile proteins and membranes. Here, we deleted by targeted mutagenesis the CNGCd gene in two Physcomitrella patens transgenic moss lines containing either the heat-inducible HSP-GUS reporter cassette or the constitutive UBI-Aequorin cassette. The stable CNGCd knockout mutation caused a hyper-thermosensitive moss phenotype, in which the heat-induced entry of apoplastic Ca(2+) and the cytosolic accumulation of GUS were triggered at lower temperatures than in wild type. The combined effects of an artificial membrane fluidizer and elevated temperatures suggested that the gene products of CNGCd and CNGCb are paralogous subunits of Ca(2+)channels acting as a sensitive proteolipid thermocouple. Depending on the rate of temperature increase, the duration and intensity of the heat priming preconditions, terrestrial plants may thus acquire an array of HSP-based thermotolerance mechanisms against upcoming, otherwise lethal, extreme heat waves.

Association of BAFF/BLyS overexpression and altered B cell differentiation with Sjögren's syndrome.

BAFF (BLyS, TALL-1, THANK, zTNF4) is a member of the TNF superfamily that specifically regulates B lymphocyte proliferation and survival. Mice transgenic (Tg) for BAFF develop an autoimmune condition similar to systemic lupus erythematosus. We now demonstrate that BAFF Tg mice, as they age, develop a secondary pathology reminiscent of Sjögren's syndrome (SS), which is manifested by severe sialadenitis, decreased saliva production, and destruction of submaxillary glands. In humans, SS also correlates with elevated levels of circulating BAFF, as well as a dramatic upregulation of BAFF expression in inflamed salivary glands. A likely explanation for disease in BAFF Tg mice is excessive survival signals to autoreactive B cells, possibly as they pass through a critical tolerance checkpoint while maturing in the spleen. The marginal zone (MZ) B cell compartment, one of the enlarged B cell subsets in the spleen of BAFF Tg mice, is a potential reservoir of autoreactive B cells. Interestingly, B cells with an MZ-like phenotype infiltrate the salivary glands of BAFF Tg mice, suggesting that cells of this compartment potentially participate in tissue damage in SS and possibly other autoimmune diseases. We conclude that altered B cell differentiation and tolerance induced by excess BAFF may be central to SS pathogenesis.

Quantitative proteomics reveals subset-specific viral recognition in dendritic cells.

Dendritic cell (DC) populations consist of multiple subsets that are essential orchestrators of the immune system. Technological limitations have so far prevented systems-wide accurate proteome comparison of rare cell populations in vivo. Here, we used high-resolution mass spectrometry-based proteomics, combined with label-free quantitation algorithms, to determine the proteome of mouse splenic conventional and plasmacytoid DC subsets to a depth of 5,780 and 6,664 proteins, respectively. We found mutually exclusive expression of pattern recognition pathways not previously known to be different among conventional DC subsets. Our experiments assigned key viral recognition functions to be exclusively expressed in CD4(+) and double-negative DCs. The CD8alpha(+) DCs largely lack the receptors required to sense certain viruses in the cytoplasm. By avoiding activation via cytoplasmic receptors, including retinoic acid-inducible gene I, CD8alpha(+) DCs likely gain a window of opportunity to process and present viral antigens before activation-induced shutdown of antigen presentation pathways occurs.

Dual role of the antioxidant enzyme peroxiredoxin 6 in skin carcinogenesis.

The antioxidant enzyme peroxiredoxin 6 (Prdx6) is a key regulator of the cellular redox balance, particularly under stress conditions. We identified Prdx6 as an important player in different phases of skin carcinogenesis. Loss of Prdx6 in mice enhanced the susceptibility to skin tumorigenesis, whereas overexpression of Prdx6 in keratinocytes of transgenic mice had the opposite effect. The tumor-preventive effect of Prdx6, which was observed in a human papilloma virus 8-induced and a chemically induced tumor model, was not due to alterations in keratinocyte proliferation, apoptosis, or in the inflammatory response. Rather, endogenous and overexpressed Prdx6 reduced oxidative stress as reflected by the lower levels of oxidized phospholipids in the protumorigenic skin of Prdx6 transgenic mice and the higher levels in Prdx6-knockout mice than in control animals. In contrast to its beneficial effect in tumor prevention, overexpression of Prdx6 led to an acceleration of malignant progression of existing tumors, revealing a dual function of this enzyme in the pathogenesis of skin cancer. Finally, we found strong expression of PRDX6 in keratinocytes of normal human skin and in the tumor cells of squamous cell carcinomas, indicating a role of Prdx6 in human skin carcinogenesis. Taken together, our data point to the potential usefulness of Prdx6 activators or inhibitors for controlling different stages of skin carcinogenesis.

Doxycycline increases neurogenesis and reduces microglia in the adult hippocampus.

Adult hippocampal neurogenesis results in the continuous formation of new neurons and is a process of brain plasticity involved in learning and memory. Although inducible-reversible transgenic mouse models are increasingly being used to investigate adult neurogenesis, transgene control requires the administration of an activator, doxycycline (Dox), with unknown effects on adult neurogenesis. Here, we tested the effect of Dox administration on adult neurogenesis in vivo. We found that 4 weeks of Dox treatment at doses commonly used for gene expression control, resulted in increased neurogenesis. Furthermore, the dendrites of new neurons displayed increased spine density. Concomitantly, Iba1-expressing microglia was reduced by Dox treatment. These results indicate that Dox treatment may interfere with parameters of relevance for the use of inducible transgenic mice in studies of adult neurogenesis or brain inflammation.

The Na+-dependent chloride-bicarbonate exchanger SLC4A8 mediates an electroneutral Na+ reabsorption process in the renal cortical collecting ducts of mice.

Regulation of sodium balance is a critical factor in the maintenance of euvolemia, and dysregulation of renal sodium excretion results in disorders of altered intravascular volume, such as hypertension. The amiloride-sensitive epithelial sodium channel (ENaC) is thought to be the only mechanism for sodium transport in the cortical collecting duct (CCD) of the kidney. However, it has been found that much of the sodium absorption in the CCD is actually amiloride insensitive and sensitive to thiazide diuretics, which also block the Na-Cl cotransporter (NCC) located in the distal convoluted tubule. In this study, we have demonstrated the presence of electroneutral, amiloride-resistant, thiazide-sensitive, transepithelial NaCl absorption in mouse CCDs, which persists even with genetic disruption of ENaC. Furthermore, hydrochlorothiazide (HCTZ) increased excretion of Na+ and Cl- in mice devoid of the thiazide target NCC, suggesting that an additional mechanism might account for this effect. Studies on isolated CCDs suggested that the parallel action of the Na+-driven Cl-/HCO3- exchanger (NDCBE/SLC4A8) and the Na+-independent Cl-/HCO3- exchanger (pendrin/SLC26A4) accounted for the electroneutral thiazide-sensitive sodium transport. Furthermore, genetic ablation of SLC4A8 abolished thiazide-sensitive NaCl transport in the CCD. These studies establish what we believe to be a novel role for NDCBE in mediating substantial Na+ reabsorption in the CCD and suggest a role for this transporter in the regulation of fluid homeostasis in mice.

Adipose tissue integrity as a prerequisite for systemic energy balance: a critical role for peroxisome proliferator-activated receptor gamma.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is an essential regulator of adipocyte differentiation, maintenance, and survival. Deregulations of its functions are associated with metabolic diseases. We show here that deletion of one PPARgamma allele not only affected lipid storage but, more surprisingly, also the expression of genes involved in glucose uptake and utilization, the pentose phosphate pathway, fatty acid synthesis, lipolysis, and glycerol export as well as in IR/IGF-1 signaling. These deregulations led to reduced circulating adiponectin levels and an energy crisis in the WAT, reflected in a decrease to nearly half of its intracellular ATP content. In addition, there was a decrease in the metabolic rate and physical activity of the PPARgamma(+/-) mice, which was abolished by thiazolidinedione treatment, thereby linking regulation of the metabolic rate and physical activity to PPARgamma. It is likely that the PPARgamma(+/-) phenotype was due to the observed WAT dysfunction, since the gene expression profiles associated with metabolic pathways were not affected either in the liver or the skeletal muscle. These findings highlight novel roles of PPARgamma in the adipose tissue and underscore the multifaceted action of this receptor in the functional fine tuning of a tissue that is crucial for maintaining the organism in good health.

Production of renewable polymers from crop plants

Plants produce a range of biopolymers for purposes such as maintenance of structural integrity, carbon storage, and defense against pathogens and desiccation. Several of these natural polymers are used by humans as food and materials, and increasingly as an energy carrier. In this review, we focus on plant biopolymers that are used as materials in bulk applications, such as plastics and elastomers, in the context of depleting resources and climate change, and consider technical and scientific bottlenecks in the production of novel or improved materials in transgenic or alternative crop plants. The biopolymers discussed are natural rubber and several polymers that are not naturally produced in plants, such as polyhydroxyalkanoates, fibrous proteins and poly-amino acids. In addition, monomers or precursors for the chemical synthesis of biopolymers, such as 4-hydroxybenzoate, itaconic acid, fructose and sorbitol, are discussed briefly

Apoplastic polyesters in Arabidopsis surface tissues--a typical suberin and a particular cutin.

Cutinized and suberized cell walls form physiological important plant-environment interfaces as they act as barriers limiting water and nutrient loss and protect from radiation and invasion by pathogens. Due to the lack of protocols for the isolation and analysis of cutin and suberin in Arabidopsis, the model plant for molecular biology, mutants and transgenic plants with a defined altered cutin or suberin composition are unavailable, causing that structure and function of these apoplastic barriers are still poorly understood. Transmission electron microscopy (TEM) revealed that Arabidopsis leaf cuticle thickness ranges from only 22 nm in leaf blades to 45 nm on petioles, causing the difficulty in cuticular membrane isolation. We report the use of polysaccharide hydrolases to isolate Arabidopsis cuticular membranes, suitable for depolymerization and subsequent compositional analysis. Although cutin characteristic omega-hydroxy acids (7%) and mid-chain hydroxylated fatty acids (8%) were detected, the discovery of alpha,omega-diacids (40%) and 2-hydroxy acids (14%) as major depolymerization products reveals a so far novel monomer composition in Arabidopsis cutin, but with chemical analogy to root suberin. Histochemical and TEM analysis revealed that suberin depositions were localized to the cell walls in the endodermis of primary roots and the periderm of mature roots of Arabidopsis. Enzyme digested and solvent extracted root cell walls when subjected to suberin depolymerization conditions released omega-hydroxy acids (43%) and alpha,omega-diacids (24%) as major components together with carboxylic acids (9%), alcohols (6%) and 2-hydroxyacids (0.1%). This similarity to suberin of other species indicates that Arabidopsis roots can serve as a model for suberized tissue in general.

Characterization of the role of AKAP-Lbc/p38 complex in the hypertrophic response of the heart

In response to chronic stress the heart undergoes an adverse remodeling process associated with cardiomyocyte hypertrophy, increased cellular apoptosis and fibrosis, which ultimately causes cardiac dysfunction and heart failure. Increasing evidence suggest the role of scaffolding and anchoring proteins in coordinating different signaling pathways that mediate the hypertrophic response of the heart. In this context, the family of Α-kinase anchoring proteins (AKAPs) emerged as important regulators of the cardiac function. During my thesis work I have conducted two independent projects, both of them aiming at elucidating the role of AKAPs in the heart. It has been shown that AKAP-Lbc, an anchoring protein that possesses an intrinsic Rho- specific exchange factor activity, organizes a signaling complex that links AKAP-Lbc- dependent activation of RhoA with the mitogen activated protein kinase (MAPK) p38. The first aim of my thesis was to study the role of this novel transduction pathway in the context of cardiac hypertrophy. Here we show that transgenic mice overexpressing in cardiomyocytes a competitor fragment of AKAP-Lbc, which specifically disrupts endogenous AKAP-Lbc / p38 complexes, developed early dilated cardiomyopathy in response to two weeks of transverse aortic constriction (TAC) as compared to controls. Interestingly, inhibition of the AKAP-Lbc / p38 transduction pathway significantly reduced the hypertrophic growth of single cardiomyocytes induced by pressure overload. Therefore, it appears that the AKAP- Lbc / p38 complex is crucially involved in the regulation of stress-induced cardiomyocyte hypertrophy and that disruption of this signaling pathway is detrimental for the heart under conditions of sustained hemodynamic stress. Secondly, in order to identify new AKAPs involved in the regulation of cardiac function, we followed a proteomic approach which allowed us to characterize AKAP2 as a major AKAP in the heart. Importantly, here we show that AKAP2 interacts with several proteins known to be involved in the control of gene transcription, such as the nuclear receptor coactivator 3 (NCoA3) or the ATP-dependent SWI/SNF chromatin remodeling complex. Thus, we propose AKAP2 as a novel mediator of cardiac gene expression through its interaction with these transcriptional regulators.

cFLIP protein prevents tumor necrosis factor-alpha-mediated induction of caspase-8-dependent apoptosis in insulin-secreting betaTc-Tet cells.

Type 1 diabetes is characterized by the infiltration of activated leukocytes within the pancreatic islets, leading to beta-cell dysfunction and destruction. The exact role played by interferon-gamma, tumor necrosis factor (TNF)-alpha, and interleukin-1beta in this pathogenic process is still only partially understood. To study cytokine action at the cellular level, we are working with the highly differentiated insulin-secreting cell line, betaTc-Tet. We previously reported that it was susceptible to apoptosis induced by TNF-alpha, in combination with interleukin-1beta and interferon-gamma. Here, we report that cytokine-induced apoptosis was correlated with the activation of caspase-8. We show that in betaTc-Tet cells, overexpression of cFLIP, the cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein, completely abolished cytokine-dependent activation of caspase-8 and protected the cells against apoptosis. Furthermore, cFLIP overexpression increased the basal and interleukin-1beta-mediated transcriptional activity of nuclear factor (NF)-kappaB, whereas it did not change cytokine-induced inducible nitric oxide synthase gene transcription and nitric oxide secretion. The presence of cFLIP prevented the weak TNF-alpha-induced reduction in cellular insulin content and secretion; however, it did not prevent the decrease in glucose-stimulated insulin secretion induced by the combined cytokines, in agreement with our previous data demonstrating that interferon-gamma alone could induce these beta-cell dysfunctions. Together, our data demonstrate that overexpression of cFLIP protects mouse beta-cells against TNF-alpha-induced caspase-8 activation and apoptosis and is correlated with enhanced NF-kappaB transcriptional activity, suggesting that cFLIP may have an impact on the outcome of death receptor-triggered responses by directing the intracellular signals from beta-cell death to beta-cell survival.

Intracisternal delivery of NFκB-inducible scAAV2/9 reveals locoregional neuroinflammation induced by systemic kainic acid treatment.

We have previously demonstrated disease-dependent gene delivery in the brain using an AAV vector responding to NFκB activation as a probe for inflammatory responses. This vector, injected focally in the parenchyma prior to a systemic kainic acid (KA) injection mediated inducible transgene expression in the hippocampus but not in the cerebellum, regions, respectively, known to be affected or not by the pathology. However, such a focal approach relies on previous knowledge of the model parameters and does not allow to predict the whole brain response to the disease. Global brain gene delivery would allow to predict the regional distribution of the pathology as well as to deliver therapeutic factors in all affected brain regions. We show that self-complementary AAV2/9 (scAAV2/9) delivery in the adult rat cisterna magna allows a widespread but not homogenous transduction of the brain. Indeed, superficial regions, i.e., cortex, hippocampus, and cerebellum were more efficiently transduced than deeper regions, such as striatum, and substantia nigra. These data suggest that viral particles penetration from the cerebrospinal fluid (CSF) into the brain is a limiting factor. Interestingly, AAV2/9-2YF a rationally designed capsid mutant (affecting surface tyrosines) increased gene transfer efficiency approximately fivefold. Neurons, astrocytes, and oligodendrocytes, but not microglia, were transduced in varying proportions depending on the brain region and the type of capsid. Finally, after a single intracisternal injection of scAAV2/9-2YF using the NFκB-inducible promoter, KA treatment induced transgene expression in the hippocampus and cortex but not in the cerebellum, corresponding to the expression of the CD11b marker of microglial activation. These data support the use of disease-inducible vectors administered in the cisterna magna as a tool to characterize the brain pathology in systemic drug-induced or transgenic disease models. However, further improvements are required to enhance viral particles penetration into the brain.