Transient prenatal expression of NPY-Y1 receptor in trigeminal axons innervating the mystacial vibrissae.

Using immunohistochemistry in combination with confocal laser scanning microscopy, we studied the ontogeny of neuropeptide Y-Y1 receptor (Y1-R) expression in the trigeminal system of the rat. The study was limited to the nerve fibers innervating the mystacial pad and the trigeminal ganglia. In the trigeminal ganglia, Y1-R-immunoreactive (IR) neurons were first observed at E16.5. At this same stage some nerve fibers in the trigeminal ganglia also exhibited Y1-R-like immunoreactivity (LI). Strongly Y1-R-IR nerve fibers innervating the follicles of the mystacial vibrissae were first observed at E18. After double labeling, the Y1-R-LI was found to be colocalized with the neuronal marker protein gene product 9.5. At P1 only weak labeling for the Y1-R was found around the vibrissae follicles, whereas the neurons in the trigeminal ganglia were intensely labeled. The same was true for the adult rat, but at this stage no Y1-R labeling at all was observed in nerve fibers around the vibrissal follicles. These results strongly support an axonal localization of the Y1-R at this developmental stage. The transient expression of the Y1-R during prenatal mystacial pad development suggests a role for the Y1-R in the functional development of the vibrissae.

Crosstalk between peroxisome proliferator-activated receptor delta and VEGF stimulates cancer progression.

Peroxisome proliferator-activated receptor (PPAR) delta is a member of the nuclear hormone receptor superfamily. PPARdelta may ameliorate metabolic diseases such as obesity and diabetes. However, PPARdelta's role in colorectal carcinogenesis remains controversial. Here, we present genetic and pharmacologic evidence demonstrating that deletion of PPARdelta decreases intestinal adenoma growth in Apc(Min/+) mice and inhibits tumor-promoting effects of a PPARdelta agonist GW501516. More importantly, we found that activation of PPARdelta up-regulated VEGF in colon carcinoma cells. VEGF directly promotes colon tumor epithelial cell survival through activation of PI3K-Akt signaling. These results not only highlight concerns about the use of PPARdelta agonists for treatment of metabolic disorders in patients who are at high risk for colorectal cancer, but also support the rationale for developing PPARdelta antagonists for prevention and/or treatment of cancer.

Covalent assembly of a soluble T cell receptor-peptide-major histocompatibility class I complex.

We used stepwise photochemical cross-linking for specifically assembling soluble and covalent complexes made of a T-cell antigen receptor (TCR) and a class I molecule of the major histocompatibility complex (MHC) bound to an antigenic peptide. For that purpose, we have produced in myeloma cells a single-chain Fv construct of a TCR specific for a photoreactive H-2Kd-peptide complex. Photochemical cross-linking of this TCR single-chain Fv with a soluble form of the photoreactive H-2Kd-peptide ligand resulted in the formation of a ternary covalent complex. We have characterized the soluble ternary complex and showed that it reacted with antibodies specific for epitopes located either on the native TCR or on the Kd molecules. By preventing the fast dissociation kinetics observed with most T cell receptors, this approach provides a means of preparing soluble TCR-peptide-MHC complexes on large-scale levels.

Mineralocorticoid receptor degradation is promoted by Hsp90 inhibition and the ubiquitin-protein ligase CHIP.

The mineralocorticoid receptor (MR) plays a crucial role in the regulation of Na(+) balance and blood pressure, as evidenced by gain of function mutations in the MR of hypertensive families. In the kidney, aldosterone binds to the MR, induces its nuclear translocation, and promotes a transcriptional program leading to increased transepithelial Na(+) transport via the epithelial Na(+) channel. In the unliganded state, MR is localized in the cytosol and part of a multiprotein complex, including heat shock protein 90 (Hsp90), which keeps it ligand-binding competent. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a benzoquinone ansamycin antibiotic that binds to Hsp90 and alters its function. We investigated whether 17-AAG affects the stability and transcriptional activity of MR and consequently Na(+) reabsorption by renal cells. 17-AAG treatment lead to reduction of MR protein level in epithelial cells in vitro and in vivo, thereby interfering with aldosterone-dependent transcription. Moreover, 17-AAG inhibited aldosterone-induced Na(+) transport, possibly by interfering with MR availability for the ligand. Finally, we identified the ubiquitin-protein ligase, COOH terminus of Hsp70-interacting protein, as a novel partner of the cytosolic MR, which is responsible for its polyubiquitylation and proteasomal degradation in presence of 17-AAG. In conclusion, 17-AAG may represent a novel pharmacological tool to interfere with Na(+) reabsorption and hypertension.

Species-Specific Recognition of Aspergillus fumigatus by Toll-like Receptor 1 and Toll-like Receptor 6.

Background. Aspergillus fumigatus causes invasive aspergillosis, a potentially fatal infection in oncohematological patients. Innate immune detection of A. fumigatus involves Toll-like receptor (TLR) 4 and TLR2, which forms a heterodimer with either TLR1 or TLR6. The role of those coreceptors in Aspergillus sensing is unknown. Methods. Cytokine production was measured in bone marrow-derived macrophages (BMDMs) from wild-type (WT) and TLR-deficient mice after incubation with a WT and an immunogenic RodA-deficient (ΔrodA-47) strain of A. fumigatus and in lungs from these mice after intranasal mold inoculation. Aspergillus fumigatus-mediated NF-κB activation was measured in HEK293T cells transfected with plasmids expressing mouse or human TLRs. Results. Bone marrow-derived macrophages from TLR1- and TLR6-deficient mice produced lower amounts of interleukin 12p40, CXCL2, interleukin 6, and tumor necrosis factor α than BMDMs from WT mice after stimulation with A. fumigatus. Lungs from TLR1- and TLR6-deficient mice had diminished CXCL1 and CXCL2 production and increased fungal burden after intranasal inoculation of ΔrodA A. fumigatus compared with lungs from WT mice. ΔrodA strain-mediated NF-κB activation was observed in HEK293T cells expressing mouse TLR2/1, mouse TLR2/6, and human TLR2/1 but not human TLR2/6. Conclusions. Innate immune detection of A. fumigatus is mediated by TLR4 and TLR2 together with TLR1 or TLR6 in mice and TLR1 but not TLR6 in humans.

Comparative effects of losartan and irbesartan on serum uric acid in hypertensive patients with hyperuricaemia and gout.

OBJECTIVE: Losartan has been shown to increase urinary uric acid excretion and hence to lower serum uric acid levels. The purposes of the present study were: (1) to evaluate the effects of losartan on serum uric acid in hypertensive patients with hyperuricemia and gout, (2) to compare the effects of losartan with those of irbesartan, another angiotensin II receptor antagonist and (3) to evaluate whether losartan 50 mg b.i.d. has a greater impact on serum uric acid levels than losartan 50 mg once a day. METHODS: Thirteen hypertensive patients with hyperuricaemia and gout completed this prospective, randomized, double-blind, cross-over study. Uric acid-lowering drugs were stopped 3 weeks before the beginning of the study. Patients were randomized to receive either losartan 50 mg or irbesartan 150 mg once a day, for 4 weeks. During this phase, a placebo was given in the evening. After 4 weeks, the dose was increased to losartan 50 mg b.i.d., or irbesartan 150 mg b.i.d. for another 4 week period. Subsequently, the patients were switched to the alternative treatment modality. Enalapril (20 mg o.d.) was given during the run-in period and between the two treatment phases. Serum and urinary uric acid were measured at the beginning and at the end of each treatment phase. RESULTS: Our results show that losartan 50 mg once daily decreased serum uric acid levels from 538 +/- 26 to 491 +/- 20 micromol/l (P < 0.01). Irbesartan had no effect on serum uric acid. Increasing the dose of losartan from 50 mg o.d. to 50 mg twice a day, did not further decrease serum uric acid. This may in part be due to a low compliance to the evening dose as measured with an electronic device. Indeed, whatever the prescribed drug, the mean compliance of the evening dose was always significantly lower than that of the morning dose. The uricosuric effect of losartan appears to decrease with time when a new steady state of lower serum uric acid is reached. CONCLUSIONS: In contrast to irbesartan, losartan was uricosuric and decreased serum uric acid levels. Losartan 50 mg b.i.d. did not produce a greater fall in serum uric acid than losartan once a day. Losartan might be a useful therapeutic tool to control blood pressure and reduce serum uric acid levels in hypertensive patients with hyperuricaemia and gout.

Severe hyperlipidemia causes impaired renin-angiotensin system function in apolipoprotein E deficient mice.

Dyslipidemia is a known risk factor for cardiovascular diseases and may associate with renal injury. Using mouse models with various degrees of hypercholesterolemia and hypertryliceridemia, we investigated the effects of lipids on the renin-angiotensin system (RAS). ApoE-/- mice were fed either a high fat diet (HF-ApoE-/-; mice developed hypertriglyceridemia and severe hypercholesterolemia) or regular chow (R-ApoE(-/-); mice developed less severe hypercholesterolemia only). Renal histopathology in the HF-ApoE-/- revealed massive lipid accumulation especially at the glomerular vascular pole. In these mice plasma renin concentration was significantly reduced (489+/-111 ng/(ml h) versus 1023+/-90 ng/(ml h) in R-ApoE-/- mice) and blood pressure was consequently significantly lower than in R-ApoE-/- (104+/-2 mmHg versus 115+/-2 mmHg, respectively). A model of renin-dependent renovascular hypertension (two-kidney, one clip) was generated and HF-ApoE-/- mice proved unable to increase renin secretion, and blood pressure, in response to diminished renal perfusion as compared to regular chow fed mice (665+/-90 ng/(ml h) versus 2393+/-372 ng/(ml h), respectively and 106+/-3 mmHg versus 140+/-2 mmHg, respectively). Hypertriglyceridemia and severe hypercholesterolemia are associated with renal lipid deposition and impaired renin secretion in ApoE-/- mice exposed to high fat diet. These observations further characterize the phenotype of this widely used mouse model and provide a rationale for the use of these mice to study lipid induced organ damage.

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.

TACI, an enigmatic BAFF/APRIL receptor, with new unappreciated biochemical and biological properties.

BAFF is a B cell survival factor that binds to three receptors BAFF-R, TACI and BCMA. BAFF-R is the receptor triggering naïve B cell survival and maturation while BCMA supports the survival of plasma cells in the bone marrow. Excessive BAFF production leads to autoimmunity, presumably as the consequence of inappropriate survival of self-reactive B cells. The function of TACI has been more elusive with TACI(-/-) mice revealing two sides of this receptor, a positive one driving T cell-independent immune responses and a negative one down-regulating B cell activation and expansion. Recent work has revealed that the regulation of TACI expression is intimately linked to the activation of innate receptors on B cells and that TACI signalling in response to multimeric BAFF and APRIL provides positive signals to plasmablasts. How TACI negatively regulates B cells remains elusive but may involve an indirect control of BAFF levels. The discovery of TACI mutations associated with common variable immunodeficiency (CVID) in humans not only reinforces its important role for humoral responses but also suggests a more complex role than first anticipated from knockout animals. TACI is emerging as an unusual TNF receptor-like molecule with a sophisticated mode of action.

Differential T cell receptor photoaffinity labeling among H-2Kd restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative. Labeling of the alpha-chain correlates with J alpha segment usage.

Using a direct binding assay based on photoaffinity labeling, we studied the interaction of T cell receptor (TCR) with a Kd-bound photoreactive peptide derivative on living cells. The Kd-restricted Plasmodium berghei circumsporozoite (PbCS) peptide 253-260 (YIPSAEKI) was reacted NH2-terminally with biotin and at the TCR contact residue Lys259 with photoreactive iodo, 4-azido salicylic acid (IASA) to make biotin-YIPSAEK(IASA)I. Cytotoxic T lymphocyte (CTL) clones derived from mice immunized with this derivative recognized this conjugate, but not a related one lacking the IASA group nor the parental PbCS peptide. The clones were Kd restricted. Recognition experiments with variant conjugates, lacking substituents from IASA, revealed a diverse fine specificity pattern and indicated that this group interacted directly with the TCR. The TCR of four clones could be photoaffinity labeled by biotin-YIPSAEK(125IASA)I. This labeling was dependent on the conjugates binding to the Kd molecule and was selective for the TCR alpha (2 clones) or beta chain (1 clone), or was common for both chains (1 clone). TCR sequence analysis showed a preferential usage of J alpha TA28 containing alpha chains that were paired with V beta 1 expressing beta chains. The TCR that were photoaffinity labeled at the alpha chain expressed these J alpha and V beta segments. The tryptophan encoded by the J alpha TA28 segment is rarely found in other J alpha segments. Moreover, we show that the IASA group interacts preferentially with tryptophan in aqueous solution. We thus propose that for these CTL clones, labeling of the alpha chain occurs via the J alpha-encoded tryptophan residue.

Altered growth in male peroxisome proliferator-activated receptor gamma (PPARgamma) heterozygous mice: involvement of PPARgamma in a negative feedback regulation of growth hormone action.

The peroxisome proliferator-activated receptor gamma (PPARgamma) plays a major role in fat tissue development and physiology. Mutations in the gene encoding this receptor have been associated to disorders in lipid metabolism. A thorough investigation of mice in which one PPARgamma allele has been mutated reveals that male PPARgamma heterozygous (PPARgamma +/-) mice exhibit a reduced body size associated with decreased body weight, reflecting lean mass reduction. This phenotype is reproduced when treating the mice with a PPARgamma- specific antagonist. Monosodium glutamate treatment, which induces weight gain and alters body growth in wild-type mice, further aggravates the growth defect of PPARgamma +/- mice. The levels of circulating GH and that of its downstream effector, IGF-I, are not altered in mutant mice. However, the IGF-I mRNA level is decreased in white adipose tissue (WAT) of PPARgamma +/- mice and is not changed by acute administration of recombinant human GH, suggesting an altered GH action in the mutant animals. Importantly, expression of the gene encoding the suppressor of cytokine signaling-2, which is an essential negative regulator of GH signaling, is strongly increased in the WAT of PPARgamma +/- mice. Although the relationship between the altered GH signaling in WAT and reduced body size remains unclear, our results suggest a novel role of PPARgamma in GH signaling, which might contribute to the metabolic disorder affecting insulin signaling in PPARgamma mutant mice.

Determinants of persistence in hypertensive patients treated with irbesartan: results of a postmarketing survey.

BACKGROUND: Persistence is a key factor for long-term blood pressure control, which is of high prognostic importance for patients at increased cardiovascular risk. Here we present the results of a post-marketing survey including 4769 hypertensive patients treated with irbesartan in 886 general practices in Switzerland. The goal of this survey was to evaluate the tolerance and the blood pressure lowering effect of irbesartan as well as the factors affecting persistence in a large unselected population. METHODS: Prospective observational survey conducted in general practices in all regions of Switzerland. Previously untreated and uncontrolled pre-treated patients were started with a daily dose of 150 mg irbesartan and followed up to 6 months. RESULTS: After an observation time slightly exceeding 4 months, the average reduction in systolic and diastolic blood pressure was 20 (95% confidence interval (CI) -19.6 to -20.7 mmHg) and 12 mmHg (95% CI -11.4 to -12.1 mmHg), respectively. At this time, 26% of patients had a blood pressure < 140/90 mmHg and 60% had a diastolic blood pressure < 90 mmHg. The drug was well tolerated with an incidence of adverse events (dizziness, headaches,...) of 8.0%. In this survey more than 80% of patients were still on irbesartan at 4 month. The most important factors predictive of persistence were the tolerability profile and the ability to achieve a blood pressure target < or = 140/90 mmHg before visit 2. Patients who switched from a fixed combination treatment tended to discontinue irbesartan more often whereas those who abandoned the previous treatment because of cough (a class side effect of ACE-Inhibitors) were more persistent with irbesartan. CONCLUSION: The results of this survey confirm that irbesartan is effective, well tolerated and well accepted by patients, as indicated by the good persistence. This post-marketing survey also emphasizes the importance of the tolerability profile and of achieving an early control of blood pressure as positive predictors of persistence.

Molecular insights for optimizing T cell receptor specificity against cancer.

Cytotoxic CD8 T cells mediate immunity to pathogens and they are able to eliminate malignant cells. Immunity to viruses and bacteria primarily involves CD8 T cells bearing high affinity T cell receptors (TCRs), which are specific to pathogen-derived (non-self) antigens. Given the thorough elimination of high affinity self/tumor-antigen reactive T cells by central and peripheral tolerance mechanisms, anti-cancer immunity mostly depends on TCRs with intermediate-to-low affinity for self-antigens. Because of this, a promising novel therapeutic approach to increase the efficacy of tumor-reactive T cells is to engineer their TCRs, with the aim to enhance their binding kinetics to pMHC complexes, or to directly manipulate the TCR-signaling cascades. Such manipulations require a detailed knowledge on how pMHC-TCR and co-receptors binding kinetics impact the T cell response. In this review, we present the current knowledge in this field. We discuss future challenges in identifying and targeting the molecular mechanisms to enhance the function of natural or TCR-affinity optimized T cells, and we provide perspectives for the development of protective anti-tumor T cell responses.