Altered contractile response due to increased beta3-adrenoceptor stimulation in diabetic cardiomyopathy: the role of nitric oxide synthase 1-derived nitric oxide

BACKGROUND: In the diabetic heart, the positive inotropic response to beta-adrenoceptor stimulation is altered and beta1 and beta2 adrenoceptors are down-regulated, whereas beta3 adrenoceptor is up-regulated. In heart failure, beta3-adrenoceptor stimulation induces a negative inotropic effect that results from endothelial nitric oxide synthase (NOS3)-derived nitric oxide production. The objective of our study was to investigate the role of beta3-adrenoceptor in diabetic cardiomyopathy. METHODS: beta-Adrenergic responses were investigated in vivo (dobutamine echocardiography) and in vitro (left ventricular papillary muscle) in healthy and streptozotocin-induced diabetic rats. The effect of beta3-adrenoceptor inhibition on the inotropic response was studied in vitro. Immunoblots and NOS activities were performed in heart homogenates (electron paramagnetic resonance) and isolated cardiomyocytes. Data are mean percentage of baseline +/- SD. RESULTS: The impaired positive inotropic effect was confirmed in diabetes both in vivo (121 +/- 15% vs. 160 +/- 16%; P < 0.05) and in vitro (112 +/- 5% vs. 179 +/- 15%; P < 0.05). In healthy rat, the positive inotropic effect was not significantly modified in presence of beta3-adrenoceptor antagonist (174 +/- 20%), nonselective NOS inhibitor (N -nitro-l-arginine methylester [l-NAME]; 183 +/- 19%), or selective NOS1 inhibitor (vinyl-l-N-5-(1-imino-3-butenyl)-l-ornithine [l-VNIO]; 172 +/- 13%). In diabetes, in parallel with the increase in beta3-adrenoceptor protein expression, the positive inotropic effect was partially restored by beta3-adrenoceptor antagonist (137 +/- 8%; P < 0.05), l-NAME (133 +/- 11%; P < 0.05), or l-VNIO (130 +/- 13%; P < 0.05). Nitric oxide was exclusively produced by NOS1 within diabetic cardiomyocytes. NOS2 and NOS3 proteins were undetectable. CONCLUSIONS: beta3-Adrenoceptor is involved in altered positive inotropic response to beta-adrenoceptor stimulation in diabetic cardiomyopathy. This effect is mediated by NOS1-derived nitric oxide in diabetic cardiomyocyte.     

Anterior mitral leaflet augmentation with autologous pericardium

The results of mitral repair for rheumatic valve insufficiency are still suboptimal. Anterior leaflet augmentation with autologous pericardium is a useful adjunct to compensate leaflet and chordae retraction. The technique and its indication are described in this article.     

Traumatic pancreatic duct injury in children: minimally invasive approach to management

Background

The management of children with main pancreatic duct injuries is controversial. We report a series of patients with pancreatic trauma who were treated using minimally invasive techniques.

Methods

Retrospective review of children with pancreatic trauma treated at a UK tertiary referral institution between 1999 and 2004.

Results

Fifteen children (11 boys) were admitted with pancreatic trauma. Twelve (80%) were less than 50th centile for body weight. Contrast-enhanced computed tomography (CT) scans were used to define organ injury, supplemented by magnetic resonance cholangiopancreatography (MRCP) in 7. Twelve (80%) underwent diagnostic endoscopic retrograde cholangiopancreatography (ERCP) with a median time after injury of 11 (range, 6-29) days. The degree of pancreatic injury was defined by ERCP and CT/MRCP as grade II (n = 2), grade III (n = 4), grade IV (n = 9) (American Association for the Surgery of Trauma grades). Nine children had a transductal pancreatic stent inserted endoscopically. Computed tomography/ultrasound-guided drainage was performed in 4 children for acute fluid collections. Two children later underwent endoscopic cyst-gastrostomy, one of whom later required conversion to an open cyst-gastrostomy.

Conclusion

Body habitus may predispose to pancreatic duct trauma. Contrast-enhanced CT scan (and MRCP) should dictate the need for ERCP. Transductal pancreatic stenting allows internal drainage of peripancreatic collections and may reestablish duct continuity, although a proportion still requires percutaneous or endoscopic cyst-gastrostomy drainage. Open surgery for pancreatic trauma should now be an exception.     

High levels of molecular polymorphism at the KIR2DL4 locus in French and Congolese populations: Impact for anthropology and clinical studies

To characterize KIR2DL4 molecular polymorphism, a cloning-sequencing protocol was performed in 49 French and 52 Teke Congolese individuals. These two populations exhibited high levels of genetic diversity for KIR2DL4, possibly under the influence of natural selection. The most frequent alleles in French individuals (i.e., *00801 and *00802 with a cumulated frequency of 43%) were not the same in Congolese individuals (i.e., *00103 at 47%). In the latter population, four new allelic variants were detected, three of them harboring nonsynonymous substitutions leading to amino acid changes in the extracellular and cytoplasmic domains of the protein. Expression patterns of KIR2DL4 were tightly linked with 9 and 10 poly-adenine polymorphism in exon 7 (i.e., 9A and 10A type alleles). French individuals exhibited a majority of 9A alleles (62%), whereas Congolese individuals had a dominant subset of 10A alleles (72%), suggesting that KIR2DL4 polymorphism could be under the influence of various environmental and pathogenic backgrounds. We conclude that KIR2DL4 might be a good candidate to study for anthropology. In addition, the discovery of its intrinsic variability is shedding light on potential differences among human populations in relation to immunologic functions.     

Relationship between muscle coordination and racket mass during forehand drive in tennis

This study aimed at investigating the relationship between the trunk and upper limb muscle coordination and mass of the tennis racket during forehand drive. A total of 15 male tennis players performed seven series of ten crosscourt forehand drives, both with their personal racket and six rackets with increased mass ranging from 6 to 16% (step = 2%) of their personal racket mass. The electromyographic (EMG) activity was recorded from nine trunk and upper limb muscles. The onset before impact and EMGrms values of the bursts were individually calculated. Results showed that the ball speed and the muscle activation temporal sequences were similar, whatever the increase in racket mass. Interestingly, in all participants, the activation level of the pectoralis major, latissimus dorsi and biceps brachii decreased when the racket mass increased, while the variations in the anterior deltoid activation level were correlated to the individual personal racket mass. These findings strongly suggest that the study of muscle activity during tennis practice should be considered as a complementary technique to determine a better adequacy of the racket characteristics to those of the player.     

Evidence for Multiple B- and T-Cell Epitopes in Plasmodium falciparum Liver-Stage Antigen 3

Liver-stage antigen 3 (LSA-3) is a new vaccine candidate that can induce protection against Plasmodium falciparum sporozoite challenge. Using a series of long synthetic peptides (LSP) encompassing most of the 210-kDa LSA-3 protein, a study of the antigenicity of this protein was carried out in 203 inhabitants from the villages of Dielmo (n = 143) and Ndiop (n = 60) in Senegal (the level of malaria transmission differs in these two villages). Lymphocyte responses to each individual LSA-3 peptide were recorded, some at high prevalences (up to 43%). Antibodies were also detected to each of the 20 peptides, many at high prevalence (up to 84% of responders), and were directed to both nonrepeat and repeat regions. Immune responses to LSA-3 were detectable even in individuals of less than 5 years of age and increased with age and hence exposure to malaria, although they were not directly related to the level of malaria transmission. Thus, several valuable T- and B-cell epitopes were characterized all along the LSA-3 protein, supporting the antigenicity of this P. falciparum vaccine candidate. Finally, antibodies specific for peptide LSP10 located in a nonrepeat region of LSA-3 were found significantly associated with a lower risk of malaria attack over 1 year of daily clinical follow-up in children between the ages of 7 and 15 years, but not in older individuals.Preerythrocytic malaria antigens are critical targets of protective immune responses induced by irradiated sporozoites in humans (9, 22). The demonstration of T-cell-mediated protection in mice immunized by this means (10, 16), the acquisition of a significant level of protection against homologous Plasmodium falciparum challenge in human volunteers (22), and the induction by liver-stage antigens of a high level of protection against P. falciparum infection in chimpanzees (19, 31) all point to a major role for preerythrocytic stage antigens as vaccine candidates.Liver-stage antigen 3 (LSA-3) is a novel antigen expressed at the preerythrocytic stages (4). LSA-3 was selected by the differential immune response found between protected and nonprotected volunteers, both similarly immunized with irradiated sporozoites (4, 9). The gene encoding LSA-3 is unusually well-conserved (4), in contrast with many other malaria vaccine candidates (11, 19, 23). More than 10 dominant T-helper (Th), cytotoxic T-lymphocyte, and B-cell epitopes have already been characterized in LSA-3 (20), some of them displaying cross-reactivity with an homologous antigen in Plasmodium yoelii (2). The protective potential of LSA-3 was demonstrated by a series of experiments in chimpanzees and Aotus monkeys challenged with P. falciparum (4, 21) and in mice challenged by P. yoelii following immunization either by recombinant proteins with adjuvant or by formulations without adjuvant, such as recombinant proteins adsorbed on microparticles or lipopeptides in phosphate-buffered saline (PBS) (4), or DNA-based immunization (29). These convergent results stress the potential of LSA-3 as a prime vaccine candidate.We therefore decided to further analyze the antigenicity of LSA-3 and to investigate immune responses to discrete regions of the protein in exposed individuals living in areas where malaria is endemic.T- and B-cell responses were evaluated in subjects living in two villages in Senegal, West Africa, where malaria is endemic, using three small synthetic peptides and a series of 17 overlapping long synthetic peptides (LSP) encompassing most of the LSA-3 protein. In keeping with preliminary results (20), we found a high prevalence of responses to most regions of this preerythrocytic stage antigen in individuals of different age groups. These results bring additional arguments in favor of the potential of the LSA-3 protein for vaccine development.     

A Multifunctional,Synthetic Gaussia princeps Luciferase Reporter for Live Imaging of Candida albicans Infections

Real-time monitoring of the spatial and temporal progression of infection/gene expression in animals will contribute greatly to our understanding of host-pathogen interactions while reducing the number of animals required to generate statistically significant data sets. Sensitive in vivo imaging technologies can detect low levels of light emitted from luciferase reporters in vivo, but the existing reporters are not optimal for fungal infections. Therefore, our aim was to develop a novel reporter system for imaging Candida albicans infections that overcomes the limitations of current luciferase reporters for this major fungal pathogen. This luciferase reporter was constructed by fusing a synthetic, codon-optimized version of the Gaussia princeps luciferase gene to C. albicans PGA59, which encodes a glycosylphosphatidylinositol-linked cell wall protein. Luciferase expressed from this PGA59-gLUC fusion (referred to as gLUC59) was localized at the C. albicans cell surface, allowing the detection of luciferase in intact cells. The analysis of fusions to strong (ACT1 and EFT3), oxidative stress-induced (TRX1, TRR1, and IPF9996), and morphogenesis-dependent (HWP1) promoters confirmed that gLUC59 is a convenient and sensitive reporter for studies of gene regulation in yeast or hyphal cells, as well as a flexible screening tool. Moreover, the ACT1-gLUC59 fusion represented a powerful tool for the imaging of disease progression in superficial and subcutaneous C. albicans infections. gLUC59 and related cell surface-exposed luciferase reporters might find wide applications in molecular biology, cell biology, pathobiology, and high-throughput screens.Candida albicans is responsible for a large fraction of fungal infections in humans (5) and, as such, has received considerable attention from the research community over the last two decades. C. albicans now represents an invaluable model for dissecting the interplay between fungal pathogens and their hosts at the molecular level (31, 32, 43, 45, 50). Studies of host-pathogen interactions have been greatly facilitated by the use of ex vivo infection models where isolated microorganisms and host cells or reconstituted tissues are brought into contact and the kinetics of pathogen and host cell responses are monitored (12, 14, 23, 36, 45). Yet, animal models remain necessary complements to ex vivo infection models, because none of these models fully reflect the development of clinical infections. Animal models allow researchers to monitor the behavior of mutant microorganisms or the expression of reporter genes in the complex environments of organs and in the presence of a fully functional or debilitated immune system (3, 20, 24).A current limitation of animal models is the need to sacrifice animals in order to image microorganisms at the site of infection. In particular, studies aimed at evaluating whether conditions known to trigger the expression of a specific C. albicans gene in vitro are encountered at sites of infection have often relied on the detection of a reporter in tissue sections. Several reporter genes are available for gene expression studies of C. albicans, such as the Streptococcus thermophilus β-galactosidase lacZ gene (46) and the sea pansy (Renilla reniliformis) luciferase gene (41), but most in vivo studies have taken advantage of derivatives of the Aequorea victoria green fluorescent protein (GFP) gene, whose product can be detected primarily through its natural fluorescence but also through immunochemistry in tissue sections (2, 7, 16, 28). In particular, GFP fusions have been used to examine the niche-specific expression of central metabolic pathways and oxidative stress responses in C. albicans during disease progression (3, 10).As the detection of GFP (and of other reporters such as β-galactosidase) is possible only in tissue sections, it is not possible to monitor in real time the spatial and temporal progression of C. albicans infection/gene expression in a single animal. Real-time monitoring would represent a significant advance because it would probably reveal meaningful variations in fungus/host responses that can be masked by the heterogeneous behavior of individual animals (24). Real-time monitoring might also reveal the spread of C. albicans to unexpected infection sites. Furthermore, real-time monitoring would reduce the number of animals required to generate statistically significant data sets (19). In this regard, in recent years in vivo imaging technologies have been developed that take advantage of sensitive charge-coupled device cameras to detect low levels of light emitted from luciferase reporters in vivo. Pioneering work by Contag et al. (6) demonstrated that bioluminescent Salmonella could be localized to specific tissues in live animals, allowing the temporal monitoring of the infection process and of the efficacy of antimicrobial treatment. This approach has now been extended to numerous pathogenic bacteria, virus, and parasites (19), and several luciferases are available for in vivo imaging, including firefly luciferase (fLUC from Photinus pyralis), which catalyzes light production from luciferin and ATP, and sea pansy luciferase (rLUC) and Gaussia princeps luciferase (gLUC), which catalyze light production from coelenterazine in an ATP-independent manner (37, 44, 47). Recently, Doyle et al. (8, 9) showed that light emitted by C. albicans strains expressing the firefly luciferase gene under the control of the strong C. albicans ENO1 promoter could be detected in animals with induced vulvovaginal candidiasis that had been subjected to a vaginal lavage with a solution containing luciferin. The efficacy of an antifungal treatment could be monitored over a period of 19 days through imaging of the same group of animals. However, this in vivo reporter system did not allow detection of C. albicans during systemic candidiasis. As pointed out by those authors, their failure to detect light in animals despite efficient kidney colonization by luminescent C. albicans might have resulted from the limited permeability of hyphal cells to luciferin and the attenuation of light emitted from the kidneys by overlying tissues (8). Furthermore, the inability to detect intracellular firefly luciferase in hyphal cells represents a major limitation for further studies of C. albicans, since the yeast-to-hypha transition is a major virulence determinant in this species (35).In the present study, we have successfully circumvented most of these limitations by engineering a luciferase that becomes exposed at the cell surface and hence is readily accessible to its substrate whether C. albicans is in the yeast or hyphal form. This was achieved by fusing a synthetic, codon-optimized version of the gene for the naturally secreted Gaussia princeps luciferase (44) to the C. albicans PGA59 gene, which encodes a glycosylphosphatidylinositol (GPI)-linked cell wall protein required for cell wall integrity (27). We confirm that the PGA59-gLUC gene fusion (referred to as gLUC59) is a convenient and powerful reporter for in vitro gene expression studies using intact yeast or hyphal C. albicans cells. Moreover, we show that bioluminescence imaging is a powerful tool for the detailed monitoring of the spatiotemporal behavior of cutaneous, subcutaneous, and vaginal C. albicans infections in live animals. The gLUC59 reporter is also useful for the analysis of systemic infections, although the uneven distribution of the G. princeps luciferase substrate, coelenterazine, in live animals prevents accurate quantitative analysis of such infections.     

Different functional basal ganglia subcircuits associated with anti-akinetic and dyskinesiogenic effects of antiparkinsonian therapies

Subthalamic nucleus high frequency stimulation (STN-HFS) efficiently alleviates l-DOPA-sensitive parkinsonian motor symptoms, but has no direct beneficial action on l-DOPA-induced dyskinesias (LID). Here, we provide evidence that anti-akinetic STN-HFS or dyskinesiogenic l-DOPA similarly reversed the dopamine lesion-induced increases in gene expression of cytochrome oxidase subunit I (CoI), a metabolic marker of neuronal activity, in the globus pallidus, STN and substantia nigra pars reticulata (SNr) in rats. In contrast, in entopeduncular nucleus (EP), STN-HFS did not modify the lesion-induced increase in CoI mRNA levels, whereas l-DOPA induced a marked decrease versus control. Combining the two treatments did not reveal significant interaction. Interestingly, CoI gene expression in EP but not in SNr was inversely correlated with striatal preprodynorphin mRNA level, a LID marker. This work suggests the existence of two functional basal ganglia subcircuits: the one, including STN and SNr, involved in antiparkinsonian action, and the other, including EP, preferentially involved in LID.