Chronic bradykinin receptor blockade modulates neonatal renal function.

Recent data indicate that bradykinin participates in the regulation of neonatal glomerular function and also acts as a growth regulator during renal development. The aim of the present study was to investigate the involvement of bradykinin in the maturation of renal function. Bradykinin beta2-receptors of newborn rabbits were inhibited for 4 days by Hoe 140. The animals were treated with 300 microg/kg s.c. Hoe 140 (group Hoe, n = 8) or 0.9% NaCl (group control, n = 8) twice daily. Clearance studies were performed in anesthetized rabbits at the age of 8-9 days. Bradykinin receptor blockade did not impair kidney growth, as demonstrated by similar kidney weights in the two groups, nor did it influence blood pressure. Renal blood flow was higher, while renal vascular resistance and filtration fraction were lower in Hoe 140-treated rabbits. No difference in glomerular filtration rate was observed. The unexpectedly higher renal perfusion observed in group Hoe cannot be explained by the blockade of the known vasodilator and trophic effect of bradykinin. Our results indicate that in intact kallikrein-kinin system is necessary for the normal functional development of the kidney.

Participation of connexin-based channels in the control of vascular function

SUMMARYIntercellular communication is achieved at specialized regions of the plasma membrane by gap junctions. The proteins constituting the gap junctions are called connexins and are encoded by a family of genes highly conserved during evolution. In adult mouse, four connexins (Cxs) are known to be expressed in the vasculature: Cx37, Cx40, Cx43 and Cx45. Several recent studies have provided evidences that vascular connexins expression and blood pressure regulation are closely linked, suggesting a role for connexins in the control of blood pressure. However, the precise function that each vascular connexin plays under physiological and pathophysiological conditions is still not elucidated. In this context, this work was dedicated to evaluate the contribution of each of the four vascular connexins in the control of the vascular function and in the blood pressure regulation.In the present work, we first demonstrated that vascular connexins are differently regulated by hypertension in the mouse aorta. We also observed that endothelial connexins play a regulatory role on eNOS expression levels and function in the aorta, therefore in the control of vascular tone. Then, we demonstrated that Cx40 plays a pivotal role in the kidney by regulating the renal levels of COX-2 and nNOS, two key enzymes of the macula densa known to participate in the control of renin secreting cells. We also found that Cx43 forms the functional gap junction involved in intercellular Ca2+ wave propagation between vascular smooth muscle cells. Finally, we have started to generate transgenic mice expressing specifically Cx40 in the endothelium to investigate the involvement of Cx40 in the vasomotor tone, or in the renin secreting cells to evaluate the role of Cx40 in the control of renin secretion.In conclusion, this work has allowed us to identify new roles for connexins in the vasculature. Our results suggest that vascular connexins could be interesting targets for new therapies caring hypertension and vascular diseases.

Recent clinical trials with omapatrilat: new developments.

Omapatrilat belongs to the vasopeptidase inhibitors, ie, drugs that possess the ability to inhibit simultaneously the membrane-bound zinc metalloproteases, angiotensin-converting enzyme (ACE), and the neutral endopeptidase EC 3.4.24.11 (NEP). Omapatrilat was targeted to treat patients with hypertension and congestive heart failure. The preclinical and early clinical studies conducted with omapatrilat were very promising. Indeed, omapatrilat appeared to be a very potent antihypertensive agent with very favorable effects on cardiac function in heart failure patients. In contrast to these early studies, the large clinical trials were more disappointing. The results of the OCTAVE trial confirmed the antihypertensive efficacy of omapatrilat, but at the price of an angioedema rate more than threefold higher than that of an ACE inhibitor in the overall population (2.17% vs 0.68%), and close to fourfold higher in the black population. In OVERTURE, a large randomized control trial in heart failure, angioedema was also more common with omapatrilat, but the incidence was much lower (0.8% with omapatrilat vs 0.5% with enalapril). However, omapatrilat was not convincingly superior to the ACE inhibitor. Because angioedema is probably a class side effect of vasopeptidase inhibitors, the higher incidence of this potentially life-threatening complication with omapatrilat has likely stopped the development of this new class of agents. The future of vasopeptidase inhibitors will depend on the ability to improve the risk/benefit ratio either by developing agents that produce less angioedema, or by defining more precisely a high-risk population that could take advantage of dual ACE/NEP inhibition.

New insights into the long-term evolutionary history of the avian MHC

SummaryGene duplication and neofunctidnalization are important processes in the evolution of phenotypic complexity. They account for important evolutionary novelties that confer ecological adaptation, such as the major histocompatibility complex (MHC), a multigene family with a central role in vertebrates' adaptive immune system. Multigene families, which evolved in large part through duplication, represent promising systems to study the still strongly depbated relative roles of neutral and adaptive processes in the evolution of phenotypic complexity. Detailed knowledge on ecological function and a well-characterized evolutionary history place the mammals' MHC amongst ideal study systems. However mammalian MHCs usually encompass several million base pairs and hold a large number of functional and non-functional duplicate genes, which makes their study complex. Avian MHCs on the other hand are usually way more compact, but the reconstruction of. their evolutionary history has proven notoriously difficult. However, no focused attempt has been undertaken so far to study the avian MHC evolutionary history in a broad phylogenetic context and using adequate gene regions.In the present PhD, we were able to make important contributions to the understanding of the long-term evolution of the avian MHC class II Β (MHCI1B). First, we isolated and characterized MHCIIB genes in barn owl (Tyto alba?, Strigiformes, Tytonidae), a species from an avian lineage in which MHC has not been studied so far. Our results revealed that with only two functional MHCIIB genes the MHC organization of barn owl may be similar to the 'minimal essential' MHC of chicken (Gallus gallus), indicating that simple MHC organization may be ancestral to birds. Taking advantage of the sequence information from barn owl, we studied the evolution of MHCIIB genes in 13 additional species of 'typical' owls (Strigiformes, Strigidae). Phylogenetic analyses revealed that according to their function, in owls the peptide-binding region (PBR) encoding exon 2 and the non-PBR encoding exon 3 evolve by different patterns. Exon 2 exhibited an evolutionary history of positive selection and recombination, while exon 3 traced duplication history and revealed two paralogs evolving divergently from each other in owls, and in a shorebird, the great snipe {Gallinago media). The results from exon 3 were the first ever from birds to demonstrate gene orthology in species that diverged tens of millions of years ago, and strongly questioned whether the taxa studied before provided an adequate picture of avian MHC evolution. In a follow-up study, we aimed at explaining a striking pattern revealed by phylogenetic trees analyzing the owl sequences along with MHCIIB sequences from other birds: One owl paralog (termed DAB1) grouped with sequences of passerines and falcons, while the other (DAB2) grouped with wildfowl, penguins and birds of prey. This could be explained by either a duplication event preceding the evolution of these bird orders, or by convergent evolution of similar sequences in a number of orders. With extensive phylogenetic analyses we were able to show, that indeed a duplication event preceeded the major avian radiation -100 my ago, and that following this duplication, the paralogs evolved under positive selection. Furthermore, we showed that the divergently evolving amino acid residues in the MHCIIB-encoded β-chain potentially interact with the MHCI I α-chain, and that molecular coevolution of the interacting residues may have been involved in the divergent evolution of the MHCIIB paralogs.The findings of this PhD are of particular interest to the understanding of the evolutionary history of the avian MHC and, by providing essential information on long-term gene history in the avian MHC, open promising perspectives for advances in the understanding of the evolution of multigene families in general, and for avian MHC organization in particular. Amongst others I discuss the importance of including protein structure in the phylogenetic study of multigene families, and the roles of ecological versus molecular selection pressures. I conclude by providing a population genomic perspective on avian MHC, which may serve as a basis for future research to investigate the relative roles of neutral processes involving effective population size effects and of adaptation in the evolution of avian MHC diversity and organization.RésuméLa duplication de gènes et leur néo-fonctionnalisation sont des processus importants dans l'évolution de la complexité phénotypique. Ils sont impliqués dans l'apparition d'importantes nouveautés évolutives favorisant l'adaptation écologique, comme c'est le cas pour le complexe majeur d'histocompatibilité classe II Β (CMH II Β). Nous avons isolé et caractérisé les gènes CMH I IB de la chouette effraie (Tyto alba; Strigiformes, Tytonidae), une espèce appartenant à une lignée aviaire dont le CMH n'avait pas été étudié jusqu'ici. Avec uniquement deux gènes CMHIIB fonctionnels, l'organisation CMH de la chouette effraie semble être comparable à celle "minimale" du poulet (Gallus gallus), ce qui suggère qu'une organisation simple du CMH pourrait être ancestrale chez les oiseaux. En se basant sur l'information obtenue pour cette espèce, nous avons ensuite étudié l'évolution des gènes CMHIIB chez 13 espèces de chouettes "typiques" (Strigiformes, Strigidae). Les analyses phylogénétiques ont révélé que chez les chouettes, selon leur fonction, l'exon 2 codant pour la 'peptide-binding region' (PBR) et l'exon 3 ne codant pas pour la région PBR évoluent de manière distincte. L'exon 2 montre une histoire évolutive de sélection positive et recombinaison, tandis que l'exon 3 retrace l'histoire de duplication et révèle deux paralogues évoluant de façon divergente chez les chouettes, ainsi que chez un limicole, la bécassine double (Gallinago media). Les résultats découlant de l'analyse de l'exon 3 ont montré pour la première fois chez les oiseaux une orthologie de gènes chez des espèces ayant divergé il y a des dizaines de millions d'années. Dans une deuxième étude, nous avons voulu expliquer le pattern surprenant révélé par les arbres phylogénétiques lorsque les séquences CMHIIB des chouettes sont analysées avec les séquences d'autres oiseaux: un paralogue de chouette (appelé DABI) forme un groupe avec les séquences de passereaux et faucons, tandis que l'autre (DAB2) se regroupe avec les gallo-ansériformes, les manchots et les rapaces diurnes. Ceci pourrait être expliqué soit par une duplication qui a précédé l'évolution de ces ordres aviaires soit par évolution convergente dans un certain nombre d'ordres. Grâce à des analyses phylogénétiques approfondies, nous avons pu montrer qu'un événement de duplication a effectivement précédé la radiation aviaire principale d'il y a environ 100 millions d'années, et qu'après cette duplication les paralogues ont évolué sous sélection positive. De plus, les résidus d'acides aminés évoluant de façon divergente dans la chaîne β codée par le CMHIIB interagissent potentiellement avec la chaîne α codée par le CMHII, et que la coévolution moléculaire de ces résidus pourrait être à la base de l'évolution divergente des paralogues CMHIIB.Dans cette thèse sont discutés entre autres l'importance d'inclure la structure des protéines dans l'étude phylogénétique des familles multigéniques, ainsi que les rôles respectifs des pressions sélectives écologique et moléculaire. La conclusion comporte une perspective sur la génomique des populations du CMH aviaire, qui pourrait servir de base pour de futures recherches sur les rôles relatifs joués par Îes processus neutres comprenant les effets de la taille efficace des populations et l'adaptation dans l'évolution de la diversité et l'organisation du MHC aviaire.

Loss of single HLA class I allospecificities in melanoma cells due to selective genomic abbreviations.

Expression of human leucocyte antigen (HLA) Class I molecules is essential for the recognition of malignant melanoma (MM) cells by CD8(+) T lymphocytes. A complete or partial loss of HLA Class I molecules is a potent strategy for MM cells to escape from immunosurveillance. In 2 out of 55 melanoma cell cultures we identified a complete phenotypic loss of HLA allospecificities. Both patients have been treated unsuccessfully with HLA-A2 peptides. To identify the reasons underlying the loss of single HLA-A allospecificities, we searched for genomic alterations at the locus for HLA Class I alpha-chain on chromosome 6 in melanoma cell cultures established from 2 selected patients with MM in advanced stage. This deficiency was associated with alterations of HLA-A2 gene sequences as determined by polymerase chain reaction-sequence specific primers (PCR-SSP). Karyotyping revealed a chromosomal loss in Patient 1, whereas melanoma cell cultures established from Patient 2 displayed 2 copies of chromosome 6. Loss of heterozygosity (LOH) using markers located around position 6p21 was detected in both cases. By applying group-specific primer-mixes spanning the 5'-flanking region of the HLA-A2 gene locus the relevant region was amplified by PCR and subsequent sequencing allowed alignment with the known HLA Class I reference sequences. Functional assays using HLA-A2-restricted cytotoxic T-cell clones were performed in HLA-A2 deficient MM cultures and revealed a drastically reduced susceptibility to CTL lysis in HLA-A2 negative cells. We could document the occurrence of selective HLA-A2 deficiencies in cultured advanced-stage melanoma metastases and identify their molecular causes as genomic alterations within the HLA-A gene locus.

Roles of mGluR5 in synaptic function and plasticity of the mouse thalamocortical pathway.

The group I metabotropic glutamate receptor 5 (mGluR5) has been implicated in the development of cortical sensory maps. However, its precise roles in the synaptic function and plasticity of thalamocortical (TC) connections remain unknown. Here we first show that in mGluR5 knockout (KO) mice bred onto a C57BL6 background cytoarchitectonic differentiation into barrels is missing, but the representations for large whiskers are identifiable as clusters of TC afferents. The altered dendritic morphology of cortical layer IV spiny stellate neurons in mGluR5 KO mice implicates a role for mGluR5 in the dendritic morphogenesis of excitatory neurons. Next, in vivo single-unit recordings of whisker-evoked activity in mGluR5 KO adults demonstrated a preserved topographical organization of the whisker representation, but a significantly diminished temporal discrimination of center to surround whiskers in the responses of individual neurons. To evaluate synaptic function at TC synapses in mGluR5 KO mice, whole-cell voltage-clamp recording was conducted in acute TC brain slices prepared from postnatal day 4-11 mice. At mGluR5 KO TC synapses, N-methyl-D-aspartate (NMDA) currents decayed faster and synaptic strength was more easily reduced, but more difficult to strengthen by Hebbian-type pairing protocols, despite a normal developmental increase in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated currents and presynaptic function. We have therefore demonstrated that mGluR5 is required for synaptic function/plasticity at TC synapses as barrels are forming, and we propose that these functional alterations at the TC synapse are the basis of the abnormal anatomical and functional development of the somatosensory cortex in the mGluR5 KO mouse.

Unmodified self antigen triggers human CD8 T cells with stronger tumor reactivity than altered antigen

Human cancer vaccines are often prepared with altered "analog" or "heteroclitic" antigens that have been optimized for HLA class I binding, resulting in enhanced immunogenicity. Here, we take advantage of CpG oligodeoxynucleotides as powerful vaccine adjuvants and demonstrate the induction of high T cell frequencies in melanoma patients, despite the use of natural (unmodified) tumor antigenic peptide. Compared with vaccination with analog peptide, natural peptide induced T cell frequencies that were approximately twofold lower. However, T cells showed superior tumor reactivity because of (i) increased functional avidity for natural antigen and (ii) enhancement of T cell activation and effector function. Thus, novel vaccine formulations comprising potent immune stimulators may allow to circumvent the need for modified antigens and can induce highly functional T cells with precise antigen specificity

Education of Murine NK Cells Requires Both cis and trans Recognition of MHC Class I Molecules.

Although NK cells use invariant receptors to identify diseased cells, they nevertheless adapt to their environment, including the presence of certain MHC class I (MHC-I) molecules. This NK cell education, which is mediated by inhibitory receptors specific for MHC-I molecules, changes the responsiveness of activating NK cell receptors (licensing) and modifies the repertoire of MHC-I receptors used by NK cells. The fact that certain MHC-I receptors have the unusual capacity to recognize MHC-I molecules expressed by other cells (trans) and by the NK cell itself (cis) has raised the question regarding possible contributions of the two types of interactions to NK cell education. Although the analysis of an MHC-I receptor variant suggested a role for cis interaction for NK cell licensing, adoptive NK cell transfer experiments supported a key role for trans recognition. To reconcile some of these findings, we have analyzed the impact of cell type-specific deletion of an MHC-I molecule and of a novel MHC-I receptor variant on the education of murine NK cells when these mature under steady-state conditions in vivo. We find that MHC-I expression by NK cells (cis) and by T cells (trans), and MHC-I recognition in cis and in trans, are both needed for NK cell licensing. Unexpectedly, modifications of the MHC-I receptor repertoire are chiefly dependent on cis binding, which provides additional support for an essential role for this unconventional type of interaction for NK cell education. These data suggest that two separate functions of MHC-I receptors are needed to adapt NK cells to self-MHC-I.

A monoclonal antibody which blocks the function of factor D of human complement.

Factor D is an essential enzyme for activation of complement by the alternative pathway (AP). It has been difficult to obtain mouse monoclonal antibodies (Mabs) which block the function of factor D. We have developed a strategy to obtain such Mabs using a double screening procedure of the initial clones. We selected the clone whose supernatant had the lowest level of anti-factor D Ab by ELISA and abolished factor D haemolytic activity. Addition of this Mab to human serum was shown to abolish conversion of C3 by cobra venom factor, haemolysis of rabbit erythrocytes, and activation of C3 and C5 by cuprophane dialysis membranes.

Alteration in neuromuscular function after a 5 km running time trial.

The aim of this study was to characterize the effect of a 5 km running time trial on the neuromuscular properties of the plantar flexors. Eleven well-trained triathletes performed a series of neuromuscular tests before and immediately after the run on a 200 m indoor track. Muscle activation (twitch interpolation) and normalized EMG activity were assessed during maximal voluntary contraction (MVC) of plantar flexors. Maximal soleus H-reflexes and M-waves were evoked at rest (i.e. H (MAX) and M (MAX), respectively) and during MVC (i.e. H (SUP) and M (SUP), respectively). MVC significantly declined (-27%; P < 0.001) after the run, due to decrease in muscle activation (-8%; P < 0.05) and M (MAX)-normalized EMG activity (-13%; P < 0.05). Significant reductions in M-wave amplitudes (M (MAX): -13% and M (SUP): -16%; P < 0.05) as well as H (MAX)/M (MAX) (-37%; P < 0.01) and H (SUP)/M (SUP) (-25%; P < 0.05) ratios occurred with fatigue. Following exercise, the single twitch was characterized by lower peak torque (-16%; P < 0.001) as well as shorter contraction (-19%; P < 0.001) and half-relaxation (-24%; P < 0.001) times. In conclusion, the reduction in plantar flexors strength induced by a 5 km running time trial is caused by peripheral adjustments, which are attributable to a failure of the neuromuscular transmission and excitation-contraction coupling. Fatigue also decreased the magnitude of efferent motor outflow from spinal motor neurons to the plantar flexors and part of this suboptimal neural drive is the result of an inhibition of soleus motoneuron pool reflex excitability.

CD4+ T cell count recovery in HIV type 1-infected patients is independent of class of antiretroviral therapy.

BACKGROUND: In recent years, treatment options for human immunodeficiency virus type 1 (HIV-1) infection have changed from nonboosted protease inhibitors (PIs) to nonnucleoside reverse-transcriptase inhibitors (NNRTIs) and boosted PI-based antiretroviral drug regimens, but the impact on immunological recovery remains uncertain. METHODS: During January 1996 through December 2004 [corrected] all patients in the Swiss HIV Cohort were included if they received the first combination antiretroviral therapy (cART) and had known baseline CD4(+) T cell counts and HIV-1 RNA values (n = 3293). For follow-up, we used the Swiss HIV Cohort Study database update of May 2007 [corrected] The mean (+/-SD) duration of follow-up was 26.8 +/- 20.5 months. The follow-up time was limited to the duration of the first cART. CD4(+) T cell recovery was analyzed in 3 different treatment groups: nonboosted PI, NNRTI, or boosted PI. The end point was the absolute increase of CD4(+) T cell count in the 3 treatment groups after the initiation of cART. RESULTS: Two thousand five hundred ninety individuals (78.7%) initiated a nonboosted-PI regimen, 452 (13.7%) initiated an NNRTI regimen, and 251 (7.6%) initiated a boosted-PI regimen. Absolute CD4(+) T cell count increases at 48 months were as follows: in the nonboosted-PI group, from 210 to 520 cells/muL; in the NNRTI group, from 220 to 475 cells/muL; and in the boosted-PI group, from 168 to 511 cells/muL. In a multivariate analysis, the treatment group did not affect the response of CD4(+) T cells; however, increased age, pretreatment with nucleoside reverse-transcriptase inhibitors, serological tests positive for hepatitis C virus, Centers for Disease Control and Prevention stage C infection, lower baseline CD4(+) T cell count, and lower baseline HIV-1 RNA level were risk factors for smaller increases in CD4(+) T cell count. CONCLUSION: CD4(+) T cell recovery was similar in patients receiving nonboosted PI-, NNRTI-, and boosted PI-based cART.

Disorders of pupillary structure and function.

Neurologists are frequently consulted because of a pupillary abnormality. An unequal size of the pupils, an unusual shape, white colored pupils, or a poorly reactive pupil are common reasons for referral. A directed history and careful observation of the iris and pupil movements can bear out ocular pathology such as congenital or structural anomalies as the cause of abnormal pupils. Thereafter, it is important to evaluate the neurologic causes of anisocoria and poor pupil function. The first part of this article emphasizes pupillary abnormalities frequently encountered in infants and children and discusses some of the more common acquired iris structural defects. The second part focuses on evaluation of lesions in the neural pathways that result in pupillary dysfunction, with particular attention to those conditions having neurologic, systemic, or visual implications.

Protection from radiation-induced colitis requires MHC class II antigen expression by cells of hemopoietic origin.

Ulcerative colitis, an inflammatory bowel disease, is believed to result from a breakdown of dominant tolerance mechanisms that normally control intestinal immunity. Although CD4+ T lymphocyte subpopulations and expression of MHC class II molecules have been shown to play a role in the pathogenesis of the disease, the nature of the responsible mechanisms remains unclear. In this paper we describe a novel mouse model for inflammatory bowel disease, radiation-induced colitis, that occurs with complete penetrance 6-8 wk postinduction. A combination of high dose gamma-irradiation and lack of MHC class II expression on cells of hemopoietic origin results in development of colitis in C57BL/6 mice. Because of its versatility (due to susceptibility of mice of the widely genetically manipulated C57BL/6 background), high reproducibility, and 100% penetrance, radiation-induced colitis will be a useful mouse model for colitis and a significant tool to study dominant immunological tolerance mechanisms. Moreover, our data imply that tolerization to enteric Ags requires MHC class II mediated presentation by APC of hemopoietic origin.

Pulmonary artery pressure and cardiac function in children and adolescents after rapid ascent to 3,450 m.

High-altitude destinations are visited by increasing numbers of children and adolescents. High-altitude hypoxia triggers pulmonary hypertension that in turn may have adverse effects on cardiac function and may induce life-threatening high-altitude pulmonary edema (HAPE), but there are limited data in this young population. We, therefore, assessed in 118 nonacclimatized healthy children and adolescents (mean ± SD; age: 11 ± 2 yr) the effects of rapid ascent to high altitude on pulmonary artery pressure and right and left ventricular function by echocardiography. Pulmonary artery pressure was estimated by measuring the systolic right ventricular to right atrial pressure gradient. The echocardiography was performed at low altitude and 40 h after rapid ascent to 3,450 m. Pulmonary artery pressure was more than twofold higher at high than at low altitude (35 ± 11 vs. 16 ± 3 mmHg; P < 0.0001), and there existed a wide variability of pulmonary artery pressure at high altitude with an estimated upper 95% limit of 52 mmHg. Moreover, pulmonary artery pressure and its altitude-induced increase were inversely related to age, resulting in an almost twofold larger increase in the 6- to 9- than in the 14- to 16-yr-old participants (24 ± 12 vs. 13 ± 8 mmHg; P = 0.004). Even in children with the most severe altitude-induced pulmonary hypertension, right ventricular systolic function did not decrease, but increased, and none of the children developed HAPE. HAPE appears to be a rare event in this young population after rapid ascent to this altitude at which major tourist destinations are located.

Obesity in an ageing society implications for health, physical function and health service utilisation.

Key Findings • Based on body mass index (BMI) measurements, 36% of Irish over 50s are obese and a further 43% are overweight. • Based on waist circumference measurements, 52% of Irish over 50s are ‘centrally obese’, i.e., with a ‘substantially increased’ waist circumference, while a further 25% have an ‘increased’ waist circumference. • Using BMI as an indicator of obesity, a higher proportion of men (38%) are obese than women (33%); however, using waist circumference as an indicator of obesity, a higher proportion of women (56%) have a ‘substantially increased’ waist circumference than men (48%). • The prevalence of obesity in Irish men over 50 is comparable with US men over 50 (while English rates are much lower).     .This resource was contributed by The National Documentation Centre on Drug Use.