Modulation of human cardiac function through 4 beta-adrenoceptor populations

In human heart there is now evidence for the involvement of four beta-adrenoceptor populations, three identical to the recombinant beta(1)-, beta(2)- and beta(3)-adrenoceptors, and a fourth as yet uncloned putative beta-adrenoceptor population, which we designate provisionally as the cardiac putative beta(4)-adrenoceptor. This review described novel features of beta-adrenoceptors as modulators of cardiac systolic and diastolic function. We also discuss evidence for modulation by unoccupied beta(1)- and beta(2)-adrenoceptors. Human cardiac and recombinant beta(1)- and beta(2)-adrenoceptors are both mainly coupled to adenylyl cyclase through Gs protein, the latter more tightly than the former. Activation of both human beta(1)- and beta(2)-adrenoceptors not only increases cardiac force during systole but also hastens relaxation through cyclic AMP-dependent phosphorylation of phospholamban and troponin I, thereby facilitating diastolic function. Furthermore, both beta(1) and beta(2)-adrenoceptors can mediate experimental arrhythmias in human cardiac preparations elicited by noradrenaline and adrenaline. Human ventricular beta(3)-adrenoceptors appear to be coupled to a pertussis toxin-sensitive protein (Gi?). beta(3)-Adrenoceptor-selective agonists shorten the action potential and cause cardiodepression, suggesting direct coupling of a Gi protein to a K+ channel. In a variety of species, including man, cardiac putative beta(4)-adrenoceptors mediate cardiostimulant effects of non-conventional partial agonists, i.e. high affinity beta(1)- and beta(2)-adrenoceptor blockers that cause agonist effects at concentrations considerably higher than those that block these receptors. Putative beta(4)-adrenoceptors appear to be coupled positively to a cyclic AMP-dependent cascade and can undergo some desensitisation.

Quantitative and qualitative influences of tapasin on the class I peptide repertoire

Tapasin is critical for efficient loading and surface expression of most HLA class I molecules. The high level surface expression of HLA-B*2705 on tapasin-deficient 721.220 cells allowed the influence of this chaperone on peptide repertoire to be examined. Comparison of peptides bound to HLA-B*2705 expressed on tapasin-deficient and -proficient cells by mass spectrometry revealed an overall reduction in the recovery of B*2705-bound peptides isolated from tapasin-deficient cells despite similar yields of B27 heavy chain and beta (2)-microglobulin. This indicated that a proportion of suboptimal ligands were associated with B27, and they were lost during the purification process. Notwithstanding this failure to recover these suboptimal peptides, there was substantial overlap in the repertoire and biochemical properties of peptides recovered from B27 complexes derived from tapasin-positive and -negative cells. Although many peptides were preferentially or uniquely isolated from B*2705 in tapasin-positive cells, a number of species were preferentially recovered in the absence of tapasin, and some of these peptide ligands have been sequenced. In general, these ligands did not exhibit exceptional binding affinity, and we invoke an argument based on lumenal availability and affinity to explain their tapasin independence. The differential display of peptides in tapasin-negative and -positive cells was also apparent in the reactivity of peptide-sensitive alloreactive CTL raised against tapasin-positive and -negative targets, demonstrating the functional relevance of the biochemical observation of changes in peptide repertoire in the tapasin-deficient APC. Overall, the data reveal that tapasin quantitatively and qualitatively influences ligand selection by class I molecules.

FXYD7 is a brain-specific regulator of Na,K-ATPase alpha 1-beta isozymes.

Recently, corticosteroid hormone-induced factor (CHIF) and the gamma-subunit, two members of the FXYD family of small proteins, have been identified as regulators of renal Na,K-ATPase. In this study, we have investigated the tissue distribution and the structural and functional properties of FXYD7, another family member which has not yet been characterized. Expressed exclusively in the brain, FXYD7 is a type I membrane protein bearing N-terminal, post-translationally added modifications on threonine residues, most probably O-glycosylations that are important for protein stabilization. Expressed in Xenopus oocytes, FXYD7 can interact with Na,K-ATPase alpha 1-beta 1, alpha 2-beta 1 and alpha 3-beta 1 but not with alpha-beta 2 isozymes, whereas, in brain, it is only associated with alpha 1-beta isozymes. FXYD7 decreases the apparent K(+) affinity of alpha 1-beta 1 and alpha 2-beta 1, but not of alpha 3-beta1 isozymes. These data suggest that FXYD7 is a novel, tissue- and isoform-specific Na,K-ATPase regulator which could play an important role in neuronal excitability.

Selective absence of CD8+ TCRalpha beta+ intestinal epithelial cells in transgenic mice expressing beta2-microglobulin-associated ligands exclusively on thymic cortical epithelium.

Whereas interactions between the TCRalpha beta and self MHC:peptide complexes are clearly required for positive selection of mature CD4(+) and CD8(+) T cells during intrathymic development, the role of self or foreign ligands in maintaining the peripheral T cell repertoire is still controversial. In this report we have utilized keratin 14-beta2-microglobulin (K14-beta2m)-transgenic mice expressing beta2m-associated ligands exclusively on thymic cortical epithelial cells to address the possible influence of TCR:ligand interactions in peripheral CD8(+) T cell homeostasis. Our data indicate that CD8(+) T cells in peripheral lymphoid tissues are present in normal numbers in the absence of self MHC class I:peptide ligands. Surprisingly, however, steady state homeostasis of CD8(+) T cells in the intestinal epithelium is severely affected by the absence of beta2m-associated ligands. Indeed TCRalpha beta(+) IEL subsets expressing CD8alpha beta or CD8alpha alpha are both dramatically reduced in K14-beta2m mice, suggesting that the development, survival or expansion of CD8(+) IEL depends upon interaction of the TCR with MHC class I:peptide or other beta2m-associated ligands elsewhere than on thymic cortical epithelium. Collectively, our data reveal an unexpected difference in the regulation of CD8(+) T cell homeostasis by beta2m-associated ligands in the intestine as compared to peripheral lymphoid organs.

Positive and negative roles of the trans-acting T cell factor-1 for the acquisition of distinct Ly-49 MHC class I receptors by NK cells.

Members of the Ly-49 gene family code for class I MHC-specific receptors that regulate NK cell function. Due to a combinatorial distribution of Ly-49 receptors, NK cells display considerable clonal heterogeneity. The acquisition of one Ly-49 receptor, Ly-49A is strictly dependent on the transcriptional trans-acting factor T cell-specific factor-1 (TCF-1). Indeed, TCF-1 binds to two sites in the Ly-49a promoter and regulates its activity, suggesting that the Ly-49a gene is a direct TCF-1 target. TCF-1 deficiency resulted in the altered usage of additional Ly-49 receptors. We show in this study, using TCF-1 beta(2)-microglobulin double-deficient mice, that these repertoire alterations are not due to Ly-49/MHC class I interactions. Our findings rather suggest a TCF-1-dependent, cell autonomous effect on the acquisition of multiple Ly-49 receptors. Besides reduced receptor usage (Ly-49A and D), we also observed no effect (Ly-49C) and significantly expanded (Ly-49G and I) receptor usage in the absence of TCF-1. These effects did not in all cases correlate with the presence of TCF binding sites in the respective proximal promoter. Therefore, besides TCF-1 binding to the proximal promoter, Ly-49 acquisition may also be regulated by TCF-1 binding to more distant cis-acting elements and/or by regulating the expression of additional trans-acting factors. Consistent with the observed differential, positive or negative role of TCF-1 for Ly-49 receptor acquisition, reporter gene assays revealed the presence of an inducing as well as a repressing TCF site in certain proximal Ly-49 promoters. These findings reveal an important role of TCF-1 for the formation of the NK cell receptor repertoire.

Nova síntesi enantioselectiva de la (R)- i (S)-4-hidroxi-2-ciclohexenona

En el present treball de recerca s’ha plantejat la síntesi de forma enantioselectiva d’ambdós enantiòmers de la 4-hidroxi-2-ciclohexenona i derivats O-protegits. Així, s’ha dut a terme una aproximació sintètica a partir de la 1,4- ciclohexandiona parcialment protegida amb etilenglicol, per obtenir els dos enantiòmers de la molècula objectiu i derivats O-protegits, reproduint metodologies ja emprades en el nostre grup de recerca. D’aquesta manera, s’han obtingut ambdós enantiòmers del derivat O-protegit en un total de 5 passos, amb un rendiment del 60% i un 92% d’excés enantiomèric. Amb aquesta síntesi enantioselectiva s’aconsegueix una millora dels resultats del nostre grup de recerca, essent també una estratègia sintètica molt competitiva amb les que ja estan publicades tant pel bons rendiments obtinguts com per l’excés enantiomèric. L’accessibilitat del producte de partida i el fàcil escalatge de les reaccions fan d’aquesta estratègia sintètica una interessant opció a tenir en compte.

Stringent V beta requirement for the development of NK1.1+ T cell receptor-alpha/beta+ cells in mouse liver.

The liver of C57BL/6 mice contains a major subset of CD4+8- and CD4-8- T cell receptor (TCR)-alpha/beta+ cells expressing the polymorphic natural killer NK1.1 surface marker. Liver NK1.1+TCR-alpha/beta+ (NK1+ T) cells require interaction with beta2-microglobulin-associated, major histocompatibility complex I-like molecules on hematopoietic cells for their development and have a TCR repertoire that is highly skewed to Vbeta8.2, Vbeta7, and Vbeta2. We show here that congenic C57BL/6.Vbeta(a) mice, which lack Vbeta8- expressing T cells owing to a genomic deletion at the Vbeta locus, maintain normal levels of liver NK1+ T cells owing to a dramatic increase in the proportion of cells expressing Vbeta7 and Vbeta2 (but not other Vbetas). Moreover, in C57BL/6 congenic TCR-V Vbeta3 and -Vbeta8.1 transgenic mice (which in theory should not express other Vbeta, owing to allelic exclusion at the TCR-beta locus), endogenous TCR-Vbeta8.2, Vbeta7, and Vbeta2 (but not other Vbetas) are frequently expressed on liver NK1+T cells but absent on lymph node T cells. Finally, when endogenous V beta expression is prevented in TCR-Vbeta3 and Vbeta8.1 transgenic mice (by introduction of a null allele at the C beta locus), the development of liver NK1+T cells is totally abrogated. Collectively, our data indicate that liver NK1+T cells have a stringent requirement for expression of TCR-Vbeta8.2, Vbeta7, or Vbeta2 for their development.

Esfingolipids i senyal•lització cel.lular: l'(E)-2-hexadecenal com a possible lípid bioactiu

L’esfingosina-1-fosfat (S1P) és un lípid bioactiu amb funcions crucials en la biologia cel•lular. Entre aquestes, la seva activitat mitogènica i citoprotectora són les més estudiades. L’S1P és catabolitzada intracel•lularment mitjançant l’esfingosina-1-fosfat liasa (SGPL1) per generar (E)-2-hexadecenal i fosforiletanolamina. L’objectiu d’aquest projecte és explorar si l’(E)-2-hexadecenal és realment un catabòlit innocu o bé si, pel seu caràcter acceptor de Michael, és capaç de reaccionar amb pèptids o proteïnes específics. Aquesta interacció podria traduïr-se en funcions biològiques determinades, algunes de les quals són possiblement atribuïdes a l’esfingosina-1-fosfat com a tal. Per poder explorar el potencials adductes proteïcs amb l’aldehid, s’han emprat, sobre cèl•lules HeLa que sobreexpressen SGPL1, sondes anàlegs a esfingosina i esfinganina (i els seus derivats fosforil•lats) que presenten una funció azida en la posició omega de la cadena esfingoide. Aquestes, mitjançant química click sense coure, s’han fet reaccionar amb una molècula que presenta un dibenzociclooctí unit a biotina DBCObiotina). Després d’aïllar les proteïnes així biotinilades amb una reïna d’estreptavidina, aquestes es van separar per electroforesi. Les bandes proteïques observades es van extreure del gel i es van digerir amb tripsina, per posteriorment analitzar els pèptids per MALDI-TOF, el que permetria l’identificació de proteïnes a partir de “peptide mass fingerprinting”. Lamentablement, a la fi d’aquest contracte, encara no s’ha pogut identificar cap proteïna que s’uneixi a l’aldehid alliberat per la reacció de l’esfingosina-1- fosfat liasa. No obstant, durant aquest temps s’ha millorat el mètode per detectar aquests adductes proteïcs. Per això, si la recerca continua en aquesta línia, properament es podria saber amb certesa si existeixen o no aquestes interaccions covalents entre determinades proteïnes i l’(E)-2-hexadecenal.

Beta(1)-selective agonist (-)-1-(3,4-dimethoxyphenetylamino)-3-(3,4-dihydroxy)-2-propanol [(-)-RO363] differentially interacts with key amino acids responsible for beta(1)-selective binding in resting and active states.

(-)-1-(3,4-Dimethoxyphenetylamino)-3-(3,4-dihydroxy)-2-propanol [(-)-RO363] is a highly selective beta(1)-adrenergic receptor (beta(1)AR) agonist. To study the binding site of beta(1)-selective agonist, chimeric beta(1)/beta(2)ARs and Ala-substituted beta(1)ARs were constructed. Several key residues of beta(1)AR [Leu(110) and Thr(117) in transmembrane domain (TMD) 2], and Phe(359) in TMD 7] were found to be responsible for beta(1)-selective binding of (-)-RO363, as determined by competitive binding. Based on these results, we built a three-dimensional model of the binding domain for (-)-RO363. The model indicated that TMD 2 and TMD 7 of beta(1)AR form a binding pocket; the methoxyphenyl group of N-substituent of (-)-RO363 seems to locate within the cavity surrounded by Leu(110), Thr(117), and Phe(359). The amino acids Leu(110) and Phe(359) interact with the phenyl ring of (-)-RO363, whereas Thr(117) forms hydrogen bond with the methoxy group of (-)-RO363. To examine the interaction of these residues with beta(1)AR in an active state, each of the amino acids was changed to Ala in a constitutively active (CA)-beta(1)AR mutant. The degree of decrease in the affinity of CA-beta(1)AR for (-)-RO363 was essentially the same as that of wild-type beta(1)AR when mutated at Leu(110) and Thr(117). However, the affinity was decreased in Ala-substituted mutant of Phe(359) compared with that of wild-type beta(1)AR. These results indicated that Leu(110) and Thr(117) are necessary for the initial binding of (-)-RO363 with beta(1)-selectivity, and interaction of Phe(359) with the N-substituent of (-)-RO363 in an active state is stronger than in the resting state.

Reduced development of CD4-8-B220+ T cells but normal autoantibody production in lpr/lpr mice lacking major histocompatibility complex class I molecules.

The lpr gene has recently been shown to encode a functional mutation in the Fas receptor, a molecule involved in transducing apoptotic signals. Mice homozygous for the lpr gene develop an autoimmune syndrome accompanied by massive accumulation of double-negative (DN) CD4-8-B220+ T cell receptor-alpha/beta+ cells. In order to investigate the origin of these DN T cells, we derived lpr/lpr mice lacking major histocompatibility complex (MHC) class I molecules by intercrossing them with beta 2-microglobulin (beta 2m)-deficient mice. Interestingly, these lpr beta 2m-/- mice develop 13-fold fewer DNT cells in lymph nodes as compared to lpr/lpr wild-type (lprWT) mice. Analysis of anti-DNA antibodies and rheumatoid factor in serum demonstrates that lpr beta 2m-/- mice produce comparable levels of autoantibodies to lprWT mice. Collectively our data indicate that MHC class I molecules control the development of DN T cells but not autoantibody production in lpr/lpr mice and support the hypothesis that the majority of DN T cells may be derived from cells of the CD8 lineage.

Dissociation of thymic positive and negative selection in transgenic mice expressing major histocompatibility complex class I molecules exclusively on thymic cortical epithelial cells.

Thymic positive and negative selection of developing T lymphocytes confronts us with a paradox: How can a T-cell antigen receptor (TCR)-major histocompatibility complex (MHC)/peptide interaction in the former process lead to transduction of signals allowing for cell survival and in the latter induce programmed cell death or a hyporesponsive state known as anergy? One of the hypotheses put forward states that the outcome of a TCR-MHC/peptide interaction depends on the cell type presenting the selecting ligand to the developing thymocyte. Here we describe the development and lack of self-tolerance of CD8(+) T lymphocytes in transgenic mice expressing MHC class I molecules in the thymus exclusively on cortical epithelial cells. Despite the absence of MHC class I expression on professional antigen-presenting cells, normal numbers of CD8(+) cells were observed in the periphery. Upon specific activation, transgenic CD8(+) T cells efficiently lysed syngeneic MHC class I(+) targets in vitro and in vivo, indicating that thymic cortical epithelium (in contrast to medullary epithelium and antigen-presenting cells of hematopoietic origin) is incapable of tolerance induction. Thus, compartmentalization of the antigen-presenting cells involved in thymic positive selection and tolerance induction can (at least in part) explain the positive/negative selection paradox.

Impaired natural killing of MHC class I-deficient targets by NK cells expressing a catalytically inactive form of SHP-1.

NK cell function is negatively regulated by MHC class I-specific inhibitory receptors. Transduction of the inhibitory signal involves protein tyrosine phosphatases such as SHP-1 (SH2-containing protein tyrosine phosphatase-1). To investigate the role of SHP-1 for NK cell development and function, we generated mice expressing a catalytically inactive, dominant-negative mutant of SHP-1 (dnSHP-1). In this paper we show that expression of dnSHP-1 does not affect the generation of NK cells even though MHC receptor-mediated inhibition is partially impaired. Despite this defect, these NK cells do not kill syngeneic, normal target cells. In fact dnSHP-1-expressing NK cells are hyporesponsive toward MHC-deficient target cells, suggesting that non-MHC-specific NK cell activation is significantly reduced. In contrast, these NK cells mediate Ab-dependent cell-mediated cytotoxicity and prevent the engraftment with beta2-microglobulin-deficient bone marrow cells. A similar NK cell phenotype is observed in viable motheaten (mev) mice, which show reduced SHP-1 activity due to a mutation in the Shp-1 gene. In addition, NK cells in both mouse strains show a tendency to express more inhibitory MHC-specific Ly49 receptors. Our results demonstrate the importance of SHP-1 for the generation of functional NK cells, which are able to react efficiently to the absence of MHC class I molecules from normal target cells. Therefore, SHP-1 may play an as-yet-unrecognized role in some NK cell activation pathways. Alternatively, a reduced capacity to transduce SHP-1-dependent inhibitory signals during NK cell development may be compensated by the down-modulation of NK cell triggering pathways.

T cell receptor V beta repertoire in mice lacking endogenous mouse mammary tumor provirus.

When endogenous mouse mammary tumor virus (MMTV) superantigens (SAg) are expressed in the first weeks of life an efficient thymic deletion of T cells expressing MMTV SAg-reactive T cell receptor (TcR) V beta segments is observed. As most inbred mouse strains and wild mice contain integrated MMTV DNA, knowing the precise extent of MMTV influence on T cell development is required in order to study T cell immunobiology in the mouse. In this report, backcross breeding between BALB.D2 (Mtv-6, -7, -8 and -9) and 38CH (Mtv-) mice was carried out to obtain animals either lacking endogenous MMTV or containing a single MMTV locus, i.e. Mtv-6, -7, -8 or -9. The TcR V beta chain (TcR V beta) usage in these mice was analyzed using monoclonal antibodies specific for TcR V beta 2, V beta 3, V beta 4, V beta 5, V beta 6, V beta 7, V beta 8, V beta 11, V beta 12 and V beta 14 segments. Both Mtv-8+ mice and Mtv-9+ mice deleted TcR V beta 5+ and V beta 11+ T cells. Moreover, we also observed the deletion of TcR V beta 12+ cells by Mtv-8 and Mtv-9 products. Mtv-6+ and Mtv-7+ animals deleted TcR V beta 3+ and V beta 5+ cells, and TcR V beta 6+, V beta 7+ and V beta 8.1+ cells, respectively. Unexpectedly, TcR V beta 8.2+ cells were also deleted in some backcross mice expressing Mtv-7. TcR V beta 8.2 reactivity to Mtv-7 was shown to be brought by the 38CH strain and to result from an amino acid substitution (Asn-->Asp) in position 19 on the TcR V beta 8.2 fragment. Reactivities of BALB.D2 TcR V beta 8.2 and 38CH TcR V beta 8.2 to the exogenous infectious viruses, MMTV(SW) and MMTV(SHN), were compared. Finally, the observation of increased frequencies of TcR V beta 2+, V beta 4+ and V beta 8+ CD4+ T cell subsets in Mtv-8+ and Mtv-9+ mice, and TcR V beta 4+ CD4+ T cells in Mtv-6+ and Mtv-7+ mice, when compared with the T cell repertoire of Mtv- mice, is consistent with the possibility that MMTV products contribute to positive selection of T cells.

Tissue-based immune monitoring I: tumor core needle biopsies allow in-depth interrogation of the tumor microenvironment.

We sought to assess the feasibility and reproducibility of performing tissue-based immune characterization of the tumor microenvironment using CT-compatible needle biopsy material. Three independent biopsies were obtained intraoperatively from one metastatic epithelial ovarian cancer lesion of 7 consecutive patients undergoing surgical cytoreduction using a 16-gauge core biopsy needle. Core specimens were snap-frozen and subjected to immunohistochemistry (IHC) against human CD3, CD4, CD8, and FoxP3. A portion of the cores was used to isolate RNA for 1) real-time quantitative (q)PCR for CD3, CD4, CD8, FoxP3, IL-10 and TGF-beta, 2) multiplexed PCR-based T cell receptor (TCR) CDR3 Vβ region spectratyping, and 3) gene expression profiling. Pearson's correlations were examined for immunohistochemistry and PCR gene expression, as well as for gene expression array data obtained from different tumor biopsies. Needle biopsy yielded sufficient tissue for all assays in all patients. IHC was highly reproducible and informative. Significant correlations were seen between the frequency of CD3+, CD8+ and FoxP3+ T cells by IHC with CD3ε, CD8A, and FoxP3 gene expression, respectively, by qPCR (r=0.61, 0.86, and 0.89; all p< 0.05). CDR3 spectratyping was feasible and highly reproducible in each tumor, and indicated a restricted repertoire for specific TCR Vβ chains in tumor-infiltrating T cells. Microarray gene expression revealed strong correlation between different biopsies collected from the same tumor. Our results demonstrate a feasible and reproducible method of immune monitoring using CT-compatible needle biopsies from tumor tissue, thereby paving the way for sophisticated translational studies during tumor biological therapy.

Biases on the administered parenteral doses of an experimental drug during phase I clinical trials

Introduction: The pharmaceutical aspects of drug administration in clinical trials receive poor consideration compared with the important attention devoted to the analytical and mathematical aspects of biological sample exploitation. During PK calculations, many researchers merely use for dose the nominal amount declared, overlooking the noticeable biases that may result in the assessment of PK parameters. The aim of this work was to evaluate the biases related to doses injected of a biosimilar drug in 2 Phase I clinical trials. Patients (or Materials) and Methods: In trial A, 12 healthy volunteers received different doses of a biosimilar of interferon beta-1a by either subcutaneous (SC) or intravenous (IV) injection. The doses were prepared by partially emptying 0.5-mL syringes supplied by the manufacturer (drop count procedure). In trial B, 12 healthy volunteers received 3 different formulations of the drug by IV injection (biosimilar without albumin [HSA], biosimilar with HSA and original brand [Rebif®]) and 2 different formulations as multiple SC injections (biosimilar HSA-free and original brand). In both trials, the actual dose administered was calculated as: D = C·V - losses. The product titer C was assessed by ELISA. The volume administered IV was assessed by weighting. Losses were evaluated by in vitro experiments. Finally, the binding of 125I-interferon to HSA was evaluated by counting the free and HSA complexed molecule fractions separated by gel filtration. Results: Interferon was not significantly adsorbed onto the lines used for its IV administration. In trial A, the titer was very close to the one declared (96 ± 7%). In trial B, it differed significantly (156 ± 10% for biosimilar with/without HSA and 123 ± 5% for original formulation). In trial A, the dose actually administered showed a large variability. The real injected volume could be biased up to 75% compared with the theoretical volume (for the lower dose administered [ie, 0.03 mL]). This was mainly attributed to a partial re-aspiration of the drug solution before withdrawing the syringe needle. A strict procedure was therefore applied in trial B to avoid these inaccuracies. Finally, in trial B, 125I-Interferon beta-1a binding to HSA appeared time dependent and slow, reaching 50% after 16-hour incubation, which is close to steady state reported for the comparator Rebif®. Conclusion: These practical examples (especially biases on actual titer and volume injected) illustrate that actual dose assessment deserves attention to ensure accuracy for estimates of clearance and distribution volume in the scientific literature and for registration purposes, especially for bioequivalence studies.