Adaptation of left ventricular morphology to long−term training in sprint− and endurance−trained elite runners

Long−term studies on left ventricular (LV) adaptation have not been reported. The echocardiograms of 41 top−class runners (8 males and 6 females sprint−trained, 15 males and 12 females endurance−trained) were recorded at the beginning and after 1, 2, and 3 years of training. A one−way ANOVA and a linear regression analysis were conducted to determine changes and association between performance and LV values. Training resulted in an increase in performance and LV internal diameter at end−diastole (LVIDd) and decreases in end−diastolic interventricular septal wall thickness, and posterior wall thickness (PWTd). There were no significant differences in LV mass and LV ejection fraction (LVEF, %). The changes in PWTd were linked to enlargement of the LV. In athletes with unusual LV dilatation (>60 mm), LVIDd was related to performance and LVEF was >50%. Maximal wall thickness was <13 mm in all athletes. LV adaptations were independent of sex and type of training and related to the initial level of performance. We believe that LV enlargement in elite runners is a physiological adaptation and that the LVIDd is a predictor of running performance.     

Requirement of HCO 3 − for Cl−-absorption in seawater-adapted eel intestine

The role of HCO ₃ ^– /CO₂ buffer in Cl^– absorption was examined in the in vitro perfused eel intestine adapted to seawater. Cl^– absorption, expressed as short/circuit current (I _sc), was measured in either 20 mM HCO ₃ ^– /1% CO₂ Ringer or HEPES Ringer, pH 8.0. Unilateral (mucosal or serosal) substitution of HCO ₃ ^– /CO₂ with HEPES/O₂ was without effect on I _sc and transepithelial voltage (V _t), whereas bilateral removal of HCO ₃ ^– /CO₂ reduced I _sc and V _t by 50%, indicating that the presence of HCO ₃ ^– /CO₂ buffer at one side of the epithelium is sufficient to keep Cl^– absorption at the maximum rate. We examined in further detail the individual components of the HCO ₃ ^– /CO₂ system that stimulates Cl^– absorption. We found that, in tissues bathed with HEPES Ringer, addition of 1% CO₂ to the luminal or serosal solution (final pH=7.6 in the chamber) had no effect on I _sc and V _t, while both electrical parameters could be restored to control values by unilateral (luminal or serosal) substitution of HEPES Ringer with 20 mM HCO ₃ ^– /1% CO₂ Ringer or 20 mM HCO ₃ ^– alone. Stimulation of I _sc induced by unilateral (luminal or serosal) HCO ₃ ^– /CO₂ was inhibited by luminal or serosal 4-acetamido-4-isothiocyanostilbene-2,2-disulphonic acid (SITS) (0,25 mM) or by serosal Na⁺ removal, whereas amiloride (1 mM), luminal or serosal, had no effect. Acetazolamide (0.1 mM, both sides) inhibited stimulation of I _sc induced by luminal addition of HCO ₃ ^– /CO₂, whereas it was without effect when HCO ₃ ^– /CO₂ was added serosally or bilaterally. We reached the following conclusions, (a) Cl^– absorption is stimulated by HCO ₃ ^– /CO₂ buffer via an increase in intracellular HCO ₃ ^– concentration and/or pH_i changes consequent to the HCO ₃ ^– uptake mediated by HCO ₃ ^– transport systems operating on both cell membranes, (b) A Na⁺-dependent SITS-inhibitable HCO ₃ ^– transport mechanism operates at the basolateral membrane, (c) The transfer of HCO ₃ ^– through the luminal membrane is mediated by the carbonic anhydrase enzyme located on the brush-border membranes of the enterocyte: the movement of HCO ₃ ^– , via a SITS-sensitive transport system, occurs most likely in form of OH^–, which originates from the dehydration reaction of HCO ₃ ^– catalysed by the carbonic anhydrase. (d) There is no apparent amiloride-sensitive Na⁺/H⁺ antiporter on either cell membrane.This work was supported by a research grant from Ministero dell'Università e della Ricerca Scientifica e Tecnologica — Progetto di interesse nazionale e di rilevante interesse per lo sviluppo della Scienza     

CD4−8− T-cells increase in MRL/lpr mice treated with thymic factors

The in vivo effect of thymic factors on immature lymphocytes was analysed in MRL/lpr mice. This strain carries a genetic defect that causes during their life cycle a block of T-cell differentiation and abnormal proliferation of CD4⁻8⁻ (double-negative, DN) T-lymphocytes. In vivo administration of four preparations of thymic factors, thymopentin (TP-1), thymopoietin (TP-5), thymolymphotropin (TLT), and thymomodulin (TMD) into young (2-month-old) MRL/lpr mice induced a significant increase of DN T-cells both in the thymus and in the peripheral lymph nodes, with a concomitant decrease of double-positive (DP) T-cells in the thymus and of single-positive (SP) T-cells in the lymph nodes. The level of DNA fragmentation measured as propidium iodide fluorescence was increased in the thymus population of young mice and in the lymph node population of old mice treated with TLT. SCID mice transplanted with lymph node cells from MRL/lpr donors (MRL→SCID) developed graft versus host (GvH) reaction due to the activation of MRL CD8⁺ alloreactive T-cells. This model was used to analyse the effect of TMD/TLT in vivo on MRL cell proliferation and expansion; in fact, spleen cells from MRL→SCID mice after treatment with TMD/TLT showed an increased cell proliferation, and an expansion of DN T-cells with a concomitant decrease of SP cells (both CD4⁺ and CD8⁺ cells). Decreased SP cell numbers in this context could explain why TMD/TLT treatment of SCID mice engrafted with MRL cells increased their survival compared to untreated MRL→SCID mice.     

The outwardly rectifying Cl− channel is not involved in cAMP-mediated Cl− secretion in HT-29 cells: evidence for a very-low-conductance Cl− channel

The patch-clamp technique and transepithelial current measurements in conjunction with analysis of transepithelial current noise were employed in order to clarify the role of the outwardly rectifying, depolarization-induced Cl^– channel (ORDIC) during cAMP-mediated Cl^– secretion in HT-29/B6 cells. Confluent monolayers growing on permeable supports were used in order to ensure the apical location of measured Cl^– channels. The ORDIC needed to be activated by excision and/or depolarization, and was found in both cAMP-stimulated and non-stimulated cells. Both 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and 4,4-dinitro-2,2-stilbenedisulphonate (DNDS) induced fast flickery-type blocks of the ORDIC at low, micromolar blocker concentrations and were used as a probe for ORDIC. However, these substances were ineffective in blocking transepithelial forskolin-induced Cl^– secretion of monolayers in Ussing chambers. No inhibitory effect at all was detected for DNDS up to 1 mmol/l. NPPB blocked the ORDIC at low concentrations (IC₅₀=0.5±0.3 mol/l) by reducing its open probability, but NPPB did not block forskolin-induced Cl^– secretion unless high concentrations were used (IC₅₀=240±10 mol/l). In order to exclude effects of NPPB other than on the apical Cl^– channel, trans-epithelial measurements were performed in basolaterally amphotericin-permeabilized, forskolin-stimulated preparations, and a serosal-to-mucosal Cl^– gradient was applied as a driving force. Under these conditions, NPPB's inhibitory effects were also very small. Noise analysis of this gradient-driven Cl^– current showed a very-low-frequency Lorentzian noise component (f _c=1.4±0.2 Hz), which was not compatible with Lorentzians predicted from single-channel gating of ORDIC. As revealed from fura-2 fluorescence measurements, forskolin-stimulated Cl^– secretion occurred in the absence of changes in intracellular Ca²⁺. Thus, we conclude that there is an apical Cl^– channel in HT-29/B6 that is activated through the cAMP-mediated pathway and is insensitive to NPPB and DNDS, and the kinetics of which are incompatible with ORDIC kinetics. Therefore, despite its prevalence in isolated patches and even in cell-attached recordings, the ORDIC appears not to be involved in cAMP-mediated Cl^– secretion by HT-29/B6 cells. From noise analysis, a very-small-conductance (probably below 1 pS), slow-gating Cl^– channel was calculated as the conductive site in the apical membrane during forskolin stimulation.     

Peripheral CD27−CD21− B-cells represent an exhausted lymphocyte population in hepatitis C cirrhosis

Hepatitis C cirrhosis is associated with a profound disappearance of memory B-cells. We sought to determine if this loss is associated with the expansion of the CD27⁻CD21⁻ tissue-like memory B-cells with features of B-cell exhaustion. To this end, we quantified the frequency of CD27⁻CD21⁻ B-cells in healthy, non-cirrhotic HCV-infected, and cirrhotic patients. We examined the expression of putative inhibitory receptors, the proliferative and immunoglobulin-secreting capacity of CD27/CD21-defined B-cell subsets upon B-cell receptor and/or CD40 stimulation. We found that CD27⁻CD21⁻ B-cells are significantly increased in frequency relative to healthy donors in HCV-infected patients. CD27⁻CD21⁻ B-cells were hypoproliferative relative to naïve and resting memory B-cells upon agonistic stimulation, but retained similar capacity for antibody secretion. Conclusion: CD27⁻CD21⁻ tissue-like memory B-cells with exhausted proliferation circulate at increased frequency in cirrhotic and non-cirrhotic HCV-infected patients. This B-cell subset does not appear anergic, exhibiting immunoglobulin-secreting capacity on CD40 agonism indistinguishable from other CD27/CD21-defined B-cell subsets.     

Hyperbranched lysine−arginine copolymer for gene delivery

Based on the reactivity of amine groups and carboxyl groups of L-lysine and L-arginine, thermal polymerization of these two natural amino acids results in hyperbranched lysine–arginine copolymers (P-lys-arg_X, where X refers to the relevant molar ratio of arginine to lysine). Hyperbranched polylysine (P-lys) and two derivatives (P-lys-arg_0.10 and P-lys-arg_0.20) have been prepared. The arginine-rich hyperbranched polymers can interact with plasmid DNA to form nano-sized particles. The polyplexes were physicochemically analyzed by agarose gel electrophoresis, dynamic light scattering, and zeta potential measurements. Furthermore, their transfection efficiency was assessed, employing COS-7, 293T, and HeLa cell lines. It was found that P-lys showed poorly in its ability of condensation with DNA and transfection efficiency. On the other hand, arginine-rich products resulted to significant enhancement of its transfection efficiency, which is dependent on the content of arginine in the polymers, and the cell line used. P-lys-arg_0.20 exhibited better transfection efficiency under all the condition studied. Besides, P-lys-arg_0.20 showed lower toxicity in COS-7 cells.     

Effects of PGE2 upon differentiation and programmed cell death of suspension cultured CD4− CD8− thymocytes

Recently, several works have focused on the modulation of the immune response by arachidonic acid metabolites. Some of these metabolites, such as prostaglandins, have been shown to influence thymocyte “education” in vitro. However, the effect of one of them, prostaglandin E₂ (PGE₂), in the education of CD4⁻ CD8⁻ double negative immature thymocytes (DN cells) remained unclear. Using a flow cytometry analysis of DN cells cultured for 24 h in the presence of PGE₂, we observed, compared with DN thymocytes cultured without PGE₂, an increase in the CD4⁺ CD8⁻ CD3⁻ immature thymocytes and in the CD4⁺ CD8⁻ and CD8⁺ CD4⁻ mature single positive thymocytes and a decrease in the DN and CD4^high CD8^high double positive thymocytes. Other differentiation thymocyte surface markers, such as CD3, CD5, TCRαß, TCRδγ and HSAg, revealed an increasing number of thymocytes bearing these first four markers and a lower expression of the HSAg. Furthermore, we observed an accumulation of CD4^low CD8^low thymocytes and an increasing proportion of hypodiploid nuclei. These two findings have been shown to be markers of the programmed cell death process. These findings suggest that PGE₂ probably acts on thymocyte differentiation through at least two distinct pathways. On the one hand, PGE₂ seems to promote differentiation of DN cells into CD4⁺ CD8⁻ CD3⁻ immature cells and drive CD4⁺ CD8⁺ CD3⁺ thymocyte to a CD4⁺ CD8⁻ and CD8⁺ CD4⁻ mature phenotype. On the other hand, PGE₂ is probably implicated directly or indirectly in the increase or the acceleration of the programmed cell death process of immature CD4⁺ CD8⁺ CD3⁺ thymocytes, which is linked to the positive and/or negative selection.     

TCRαβ+CD3+CD4−CD8− (double negative) T cells in autoimmunity

TCRαβ⁺CD3⁺CD4^?CD8^? “double negative” (DN) T cells comprise a small subset of mature peripheral T cells. The origin and function of DN T cells are somewhat unclear and discussed controversially. While DN T cells resemble a rare and heterogeneous T cell subpopulation in healthy individuals, numbers of TCRαβ⁺ DN T cells are expanded in several inflammatory conditions, where they also exhibit distinct effector phenotypes and infiltrate inflamed tissues. Thus, DN T cells may be involved in systemic inflammation and tissue damage in autoimmune/inflammatory conditions, including SLE, Sjögren's syndrome, and psoriasis. Here, the current understanding of the origin and phenotype of DN T cells, and their role in the instruction of immune responses, autoimmunity and inflammation will be discussed in health and disease.     

The SLC4 family of HCO3 − transporters

The SLC4 family consists of ten genes. All appear to encode integral membrane proteins with very similar hydropathy plots-consistent with the presence of 10-14 transmembrane segments. At least eight SLC4 members encode proteins that transport HCO(3)(-) (or a related species, such as CO(3)(2-)) across the plasma membrane. Functionally, these eight proteins fall into two major groups: three Cl-HCO(3) exchangers (AE1-3) and five Na(+)-coupled HCO(3)(-) transporters (NBCe1, NBCe2, NBCn1, NDCBE, NCBE). Two of the Na(+)-coupled HCO(3)(- )transporters (NBCe1, NBCe2) are electrogenic; the other three Na(+)-coupled HCO(3)(-) transporters and all three AEs are electroneutral. At least NDCBE transports Cl(-) in addition to Na(+) and HCO(3)(-). Whether NCBE transports Cl(-)-in addition to Na(+) and HCO(3)(-)-is unsettled. In addition, two other SLC4 members (AE4 and BTR1) do not yet have a firmly established function; on the basis of homology, they fall between the two major groups. A characteristic of many, though not all, SLC4 members is inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). SLC4 gene products play important roles in the carriage of CO(2) by erythrocytes, the absorption or secretion of H(+) or HCO(3)(-) by several epithelia, as well as the regulation of cell volume and intracellular pH.     

CD4−CD8− T-cells in primary Sjögren's syndrome: Association with the extent of glandular involvement

ObjectivesGrowing evidence suggests that IL-17-producing T cells, lacking both CD4 and CD8 molecules and defined as double negative (DN) cells, play a pivotal role in the pathogenesis of a number of systemic autoimmune disorders. We recently demonstrated that this T-cell subset is expanded in the peripheral blood (PB) of patients with primary Sjögren's syndrome (pSS), produces IL-17 and accumulates in minor salivary glands (MSGs). We aimed to investigate glandular and PB DN T cells in early pSS in order to verify a possible correlation with MSGs histological patterns and clinical parameters.MethodsPaired samples of PB mononuclear cells and MSGs from pSS patients were evaluated at the diagnosis by flow cytometry and immunofluorescence staining respectively. Histological analysis to identify histological scores, B/T cell segregation and the presence of germinal center (GC)-like structures was also performed.ResultsIn early stages of pSS, circulating DN T cells appear to be not yet expanded and inversely correlated with circulating CD4⁺Th17 cells. The number of infiltrating DN T cells were associated with extent of glandular involvement, presence of GC-like structures and dryness symptoms and were inversely correlated with circulating DN T cells.ConclusionsOur findings suggest that DN T cells are actively involved in the pathogenic mechanisms leading to glandular dysfunction and damage in pSS and may play a role in ectopic lymphoneogenesis development occurring during the disease.     

Regulation of Cl− secretion by AMPK in vivo

Previous in vitro studies suggested that Cl(-) currents produced by the cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7) are inhibited by the alpha1 isoform of the adenosine monophosphate (AMP)-stimulated kinase (AMPK). AMPK is a serine/threonine kinase that is activated during metabolic stress. It has been proposed as a potential mediator for transport-metabolism coupling in epithelial tissues. All previous studies have been performed in vitro and thus little is known about the regulation of Cl(-) secretion by AMPK in vivo. Using AMPKalpha1(-/-) mice and wild-type littermates, we demonstrate that phenformin, an activator of AMPK, strongly inhibits cAMP-activated Cl(-) secretion in mouse airways and colon, when examined in ex vivo in Ussing chamber recordings. However, phenformin was equally effective in AMPKalpha1(-/-) and wild-type animals, suggesting additional AMPK-independent action of phenformin. Phenformin inhibited CFTR Cl(-) conductance in basolaterally permeabilized colonic epithelium from AMPKalpha1(+/+) but not AMPKalpha1(-/-) mice. The inhibitor of AMPK compound C enhanced CFTR-mediated Cl(-) secretion in epithelial tissues of AMPKalpha1(-/-) mice, but not in wild-type littermates. There was no effect on Ca(2+)-mediated Cl(-) secretion, activated by adenosine triphosphate or carbachol. Moreover CFTR-dependent Cl(-) secretion was enhanced in the colon of AMPKalpha1(-/-) mice, as indicated in Ussing chamber ex vivo and rectal PD measurements in vivo. Taken together, these data suggest that epithelial Cl(-) secretion mediated by CFTR is controlled by AMPK in vivo.     

Analysis of the inflammatory response in HY-TCR transgenic mice highlights the pathogenic potential of CD4− CD8− T cells

Transgenic mice expressing a rearranged T cell receptor (TCR)-αβ prematurely at the double-negative stage develop an abnormal population of peripheral T cells that lack CD4 and CD8 expression and are hyper-reactive to anti-TCR antibody stimulation. One such example is the HY-TCR transgenic mice. These mice express a TCR transgenic specific for the HY antigen that is expressed in male but not in female mice. As a result, male mice have an abnormal population of HY⁺/CD4^? 8^? or HY⁺/CD4^? CD8^low T cells that are much lower in female mice. In this study, we investigated the potential patho/physiological function of these cells in vivo using a model of delayed-type hypersensitivity (DTH) reaction: the λ-carrageenan-induced paw edema. Interestingly, while both male and female HY-TCR mice develop a classical biphasic inflammatory response to λ-carrageenan, the degree of inflammation in the former was much higher than that in the latter. This was accompanied by a selective expansion of HY⁺/CD4^? 8^? and HY⁺/CD4^? CD8^low T cells in male mice and by a markedly increased production of typical DTH cytokines compared with cells from female mice. These results were specific since analysis of the inflammatory response of HY-TCR transgenic mice subjected to zymosan-induced peritonitis showed no differences between male and female mice. Together, these findings provide novel evidence for the pathological role of self-reactive CD4^? CD8^? T cells, previously described in several autoimmune strains and recently identified in patients suffering from autoimmune diseases such as systemic lupus erythematosus.     

Blockers of volume-activated Cl− currents inhibit endothelial cell proliferation

Volume-activated Cl^– currents (I_Cl,vol) and cell growth have been measured in cultured endothelial cells from bovine pulmonary artery (CPAE) in the absence and presence of compounds which block these currents. The anti-oestrogen drug tamoxifen, which efficiently arrests the growth of breast cancer cells (l), inhibits both I_Cl,vol and cell proliferation with IC₅₀ of 3.8 and 4.8 mol/l respectively. NPPB and quinine, which also block I_Cl,vol, inhibit the growth of CPAE cells as well. Current and cell growth were closely correlated under all these conditions. We conclude that I_Cl,vol might be involved in the control of endothelial cell growth and thus might be important for the modulation of vascularisation and vascular remodelling.     

Temperature Dependence of H+ Transport Across Erythrocyte Membrane of Rana temporaria Grass Frog in Media Containing Cl− and SO 4 2−

H⁺ transport across the erythrocyte membrane was studied in Rana temporaria grass frog. The temperature coefficients and activation energy of H⁺ transport were calculated in media containing Cl⁻ and SO ₄ ²⁻ . Our results show that kinetic characteristics of H⁺ transport depend on function of band 3 protein in the erythrocyte membrane. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 140, No. 10, pp. 371–373, October, 2005     

Ca2+-activated Cl− currents are dispensable for olfaction

Canonical olfactory signal transduction involves the activation of cyclic AMP-activated cation channels that depolarize the cilia of receptor neurons and raise intracellular calcium. Calcium then activates Cl(-) currents that may be up to tenfold larger than cation currents and are believed to powerfully amplify the response. We identified Anoctamin2 (Ano2, also known as TMEM16B) as the ciliary Ca(2+)-activated Cl(-) channel of olfactory receptor neurons. Ano2 is expressed in the main olfactory epithelium (MOE) and in the vomeronasal organ (VNO), which also expresses the related Ano1 channel. Disruption of Ano2 in mice virtually abolished Ca(2+)-activated Cl(-) currents in the MOE and VNO. Ano2 disruption reduced fluid-phase electro-olfactogram responses by only ～40%, did not change air-phase electro-olfactograms and did not reduce performance in olfactory behavioral tasks. In contrast with the current view, cyclic nucleotide-gated cation channels do not need a boost by Cl(-) channels to achieve near-physiological levels of olfaction.     

Mitochondrial membrane dynamics are altered in cluA − mutants of Dictyostelium

In cluA ^– mutants of Dictyostelium, mitochondria are clustered near the cell center rather than being dispersed throughout the cytoplasm. We have examined two possible mechanisms that could account for this phenotype. First, we sought evidence that the cytoskeleton or a presumptive mitochondrion–cytoskeleton linkage was altered in mutant cells. We found that cytoskeletal structures in cluA ^– cells appeared normal by immunostaining, and that the distribution of peroxisomes in mutant cells was indistinguishable from that in wild type cells. Treatment of wild type cells with drugs that disrupted microtubules or actin filaments did not mimic the cluA ^– phenotype. Thus, cytoskeletal defects seemed unlikely to account for the mitochondrial clustering in cluA ^– cells. Observation of the movement of GFP-tagged mitochondria in wild type cells suggested that mitochondria are transported along microtubules, as in mammalian cells, rather than along actin filaments, as in budding yeast. Therefore, the similar phenotypes of cluA ^– Dictyostelium cells and clu1 yeast cells argued against CluA/Clu1p acting as a mitochondrion–cytoskeleton linker. We next examined the ultrastructure of mitochondria in freeze-substituted, thin-sectioned cells. We found that the clustered mitochondria in cluA ^– cells are interconnected. Often, adjacent mitochondria are linked by narrow membranous strands, although sometimes the mitochondria are partially merged. The presence of narrow constrictions at presumptive division sites argues that the constriction step of division proceeds normally. Our data suggest that cluA ^– cells may be blocked at a very late step in fission of the outer mitochondrial membrane.