Aminophylline increases ventilation and diaphragm contractility in awake canines

Traditional theophylline bronchodilators are still used clinically, especially in COPD. However, the effect of theophyllines on ventilation and respiratory muscles remains uncertain and these effects have not been measured directly in any awake, intact mammal. We hypothesized that aminophylline in the usual therapeutic dosage range, would elicit in the awake mammal, a significant increase in ventilation, and a significant increase in costal diaphragm shortening and contractility as recorded directly from the muscle. Therefore, we studied 13 awake canines, which had been chronically implanted with fine-wire EMG electrodes and sonomicrometer crystals in the costal segment of the diaphragm. Ventilatory parameters, moving average muscle EMG activity and muscle length and shortening, were measured at baseline and with aminophylline, during resting and hypercapnic stimulated breathing. Experiments were carried out prior to administration of aminophylline (baseline), and 1.5 h after loading and ongoing infusion with aminophylline. Minute ventilation, tidal volume and respiratory frequency all increased significantly with aminophylline, both during resting breathing and at equivalent levels of hypercapnic stimulated breathing. Costal diaphragm baseline muscle length was entirely unchanged with aminophylline. Costal diaphragm shortening increased significantly with aminophylline while corresponding costal diaphragm EMG activity remained constant, consistent with increased diaphragm contractility. Thus, in awake, intact mammals, aminophylline in usual therapeutic dosage elicits increased ventilation and increased contractility of respiratory muscles.     

Effect of stimulation of the medial and lateral zones of the respiratory center on electrical activity of the diaphragm,intercostal muscles,and phrenic neurons

Experiments on cats showed that the nucleus of the tractus solitarius contains zones during stimulation of which electrical activity of the phrenic neurons and diaphragm is selectively stimulated or inhibited. Stimulation of inspiratory and expiratory zones of the nucleus ambiguus influences the electrical activity of the external intercostal muscles. During stimulation of the corresponding zones in the gigantocellular nucleus electrical activity is changed in both groups of inspiratory muscles simultaneously. It is postulated that the action of stimulation of the zones of the gigantocellular nucleus on both groups of inspiratory muscles is indirect in its mechanism, through neurons of the nucleus of the tractus solitarius and nucleus ambiguus.Academy of Medical Sciences of the USSR Group, Kuibyshev Medical Institute. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 4, pp. 392–395, April, 1978.     

Classification of β-adrenoceptors in the microcirculation of skeletal muscle

Nervous and humoral β-adrenergic, postjunctional effects on microvascular resistance, on precapillary sphincter tone, and on transcapillary fluid exchange in cat skeletal muscle (Lundvall & Järhult 1974, 1976 a, Lundvall & Hillman 1978 a, b) were evaluated with regard to the β₁-or β₂,-specificity of the adrenoceptors. Marked β₂-dilator responses but no significant β₁-effects were observed. The conclusion was therefore reached that neurogenic as well as humoral β-adrenergic control of the microcirculation in skeletal muscle is exerted via activation of β₂-adrenoceptors.     

Effect of olfactory bulb pathology on olfactory function in normal aging

Decline of olfactory function is frequently observed in aging and is an early symptom of neurodegenerative diseases. As the olfactory bulb (OB) is one of the first regions involved by pathology and may represent an early disease stage, we specifically aimed to evaluate the contribution of OB pathology to olfactory decline in cognitively normal aged individuals without parkinsonism or dementia. This clinicopathological study correlates OB tau, amyloid β (Aβ) and α‐synuclein (αSyn) pathology densities and whole brain pathology load to olfactory identification function as measured with the University of Pennsylvania Smell Identification Test (UPSIT) and clinical data measured proximate to death in a large autopsy study including 138 cases considered non‐demented controls during life. Tau pathology was frequently observed in the OB (95% of cases), while both Aβ (27% of cases) and αSyn (20% of cases) OB pathologies were less commonly observed. A weak correlation was only observed between OB tau and olfactory performance, but when controlled for age, neither OB tau, Aβ or αSyn significantly predict olfactory performance. Moreover, whole brain tau and αSyn pathology loads predicted olfactory performance; however, only αSyn pathology loads survived age correction. In conclusion, OB tau pathology is frequently observed in normally aging individuals and increases with age but does not appear to independently contribute to age‐related olfactory impairment suggesting that further involvement of the brain seems necessary to contribute to age‐related olfactory decline.     

Influence of angiotensin II, α- and β-adrenoceptors on peripheral noradrenergic neurotransmission in canine gracilis muscle in vivo

Interactions between angiotensin II and adrenoceptor-mediated effects on peripheral sympathetic neurotransmission were investigated in constant flow blood-perfused canine gracilis muscle in situ, without and with pretreatment by non-competitive α- adrenoceptor blockade. Angiotensin converting enzyme (ACE)-inhibition by benazeprilat increased nerve stimulation (2 Hz, 4 min)-evoked noradrenaline (NA) overflow (+21 ± 5 yo) with α- adrenoceptors intact, but reduced NA overflow (– 18 ± 6%) when α-adrenoceptors were blocked. Vasoconstrictor responses were slightly reduced by benazeprilat. Subsequent infusion of angiotensin II (Ang 11, 20 and 500 ng kg_-1 min_-1 i.v., raising arterial concentrations from 0.6 ± 0.2 PM to 1390 ± 240 and 25 110 ± 3980 PM, respectively) failed to increase NA overflow or to enhance stimulation-evoked vasoconstriction. Adrenaline (0.4 nmol kg_-1 min_-1 i.v.) did not change evoked NA overflow before or after benazeprilat, either with or without α-adrenoceptor blockade, despite high concentrations (± 10 nM) in arterial plasma. Following benazeprilat, propranolol reduced NA overflow (–24 ± 3 yo) only if the α-adrenoceptors were blocked. In conclusion, benazeprilat reduced evoked NA overflow in the presence of α- adrenoceptor blockade to a similar degree as previously shown in the presence of neuronal uptake inhibition in this model. However, contrasting to our previous findings, benazeprilat enhanced NA overflow and reduced the post-junctional response to nerve stimulation in the absence of α-adrenoceptor blockade. This could be related to bradykinin accumulation during ACE-inhibition, in addition to the reduction of Ang II generation. Our data are not compatible with facilitation of NA release by circulating Ang II even at pharmacological dose levels. Although activation of prejunctional β-adrenoceptors may facilitate evoked NA overflow in this model, circulating adrenaline is ineffective under physiological conditions even after α-adrenoceptor blockade. Also, β-adrenoceptor-mediated prejunctional effects do not seem to involve Ang II in canine skeletal muscle in vivo.     

The effect of increased respiratory resistance on glycogen and triglyceride levels in the respiratory muscles of the rat

Summary The effect of increased respiratory resistance (stenosis of the trachea) on glycogen and triglyceride levels in the diaphragm (D) and intercostal (external-IE, internal-II) muscles was studied in the rat. Tracheal stenosis resulted in a reduction of glycogen level in the muscles. For the fed rats the reductions were: D-45 and 79%, IE-14 and 30%, II-14 and 35%, 0.5 and 3 h after stenosis, respectively. For rats fasted for 24 h the reductions were: D-64 and 86%, IE-33 and 71%, II-40 and 82%, after 0.5 and 3 h respectively. The level of triglycerides in the muscles was stable during stenosis in the fed group, whereas in the fasted group it were reduced in the diaphragm by 50% after 0.5 h, and by 52% after 3 h. It is concluded that both endogenous and blood-born energy fuels are utilized by the respiratory muscles during increased resistance breathing. The work was supported by Polish Academy of Sciences (10.4.)     

Deciphering TGF‐β3 function in medial edge epithelium specification and fusion during mouse secondary palate development

Background: Transforming growth factor‐β3 (TGF‐β3) plays a central role in mediating secondary palate fusion along the facial midline. However, the mechanisms by which TGF‐β3 functions during secondary palate fusion are still poorly understood. Results : We found that mouse cytokeratin 6α and 17 mRNAs were expressed exclusively in the palate medial edge epithelium on embryonic day 14.5, and this expression was completely abolished in Tgf‐β3 mutant embryos. In contrast, we found that Jagged2 was initially expressed throughout the palate epithelium, but was specifically down‐regulated in the medial edge epithelium during palatal fusion. Jagged2 down‐regulation was regulated by TGF‐β3, since Jagged2 was persistently expressed in palatal medial edge epithelium in Tgf‐β3 null mutant embryos. Moreover, addition of DAPT, a specific inhibitor of Notch signaling, partially rescued the fusion defects in Tgf‐β3 null mutant palatal shelves. Conclusions : Based on these results, together with the previous study indicating that the loss of Jagged2 function promotes embryonic oral epithelial fusion, we concluded that TGF‐β3 mediates palate fusion in part by down‐regulating Jagged2 expression in palatal medial edge epithelium. In addition, cytokeratin 6α and 17 are two TGF‐β3 downstream target genes in palate medial edge epithelium differentiation. Developmental Dynamics 243:1536–1543, 2014. © 2014 Wiley Periodicals, Inc.     

Expression of pro-inflammatory cytokines by TCRαβ+ T and TCRγδ+ T cells in an experimental model of colitis

An inflammatory bowel disease (IBD) comparable to human ulcerative colitis is induced upon transfer of T cell-depleted wild-type (F1) bone marrow into syngeneic T cell-deficient (tgε26) mice (F1 → tgε26). Previously we have shown that activated CD4⁺ T cells predominate in transplanted tgε26 mice, and adoptive transfer experiments verified the potential of these cells to cause disease in immunodeficient recipient mice. Using flow cytometry for the detection of intracellular cytokine expression, we demonstrate in the present study that large numbers of CD4⁺ and CD8⁺ TCRαβ⁺ T cells from the intraepithelial region and lamina propria of the colon of diseased, but not from disease-free mice, produced interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Large numbers of T cells from peripheral lymphoid tissues of these animals also expressed IFN-α and TNF-α, but few expressed interleukin-4, demonstrating g strong bias towards Th1-type T cell responses in these animals. TCRγδ⁺ T cells, typically minor constituents of the inflammatory infiltrate of the colon in F1 → tgε26 mice, also expressed IFN-γ at a high frequency upon CD3 stimulation. In light of these findings we examined the potential involvement of TCRγδ⁺ T cells by testing their ability to induce colitis in tgε26 mice. We report here that tgε26 mice transplanted with T cell-depleted bone marrow from TCRα^null and TCRβ^null animals developed IBD. Furthermore, disease in these mice correlated with the development of peripheral and colonic TCRαδ⁺ T cells capable of IFN-γ production. These results suggest that IFN-γ may be a common mediator of IBD utilized by pathogenic T cells of distinct phenotype.     

αIIbβ3 integrin: new allelic variants in Glanzmann thrombasthenia,effects on ITGA2B and ITGB3 mRNA splicing,expression, and structure–function

Glanzmann thrombasthenia (GT) is an autosomal recessive inherited bleeding disorder characterized by an impaired platelet aggregation due to defects in integrin αIIbβ3 (ITGA2B, ITGB3), a fibrinogen receptor. Mutations from 24 GT patients and two carriers of various origins, Caucasian, North‐African and Asian were characterized. Promoter and exon sequences of αIIb and β3 genes were amplified and directly sequenced. Among 29 identified mutations, 17 new allelic variants resulting from nonsense, missense and deletion/insertion mutations were described. RNA alterations were evaluated by using Web servers. The αIIb p.S926L, p.V903F, and β3 p.C38Y, p.M118R, p.G221D substitutions prevented complex expression at the surface of COS‐7 cells by altering the αIIb or the β3 subunit structure. As shown by free energy analyses applied on the resolved structure of αIIbβ3 and structural modeling of the mutant, the p.K253M substitution of β3 helped to define a key role of the K253 in the interaction of the αIIb β‐propeller and the β3 β‐I domains. finally, the αIIb p.Q595H substitution allowed cell surface expression of the complex but its corresponding c.2800G>T mutation is predicted to alter normal RNA splicing. In conclusion, our study yielded the discovery of 17 new GT allelic variants, revealed the key role of K253 of αIIb for the αIIbβ3 complex formation and provides an additional example of an apparently missense mutation causing a splicing defect. Hum Mutat 30:1–10, 2010. © 2010 Wiley‐Liss, Inc.     

αβ Lineage-committed thymocytes can be rescued by the γδ T cell receptor (TCR) in the absence of TCR β chain

Commitment of the αβ and γδ T cell lineages within the thymus has been studied in T cell receptor (TCR)-transgenic and TCR mutant murine strains. TCRγδ-transgenic or TCRβ knockout mice, both of which are unable to generate TCRαβ-positive T cells, develop phenotypically αβ-like thymocytes in significant proportions. We provide evidence that in the absence of functional TCRβ protein, the γδTCR can promote the development of αβ-like thymocytes, which, however, do not expand significantly and do not mature into γδ T cells. These results show that commitment to the αβ lineage can be determined independently of the isotype of the TCR, and suggest that αβ versus γδ T cell lineage commitment is principally regulated by mechanisms distinct from TCR-mediated selection. To accommodate our data and those reported previously on the effect of TCRγ and δ gene rearrangements on αβ T cell development, we propose a model in which lineage commitment occurs independently of TCR gene rearrangement.     

Autoradiographic location of β-adrenoceptor subtypes in cat colon smooth muscle

In order to localize β-adrenoceptors ¹²⁵I-(—)pindolol (IPIN) was used in binding to sections from cat colon. The binding characteristics for IPIN to β-adrenoceptors on colon sections were estimated by demonstrating reversible binding in the presence of isoprenaline and by steroselective binding to the isomers of propranolol. The binding of IPIN to both β₁-and β₂-adrenoceptors was shown by biphasic displacement curves in the presence of the selective β-adrenoceptor compounds betaxolol, ICI 118.551 and procaterol. The colon sections were found to contain proportions of β₁-adrenoceptors (30–50%) and β₂-adrenoceptors (50–70%). In the autoradiographic studies, 100% of the developed grains after exposure of IPIN to the photographic emulsion were displaced by 50 μm of isoprenaline. By microscopic counting at autoradiographic grains, 30–40% of the grains were found in the circular smooth muscle, while 60–70% of the grains were found in the longitudinal smooth muscle. A concentration of 2 nm ICI 118.551 completely displaced all grains in the circular smooth muscle and partly displaced those found in the longitudinal smooth muscle. A high concentration of ICI 118.551 (1 μm ) displaced all grains above background from the smooth muscle. It is concluded that the circular smooth muscle only contains β₂-adrenoceptors, while longitudinal smooth muscle may contain a proportion of β₁-adrenoceptors. Whether such a location of β-adrenoceptors can be related to the β₁-adrenoceptor-mediated inhibition of colon motility can not be clarified from these studies. However, it seems that β₁-adrenoceptors are located to the longitudinal smooth muscle instead of to the myenteric plexus of the colon.     

Metabolism of murine TH17 cells: Impact on cell fate and function

An effective adaptive immune response relies on the ability of lymphocytes to rapidly act upon a variety of insults. In T lymphocytes, this response includes cell growth, clonal expansion, differentiation, and cytokine production, all of which place a significant energy burden on the cell. Recent evidence shows that T‐cell metabolic reprogramming is an essential component of the adaptive immune response and specific metabolic pathways dictate T‐cell fate decisions, including the development of T_H17 versus T regulatory (Treg) cells. T_H17 cells have garnered significant attention due to their roles in the pathology of immune‐mediated inflammatory diseases. Attempts to characterize T_H17 cells have demonstrated that they are highly dynamic, adjusting their function to environmental cues, which dictate their metabolic program. In this review, we highlight recent data demonstrating the impact of cellular metabolism on the T_H17/Treg balance and present factors that mediate T_H17‐cell metabolism. Some examples of these include the differential impact of the mTOR signaling complexes on T‐helper‐cell differentiation, hypoxia inducible factor 1 alpha (HIF1α) promotion of glycolysis to favor T_H17‐cell development, and ACC1‐dependent de novo fatty acid synthesis favoring T_H17‐cell development over Treg cells. Finally, we discuss the potential therapeutic options and the implications of modulating T_H17‐cell metabolism for the treatment of T_H17‐mediated diseases.     

β2-adrenergic control of plasma volume in hemorrhage

Hemorrhage is associated with absorption of extravascular fluid from skeletal muscle to blood in order to compensate for the loss of intravascular volume. Our previous studies have shown that this fluid gain is mainly linked to β-adrenergic microvascular adjustments leading to decrease in capillary hydrostatic pressure and to precapillary ‘sphincter’ mediated increase in the capillary surface area available for fluid exchange. In the present study the importance of β-adrenergic control of plasma volume in bleeding was confirmed by measurement of changes in plasma volume after graded hemorrhage in animals with intact and blocked vascular β₂-adrenoceptors (i. v. administration of the ‘selective’β₂-blocking agent ICI 118, 551). With intact β₂-adrenoceptors plasma volume was gradually restored after bleeding so that about 50% of the shed plasma volume (about 35% of the shed blood volume) had been compensated for at two hours after exsanguination of 20% as well as 40% of the blood volume. The corresponding figures in animals with blocked β₂-adrenoceptors were only 14% of the shed plasma volume and 8% of the shed blood volume at both degrees of hemorrhage.     

CD3 expression distinguishes two γδT cell receptor subsets with different phenotype and effector function in tuberculous pleurisy

Tuberculous pleurisy is a naturally occurring site of Mycobacterium tuberculosis (Mtb) infection. Herein, we describe the expression of activation, natural killer (NK) and cell migration markers, as well as effector functions from γδT cells in peripheral blood (PB) and pleural effusion (PE) from tuberculosis patients (TB). We observed a decreased percentage of circulating γδT from TB patients and differential expression of NK as well as of chemokine receptors on PB and PE. Two subsets of γδT cells were differentiated by the CD3/γδT cell receptor (γδTCR) complex. The γδTCR^low subset had a higher CD3 to TCR ratio and was enriched in Vδ2⁺ cells, whereas most Vδ1⁺ cells belonged to the γδTCR^high subset. In PB from TB, most γδTCR^high were CD45RA⁺CCR7^‐ and γδTCR^low were CD45RA^+/?CCR7⁺CXCR3⁺. In the pleural space the proportion of CD45RA^‐CCR7⁺CXCR3⁺ cells was higher. Neither spontaneous nor Mtb‐induced interferon (IFN)‐γ production was observed in PB‐γδT cells from TB; however, PE‐γδT cells showed a strong response. Both PB‐ and PE‐γδ T cells expressed surface CD107a upon stimulation with Mtb. Notably, PE‐γδTCR^low cells were the most potent effector cells. Thus, γδT cells from PB would acquire a further activated phenotype within the site of Mtb infection and exert full effector functions. As γδT cells produce IFN‐γ within the pleural space, they would be expected to play a beneficial role in tuberculous pleurisy by helping to maintain a T helper type 1 profile.     

Liver distribution of γδ‐T‐cells in patients with chronic hepatitis of different etiology

The γδ T cells represent a minor unique T‐cell subpopulation long been considered as innate‐like immune cells. They are found in increased numbers in tissues from various inflammatory conditions. Their role in chronic hepatitis, however, is still discussed controversially. Fresh frozen tissues from 50 patients (18 cases hepatitis B infection, 25 hepatitis C, three cases with co‐infection of hepatitis B and C and four patients with autoimmune hepatitis) were investigated. Immunohistochemistry with primary antibodies detecting αβ and γδ TCR was used to evaluate their incidence and distribution in the different histological structures of the liver. The inflammatory infiltrate in all cases of chronic hepatitis was dominated by αβ T cells and was mainly localized in the portal tracts with formation of an interface hepatitis (95.3%αβ T cells; 4.7%γδ T cells). There were neither significant differences between inflammatory infiltrate nor the amount or percentage of γδ T cells between hepatitis B, C or autoimmune hepatitis. No accumulation of γδ T cells could be observed in cases of chronic hepatitis of different etiologies. The immune‐mediated phenomena in chronic hepatitis are dominated by αβ T cells. Thus, the adapted immune system is responsible for the inflammatory processes in chronic hepatitis.     

Lack of intermediate-affinity interleukin-2 receptor in mice leads to dependence on interleukin-2 receptor α, β and γ chain expression for T cell growth

An interleukin (IL)-4 dependant mouse T cell clone 8.2 derived from an IL-2-dependent T cell line was characterized. As measured by flow cytometric analysis and Northern blotting, it expresses IL-2 receptor β (IL-2Rβ) and γ (IL-2Rγ) chains, but has lost expression of IL-2 receptor α chain (IL-2Rα). To investigate the properties of the mouse IL-2Rβγ complex and the role of IL-2Rα gene expression, this clone was further studied. T cell clone 8.2 has lost the capacity to bind ¹²⁵I-labeled human IL-2 under experimental conditions able to detect intermediate-affinity IL-2R in human cells. Mouse IL-2 is unable to block the binding of mAb TMβ1 to 8.2 cells. Under the same experimental conditions, mouse IL-2 blocks the binding of TMβ1 to C30-1 cells expressing the IL-2αβγ complex. Since TMβ1 recognizes an epitope related to the IL-2 binding site of IL-2Rβ, these results can be taken as a demonstration that mouse IL-2Rβγ does not bind mouse IL-2. Furthermore, T cell clone 8.2 does not proliferate in response to recombinant mouse or human IL-2. On the other hand, T cell transfectant lines expressing heterospecific receptors made of the human IL-2Rβ and mouse IL-2Rγ chains bind ¹²⁵I-labeled human IL-2 and proliferate in response to IL-2. This establishes the difference between mouse and human IL-2Rβ chains. Transfection of T cell clone 8.2 with human IL-2Rα genes restores their capacity to proliferate in response to IL-2. In addition, all transfectants grown in IL-2 express the endogeneous mouse IL-2Rα chain. When grown in IL-4, the endogeneous mouse IL-2Rα gene remains silent in all these transfectants. These results show that, contrary to the human, the mouse does not express an intermediate-affinity IL-2R. Expression of the IL-2Rα gene is therefore required for the formation of the functional IL-2R in mice.