Sorghum stem extract modulates Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase,ecto-5′-nucleotidase,and acetylcholinesterase activities

Sorghum stem (Sorghum bicolor) has been in use in traditional medicine systems for the management of neurodegenerative conditions. However, there is dearth of information on the scientific basis for its use in the treatment of such conditions. This study sought to assess the antioxidant activity and effects of phenolic extract from sorghum stem (Sorghum bicolor) on some cholinergic (acetylcholinesterase (AChE)) and purinergic (Na⁺/K⁺-ATPase and ecto-5′-nucleotidase) enzymes associated with neurological conditions. Phenolic-rich extract was prepared using methanol: 1 N HCl (1:1, v/v) mixture and characterized using high-performance liquid chromatography-diode array detector (HPLC-DAD). In vitro tests were used to investigate the effects of the phenolic extract on AChE, Na⁺/K⁺-ATPase, and ecto-5′-nucleotidase activities. Furthermore, the hydroxyl (OH) radical scavenging and Fe²⁺-chelating abilities of the extract were investigated. HPLC-DAD analysis revealed the presence of some phenolic acids such as caffeic acid (120.58 mg/g), ferulic acid (76.45 mg/g), gallic acid (17.48 mg/g), chlorogenic acid (16.25 mg/g), and flavonoids such as kaempferol (15.98 mg/g), rutin (51.07 mg/g), quercetin (263.16 mg/g), and quercitrin (89.21 mg/g) in the phenolic extract. The extract significantly inhibited AChE and ecto-5′-nucleotidase activities in a dose dependent manner with IC₅₀?=?24.88 μg/ml and IC₅₀?=?37.49 μg/ml, respectively, and increased Na⁺/K⁺-ATPase activity in a dose dependent manner. Furthermore, the phenolic extract also scavenged OH radicals and was able to chelate Fe²⁺ in a dose dependent manner with IC₅₀?=?54.27 μg/ml and 18.47 μg/ml, respectively. This study revealed the antioxidant and modulatory effects of phenolic extracts from sorghum stem on some cholinergic and purinergic enzymes.     

Adenosine Triphosphoric Acid as a Factor of Nervous Regulation of Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>/2Cl<Superscript>−</Superscript> Cotransport in Rat Skeletal Muscle Fibers

Exogenous adenosine triphosphoric acid produces a biphasic effect on the resting membrane potential of muscle fibers in rat diaphragm. Depolarization of the sarcolemma observed 10 min after application of adenosine triphosphoric acid results from activation of Na⁺/K⁺/2Cl⁻ cotransport. The increase in chloride cotransport is related to activation of postsynaptic P2Y receptors and protein kinase C. Repolarization of the membrane develops 40 min after treatment with adenosine triphosphoric acid and after 50 min the resting membrane potential almost returns the control level. This increase in the resting membrane potential of the sarcolemma is probably associated with activation of the Na⁺/K⁺ pump and increase in membrane permeability for chlorine ions in response to longterm activity of Cl⁻ cotransport. Thus, adenosine triphosphoric acid co-secreted with acetylcholine in the neuromuscular synapse probably plays a role in the regulation resting membrane potential and cell volume of muscle fibers.     

Maternal–neonatal erythrocyte membrane Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase and Mg<Superscript>2+</Superscript>-ATPase activities in relation to the mode of delivery

Free radical production and high catecholamine levels are implicated in the modulation of Na(+), K(+)-ATPase, and Mg(2+)-ATPase activities. The aim of this study was to investigate the effect of the mode of delivery on the above-mentioned enzyme activities in maternal-neonatal erythrocyte membrane. Women with normal pregnancy (N = 30) were divided into two groups: Group A (N = 16) with normal labor and vaginal delivery, and Group B (N = 14) with scheduled cesarean section; 20 non-pregnant women were the controls. Blood was obtained from controls and mothers, pre- versus post-delivery, and from the umbilical cord (CB). Total antioxidant status (TAS), membrane enzyme activities, and catecholamine blood levels were measured with a commercial kit, spectrophotometrically, and by HPLC methods, respectively. The results showed that: TAS levels, catecholamine, and the membrane enzyme activities were similar in the two groups of mothers pre-delivery, whereas both enzyme activities were lower than those of controls. TAS levels were reduced whereas Na(+), K(+)-ATPase activities (0.35 +/- 0.03 vs. 0.65 +/- 0.06 micromol Pi/h x mg protein, P < 0.001), and catecholamine levels were increased post-delivery in mothers of Group A and unaltered in Group B (0.38 +/- 0.02 vs. 0.40 +/- 0.03 micromol Pi/h x mg protein, P > 0.05), at the same times of study. Mg(2+)-ATPase activities remained unaltered in both groups of mothers and newborns. Na(+), K(+)-ATPase activity was similarly lower in the CB of neonates than those of their mothers, pre-delivery. Our results suggest that: (a) during a normal vaginal delivery process, the low TAS and the increased levels of catecholamines may increase Na(+), K(+)-ATPase activity, post-delivery; (b) the low enzyme activities evaluated in mothers pre-delivery may be due to the high estrogen levels and those in newborns due to perinatal immaturity.     

CD3<Superscript>+</Superscript>ICOS<Superscript>+</Superscript> T cells show differences in the synthesis of nitric oxide,IFN-γ, and IL-10 in patients with pulmonary tuberculosis or in healthy household contacts

Evidence indicates that more than 90 % of infected individuals never develop active tuberculosis. This fact highlights the relevance of the immune response in tuberculosis control. The inducible co-stimulator (ICOS) is a regulator of the function, differentiation, proliferation, and activation of T cells. Moreover, T cells synthesise nitric oxide (NO), interferon gamma (IFN-γ), and interleukin (IL)-10, which help regulate the immune response to tuberculosis. Therefore, we assessed the synthesis of NO, IFN-γ, and IL-10 in CD3⁺ICOS⁺ T cells from healthy individuals, household contacts (HHC), and patients with active pulmonary tuberculosis (PTB), previously stimulated with the antigen H37Rv. Our results indicated a significant increase in both the percentage of ICOS⁺ cells and CD3⁺ICOS⁺ T cells producing NO, IFN-γ, and IL-10 in cells obtained from patients with PTB (p < 0.01). In addition, a high mitochondrial membrane potential (ΔΨ _m) in CD3⁺ICOS⁺ T cells was observed in the cells from HHC and from PTB patients, and is associated with the activation of T cells. In conclusion, results show that the CD3⁺ICOS⁺ T cells obtained from PTB patients are the main producers of NO, IFN-γ, and IL-10. In addition, our results imply that NO is a modulator of ICOS expression of T cells from PTB patients.     

T<Superscript>+</Superscript> NK<Superscript>+</Superscript> IL-2 Receptor γ Chain Mutation: a Challenging Diagnosis of Atypical Severe Combined Immunodeficiency

Purpose

All reported patients with hypomorphic X-linked severe combined immunodeficiency (X-SCID) due to c.664C>T (p.R222C) mutations in the gene (IL2RG) encoding the common γ chain (γc) have presented with opportunistic infections within the first year of life, despite the presence of nearly normal NK and T cell numbers. Reporting five children of one extended family with hemizygous mutations in IL2RG, we explore potential diagnostic clues and extend our comprehension of the functional impact of this mutation.

Methods

Whole exome sequencing (WES); detailed immune phenotyping; cytokine-induced STAT phosphorylation; B, T, and NK cell activation; and quantification of sjTRECs in five Arab children with c.664C>T (p.R222C) IL2RG mutation.

Results

The mean age at clinical presentation with respiratory tract infection or diarrhea was 6.8 (range: 2–12) months. None of the children presented with opportunistic infections. Diagnostic clues were early onset in the first year of life, and a suggestive family history associated with reduced naïve CD4 T cells and absent switched memory B cells. Number and phenotype of NK cells and innate-like lymphocytes were normal. The diagnosis was made by WES and corroborated by absent STAT phosphorylation and reduced functional response after IL-2 and IL-21 stimulation. Four patients underwent successful hematopoietic stem cell transplantation.

Conclusions

As early diagnosis and treatment are important, a high index of suspicion in the diagnosis of c.664C>T (p.R222C) X-SCID is needed. This requires prompt genetic testing by next generation sequencing in order to avoid unnecessary delays in the definite diagnosis since immunological work up may not be discriminating. Assays directly testing cytokine signaling or cytokine-dependent functions are helpful in confirming the functional impact of the identified hypomorphic variants.

     

Use of a <Emphasis Type="Italic">ura5</Emphasis><Superscript>+</Superscript>–<Emphasis Type="Italic">lys7</Emphasis><Superscript>+</Superscript> cassette to construct unmarked gene knock-ins in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

While the counterselectable Schizosaccharomyces pombe ura4 ⁺ gene can be used to prepare a site in the S. pombe genome to receive an unmarked mutant allele (loss of ura4 ⁺ confers 5FOA-resistant (5FOA^R) growth), the desired unmarked knock-in strains are generally outnumbered by spontaneously arising 5FOA^R mutants. Relative to the same approach using the homologous URA3 ⁺ gene in Saccharomyces cerevisiae, knock-ins in S. pombe are harder to identify due to a lower efficiency of homologous recombination and a relatively high background of spontaneous 5FOA^R colonies. To develop an improved method for identifying cells receiving unmarked mutant alleles, we first determined that 5FOA^R strains carry mutations in either of two genes; ura4 ⁺ and ura5 ⁺. We then cloned the S. pombe ura5 ⁺ orotate phosphoribosyltransferase gene and constructed a 2.1 kb cassette containing ura5 ⁺ together with the S. pombe lys7 ⁺ gene. Using this doubly marked cassette to disrupt the sck1 ⁺ kinase gene, we can distinguish between strains created by homologous knock-in of unmarked wild-type or kinase-dead alleles and spontaneously arising ura4 ⁻ and ura5 ⁻ mutants by screening 5FOA^R colonies for the loss of the lys7 ⁺ marker. The utility of this system, especially when the phenotype for the strain carrying the knock-in allele is indistinguishable from that of the disruption strain, is borne out by the fact that ~95% of 5FOA^R colonies in our studies arose from background ura4 ⁻ and ura5 ⁻ mutations.     

Increased O<Subscript>2</Subscript> consumption in excitation–contraction coupling in hypertrophied rat heart slices related to increased Na<Superscript>+</Superscript>–Ca<Superscript>2+</Superscript> exchange activity

The goal of our study was to evaluate the origin of the increased O₂ consumption in electrically stimulated left ventricular slices of isoproterenol-induced hypertrophied rat hearts with normal left ventricular pressure. O₂ consumption per minute (mVO₂) of mechanically unloaded left ventricular slices was measured in the absence and presence of 1-Hz field stimulation. Basal metabolic mVO₂, i.e., mVO₂ without electrical stimulation, was significantly smaller, but mVO₂ for the total Ca²⁺ handling in excitation–contraction coupling (E–C coupling mVO₂), i.e., delta mVO₂ (=mVO₂ with stimulation − mVO₂ without stimulation), was significantly larger in the hypertrophied heart. Furthermore, the fraction of E–C coupling mVO₂ was markedly altered in the hypertrophied heart. Namely, mVO₂ consumed by sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2) was depressed by 40%; mVO₂ consumed by the Na⁺/K⁺-ATPase (NKA)-Na⁺/Ca²⁺ exchange (NCX) coupling was increased by 100%. The depressed mVO₂ consumption by SERCA2 was supported by lower protein expressions of phosphorylated-Ser¹⁶ phospholamban and SERCA2. The increase in NKA–NCX coupling mVO₂ was supported by marked augmentation of NCX current. However, the increase in NCX current was not due to the increase in NCX1 protein expression, but was attributable to attenuation of the intrinsic inactivation mechanisms. The present results demonstrated that the altered origin of the increased E–C coupling mVO₂ in hypertrophy was derived from decreased SERCA2 activity (1ATP: 2Ca²⁺) and increased NCX activity coupled to NKA activity (1ATP: Ca²⁺). Taken together, we conclude that the energetically less efficient Ca²⁺ extrusion pathway evenly contributes to Ca²⁺ handling in E–C coupling in the present hypertrophy model.     

Altered Proportions of Naïve,Central Memory and Terminally Differentiated Central Memory Subsets among CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T Cells Expressing CD26 in Patients with Type 1 Diabetes

Type 1 diabetes is an autoimmune process predominantly T-cell mediated. CD26 plays a role in T-cell costimulation, migration, memory development, thymic maturation and emigration patterns. In peripheral blood from 55 patients with type 1 diabetes and 20 healthy controls, CD4⁺ and CD8⁺ T cells expressing CD26 were differentiated into naïve (N, CD45RA⁺CCR7⁺), central memory (CM, CD45RA^?CCR7⁺), effector memory (EM, CD45RA^?CCR7^?), and terminally differentiated effector memory (TEMRA, CD45RA⁺CCR7^?). In type 1 diabetes, CD4⁺ and CD8⁺ T cells expressing CD26 showed a distinctive differentiation profile: percentages and absolute numbers of CM and N cells were reduced, whereas those of TEMRA cells were markedly increased. The indices of intermediate- and long-term glycaemic control were associated negatively with the number of CM and N cells while positively with the number of TEMRA cells. The considerable accumulation of TEMRA T cells in our patients suggests life-long stimulation by protracted antigen exposure (viruses, other agents or residual self-antigens?) or a homeostatic defect in the regulation/contraction of immune responses.     

Effects of<Emphasis Type="Italic"> Eimeria separata</Emphasis> infections on Na<Superscript>+</Superscript> and Cl<Superscript>−</Superscript> transport in the rat large intestine

To study the pathophysiology of diarrhoea in coccidial infections, Na⁺ and Cl^– fluxes (J_Na, J_Cl), short circuit current (I_sc) and tissue conductance (g_t) were determined in stripped gut epithelia of Eimeria separata infected rats employing the Ussing chamber technique. E. separata invades enterocytes of the caecum and proximal colon. Na⁺ absorption was generally reduced in infected tissues, Cl^– absorption only in the caecum. I_sc values were increased in the caecum and reduced in the proximal colon. Tissue conductance was not affected. Values tended to normal with time after infection. Theophylline caused markedly increased I_sc and g_t values in the caecum epithelia of infected rats. In the epithelia of the distal colon, i.e. the non-infested part of the large intestine, g_t values remained unaffected but I_sc was fourfold increased. This I_sc increase was strongly sensitive to amiloride, suggesting a compensatory activation of Na⁺ channels in the distal colon of infected rats. Accordingly, serum levels of aldosterone, which activates Na⁺ channels in the distal colon, were increased to eightfold levels in infected animals. Thus compensatory Na⁺ absorption was under endocrine control.     

Inhibitory effects of the neurotensin<Superscript>8–13</Superscript> analogs Asp<Superscript>13</Superscript>-NT<Superscript>8–13</Superscript> and Asp<Superscript>12</Superscript>-NT<Superscript>8–13</Superscript> on mast cell secretion

Pretreatment of isolated mast cells with analogs of neurotensin 8–13 (NT^8–13), in which the amino acids Leu¹³ or Ile¹² are replaced with an aspartic acid (Asp¹³-NT^8–13 or Asp¹²-NT^8–13), inhibits the secretion of histamine in response to NT. A 10 min pretreatment with either analog (10 μM) inhibited NT-induced histamine release by 90% (Asp¹³-NT^8–13) or by 98% (Asp¹²-NT^8–13). At concentrations that are inhibitory, Asp¹³-NT^8–13 and Asp¹²-NT^8–13 alone elicit very little release (<5% at 10 μM). In the continued presence of the analogs, the inhibitory effect lasts for more than 45 min; removal of the analogs resulted in restoration of sensitivity to NT within 10 min. Pretreatment with analog Asp¹³-NT^8–13 resulted in a 39% inhibition of stimulation by substance P and a 52% inhibition of stimulation by histaminereleasing peptide (HRP). In contrast, pretreatment with analog Asp¹²-NT^8–13 gave no inhibition of release by SP or HRP. Neither analog inhibited histamine release in response to bradykinin (BK), NT^1–12, compound 48/80 (48/80), the calcium ionophore A23187, or anti-IgE stimulation of passively sensitized mast cells. Although Asp¹²-NT^8–13 and Asp¹³-NT^8–13 differ slightly in regard to the peptides they inhibit, both probably act at a step early in the stimulus-secretion coupling sequence; most likely before the rise in the level of free intracellular calcium that has been shown to accompany secretion in mast cells. It is suggested that these analogs exert their inhibitory effect on NT by competing with NT for a binding site on the mast cell membrane. The limited number of peptides inhibited by these analogs suggest that not all basic peptides act at the same site to stimulate secretion.     

In vitro binding evaluation of <Superscript>177</Superscript>Lu-AMBA,a novel <Superscript>177</Superscript>Lu-labeled GRP-R agonist for systemic radiotherapy in human tissues

Members of the gastrin-releasing peptide (GRP) family and its analogs bombesin (BBN) have been implicated in the biology of several human cancers including prostate, breast, colon and lung. To date, three mammalian GRP/BBN receptor subtypes have been cloned and characterized: the neuromedin B receptor (NMBR), the GRP receptor (GRPR) and the BBN-receptor subtype 3 (BB₃). The fourth BBN receptor subtype, BB₄, has only been identified in amphibian and at present no mammalian equivalent of this receptor has been described. GRPR analogs have been used as carriers to deliver drugs, radionuclides and cytotoxins to target various cancer types that are GRPR positive. We investigated the in vitro binding properties of ¹⁷⁷Lu-AMBA, a novel radiolabelled BBN analog currently undergoing clinical trial as systemic radiotherapy for hormone refractory prostate cancer (HRPC) patients. Pharmacological analyses of the ¹⁷⁷Lu-AMBA was determined using in vitro binding studies using membrane target system containing specific receptor subtypes. We investigated the distribution of binding sites for ¹⁷⁷Lu-AMBA by receptor autoradiography on human neoplastic and non-neoplastic tissues. Pharmacological characterizations of ¹⁷⁷Lu-AMBA shows, high affinity towards NMB and GRP receptors, while little or no affinity towards BB₃ receptor. Among the 40 different types of non-neoplastic tissues tested seven of them showed limited but specific binding of ¹⁷⁷Lu-AMBA. Fourteen of 17 primary prostate cancers, six of 13 primary breast cancers expressed binding sites for ¹⁷⁷Lu-AMBA. Furthermore, no apparent differences in ¹⁷⁷Lu-AMBA-binding sites expression were observed between matched pairs (primary vs. secondary) of prostate and breast cancer tissues. These data represent the molecular basis for clinical applications of ¹⁷⁷Lu-AMBA for diagnosis and treatment of GRP-R and NMB-R positive tumors.     

Evaluation of the Safety,Tolerability, and Pharmacokinetics of Gammaplex<Superscript>®</Superscript> 10% Versus Gammaplex<Superscript>®</Superscript> 5% in Subjects with Primary Immunodeficiency

Purpose

This phase 3, multicenter, open-label, randomized, two-period, crossover bioequivalence trial evaluated the safety, tolerability, and pharmacokinetics of intravenous immunoglobulins (IVIGs) Gammaplex 5% and Gammaplex 10% in 33 adults and 15 children with primary immunodeficiency diseases (PIDs).

Methods

Eligible adults received five Gammaplex 5% infusions followed by five Gammaplex 10% infusions, or vice versa, stratified by a 21- or 28-day dosing regimen. Pediatric subjects received five Gammaplex 10% infusions only.

Results

The primary objective, to demonstrate the bioequivalence of Gammaplex 10% and Gammaplex 5% at the 28-day dosing interval, was met based on the Gammaplex 10%/Gammaplex 5% ratio of area under the concentration versus time curve (AUC_0–28) values. Throughout the study, total immunoglobulin G trough levels were well maintained, with total values generally ≥600 mg/dL (minimum level for study inclusion). At the dosing schedules and infusion rates used in this study, safety and tolerability were comparable and acceptable in adult and pediatric PID subjects treated with Gammaplex 10% and 5%.

Conclusions

In this study, the first direct comparison of 5% IVIG and 10% IVIG products in PID subjects, the pharmacokinetic analysis demonstrated bioequivalence of Gammaplex 10% and Gammaplex 5% at the 28-day dosing interval. The Gammaplex 10% formulation was safe and well tolerated in pediatric and adult PID subjects. Based on the results from this bridging study in PID subjects, Gammaplex 10% could be expected to have a therapeutic effect similar to the licensed Gammaplex 5%, which has demonstrated efficacy and tolerability in patients with PID and idiopathic thrombocytopenic purpura.

     

HCO<Subscript>3</Subscript><Superscript>−</Superscript>-dependent transient acidification induced by ionomycin in rat submandibular acinar cells

Ionomycin (IM, 5 μM), which exchanges 1 Ca²⁺ for 1 H⁺, changed intracellular pH (pH_i) with Ca²⁺ entry into rat submandibular acinar cells. IM-induced changes in pH_i consisted of two components: the first is an HCO₃ ⁻-dependent transient pH_i decrease, and the second is an HCO₃ ⁻-independent gradual pH_i increase. IM (1 μM), which activates store-operated Ca²⁺ channels, induced an HCO₃ ⁻-dependent and transient pH_i decrease without any HCO₃ ⁻-independent pH_i increase. Thus, a gradual pH_i increase was induced by the Ca²⁺/H⁺ exchange. The HCO₃ ⁻-dependent and transient pH_i decrease induced by IM was abolished by acetazolamide, but not by methyl isobutyl amiloride (MIA) or diisothiocyanatostilbene disulfonate (DIDS), suggesting that the Na⁺/H⁺ exchange, the Cl⁻/HCO₃ ⁻ exchange, or the Na⁺-HCO₃ ⁻ cotransport induces no transient pH_i decrease. Thapsigargin induced no transient pH_i decrease. Thus, IM, not Ca²⁺ entry, reduced pH_i transiently. IM reacts with Ca²⁺ to produce H⁺ in the presence of \textCO ₂ /\textHCO ₃ ^- :  [ \textH - \textIM ] ^- + \text Ca ²⁺  + \textCO ₂ \rightleftarrows [ \textH-\textCa - \textIM ] ⁺ ·\textHCO ₃ ^- + \textH ⁺ {\text{CO}}_{ 2} /{\text{HCO}}_{ 3}{^{ - }} : \, \left[ {{\text{H}} - {\text{IM}}} \right]^{ - } + {\text{ Ca}}^{ 2+ } \,+ {\text{CO}}_{ 2} \rightleftarrows \left[ {{\text{H}}-{\text{Ca}} - {\text{IM}}} \right]^{ + } \cdot {\text{HCO}}_{ 3}{^{ - } }+ {\text{H}}^{ + } . In this reaction, a monoprotonated IM reacts with Ca²⁺ and CO₂ to produce an electroneutral IM complex and H⁺, and then H⁺ is removed from the cells via CO₂ production. Thus, IM transiently decreased pH_i. In conclusion, in rat submandibular acinar cells IM (5 μM) transiently reduces pH_i because of its chemical characteristics, with HCO₃ ⁻ dependence, and increases pH_i by exchanging Ca²⁺ for H⁺, which is independent of HCO₃ ⁻.     

Chronotropic effect of D-Ala<Superscript>2</Superscript>,Leu<Superscript>5</Superscript>,Arg<Superscript>6</Superscript>-enkephalin (dalargin) is associated with activation of peripheral κ-opioid receptors

Intravenous infusion of D-Ala²,Leu⁵,Arg⁶-enkephalin (dalargin) caused bradycardia in narcotized rats. This effect was not observed during opioid receptor blockade with naloxone, naloxone methiodide, and norbinaltorphimine. Dalargin and (-)-U-50,488 added to Krebs—Henseleit perfusion solution for isolated rat heart decreased heart rate. Ganglionic blocker hexamethonium potentiated the negative chronotropic effect of dalargin. The negative chronotropic effect of dalargin is probably associated with activation of cardiac κ-opioid receptors. It should be noted that dalargin caused tachycardia in some animals. This reaction was not observed after treatment with hexamethonium. The positive chronotropic effect of dalargin is probably related to modulation of the parasympathetic autonomic nervous system. Agonists and antagonists of δ-opioid receptors caused persistent bradycardia. We hypothesized that selective δ-opioid antagonists exhibit properties of partial δ-receptor agonists. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 140, No. 12, pp. 633–638, December, 2005     

β2-adrenergic stimulation of dendritic cells favors IL-10 secretion by CD4<Superscript>+</Superscript> T cells

Adrenergic receptor agonists and antagonists are extensively used as drugs in medicine for a broad spectrum of indications. We examined the consequences of β2-adrenergic stimulation of murine dendritic cells (DCs) on CD4⁺ T cell activation. We demonstrated in vitro that treatment of LPS-matured DCs with the β2-agonist salbutamol reduced their ability to trigger OT-II T cell proliferation specific for ovalbumin antigen. Salbutamol also induced a decrease in MHC class II molecule expression by DC through Gi protein activation. Co-culture of CD4⁺ T cells with salbutamol-conditioned mature DC impaired TNFα and IL-6 secretion while preserving IL-10 production by T cells. Using a vaccination protocol in mice, we showed that salbutamol favored IL-10-producing CD4⁺ T cells. None of these effects was observed when working with β2-adrenoreceptor deficient mice. Finally, we suggest that β2-adrenergic stimulation of DC could be an interesting way to shape CD4⁺ T cell responses for the purposes of immunotherapy.     

Effect of interleukin-1β on NMDA-induced <Superscript>45</Superscript>Ca<Superscript>2+</Superscript> uptake by synaptosomes of rat brain cortex

The effect of interleukin-1β on presynaptic NMDA receptors was evaluated by studying NMDA-induced ⁴⁵Ca²⁺ uptake by synaptosomes from rat brain cortex. Interleukin-1β inhibited ⁴⁵Ca²⁺ uptake by synaptosomes. Our results indicate that interleukin-1β modulates presynaptic NMDA receptors and is probably involved in the regulation of synaptic transmission in the central nervous system. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 140, No. 12, pp. 645–646, December, 2005     

Nodal peripheral T-cell lymphomas and,in particular,their lymphoepithelioid (Lennert’s) variant are often derived from CD8<Superscript>+</Superscript> cytotoxic T-cells

Nodal peripheral T-cell lymphomas are not well understood, and most of them are classified in the not otherwise specified group (PTCL-NOS). Data on their normal cellular derivation are ambiguous. Most peripheral T-cell lymphomas are composed of tumor cells and a (sometimes dominant) reactive background, which also includes resting and activated T-lymphocytes. We defined the phenotype of the tumor cells in 101 PTCL-NOS based on their cytological atypia and using immunohistochemical double stains on paraffin sections with CD4/Ki67 and CD8/Ki67. The results were correlated to clinical presentation and outcome. Lineage could be defined in 98 cases (97%). Tumor cells were CD4⁺ in 43 cases and CD8⁺ in 38. These presented at a younger age but a higher clinical stage compared with the CD4⁺ lymphomas. In 15 cases, the atypical cells were CD4^–CD8^–; two cases were CD4⁺CD8⁺. Of 17 lymphoepithelioid (Lennerts) lymphomas, 15 expressed CD8, one each was CD4⁺ and CD4^–CD8^–.     

Mice deficient in Vβ8<Superscript>+</Superscript>NKT cells are resistant to experimental hepatitis but are partially susceptible to generalised Shwartzman reaction

Abstract NKT cells are responsible for hepatitis induced either by concanavalin A (Con-A) or α-galactosylceramide (α-GalCer), and they are also profoundly involved in the generalised Shwartzman reaction (GSR) induced by consecutive injections of interleukin (IL)-12 and lipopolysaccharide (LPS). In the present study, using NC/Nga (NC) mice and SJL mice lacking the Vβ⁺8 gene, we examined the role of Vβ⁺8+NKT cells in hepatitis models and in the GSR. The absence of Vβ⁺8+NKT cells in the liver mononuclear cells (MNC) was confirmed by the α-GalCer/CD1d/Ig dimer. Unexpectedly, other dimer+NKT cells including Vβ7⁺NKT cells in these mice were found to decrease in comparison to that of C57BL/6 mice. No significant hepatocyte injury was observed after α-GalCer or Con-A administration in either mice. The serum interferon (IFN)-γ, IL-4 and tumour necrosis factor (TNF) levels did not increase in these mice after α-GalCer injection, however these cytokines substantially increased after Con-A administration, thus suggesting that the roles of NKT cells differ between the two hepatitis models. However, in GSR, although neither mice showed lower IFN-γ levels after a priming IL-12 injection, they showed TNF levels comparable to those in normal mice after LPS injection, and thus resulted in a decreased but substantial mortality. Although liver MNC from IL-12-injected SJL mice showed an impaired antitumour cytotoxicity, liver MNC of NC mice exhibited a greater antitumour cytotoxicity than that of C57BL/6 mice because liver NK cells proportionally increased in NC mice. These results confirm the critical role that Vβ8⁺NKT cells play in both liver and multi-organ injury.     

Remodelling of human atrial K<Superscript>+</Superscript> currents but not ion channel expression by chronic β-blockade

Chronic β-adrenoceptor antagonist (β-blocker) treatment in patients is associated with a potentially anti-arrhythmic prolongation of the atrial action potential duration (APD), which may involve remodelling of repolarising K⁺ currents. The aim of this study was to investigate the effects of chronic β-blockade on transient outward, sustained and inward rectifier K⁺ currents (I_TO, I_KSUS and I_K1) in human atrial myocytes and on the expression of underlying ion channel subunits. Ion currents were recorded from human right atrial isolated myocytes using the whole-cell-patch clamp technique. Tissue mRNA and protein levels were measured using real time RT-PCR and Western blotting. Chronic β-blockade was associated with a 41% reduction in I_TO density: 9.3 ± 0.8 (30 myocytes, 15 patients) vs 15.7 ± 1.1 pA/pF (32, 14), p < 0.05; without affecting its voltage-, time- or rate dependence. I_K1 was reduced by 34% at −120 mV (p < 0.05). Neither I_KSUS, nor its increase by acute β-stimulation with isoprenaline, was affected by chronic β-blockade. Mathematical modelling suggested that the combination of I_TO- and I_K1-decrease could result in a 28% increase in APD₉₀. Chronic β-blockade did not alter mRNA or protein expression of the I_TO pore-forming subunit, Kv4.3, or mRNA expression of the accessory subunits KChIP2, KChAP, Kvβ1, Kvβ2 or frequenin. There was no reduction in mRNA expression of Kir2.1 or TWIK to account for the reduction in I_K1. A reduction in atrial I_TO and I_K1 associated with chronic β-blocker treatment in patients may contribute to the associated action potential prolongation, and this cannot be explained by a reduction in expression of associated ion channel subunits.