Positron emission tomography imaging of mu- and delta-opioid receptor binding in alcohol-dependent and healthy control subjects

Background: The endogenous opioid system plays a significant role in alcohol dependence. The goal of the current study was to investigate regional brain mu‐opioid receptor (MOR) and delta‐opioid receptor (DOR) availability in recently abstinent alcohol‐dependent and age‐matched healthy control men and women with positron emission tomography (PET) imaging. Methods: Alcohol‐dependent subjects completed an inpatient protocol, which included medically supervised withdrawal and PET imaging on day 5 of abstinence. Control subjects completed PET imaging following an overnight stay. PET scans with the MOR‐selective ligand [¹¹C]carfentanil (CFN) were completed in 25 alcohol‐dependent and 30 control subjects. Most of these same subjects (20 alcohol‐dependent subjects and 18 controls) also completed PET scans with the DOR‐selective ligand [¹¹C]methylnaltrindole (MeNTL). Results: Volumes of interest and statistical parametric mapping analyses indicated that alcohol‐dependent subjects had significantly higher [¹¹C]CFN binding potential (BP_ND) than healthy controls in multiple brain regions including the ventral striatum when adjusting for age, gender, and smoking status. There was an inverse relationship between [¹¹C]CFN BP_ND and craving in several brain regions in alcohol‐dependent subjects. Groups did not differ in [¹¹C]MeNTL BP_ND; however, [¹¹C]MeNTL BP_ND in caudate was positively correlated with recent alcohol drinking in alcohol‐dependent subjects. Conclusions: Our observation of higher [¹¹C]CFN BP_ND in alcohol‐dependent subjects can result from up‐regulation of MOR and/or reduction in endogenous opioid peptides following long‐term alcohol consumption, dependence, and/or withdrawal. Alternatively, the higher [¹¹C]CFN BP_ND in alcohol‐dependent subjects may be an etiological difference that predisposed these individuals to alcohol dependence or may have developed as a result of increased exposure to childhood adversity, stress, and other environmental factors known to increase MOR. Although the direction of group differences in [¹¹C]MeNTL BP_ND was similar in many brain regions, differences did not achieve statistical significance, perhaps as a result of our limited sample size. Additional research is needed to further clarify these relationships. The finding that alcohol‐dependent subjects had higher [¹¹C]CFN BP_ND is consistent with a prominent role of the MOR in alcohol dependence.     

Plasma Exchange Downregulates Activated Monocytes and Restores Regulatory T Cells in Kawasaki Disease

In Kawasaki disease (KD), the effect of plasma exchange (PE) on immune cells has not been fully elucidated. Therefore, we examined the changes in the number of CD14⁺ CD16⁺ activated monocytes, regulatory T (T_reg), and T‐helper type 17 (Th17) cells in KD patients treated with PE. The percentage of total monocytes and subclasses of lymphocytes, including CD4⁺ and CD8⁺ T cells, and CD19⁺ B cells, showed no significant difference before and after PE. However, the percentage of CD14⁺ CD16⁺ monocytes in total leukocytes decreased significantly after PE (1.1% ± 1.5% vs. 2.1% ± 2.3%, P < 0.05). Furthermore, while the percentage of Th17 cells in CD4⁺ T cells did not change, the percentage of T_reg cells in CD4⁺ T cells increased significantly after PE (11.1% ± 5.1% vs. 8.0% ± 4.4%, P < 0.05). Therefore, PE downregulates activated monocytes and upregulates T_reg cells toward normal levels and thus attenuates inflammation in KD.     

Brain-derived neurotrophic factor serum levels in alcohol-dependent subjects 6 months after alcohol withdrawal

Background: Diagnosing alcohol dependence is based on clinical signs and on the measured levels of biological markers of alcohol consumption. However, these markers are neither sufficiently sensitive and nor specific enough to definitively determine alcohol dependence. The neuroadaptive changes associated with alcohol dependence involve markers such as brain‐derived neurotrophic factor (BDNF), which regulate neuronal plasticity. Serum levels of BDNF have been reported to decrease during alcohol dependence and may be restored to normal soon after alcohol is withdrawn. However, the long‐term relationship between serum BDNF levels and abstinence status is unknown. Methods: We investigated serum BDNF levels in 101 abstinent and relapsing alcohol‐dependent subjects at the moment of hospitalization for alcohol withdrawal (M0) and 6 months later (M6) and compared them to the serum BDNF levels of 41 nondependent subjects. The BDNF levels of the alcohol‐dependent subjects were compared to their serum gamma glutamyl transferase (GGT) levels, mean corpuscular volume (MCV) values, and their score on the Beck Depression Inventory (BDI) questionnaire. Results: Forty‐four percent of the alcohol‐dependent participants remained abstinent during the 6 months following alcohol detoxification. Serum BDNF levels of the abstinent group at M6 were significantly higher than those of the original group of alcohol‐dependent subjects at M0 (p = 0.034). Only the abstinent group had higher BDNF levels than the control group (p < 0.001). Serum BDNF levels increased to a greater extent in the abstinent group than in the nonabstinent group (p = 0.016). No correlations were found between serum BDNF levels and GGT level, MCV value, or BDI score. Conclusions: Our data confirm that serum BDNF levels do not correlate with either chronic alcohol consumption or peripheral toxicity but may be linked to neuronal aspects of alcohol consumption and dependence. The increased serum levels of BDNF may reflect the concomitant activation of BDNF synthesis that accompanies the neuronal remodeling triggered by alcohol withdrawal and suggests that BDNF synthesis may have a role in the long‐term maintenance of abstinence. Monitoring the serum BDNF levels of alcoholics undergoing treatment could help to characterize alcohol dependence profiles and predict relapse.     

Reduced dopamine D4 receptor mRNA expression in lymphocytes of long-term abstinent alcohol and heroin addicts

Aim It has been repeatedly suggested that dopamine receptor expression in peripheral blood lymphocytes reflects, to some extent, brain status. The aim of the present study was to investigate dopamine receptor expression in peripheral blood lymphocytes of long‐term abstinent alcohol and heroin addicts against the background of the hypothesis, that a persisting dysfunction of the dopaminergic system contributes a biological cause to the chronic character of addiction. Design Dopamine D3 and D4 receptor mRNA expression in peripheral blood lymphocytes was measured by real‐time polymerase chain reaction (PCR) in 19 alcohol addicts, abstinent for 6.2 ± 4.7 months (mean ± SD), and 20 heroin addicts, abstinent for 6.7 ± 3.7 months (mean ± SD), and compared to a control group of 29 age‐ and sex‐matched individuals with no life‐time history of substance abuse. Findings One‐way anova showed significant differences in D4 mRNA expression between the groups (P = 0.005): both groups of addicts showed an approximately 50% reduction in D4 receptor mRNA expression in peripheral blood lymphocytes (PBL) compared to controls. No differences were found for D3 mRNA expression between the groups. Conclusion The results of the present study indicate a withdrawal‐persisting dopaminergic imbalance in abstinent addicts as measured by a suggested peripheral marker.     

Melatonin treatment mimics the antidepressant action in chronic corticosterone‐treated mice

Abstract: Melatonin, involved in circadian cycle, provides protective effects on neuronal cells and acts as antidepressant by restoration of corticosterone levels. A mouse model of anxiety/depressive‐like behavior, induced by chronic corticosterone treatment, has been used to evaluate behavior and adult hippocampal neurogenesis in mice and their possible modulation under melatonin. With this aim, CD1 mice were subjected to 7 wk of corticosterone administration, and then behavioral tests as novelty‐suppressed feeding, open field and a forced swim test were performed. Cell proliferation in hippocampal dentate gyrus (DG) was investigated by 5‐bromo‐2′‐deoxyuridine and doublecortin immunohistochemistry techniques, and stereological procedure was used to quantify labeled cells. Golgi‐impregnated method was used to evaluate the changes of dendritic spines in DG neurons. A new therapeutic approach with antidepressant‐like substances (3 wk) such as melatonin (8 mg/kg) was employed to possibly modulate neural development in the adult hippocampus and the behavioral changes. The depressive‐like state caused by chronic corticosterone treatment was reversed by exogenous administration of melatonin; the proliferation of progenitor cells in mice hippocampus was significantly reduced under chronic corticosterone treatment (cort? 83.7 ± 20.3 versus cort+ 60.5 ± 18.2; P < 0.05), whereas long‐term treatment with melatonin prevented the corticosterone‐induced reduction in hippocampal cell proliferation (cort? 60.5 ± 18.2 versus mel 133.4 ± 26.9; P < 0.05). Corticosterone‐treated mice exhibited a reduced spine density, which was ameliorated by melatonin administration. These findings suggest a strong correspondence between behavior and neurogenesis, strengthening the hypothesis that neurogenesis contributes to the effects of melatonin as an antidepressant.     

Aldosterone increases Na+ -K+ -ATPase activity in skeletal muscle of patients with Conn's syndrome

Objective In Conn’s syndrome, hypokalaemia normally results from renal potassium loss because of the effect of excess aldosterone on Na⁺‐K⁺‐ATPase in principal cells. Little is known about the effect of aldosterone on cellular potassium redistribution in skeletal muscle. Our study determined the effect of aldosterone on muscle Na⁺‐K⁺‐ATPase. Design Muscle biopsies were taken from six patients immediately before and 1 month after adrenalectomy. Ten age‐matched subjects with normal levels of circulating aldosterone served as controls. Results Average plasma aldosterone was significantly higher in presurgery (235·0 ± 51·1 pg/ml) than postsurgery (64·5 ± 25·1 pg/ml) patients. Similarly, Na⁺‐K⁺‐ATPase activity, relative mRNA expression of α₂ (not α₁ or α₃) and β₁ (not β₂ or β₃), and protein abundance of α₂ and β₁ subunits were greater in pre‐ than postsurgery samples (128·7 ± 12·3 vs 79·4 ± 13·3 nmol·mg/protein/h, 2·45 ± 0·31 vs 1·04 ± 0·17, 1·92 ± 0·22 vs1·02 ± 0·14, 2·17 ± 0·33 vs 0·98 ± 0·09 and 1·70 ± 0·17 vs 0·90 ± 0·17, respectively, all P < 0·05). The activity and mRNA expression of the α₂ and β₁ subunits correlated well with plasma aldosterone levels (r = 0·71, r = 0·75 and r = 0·78, respectively, all P < 0·01). Conclusions Our study provides the first evidence in human skeletal muscle that increased plasma aldosterone leads to increased Na⁺‐K⁺‐ATPase activity via increases in α₂ and β₁ subunit mRNAs and their protein expressions. The increased activity may contribute in part to the induction of hypokalaemia in patients with Conn’s syndrome.     

Efficacy and Safety of Baclofen for Alcohol Dependence: A Randomized,Double‐Blind,Placebo‐Controlled Trial

Background: Recent clinical trials and case‐reports indicate that baclofen, a GABA_B agonist, may have efficacy for alcohol dependence. Baclofen has been shown to enhance abstinence, to reduce drinking quantity, to reduce craving, and to reduce anxiety in alcohol‐dependent individuals in 2 placebo‐controlled trials in Italy. However, the clinical trial data with baclofen is limited. The purpose of the present study was to test the efficacy and tolerability of baclofen in alcohol dependence in the United States. Methods: The study was a double‐blind, placebo‐controlled, randomized study comparing 30 mg/d of baclofen to placebo over 12 weeks of treatment and utilizing 8 sessions of BRENDA, a low‐intensity psychosocial intervention. One hundred and twenty‐one subjects were screened to yield 80 randomized subjects (44 men) with randomization balanced for gender. Percent heavy drinking days was the primary outcome measure with other drinking outcomes, anxiety levels, and craving as secondary outcomes. Tolerability was examined. Results: Seventy‐six percent of subjects completed the study. No difference by drug condition was seen in percentage of heavy drinking days where on‐average rates were 25.5% (±23.6%) for placebo and 25.9% (±23.2%) for baclofen during treatment (t₇₃ = 0.59, p = 0.56). Similarly, no differences were seen by drug condition in percentage of days abstinent, time to first drink, or time to relapse to heavy drinking. Baclofen was associated with a significant reduction in state anxiety (F_1,73 = 5.39, p = 0.02). Baclofen was well tolerated with only 2 individuals stopping baclofen because of adverse events. There were no serious adverse events. Conclusions: Baclofen, a GABA_B agonist, represents a possible new pharmacotherapeutic approach to alcohol dependence. Despite encouraging preclinical data and prior positive clinical trials with baclofen in Italy, the current trial did not find evidence that baclofen is superior to placebo in the treatment of alcohol dependence. Additional clinical trial work is necessary to establish whether baclofen does or does not have therapeutic efficacy in alcohol dependence and, if it does, what factors are predictive of response.     

Ex vivo amplification of human hematopoietic stem and progenitor cells in an alginate three-dimensional culture system

Introduction: One of the key factors critical for a successful human cord blood transplantation in treating patients with hematopoietic disorders is the number of hematopoietic stem/progenitor cells derived from human cord blood. Here, we report an alginate three‐dimensional (3D) culture system for the expansion of CD34⁺ cells in cord blood mononuclear cells (CBMCs). Methods: Cord blood mononuclear cells were isolated from human cord blood and encapsulated in 3D alginate beads. The cells were grown with different concentrations of cytokines. At day 0, 3, 6, 9, and 12, respectively, the percentage of CD34⁺ cells was quantified by flow cytometry. Colony‐forming cell assay was performed to determine the potential of hematopoietic reconstruction of the amplified cells under the 3D culture system. Results: After culturing for 12 days, the CBMCs encapsulated in the 3D alginate beads were amplified 5.89 ± 0.72 fold, CD34⁺ cells increased from 2.60 ± 0.52% to 13.27 ± 2.65%, and the colony‐forming assay showed that the colony‐forming unit‐granulocyte/granulocyte‐macrophage (CFU‐G/GM) increased from 363.34 ± 34.47/10⁵ cells to 3423.33 ± 645.14/10⁵ cells (P < 0.001). In comparison, the conventional two‐dimensional (2D) culture system showed that the CBMCs, CD34⁺ cells and the CFU‐G/GM were 0.68 ± 0.16 fold, 0.45 ± 0.17%, and 532.92 ± 82.97/10⁵ cells, respectively. Conclusion: This study demonstrates a new and efficient method to amplify the CD34⁺ human cord blood hematopoietic stem/progenitor cells in a 3D alginate culture system ex vivo for extended periods while retaining the hematopoietic reconstruction capacity.     

CCL17 and CCL22 chemokines within tumor microenvironment are related to infiltration of regulatory T cells in esophageal squamous cell carcinoma

It has been reported that an increased population of regulatory T cells (T‐regs) is one of the reasons for impaired anti‐tumor immunity. We investigated the frequency of Foxp3⁺ T‐regs in tumor‐infiltrating lymphocytes (TILs) and peripheral blood lymphocytes (PBLs) of patients with esophageal squamous cell carcinoma (ESCC). Furthermore, in order to elucidate the mechanisms behind T‐regs accumulation within tumors, we evaluated the relationship between CCL17 or CCL22 expression and the frequency of Foxp3⁺ T‐regs. CD4⁺CD25⁺Foxp3⁺ T‐regs as a percentage of CD4⁺ cells were counted by flow cytometry. The frequency of CCL17⁺ or CCL22⁺ cells among CD14⁺ cells in tumors was also evaluated by flow cytometry. Moreover, an in vitro migration assay using T‐regs derived from ESCC was performed in the presence of CCL17 or CCL22. The frequency of Foxp3⁺ T‐regs in TILs was significantly higher than that in the normal esophageal mucosa (24.6 ± 10.0 vs 7.1 ± 5.9%, P < 0.01). The frequency of Foxp3⁺ T‐regs in PBLs of ESCC patients was significantly higher than that in normal healthy donors (7.0 ± 4.2 vs 2.5 ± 1.0%, P < 0.01). Furthermore, the frequency of CCL17⁺ or CCL22⁺ cells among CD14⁺ cells within tumors was significantly higher than that of normal esophageal mucosa, and there was a significant correlation between the frequency of CCL17⁺ or CCL22⁺ cells and Foxp3⁺ T‐regs in TILs. In addition, the in vitro migration assay indicated that T‐regs were significantly induced to migrate by CCL17 or CCL22. In conclusion, CCL17 and CCL22 within the tumor are related to the increased population of Foxp3⁺ T‐regs in ESCC.     

Effect of a Comprehensive Lifestyle Modification Program on the Bone Density of Male Heavy Drinkers

Background: Heavy alcohol drinking is implicated in osteoporosis. Although abstinence is rapidly followed by a restoration of osteoblastic activity, little is known about the contributions of alcohol‐related factors or the effectiveness of a lifestyle modification program (LMP) on bone density. Methods: We conducted a study of 138 male alcoholic patients to investigate whether drinking history and concurrent factors were associated with the bone density of the calcaneus. A 2.5‐months LMP in an institutionalized setting was completed by 20 of them, and its effect on bone density, serum parathyroid hormone (PTH), and 1.25‐(OH)₂ vitamin D levels were assessed. Results: The patients had a high prevalence of daytime drinking (93.5%), continuous drinking (84.1%), and current smoking (82.0%) with mean duration of alcohol abuse of 30.0 ± 12.8 years. The patients had lower bone density than a reference control group (Z‐scores: ?0.45 ± 1.02). Multiple stepwise regression analysis identified age, poor activities of daily living (ADL), continuous drinking, absence of liver cirrhosis, depression, and dementia as determinants of low bone density. The bone density of the 20 participants in the LMP improved 2.3% (p = 0.0003) with a more ameliorating effect on bone density than a conventional abstinence therapy (p = 0.014 for interventional effect). The upper normal range of PTH levels at baseline were significantly decreased, and 1.25‐(OH)₂ vitamin D levels also had a trend toward decrease during the abstinence. Conclusions: Alcoholic patients may have many complications such as poor ADL and dementia, which are independently associated with decreased bone density. The results of this study support the idea that comprehensive approach to lifestyle factors to minimize risk of osteoporosis is the best way to improve bone density.     

Effect of detoxification on liver stiffness assessed by Fibroscan® in alcoholic patients

Aims: The aims of this study were to determine whether alcohol consumption or cessation influences transient elastography (TE) measurements and whether TE is a useful tool to monitor alcoholic patients. Patients: Twenty‐three consecutive heavy drinkers (20 men and 3 women; mean age 47.2 years) admitted for a 7‐day hospitalization for alcohol detoxification were included. On admission (D0), a detailed medical history was taken and the following laboratory tests were performed [aspartate aminotransferase (ASAT), gamma glutamyltransferase (γGT), and carbohydrate‐deficient transferrin (CDT) levels, and Fibroscan^®]. All examinations were repeated on D8, D30, and D60. Variation in the median Fibroscan value of >20% was considered significant. Results: After 1 week of detoxification, the % variation in TE was ?21.67 ± ?27.6%. The median variation in TE between D8 and D60 was ?20% in the abstinent group and 32% in the relapse group (p = 0.007). An increase in proportion of patients with a significant decrease in TE was observed with an increased duration of abstinence: 41.7% at D8 and 66.7% at D60. TE values were significantly correlated with ASAT, γGT, and CDT at D0 and D8, and with ASAT and γGT at D60. Conclusions: TE in alcoholics is influenced by major variations in the biochemical activity of the disease. The kinetics of variation of TE suggest that this method may be useful to assess alcohol abuse and control.     

Alcohol Exposure Impairs Myeloid Dendritic Cell Function in Rhesus Macaques

Background: Alcohol intoxication suppresses both the innate and adaptive immunities. Dendritic cells (DCs) are the major cell type bridging the innate and acquired immune responses. At the present time, the effects of alcohol on DC development in hematopoietic tissues and the functional activities of DCs are incompletely elucidated. This study investigated the impact of chronic alcohol exposure on the alteration of hematopoietic precursor cell and DC populations in the bone marrow and peripheral blood of rhesus macaques. Methods: Rhesus macaques were administered alcohol or isocaloric sucrose daily for a period of 3 months through surgically implanted gastric catheters. Peripheral blood mononuclear cells (PBMCs) and bone marrow cells (BMCs) were isolated for flow cytometric analysis after 3 months. Monocytes were cultured with human IL‐4 (10 ng/ml) and GM‐CSF (50 ng/ml) in the absence and presence of alcohol (50 mM). On day 6 of the culture, a cocktail of stimulants including IL‐1β (18 ng), IL‐6 (1800 U), TNF‐α (18 ng), and PGE₂ (1.8 μg) were added to the designated wells for transformation of immature dendritic cells (iDCs) to mature myeloid DCs. The cells were analyzed on day 8 by flow cytometry for expression of DC costimulatory molecule expression. Results: EtOH‐treated animals had significantly lower numbers of myeloid DCs (lineage‐HLA‐DR+CD11c+CD123?) in both the PBMCs and BMCs compared to controls (5,654 ± 1,273/10⁶ vs. 2,353 ± 660/10⁶ PBMCs and 503 ± 34 vs. 195 ± 44/10⁶ BMCs). Under culture conditions, the number of lineage‐HLA‐DR+CD83+ cells was low in control wells (0.38 ± 0.08%). Alcohol inhibited the increase in the number of lineage‐HLA‐DR+CD83+ cells in iDC wells (2.30 ± 0.79% vs. 5.73 ± 1.40%). Alcohol also inhibited the increase in the number of lineage‐HLA‐DR+CD83+ cells in mature DC wells (1.23 ± 0.15% vs. 4.13 ± 0.62%). Conclusions: Chronic EtOH decreases the bone marrow and circulating pools of myeloid DCs. Additionally, EtOH suppresses costimulatory molecule CD83 expression during DC transformation, which may attenuate the ability of DCs to initiate T‐cell expansion.     

Persistent impairment of hippocampal neurogenesis in young adult rats following early postnatal alcohol exposure

Background: Prenatal alcohol exposure can cause damage to the developing fetus with outcomes including growth deficiency, facial dysmorphology, brain damage, and cognitive and behavioral deficits. Smaller brains in children with FASD have been linked both with reduced cell proliferation in the developing CNS and with apoptotic cell loss of postmitotic neurons. Prenatal alcohol exposure in rodents during the period of brain development comparable to that of the first and second trimesters of human pregnancy persistently alters adult neurogenesis. Long‐term effects of alcohol exposure during the third trimester equivalent, which occurs postnatally in the rat, on adult neurogenesis have not been previously reported. The goal of this study was to examine the effect of postnatal binge‐like alcohol exposure on cell proliferation and neurogenesis in hippocampal dentate gyrus during adolescence and young adulthood. Methods: Male Long‐Evans rat pups were assigned to 3 groups: alcohol‐exposed (AE), sham‐intubated (SI) or suckle control (SC). AE pups received ethanol in a milk formula in a binge manner (2 feedings, 2 hours apart, total dose 5.25 g/kg/day) on postnatal days (PD) 4–9. BrdU was injected every other day on PD30–50. Animals were perfused either on PD50 to examine cytogenesis and neurogenesis in hippocampal dentate gyrus at the end of BrdU injections or on PD80 to evaluate new cell survival. Dorsal hippocampal sections were immunostained for BrdU, a marker for proliferating cells, Ki67, endogenous marker of proliferation, and NeuN, a marker for mature neurons. Results: Binge‐like alcohol exposure on PD4–9 significantly reduced the number of mature neurons in adult hippocampal dentate gyrus (DG) both on PD50 and PD80, without altering cumulative cytogenesis on PD50. In addition, the number of new neurons, that were generated between PD30 and 50, was further reduced after 30 days of survival in all 3 groups (SC, SI, and AE). Conclusions: These observations suggest that early postnatal binge alcohol exposure results in long‐term deficits of adult hippocampal neurogenesis, providing a potential basis for the deficits of hippocampus‐dependent behaviors reported for this model.     

T-lymphocytes expressing CC chemokine receptor-5 are increased in frail older adults

OBJECTIVES: To evaluate the frequencies of T‐lymphocytes expressing CC chemokine receptor‐5 (CCR5⁺ T‐cells) and their relationship with frailty in older adults. DESIGN: Case‐control study with an age‐, race‐, and sex‐matched design. SETTING: General Clinical Research Center. PARTICIPANTS: Community‐dwelling adults aged 72 and older from Baltimore, Maryland. METHODS: Frailty was determined using five validated criteria: weakness, slow walking speed, fatigue, low physical activity, and weight loss. Those meeting three or more of these five criteria were defined as frail and those with none as nonfrail. Complete blood counts were performed to obtain peripheral lymphocyte counts using an automated (Coulter) counter. Peripheral blood was collected for surface immunofluorescent staining of CCR5 and other T‐cell markers. RESULTS: Twenty‐six frail and matched nonfrail participants (mean age±standard deviation 83.8±5.3, range 72–94) completed the study. Frail participants had higher CCR5⁺, CCR5⁺CD8⁺, and CCR5⁺CD45RO^? T‐cell counts than matched nonfrail controls (349±160/mm³ vs 194±168/mm³, P=.02; 208±98/mm³ vs 105±62/mm³, P=.02; and 189±149/mm³ vs 52±36/mm³, P=.01; respectively). Furthermore, there was a trend toward graded increase in these T‐cell counts across the frailty scores in frail participants (e.g., CCR5⁺CD8⁺ counts of 123±52/mm³, 248±115/mm³, and 360±215/mm³ for those with frailty scores of 3, 4, and 5, respectively). CONCLUSION: These initial results suggest an expansion of the CCR5⁺ T‐cell subpopulation in frailty. They provide a basis for further characterization of CCR5⁺ T‐cells and their role in frailty, with potential therapeutic implications.     

Reduction of Ventricular Sodium Current in a Mouse Model of HIV

Effect of HIV on Cardiac Sodium Current. Introduction: Cardiac arrhythmias have been reported in AIDS patients. Arrhythmias can arise from alterations in ventricular Na⁺ channel function. However, it is unknown whether HIV affects cardiac Na⁺ channel function. Therefore, the purpose of this study was to characterize the effect of HIV on ventricular Na⁺ current (I_Na) in a transgenic model of HIV (CD4C/HIV mice), which exhibit a severe AIDS‐like disease. Methods and Results: Patch‐clamp techniques were used to examine I_Na and action potentials (AP) in ventricular myocytes isolated from HIV and wild‐type (WT) mice. In HIV myocytes peak I_Na was reduced (at ?50 mV: HIV, ?55.3 ± 4.3 pA/pF, n = 15; WT, ?79.4 ± 5.2 pA/pF, n = 16, P < 0.05), whereas late I_Na was similar in both groups (HIV, ?4.3 ± 0.4 pA/pF; WT, ?4.4 ± 0.4 pA/pF, n = 22/group). AP amplitude (HIV 91.5 ± 4.7 mV, n = 12; WT 104.4 ± 3.1 mV, n = 15, P < 0.05) and the maximal velocity of the AP upstroke (V_max; HIV, 57.2 ± 9.3 mV/ms, n = 12; WT, 113.5 ± 8 mV/ms, n = 15, P < 0.05) were decreased in HIV myocytes. ECG recordings revealed that the QRS complex was prolonged in HIV mice (HIV, 15.7 ± 0.2 ms, n = 22; WT, 14.1 ± 0.5 ms, n = 10, P < 0.05). The serum levels of interleukin‐1β were elevated in HIV mice (HIV, 18.1 ± 3.1 pg/mL, n = 3; WT, 5.1 ± 1.1 pg/mL, n = 4, P < 0.05) in line with previous evidence that suggests that elevated levels of cytokines can affect cardiac ion currents. Conclusion: Overall, our observations suggest that elevated levels of proinflammatory cytokines in CD4C/HIV mice could alter Na⁺ channel function, thus altering cardiac depolarization and contribute to the generation of arrhythmias. (J Cardiovasc Electrophysiol, Vol. 21, pp. 916‐922, August 2010)     

Expansion of CD4+CD25+FoxP3+ regulatory T cells in hepatitis C virus‐related chronic hepatitis,cirrhosis and hepatocellular carcinoma

Aim: Regulatory T (Treg) cells may play a pivotal role in the persistence of hepatitis C virus (HCV) infection and the development of hepatocellular carcinoma (HCC). Therefore, we examined their frequency in peripheral blood from patients with HCV‐positive chronic hepatitis (CH), cirrhosis (LC) and HCC. Methods: Treg cells were identified as CD4⁺, CD25⁺ and FoxP3⁺ T lymphocytes using three‐color FACS. The frequency of Treg cells was expressed as a percentage of the total CD4⁺ T lymphocytes, and the phenotype of Treg cells was examined using CD45RA. Results: Treg cells were significantly increased in CH (5.88 ± 0.19%, n = 76; P < 0.01), LC (6.10 ± 0.28%, n = 40; P < 0.001) and HCC (6.80 ± 0.30%, n = 57; P < 0.0001) compared to healthy control (5.13 ± 0.25%, n = 31). However, Treg cells were not increased with the progression of fibrosis or the grade of inflammations. Treg cells were slightly increased in early‐stage HCC (6.91 ± 0.40%) compared with advanced‐stage HCC (6.58 ± 0.39%), but these results were not statistically significant. In a serial examination, a distinct increase in Treg cells after local therapy for early‐stage HCC was a hallmark of early recurrence. Most expanded Treg cells in HCC were CD45RA^‐, suggesting that a memory‐type Treg population had differentiated in the periphery and not in the thymus. Conclusion: We observed an increase in Treg cells in HCV‐related chronic liver disease, particularly in HCC, and these cells were shown to be memory‐type Treg cells.     

Involvement of primary mesenchymal precursors and hematopoietic bone marrow cells from chronic myeloid leukemia patients by BCR‐ABL1 fusion gene

For decades now, it is well established that chronic myeloid leukemia (CML) is a hematopoietic stem cell (HPC) disorder. However, it remains to be determined whether BCR‐ABL1 gene rearrangement occurs in a HPC or at an earlier stem cell and whether the degree of involvement of hematopoiesis by the BCR‐ABL1 fusion gene relates to the response to therapy. Here, we have investigated by interphase fluorescence in situ hybridization (iFISH) the distribution of BCR‐ABL1 fusion gene in FACS‐sorted bone marrow (BM) populations of mesenchymal precursor cells (MPC) and other hematopoietic cell populations from 18 newly diagnosed CML patients. Overall, our results showed systematic involvement at relatively high percentages of BM maturing neutrophils (97% ± 15%), basophils (95% ± 12%), eosinophils (90% ± 8%), CD34⁺ precursors cells (90% ± 7%), monocytes (84% ± 30%), nucleated red blood cells (87% ± 24%), and mast cells (77% ± 33%). By contrast, MPC (30% ± 34%), B‐cells (15% ± 27%), T‐lymphocytes (50% ± 26%), and NK‐cells (35% ± 34%) were involved at lower percentages. In 8/18 CML patients, ≥2 tumor BCR‐ABL1⁺ subclones were detected by iFISH. Of note, all tumor cell subclones were systematically detected in CD34⁺ cells, whereas MPC were only involved by the ancestral tumor cell subclone. In summary, here we confirm the presence at diagnosis of the BCR‐ABL1 fusion gene in MPC, CD34⁺ precursors, and other different BM hematopoietic myeloid cell lineages from CML patients, including also in a significant fraction of cases, a smaller percentage of T, B, and NK lymphocytes. Interestingly, involvement of MPC was restricted to the ancestral BCR‐ABL1⁺ subclone. Am. J. Hematol. 89:288–294, 2014. © 2013 Wiley Periodicals, Inc.     

Alcohol-Induced Electrical Remodeling: Effects of Sustained Short-Term Ethanol Infusion on Ion Currents in Rabbit Atrium

Background: In some patients, above‐average alcohol consumption before occurrence of atrial fibrillation (AF) in terms of a “holiday heart syndrome” (HHS) can be determined. There is evidence that long before development of apparent alcohol‐induced cardiomyopathy, above‐average alcohol consumption generates an arrhythmogenic substrate which abets the onset of AF. Changes of atrial current densities in terms of an electrical remodeling after sustained short‐term ethanol infusion in rabbits as a potential part of HHS pathophysiology were examined in this study. Methods: Rabbits of the ethanol group (EG) received sustained short‐term intravenous alcohol infusion for 120 hours (during infusion period, blood alcohol level did not fall below 158 mg/dl), whereas NaCl 0.9% was infused in the placebo group (PG). Using patch clamp technique in whole‐cell mode, atrial current densities were measured and compared between both groups. Results: Ethanol infusion did not alter current densities of I_to [58.7 ± 5.0 pA/pF (PG, n = 20 cells) vs. 53.9 ± 5.0 pA/pF (EG, n = 24)], I_sus [11.3 ± 1.4 pA/pF (PG, n = 20) vs. 10.2 ± 1.0 pA/pF (EG, n = 24)], and I_K1 [?1.6 ± 0.3 pA/pF (PG, n = 17) vs. ?2.0 ± 0.3 pA/pF (EG, n = 22)]. However, alcohol infusion resulted in a remarkable reduction of I_Ca,L current densities [?28.4 ± 1.8 pA/pF (PG, n = 20) vs. ?15.2 ± 1.4 pA/pF (EG, n = 22)] and I_Na [?75.4 ± 3.6 pA/pF (PG, n = 17) vs. ?35.4 ± 4.4 pA/pF (EG, n = 21)], respectively. Conclusion: Sustained short‐term ethanol infusion in rabbits alters atrial current densities. HHS might be favored by alcohol‐induced atrial electrical remodeling.     

23Na NMR demonstrates prolonged increase of intracellular sodium following transient regional ischemia in the in situ pig heart

This study tests the hypothesis that Na⁺ _i increases during regional ischemia in the in situ pig heart. An extracorporeal shunt was created between the carotid artery and the left anterior descending artery of 14 open chest pigs. ²³Na and ³¹P NMR spectroscopy measured myocardial Na⁺ _i and high energy phosphates (HEPs). The protocol consisted of three 40 min periods: pre-ischemia (shunt pressure, 76±23 mmHg (S.D.)), ischemia (shunt pressure, 25±7 mmHg), and post-ischemia (shunt pressure, 53±11 mmHg). The pre-ischemia Na⁺ _i concentration was 6.7±4.2 mM. Phosphocreatine (PCr) was 15.3±0.5 mM, ATP 9.4±0.4 mM, inorganic phosphate (Pi) 1.5±0.2 mM, and pH_i 7.16±0.09. At the end of ischemia Na⁺ _i had increased to 10.5±2.8 mM (p<0.0002); PCr decreased to 5.9±2.1 mM (p<0.0002); ATP was 6.5±0.5 mM (p<0.003); Pi had increased to 6.3±1.0 mM (p<0.0002), and pH_i was 6.41±0.06 (p<0.0002). During the first 10 min of the reperfusion, Na⁺ _i increased further to 12.4±2.8 mM (p<0.025), whereas HEPs all returned to pre-ischemic values. Na⁺ _i increases during regional ischemia in the in situ pig heart, suggesting reduced Na⁺/K⁺ ATPase activity. While ATP probably does not limit Na⁺/K⁺ ATPase activity, increases in Pi and decreases in pH_i may reduce Na⁺/K⁺ ATPase activity. Additional Na⁺ _i increases during reperfusion suggest either augmented Na⁺ influx or decreased Na⁺ efflux. Received: 25 May 1998, Returned for revision: 22 June 1998, Revision received: 20 August 1998, Accepted: 15 September 1998