Alterations in the innate and adaptive immune system in a real-world cohort of multiple sclerosis patients treated with ocrelizumab.

B cell depletion by the anti-CD20 antibody ocrelizumab is effective in relapsing-remitting (RR) and primary progressive (PP) multiple sclerosis (MS). We investigated immunological changes in peripheral blood of a real-world MS cohort after 6 and 12 months of ocrelizumab. All RRMS and most PPMS patients (15/20) showed treatment response. Ocrelizumab not only reduced CD20+ B cells, but also numbers of CD20+ T cells. Absolute numbers of monocytes, dendritic cells and CD8+ T cells were increased, while CD56hi natural killer cells were reduced after ocrelizumab. The residual B cell population shifted towards transitional and activated, IgA+ switched memory B cells, double negative B cells, and antibody-secreting cells. Delaying the treatment interval by 2-3 months increased mean B cell frequencies and enhanced naive B cell repopulation. Ocrelizumab reduced plasma levels of interleukin(IL)-12p70 and interferon(IFN)-α2. These findings will contribute to understanding ineffective treatment responses, dealing with life-threatening infections and further unravelling MS pathogenesis.

Diagnostic accuracy of body mass index to identify obesity in older adults: NHANES 1999-2004.

BACKGROUND: Body composition changes with aging lead to increased adiposity and decreased muscle mass, making the diagnosis of obesity challenging. Conventional anthropometry, including body mass index (BMI), while easy to use clinically may misrepresent adiposity. We determined the diagnostic accuracy of BMI using dual-energy X-ray absorptiometry (DEXA) in assessing the degree of obesity in older adults. METHODS: The National Health and Nutrition Examination Surveys 1999-2004 were used to identify adults aged ⩾60 years with DEXA measures. They were categorized (yes/no) as having elevated body fat by gender (men: ⩾25%; women ⩾35%) and by BMI ⩾25 and ⩾30 kg m(-)(2). The diagnostic performance of BMI was assessed. Metabolic characteristics were compared in discordant cases of BMI/body fat. Weighting and analyses were performed per NHANES (National Health and Nutrition Examination Survey) guidelines. RESULTS: We identified 4984 subjects (men: 2453; women: 2531). Mean BMI and % body fat was 28.0 kg m(-2) and 30.8% in men, and 28.5 kg m(-)(2) and 42.1% in women. A BMI ⩾30 kg m(-)(2) had a low sensitivity and moderately high specificity (men: 32.9 and 80.8%, concordance index 0.66; women: 38.5 and 78.5%, concordance 0.69) correctly classifying 41.0 and 45.1% of obese subjects. A BMI ⩾25 kg m(-2) had a moderately high sensitivity and specificity (men: 80.7 and 99.6%, concordance 0.81; women: 76.9 and 98.8%, concordance 0.84) correctly classifying 80.8 and 78.5% of obese subjects. In subjects with BMI <30 kg m(-)(2), body fat was considered elevated in 67.1% and 61.5% of men and women, respectively. For a BMI ⩾30 kg m(-)(2), sensitivity drops from 40.3% to 14.5% and 44.5% to 23.4%, whereas specificity remains elevated (>98%), in men and women, respectively, in those 60-69.9 years to subjects aged ⩾80 years. Correct classification of obesity using a cutoff of 30 kg m(-)(2) drops from 48.1 to 23.9% and 49.0 to 19.6%, in men and women in these two age groups. CONCLUSIONS: Traditional measures poorly identify obesity in the elderly. In older adults, BMI may be a suboptimal marker for adiposity.

Differential effects of leptin on adiponectin expression with weight gain versus obesity.

BACKGROUND/OBJECTIVE: Adiponectin exerts beneficial effects by reducing inflammation and improving lipid metabolism and insulin sensitivity. Although the adiponectin level is lower in obese individuals, whether weight gain reduces adiponectin expression in humans is controversial. We sought to investigate the role of weight gain, and consequent changes in leptin, on altering adiponectin expression in humans. METHODS/RESULTS: Forty-four normal-weight healthy subjects were recruited (mean age 29 years; 14 women) and randomized to either gain 5% of body weight by 8 weeks of overfeeding (n=34) or maintain weight (n=10). Modest weight gain of 3.8±1.2 kg resulted in increased adiponectin level (P=0.03), whereas weight maintenance resulted in no changes in adiponectin. Further, changes in adiponectin correlated positively with changes in leptin (P=0.0085). In-vitro experiments using differentiated human white preadipocytes showed that leptin increased adiponectin mRNA and protein expression, whereas a leptin antagonist had opposite effects. To understand the role of leptin in established obesity, we compared adipose tissue samples obtained from normal-weight versus obese subjects. We noted, first, that leptin activated cellular signaling pathways and increased adiponectin mRNA in the adipose tissue from normal-weight participants, but did not do so in the adipose tissue from obese participants. Second, we noted that obese subjects had increased caveolin-1 expression, which attenuates leptin-dependent increases in adiponectin. CONCLUSIONS: Modest weight gain in healthy individuals is associated with increases in adiponectin levels, which correlate positively with changes in leptin. In vitro, leptin induces adiponectin expression, which is attenuated by increased caveolin-1 expression. In addition, the adipose tissue from obese subjects shows increased caveolin-1 expression and impaired leptin signaling. This leptin signal impairment may prevent concordant increases in adiponectin levels in obese subjects despite their high levels of leptin. Therefore, impaired leptin signaling may contribute to low adiponectin expression in obesity and may provide a target for increasing adiponectin expression, hence improving insulin sensitivity and cardio-metabolic profile in obesity.

Anti-SPAG16 antibodies in primary progressive multiple sclerosis are associated with an elevated progression index.

BACKGROUND AND PURPOSE: Sperm-associated antigen 16 (SPAG16), a sperm protein which is upregulated in reactive astrocytes in multiple sclerosis (MS) lesions, has recently been identified as a novel autoantibody target in MS. The aim of this study was to investigate whether anti-SPAG16 antibody levels differ between MS subtypes (relapsing-remitting, RR; primary or secondary progressive, PP, SP) and whether antibody positivity is associated with clinical characteristics. METHODS: Plasma anti-SPAG16 antibody levels were determined by recombinant protein enzyme-linked immunosorbent assay (ELISA) in 374 MS patients (274 RRMS, 39 SPMS and 61 PPMS) and 106 healthy controls. RESULTS: Significantly elevated anti-SPAG16 antibodies were found in 22% of MS patients with 93% specificity. Anti-SPAG16 seropositivity was associated with an increased Expanded Disability Status Scale (EDSS) in overall MS. A higher proportion of PPMS patients showed anti-SPAG16 antibody reactivity (34%) compared to RRMS (19%) and SPMS (26%), and presented with higher anti-SPAG16 antibody levels. Seropositive PPMS patients had a significantly increased progression index compared to seronegative patients. CONCLUSIONS: Anti-SPAG16 antibodies are associated with an increased EDSS in overall MS, indicating that they are linked to a worse MS disease outcome. Moreover, the presence of anti-SPAG16 antibodies may be a biomarker for a more severe disease in PPMS patients, as indicated by an increased progression index.

Diagnostic performance of body mass index to identify obesity as defined by body adiposity: a systematic review and meta-analysis.

OBJECTIVE: We performed a systematic review and meta-analysis of studies that assessed the performance of body mass index (BMI) to detect body adiposity. DESIGN: Data sources were MEDLINE, EMBASE, Cochrane, Database of Systematic Reviews, Cochrane CENTRAL, Web of Science, and SCOPUS. To be included, studies must have assessed the performance of BMI to measure body adiposity, provided standard values of diagnostic performance, and used a body composition technique as the reference standard for body fat percent (BF%) measurement. We obtained pooled summary statistics for sensitivity, specificity, positive and negative likelihood ratios (LRs), and diagnostic odds ratio (DOR). The inconsistency statistic (I2) assessed potential heterogeneity. RESULTS: The search strategy yielded 3341 potentially relevant abstracts, and 25 articles met our predefined inclusion criteria. These studies evaluated 32 different samples totaling 31 968 patients. Commonly used BMI cutoffs to diagnose obesity showed a pooled sensitivity to detect high adiposity of 0.50 (95% confidence interval (CI): 0.43-0.57) and a pooled specificity of 0.90 (CI: 0.86-0.94). Positive LR was 5.88 (CI: 4.24-8.15), I (2)=97.8%; the negative LR was 0.43 (CI: 0.37-0.50), I (2)=98.5%; and the DOR was 17.91 (CI: 12.56-25.53), I (2)=91.7%. Analysis of studies that used BMI cutoffs >or=30 had a pooled sensitivity of 0.42 (CI: 0.31-0.43) and a pooled specificity of 0.97 (CI: 0.96-0.97). Cutoff values and regional origin of the studies can only partially explain the heterogeneity seen in pooled DOR estimates. CONCLUSION: Commonly used BMI cutoff values to diagnose obesity have high specificity, but low sensitivity to identify adiposity, as they fail to identify half of the people with excess BF%.

A novel method for making human monoclonal antibodies.

We have developed a B cell immortalization method for low B cell numbers per well using simultaneous B cell stimulation by CpG2006 and B cell infection by Epstein-Barr virus (EBV), followed by an additional CpG2006 and interleukin-2 (IL-2) stimulus. Using this method, immunoglobulin G (IgG)-producing immortalized B cell lines were generated from peripheral blood IgG(+)CD22(+) B cells with an efficiency of up to 83%. Antibody can already be obtained from the culture supernatant after 3-4 weeks. Moreover, clonality analysis demonstrated monoclonality in 87% of the resulting immortalized B cell lines. Given the high immortalization efficiency and monoclonality rate, evidence is provided that no further subcloning is necessary. An important application of this B cell immortalization method is the characterization of (autoreactive) antibodies from patients with autoimmune disease. This could eventually lead to the identification of new autoantigens, disease markers or targets for therapy.

Accuracy of body mass index in diagnosing obesity in the adult general population.

BACKGROUND: Body mass index (BMI) is the most widely used measure to diagnose obesity. However, the accuracy of BMI in detecting excess body adiposity in the adult general population is largely unknown. METHODS: A cross-sectional design of 13 601 subjects (age 20-79.9 years; 49% men) from the Third National Health and Nutrition Examination Survey. Bioelectrical impedance analysis was used to estimate body fat percent (BF%). We assessed the diagnostic performance of BMI using the World Health Organization reference standard for obesity of BF%>25% in men and>35% in women. We tested the correlation between BMI and both BF% and lean mass by sex and age groups adjusted for race. RESULTS: BMI-defined obesity (> or =30 kg m(-2)) was present in 19.1% of men and 24.7% of women, while BF%-defined obesity was present in 43.9% of men and 52.3% of women. A BMI> or =30 had a high specificity (men=95%, 95% confidence interval (CI), 94-96 and women=99%, 95% CI, 98-100), but a poor sensitivity (men=36%, 95% CI, 35-37 and women=49%, 95% CI, 48-50) to detect BF%-defined obesity. The diagnostic performance of BMI diminished as age increased. In men, BMI had a better correlation with lean mass than with BF%, while in women BMI correlated better with BF% than with lean mass. However, in the intermediate range of BMI (25-29.9 kg m(-2)), BMI failed to discriminate between BF% and lean mass in both sexes. CONCLUSIONS: The accuracy of BMI in diagnosing obesity is limited, particularly for individuals in the intermediate BMI ranges, in men and in the elderly. A BMI cutoff of> or =30 kg m(-2) has good specificity but misses more than half of people with excess fat. These results may help to explain the unexpected better survival in overweight/mild obese patients.

Dimethyl fumarate induces a persistent change in the composition of the innate and adaptive immune system in multiple sclerosis patients.

The effects of dimethyl fumarate (DMF) on the immune system in multiple sclerosis (MS) are not completely elucidated. In this study, an extensive immunophenotypic analysis of innate and adaptive immune cells of DMF-treated MS patients was performed. Peripheral blood immune cell phenotypes were determined using flow cytometry in a follow-up study of 12 MS patients before, after 3 and 12 months of DMF treatment and a cross-sectional study of 25 untreated and 64 DMF-treated MS patients. Direct effects of DMF on B cells were analyzed in vitro. After 12 months of DMF treatment, percentages of monocytes, natural killer cells, naive T and B cells and transitional B cells increased. Percentages of (effector) memory T cells, (non) class-switched memory B cells and double negative B cells decreased together with CD4+ T cells expressing interferon-γ (IFN-γ), granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-17 (IL-17). DMF treatment was fully effective as of 6 months and directly induced apoptosis and decreased expression of costimulatory CD40, antigen presentation molecule MHCII and B cell activating factor receptor (BAFFR) on B cells. DMF induced a persistent change of the immune system of MS patients, directly induced apoptosis and reduced expression of functional markers on B cells.

Interaction of baroreceptor and chemoreceptor reflex control of sympathetic nerve activity in normal humans.

Animal studies have demonstrated that activation of the baroreflex by increases in arterial pressure inhibits cardiovascular and ventilatory responses to activation of peripheral chemoreceptors (PC) with hypoxia. In this study, we examined the influences of baroreflex activation on the sympathetic response to stimulation of PC and central chemoreceptors in humans. PC were stimulated by hypoxia (10% O2/90% N2) (n = 6) and central chemoreceptors by hypercapnia (7% CO2/93% O2) (n = 6). Responses to a cold pressor stimulus were also obtained as an internal reflex control to determine the selectivity of the interactive influence of baroreflex activation. Baroreflex activation was achieved by raising mean blood pressure by greater than 10 mmHg with intravenous infusion of phenylephrine (PE). Sympathetic nerve activity (SNA) to muscle was recorded from a peroneal nerve (microneurography). During hypoxia alone, SNA increased from 255 +/- 92 to 354 +/- 107 U/min (P less than 0.05). During PE alone, mean blood pressure increased and SNA decreased to 87 +/- 45 U/min (P less than 0.05). With hypoxia during baroreflex activation with PE, SNA did not increase (50 +/- 23 U/min). During hypercapnia alone, SNA increased from 116 +/- 39 to 234 +/- 72 U/min (P less than 0.01). Hypercapnia during baroreflex activation with PE increased SNA from 32 +/- 25 U/min during PE alone to 61 +/- 26 U/min during hypercapnia and PE (P less than 0.05). Like hypercapnia (but unlike hypoxia) the cold pressor test also increased SNA during PE. We conclude that baroreflex activation selectively abolishes the SNA response to hypoxia but not to hypercapnia or the cold pressor test. The inhibitory interaction of the baroreflex and the peripheral chemoreflex may be explained by convergence of baroreceptor and peripheral chemoreceptor afferents on neurons in the medulla.

Sympathetic neural mechanisms in obstructive sleep apnea.

Blood pressure, heart rate, sympathetic nerve activity, and polysomnography were recorded during wakefulness and sleep in 10 patients with obstructive sleep apnea. Measurements were also obtained after treatment with continuous positive airway pressure (CPAP) in four patients. Awake sympathetic activity was also measured in 10 age- and sex-matched control subjects and in 5 obese subjects without a history of sleep apnea. Patients with sleep apnea had high levels of nerve activity even when awake (P < 0.001). Blood pressure and sympathetic nerve activity did not fall during any stage of sleep. Mean blood pressure was 92 +/- 4.5 mmHg when awake and reached peak levels of 116 +/- 5 and 127 +/- 7 mmHg during stage II sleep (n = 10) and rapid eye movement (REM) sleep (n = 5), respectively (P < 0.001). Sympathetic activity increased during sleep (P = 0.01) especially during stage II (133 +/- 9% above wakefulness; P = 0.006) and REM (141 +/- 13%; P = 0.007). Peak sympathetic activity (measured over the last 10 s of each apneic event) increased to 299 +/- 96% during stage II sleep and to 246 +/- 36% during REM sleep (both P < 0.001). CPAP decreased sympathetic activity and blood pressure during sleep (P < 0.03). We conclude that patients with obstructive sleep apnea have high sympathetic activity when awake, with further increases in blood pressure and sympathetic activity during sleep. These increases are attenuated by treatment with CPAP.

Exploring cDNA phage display for autoantibody profiling in the serum of multiple sclerosis patients: optimization of the selection procedure.

We applied a cDNA phage display method called serological antigen selection (SAS) to identify immunogenic targets that evoke an autoantibody response in the serum of multiple sclerosis (MS) patients. This method involves the display of a cDNA expression library, in this study a MS brain library, on filamentous phage and subsequent selection using patient immunoglobulin G (IgG). To apply the SAS technology for autoantibodies in the serum of MS patients, an optimization was necessary to deplete cDNA products that encode IgG fragments derived from B cells present in the MS brain plaques. We describe a differential screening procedure in which positive selection rounds on MS serum and negative selection rounds on healthy control serum were alternated to optimize the selection procedure. As a result, a substantial decrease of IgG-displaying phage clones was observed after each negative selection round, thereby preventing an overgrowth of IgG-displaying phage clones. Our depletion strategy was therefore successful in preventing the enrichment of IgG-displaying phage clones. This approach will facilitate the identification of possible MS-related antigens.

Exonuclease enhances hybridization efficiency: improved direct cycle sequencing and point mutation detection.

Solution hybridization is an essential step in sequencing and some point mutation detection methods. In practice, this hybridization is hampered resulting in the need of additional purification of the amplification products. The use of T7 gene 6 exonuclease may lead to efficient production of single-stranded DNA. In this study, the effect of pretreatment with exonuclease on direct cycle sequencing and point mutation detection was analyzed. Exonuclease-treated products were directly cycle sequenced without further purification. This resulted in highly efficient quality improvement for sequencing allowing detection of heterozygotes. Point mutation detection by Point-EXACCT (exonuclease-amplification coupled capture technique) demonstrated detection of one cell containing a mutation in an excess of 75000 wild type cells. Exonuclease-enhanced detection methods offer simple, rapid detection strategies that are easily adaptable for widespread clinical laboratory use. With the use of exonuclease, the detection of heterozygosity using fluorescent cycle sequencing is becoming more reliable. The high sensitivity of Point-EXACCT due to the use of exonuclease makes it a highly promising method for large-scale screening of (pre)malignant changes in patients with a high risk for developing cancer.

Alcohol potentiates orthostatic hypotension : implications for alcohol-related syncope.

BACKGROUND: Alcohol consumption may be linked to syncopal events. The mechanisms by which alcohol may induce syncope are unknown. Impairment of the response to orthostatic stress may be involved. Using a double-blind, randomized, placebo-controlled study, we tested the hypothesis that short-term alcohol intake causes orthostatic hypotension because of an impairment in the vasoconstrictor response to orthostatic stress. METHODS AND RESULTS: We examined the effects of alcohol on blood pressure, heart rate, and forearm vascular resistance (FVR) during orthostatic stress achieved by stepwise increases in lower-body negative pressure (LBNP) in 14 healthy young volunteers. During the placebo session, blood pressure did not change significantly during LBNP at -5, -10, and -20 mm Hg. A significant decrease in blood pressure was evident only at -40 mm Hg. In contrast, blood pressure fell significantly at all levels of LBNP during the alcohol session. Compared with placebo, alcohol potentiated the hypotensive responses to LBNP, particularly at -40 mm Hg, when the decrease in systolic blood pressure after alcohol intake (-14 mm Hg) was double that after placebo intake (-7 mm Hg). FVR increased with LBNP after placebo. However, after alcohol intake, FVR did not increase during LBNP despite the potentiated decrease in blood pressure. FVR responses during LBNP were reduced during alcohol compared with placebo consumption (P=0.04). CONCLUSIONS: Short-term alcohol consumption elicits hypotension during orthostatic stress because of impairment of vasoconstriction. These findings have implications for the understanding of the hemodynamic effects of alcohol and, in particular, for understanding syncopal events that occur in association with alcohol intake.

Predisposition to vasovagal syncope in subjects with blood/injury phobia.

BACKGROUND: Most subjects with blood/injury phobia experience syncope or presyncope as part of the phobic response. We tested the hypothesis that these subjects have a constitutional autonomic dysregulation that predisposes them to vasovagal syncope during head-up tilt. METHODS AND RESULTS: We studied 11 subjects (9 females, 2 males) who had a history of syncope or presyncope only in response to a blood or injury stimulus and 11 healthy matched controls (10 females, 1 male) without a history of syncope. Blood pressure (BP) and heart rate (HR) were measured during a 15-minute baseline period with subjects in the supine position and then during 45 minutes of head-up tilt to 70 degrees. Measurements at rest did not differ between the blood phobic and control subjects. During tilt, 9 (82%) of the 11 blood phobic subjects experienced presyncope or syncope, leading to termination of the study after 22+/-17 minutes of tilt. Only 1 (9%) of the 11 control subjects experienced presyncope (chi(2)=11.7, P=0.001). Hemodynamic responses to tilt were consistent with a vasovagal mechanism in the blood phobic subjects, with simultaneous decreases in BP and HR during tilt. During tilt, systolic BP fell by 21+/-15 mm Hg (P=0.001), and HR fell by 22+/-25 bpm (P=0.01). By contrast, BP and HR were very stable in the control group. CONCLUSIONS: Subjects with syncope related to blood/injury phobia have an underlying autonomic dysregulation predisposing them to neurally mediated syncope, even in the absence of any blood or injury stimulus. Fainting related to these stimuli may in large part be due to dysfunction in neural circulatory control, which may secondarily lead to the phobia because of repeated syncopal events.

Sympathetic activation by sildenafil.

BACKGROUND: Sildenafil citrate is an effective and widely prescribed therapy for erectile dysfunction. Little is known about the effects of sildenafil on neural control of the circulation or about the effects of sildenafil on neurocirculatory stress responses. METHODS AND RESULTS: We studied 14 normal volunteers (age 32+/-7 years) who were randomized in a double-blind crossover fashion to receive a single oral dose of sildenafil 100 mg or placebo on 2 separate study days. Blood pressure, heart rate, forearm vascular resistance, muscle sympathetic nerve activity, and plasma catecholamines were measured at baseline and at 30 and 60 minutes after sildenafil and after placebo administration. The effects of sildenafil and placebo on neural and circulatory responses to stressful stimuli (sustained handgrip, maximal forearm ischemia, mental stress, and the cold pressor test) were also evaluated. Blood pressure, heart rate, and forearm vascular resistance after sildenafil and placebo were similar. However, muscle sympathetic nerve activity increased strikingly after sildenafil (by 141+/-26%, mean+/-SEM) compared with placebo (3+/-8%) (P=0.006); plasma norepinephrine levels also increased by 31+/-5% after sildenafil administration (P=0.004). Sympathetic nerve traffic during mental, physical, and cold stresses was 2- to 8-fold higher after sildenafil than with placebo (P<0.05). CONCLUSIONS: Sildenafil causes a marked increase in sympathetic activation, evident both at rest and during stressful stimuli. Sympathetic activation by sildenafil may have implications for understanding cardiovascular events associated with sildenafil use.

Enhanced sympathetic and ventilatory responses to central chemoreflex activation in heart failure.

BACKGROUND: Sympathetic activation and respiratory abnormalities may each be implicated in the pathophysiology of congestive heart failure (CHF). Chemoreflexes are an important mechanism regulating both sympathetic drive and breathing. We therefore tested the hypothesis that chemoreflex function is altered in CHF. METHODS AND RESULTS: We compared ventilatory, sympathetic, heart rate, and blood pressure responses to hypoxia, hypercapnia, and the cold pressor test in 9 patients with CHF and 9 control subjects matched for age and body mass index. Baseline muscle sympathetic nerve activity (MSNA) was higher in the patients with CHF compared with control subjects (47+/-8 versus 23+/-3 bursts per minute, P<0.01). During hypercapnia, patients with CHF had greater increases in minute ventilation (6.7+/-1.4 versus 2.7+/-0.9 L/min, P=0.03) and heart rate (7.0+/-2.1 versus 0.6+/-1.2 bpm, P=0.02). Despite higher ventilation, which inhibits sympathetic activity, the MSNA increase in patients with CHF was also greater than that in control subjects (58+/-12% versus 21+/-9%, P=0.03). Ventilatory, autonomic, and blood pressure responses to hypoxia and the cold pressor test in CHF patients were not different from those in control subjects. CONCLUSIONS: Chronic heart failure is characterized by a selective potentiation of ventilatory and sympathetic responses to central chemoreceptor activation by hypercapnia.

Independent association between plasma leptin levels and heart rate in heart transplant recipients.

BACKGROUND: Leptin, the protein product of the ob gene, has been linked to a faster heart rate in animal and human studies. The interaction between leptin and heart rate in the denervated heart is not known. Therefore, we studied the relationship between plasma leptin levels and heart rate in heart transplant recipients. METHODS AND RESULTS: We studied 32 male patients (mean age, 56.5+/-9.3 years; range, 41 to 74 years) after orthotopic heart transplantation. All subjects underwent a physical examination, anthropometric measurements, blood chemistry analysis, and office blood pressure measurements. A blood sample was collected from each subject while fasting. In univariate analysis, heart rate was related to leptin levels (r=0.47, P=0.007) but heart rate was not related to systolic or diastolic blood pressure, mean arterial pressure, body mass index, or catecholamines. Leptin levels were only strongly associated with heart rate and body mass index (r=0.73, P<0.0001). In multivariate analysis, heart rate was independently and positively associated with leptin levels (F=2.61, P=0.017). We also observed a strong, independent association between leptin levels and body mass index (F=5.8, P<0.00001). CONCLUSIONS: We show an independent association between leptin levels and heart rate in heart transplant recipients. We speculate that this may be due, in part, to a direct effect of leptin on heart rate, conceivably mediated through cardiac leptin receptors.

Differential characteristics of neural circulatory control: early versus late after cardiac transplantation.

BACKGROUND: Reappearance of low-frequency (LF) (+/-0.10 Hz) oscillations in RR interval (RR) after cardiac transplantation is indicative of sympathetic efferent reinnervation. We hypothesized that restored LF oscillations in RR in heart transplant recipients (HTRs) are linked to oscillations in muscle sympathetic nerve traffic (MSNA). METHODS AND RESULTS: RR, RR variability, and MSNA were recorded 5+/-2 months (n=7, short-term HTRs) and 138+/-8 months (n=7, long-term HTRs) after heart transplantation and compared with matched hypertensive patients (n=7). A coherence function determined the coupling between LF oscillations in MSNA and RR. RR variance did not differ between short-term and long-term HTRs. However, LF variability was only 1+/-0.5 ms(2) in the short-term HTRs but was 15+/-8 ms(2) in the long-term HTRs (P<0.05). Normalized LF variability was also higher in the long-term HTRs (40+/-14 normalized unites) versus the short-term HTRs (6+/-3 normalized united, P<0.05) but did not differ from the LF variability of the hypertensive patients. Long-term HTRs were taking less cyclosporine (P<0.01) but had higher MSNA than the short-term HTRs (62+/-7 versus 31+/-7 burst/min, respectively, P<0.05). Coherence between LF oscillations in MSNA and RR was similar in the long-term HTRs (0.59+/-0.11) and the hypertensive patients (0.60+/-0.07) and was 3-fold greater than in the short-term HTRs (0.20+/-0.06, P<0.05). CONCLUSIONS: Cardiac reinnervation after long-term heart transplantation is characterized by a restoration of the coherence between LF oscillations in RR and MSNA. Higher MSNA in long-term than in short-term HTRs suggests that time elapsed after cardiac transplantation may be a major determinant of sympathetic excitation in heart transplant recipients.

Impairment of endothelium-dependent vasodilation of resistance vessels in patients with obstructive sleep apnea.

BACKGROUND: Patients with obstructive sleep apnea (OSA) experience repetitive episodic hypoxemia with consequent sympathetic activation and marked blood pressure surges, each of which may impair endothelial function. We tested the hypothesis that patients with OSA have impaired endothelium-dependent vasodilation, even in the absence of overt cardiovascular disease. METHODS AND RESULTS: We studied 8 patients with OSA (age 44+/-4 years) and 9 obese control subjects (age 48+/-3 years). Patients with OSA were newly diagnosed, never treated for OSA, on no medications, and free of any other known diseases. All obese control subjects underwent complete overnight polysomnographic studies to exclude occult OSA. Resistance-vessel function was tested by use of forearm blood flow responses to intra-arterial infusions of acetylcholine (a vasodilator that stimulates endothelial release of nitric oxide), sodium nitroprusside (an exogenous nitric oxide donor), and verapamil (a calcium channel blocker). Conduit-vessel function was also evaluated by ultrasonography. Brachial artery diameter was measured under baseline conditions, during reactive hyperemia (with flow increase causing endothelium-dependent dilatation), and after sublingual administration of nitroglycerin (an endothelium-independent vasodilator). Patients with OSA had a blunted vasodilation in response to acetylcholine (P:<0.007), but responses to sodium nitroprusside and verapamil were not significantly different from those of control subjects. No significant difference in conduit-vessel dilation was evident between OSA patients and obese control subjects. CONCLUSIONS: Patients with OSA have an impairment of resistance-vessel endothelium-dependent vasodilation. This may be implicated in the pathogenesis of hypertension and heart failure in this condition.

Detection of K-ras point mutations in sputum from patients with adenocarcinoma of the lung by point-EXACCT.

PURPOSE: Kirsten ras (K-ras) point mutations are found in 30% to 56% of pulmonary adenocarcinomas by means of highly sensitive techniques. Recently, the Point-EXACCT (point mutation detection using exonuclease amplification coupled capture technique) method was described, which detected one cell with a mutation in 15,000 normal cells. The aim of this study was to examine whether K-ras point mutations could be found with this rapid method in the sputum of patients with adenocarcinoma of the lung. PATIENTS AND METHODS: DNA from paraffin-embedded adenocarcinoma and corresponding sputum samples were analyzed for mutations of the K-ras gene. Twenty-eight biopsy specimens and 54 sputum samples of 22 patients were used for amplification and K-ras codon 12 point mutation detection. RESULTS: In 11 of 22 patients (50%), a mutation in K-ras codon 12 was shown in the tumor sample. In five of 11 patients (45%) with a K-ras mutation in the tumor, the same type of mutation was identified in at least one sputum sample. A mutation could not be detected in any of the sputum samples from patients with a K-ras-negative tumor. Time between K-ras point mutation detection in sputum and clinical diagnosis of lung cancer varied from 1 month to almost 4 years. In two of the five patients with K-ras-positive sputum specimens, malignant cells were found with cytologic examination. CONCLUSION: Point-EXACCT is suitable for the detection of K-ras point mutations in sputum samples of patients with adenocarcinoma of the lung. This approach may be an important adjunct to cytology in the early diagnosis of lung cancer.