Extent and magnitude of low-voltage areas assessed by ultra-high-density electroanatomical mapping correlate with left atrial function.

INTRODUCTION: The extent of left atrial (LA) adverse remodeling as a cardiac disease marker has become increasingly important. In patients with atrial fibrillation (AF), atrial remodeling (AR) is accompanied by increased mortality. The relation between LA function and the extent of low-voltage areas (LVAs) has not yet been systematically investigated. METHODS: In patients with AF undergoing catheter-ablation, LA was studied using echocardiography and ultra-high-density mapping (Rhythmia®). Fibrosis (i.e. extent of LVAs) was estimated by quantifying areas with bipolar electrogram amplitudes of ≤0.5, ≤0.4, ≤0.3, ≤0.2 or ≤0.1 mV. RESULTS: A total of 22 patients with a mean LVEF of 53 ±â€¯2% was studied. Mean LA volume index (LAVI) was significantly increased at 39 ±â€¯3 ml/m2 indicating AR. Size of LVAs was 57 ±â€¯7 cm2 representing 47 ±â€¯5% of the total LA area (low-voltage set to ≤0.5 mV). With low-voltage set to ≤0.4, ≤0.3, ≤0.2 and ≤0.1, total area decreased to 34 ±â€¯6, 28 ±â€¯6, 22 ±â€¯5 and 12 ±â€¯3 cm2. LAVI positively correlated with the extent of LVAs at all cut-offs. Mean LA emptying fraction was 42 ±â€¯3% and showed a negative correlation with LVAs with low-voltage set to ≤0.4 mV. Moreover, mean LA strain was 13 ±â€¯2% and correlated with LVAs with low-voltage at all cut-offs further supporting the notion that the extent of LVAs impacts LA function. Notably, with low-voltage set to ≤0.2, ≤0.3 and ≤0.4 mV impaired LA strain was detected with an accuracy of >76% (p < 0.05). CONCLUSION: Structural (i.e. LAVI) and functional (i.e. LA emptying fraction and LA strain) parameters of the LA correlate with the extent of LVAs.

Pathophysiological and therapeutic implications in patients with atrial fibrillation and heart failure.

Heart failure and atrial fibrillation are common and responsible for significant mortality of patients. Both share the same risk factors like hypertension, ischemic heart disease, diabetes, obesity, arteriosclerosis, and age. A variety of microscopic and macroscopic changes favor the genesis of atrial fibrillation in patients with preexisting heart failure, altered subcellular Ca2+ homeostasis leading to increased cellular automaticity as well as concomitant fibrosis that are induced by pressure/volume overload and altered neurohumoral states. Atrial fibrillation itself promotes clinical deterioration of patients with preexisting heart failure as atrial contraction significantly contributes to ventricular filling. In addition, atrial fibrillation induced tachycardia can even further compromise ventricular function by inducing tachycardiomyopathy. Even though evidence has been provided that atrial functions significantly and independently of confounding ventricular pathologies, correlate with mortality of heart failure patients, rate and rhythm controls have been shown to be of equal effectiveness in improving mortality. Yet, it also has been shown that cohorts of patients with heart failure benefit from a rhythm control concept regarding symptom control and hospitalization. To date, amiodarone is the most feasible approach to restore sinus rhythm, yet its use is limited by its extensive side-effect profile. In addition, other therapies like catheter-based pulmonary vein isolation are of increasing importance. A wide range of heart failure-specific therapies are available with mixed impact on new onset or perpetuation of atrial fibrillation. This review highlights pathophysiological concepts and possible therapeutic approaches to treat patients with heart failure at risk for or with atrial fibrillation.

Dyssynchronous calcium removal in heart failure-induced atrial remodeling.

We tested the hypothesis that in atrial myocytes from a rabbit left ventricular heart failure (HF) model, spatial inhomogeneity and temporal dyssynchrony of Ca removal during excitation-contraction coupling together with increased Na/Ca exchange (NCX) activity generate a substrate for proarrhythmic Ca release. Ca removal occurs via Ca reuptake into the sarcoplasmic reticulum and extrusion via NCX exclusively in the cell periphery since rabbit atrial myocytes lack transverse tubules. Ca removal kinetics were assessed by the time constant τ of decay of local peripheral subsarcolemmal (SS) and central (CT) action potential (AP)-induced Ca transients (CaTs) recorded in confocal line scan mode (using Fluo-4). Spatial and temporal dyssynchrony of Ca removal was quantified by CV TAU, defined as the standard deviation of local τ along the transverse cell axis divided by mean τ. In normal cells CT CaT decline was slower compared with the SS domain, while in HF cells decline was accelerated, became equal in SS and CT regions, and a significant increase of CV TAU indicated an increased Ca removal dyssynchrony. In HF atrial cells NCX upregulation was accompanied by an overall higher incidence of spontaneous Ca waves and a higher propensity of arrhythmogenic Ca waves, defined as waves that triggered APs due to NCX-mediated membrane depolarization. NCX inhibition normalized CV TAU in HF atrial cells and decreased the propensity of Ca waves. In summary, HF atrial myocytes show accelerated but dyssynchronous diastolic Ca removal and altered sarcoplasmic reticulum Ca-ATPase (SERCA) and NCX activity that result in increased susceptibility to arrhythmia.

Socio-economic effects and cost saving potential of remote patient monitoring (SAVE-HM trial).

OBJECTIVE: In the SAVE-trial we evaluated the safety, reliability and improvements of patient management using the BIOTRONIK Home Monitoring®-System (HM) in pacemaker (PM) and implanted cardioverter defibrillator (ICD) patients. DESIGN: 115 PM (Module A) and 36 ICD-patients (Module B) were recruited 3 months after implantation. PATIENTS: 65 patients in Module A were randomised to HM-OFF and had one scheduled outpatient clinic follow-up(FU) per year, whereas patients randomised to HM-ON were equipped with the mobile transmitter and discharged without any further scheduled in-office FU. In Module B 18 patients were randomised to HM-OFF and followed by standard outpatient clinic controls every 6 months; 18 patients were randomised to HM-ON receiving remote monitoring plus one outpatient clinic visit per year; unscheduled follow-ups were performed when necessary. RESULTS: The average follow-up period was 17.1 ± 9.2 months in Module A and 26.3 ± 8.6 months in Module B. In both modules, the number of FUs per year was significantly reduced (Module A HM-ON 0.29 ± 0.6 FUs/year vs HM-OFF 0.53 ± 0.5 FUs/year; p b 0.001; Module B HM-ON 0.87 ± 0.25 vs HM-OFF 1.73 ± 0.53 FU/year,p b 0.001). Cost analysis was significantly lower in the HM-ON group compared to the HM-OFF group (18.0 ± 41.3 and 22.4 ± 26.9 € respectively; p b 0.003). 93% of the unscheduled visits in Module B were clinically indicated,whereas 55% of the routine FUs were classified as clinically unnecessary. CONCLUSION: Remote home monitoring of pacemaker and ICD devices was safe, reduced overall hospital visits, and detected events that mandated unscheduled visits.

JTV519 (K201) reduces sarcoplasmic reticulum Ca²âº leak and improves diastolic function in vitro in murine and human non-failing myocardium.

BACKGROUND AND PURPOSE: Ca²âº leak from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyR2s) contributes to cardiomyocyte dysfunction. RyR2 Ca²âº leak has been related to RyR2 phosphorylation. In these conditions, JTV519 (K201), a 1,4-benzothiazepine derivative and multi-channel blocker, stabilizes RyR2s and decrease SR Ca²âº leak. We investigated whether JTV519 stabilizes RyR2s without increasing RyR2 phosphorylation in mice and in non-failing human myocardium and explored underlying mechanisms. EXPERIMENTAL APPROACH: SR Ca²âº leak was induced by ouabain in murine cardiomyocytes. [Ca²âº]-transients, SR Ca²âº load and RyR2-mediated Ca²âº leak (sparks/waves) were quantified, with or without JTV519 (1 µmol·L⁻¹). Contribution of Ca²âº -/calmodulin-dependent kinase II (CaMKII) was assessed by KN-93 and Western blot (RyR2-Ser(2814) phosphorylation). Effects of JTV519 on contractile force were investigated in non-failing human ventricular trabeculae. KEY RESULTS: Ouabain increased systolic and diastolic cytosolic [Ca²âº](i) , SR [Ca²âº], and SR Ca²âº leak (Ca²âº spark (SparkF) and Ca²âº wave frequency), independently of CaMKII and RyR-Ser(2814) phosphorylation. JTV519 decreased SparkF but also SR Ca²âº load. At matched SR [Ca²âº], Ca²âº leak was significantly reduced by JTV519, but it had no effect on fractional Ca²âº release or Ca²âº wave propagation velocity. In human muscle, JTV519 was negatively inotropic at baseline but significantly enhanced ouabain-induced force and reduced its deleterious effects on diastolic function. CONCLUSIONS AND IMPLICATIONS: JTV519 was effective in reducing SR Ca²âº leak by specifically regulating RyR2 opening at diastolic [Ca²âº](i) in the absence of increased RyR2 phosphorylation at Ser(2814) , extending the potential use of JTV519 to conditions of acute cellular Ca²âº overload.

Filaria/Wolbachia activation of dendritic cells and development of Th1-associated responses is dependent on Toll-like receptor 2 in a mouse model of ocular onchocerciasis (river blindness).

Toll-like receptors (TLRs) regulate dendritic cell function and activate signals that mediate the nature of the adaptive immune response. The current study examined the role of TLRs in dendritic cell activation and in regulating T cell and antibody responses to antigens from the filarial parasites Onchocerca volvulus and Brugia malayi, which cause river blindness and lymphatic filariasis, respectively. Bone-marrow-derived CD11c(+) cells from C57BL/6 and TLR4(-/-) mice produced high levels of IL-6 and RANTES, and showed elevated surface CD40 expression, whereas CD11c(+) cells from myeloid differentiation factor 88(-/-) (MyD88(-/-)), TLR2(-/-) and TLR2/4(-/-) mice were not activated. Similarly, IFN-gamma production by splenocytes from immunized TLR2(-/-) mice was significantly impaired compared with splenocytes from C57BL/6 and TLR4(-/-) mice. In contrast, there was no difference among these strains in Th2-associated responses including IL-5 production by splenocytes from immunized animals, serum IgE and IgG(1), or eosinophil infiltration into the corneal stroma. Neutrophil recruitment to the cornea and CXC chemokine production was inhibited in immunized TLR2(-/-) mice compared with C57BL/6 and TLR4(-/-) mice. Taken together, these findings demonstrate an essential role for TLR2 in filaria-induced dendritic cell activation, IFN-gamma production and neutrophil migration to the cornea, but does not affect filaria-induced Th2-associated responses.

Role of connexin 43 in ischemic preconditioning does not involve intercellular communication through gap junctions.

Connexin 43 (Cx 43) has recently been implicated in protection of ischemic preconditioning. Cx 43 colocalization with protein kinase C and p38 mitogen-activated protein kinase is increased in preconditioned myocardium, Cx 43 phosphorylation is preserved in preconditioned myocardium, and hearts from Cx 43-deficient mice cannot be preconditioned. It is, however, unclear whether the important role of Cx 43 relates to intercellular communication through gap junctions or its function in volume homeostasis. To address this issue, we used isolated cardiomyocytes, which no longer-form gap junctions, from wild-type (n = 5) and heterozygous Cx 43-deficient mice (n = 8) and subjected them to 2 h simulated ischemia (hypoxia, acidosis) and an additional challenge by extracellular hypo-osmolarity (from 310 to 250 mOsm/l). Viability (trypan blue exclusion) was well maintained in normoxic wild-type cardiomyocytes (54 +/- 5% at baseline vs. 46 +/- 4 (mean +/- S.D.) % at 2 h). With simulated ischemia, viability was reduced to 17 +/- 5%. Preconditioning by a preceding exposure to 10 min simulated ischemia and 15 min reoxygenation preserved viability after 2 h simulated ischemia (36 +/- 1%, P < 0.001 vs. simulated ischemia). In Cx 43-deficient cardiomyocytes, viability was also well maintained in normoxia (56 +/- 10% vs. 44 +/- 10%). Viability was also reduced to 17 +/- 6% with 2 h simulated ischemia. In contrast to wild-type cells, preconditioning did not prevent the reduction in viability (18 +/- 8%). In conclusion, Cx 43 is essential for preconditioning in the absence of gap junctions, supporting its function through improved volume regulation.

Extrapolation to zero-flow pressure in cerebral arteries to estimate intracranial pressure.

BACKGROUND: Cerebral perfusion pressure (CPP) is commonly calculated from the difference between arterial blood pressure (AP) and intracranial pressure (ICP). ICP can be considered the effective downstream pressure of the cerebral circulation. Consequently, cerebral circulatory arrest would occur when AP equals ICP. Estimation of AP for zero-flow pressure (ZFP) may thus allow estimation of ICP. We estimated ZFP from cerebral pressure-flow velocity relationships so that ICP could be measured by transcranial Doppler sonography. METHODS: We studied 20 mechanically ventilated patients with severe head injury, in whom ICP was monitored by epidural pressure transducers. AP was measured with a radial artery cannula. Blood flow velocity in the middle cerebral artery (V(MCA)) ipsilateral to the site of ICP measurement was measured with a 2 MHz transcranial Doppler probe. All data were recorded by a microcomputer from analogue-digital converters. ZFP was extrapolated by regression analysis of AP-V(MCA) plots and compared with simultaneous measurements of ICP. RESULTS: ZFP estimated from AP-V(MCA) plots was linearly related to ICP over a wide range of values (r=0.93). There was no systematic difference between ZFP and ICP. Limit of agreement (2 SD) was 15.2 mm Hg. Short-term variations in ICP were closely followed by changes in ZFP. CONCLUSION: Extrapolation of cerebral ZFP from instantaneous AP-V(MCA) relationships enables detection of severely elevated ICP and may be a useful and less invasive method for CPP monitoring than other methods.

Isolation and morphology of single Purkinje cells from the porcine heart.

Purkinje cells were isolated from both ventricles of young adult domestic pigs and examined by transmitted light or laser scanning confocal microscopy. Purkinje cells in free running Purkinje fibres were organised in multicellular strands where individual cells were tightly connected end-to-end and closely side-to-side. After isolation, single cells gradually lost the elongated appearance and became more rounded, but the cell membrane remained smooth and undamaged. The contractile material was not very dense and was seen most clearly in the submembraneous area. Staining of the cell membrane with the lipophilic fluorescent (lye di-8-ANNEPS, and visualization with confocal microscopy, confirmed that the cell surface membrane was smooth without blebs. This staining also showed that Purkinje cells had no transversal tubules. We reconstructed the three-dimensional geometry of the Purkinje cells and determined the cell size. The average values were 62 +/- 9 microm for length, 32 +/- 3 microm for width, and 41 +/- 4 microm for depth (n = 7). Calculated cross-section area and volume were 1047 +/- 167 microm2 and 47 +/- 14 pl. Compared to ventricular cells, the morphology of the Purkinje cells reflects their specific role in impulse conduction.

Deactivation of the innate cellular immune response following endotoxic and surgical injury.

The innate cellular immune (iCMI) system provides for the rapid production of interferon-gamma (IFN gamma) by NK cells in response to microbial threats. In this review, we examine the cellular and cytokine mechanisms of innate cellular immunity as determined in murine endotoxemia. This will be contrasted to the subsequent suppression of these same responses present in the mouse model of endotoxin tolerance, which is characterized by profound deficiency in both IL-12 and IFN gamma synthesis. Transient IFN gamma deficiency due to altered iCMI function has also been described in trauma or burn patients and is termed "clinical immune paralysis." If the common pathogenesis of these entities can be better understood, immune-based interventions might be identified for restoring iCMI function. In addition to the gain in basic immunologic insight, research on this subject may deliver future forms of prophylaxis against infection that do not rely on antibiotics and that will not promote antimicrobial resistance.

Stimulation of the left ventricle through the coronary sinus with a newly developed 'over the wire' lead system--early experiences with lead handling and positioning.

AIMS: This report describes the initial clinical results with a newly designed guiding catheter and an 'over the wire' pacing lead based on angiolasty technology to stimulate the left ventricle using the transvenous route via the coronary sinus (OTW-CV lead). METHODS AND RESULTS: In 75% of the 15 patients (6 males, 9 females, mean age of 53 +/- 9 years) with congestive heart failure, access to coronary sinus required less than 2 min; in one patient. the attempt failed. Mean R wave amplitudes plus or minus the standard deviation, measured at apical, mid-ventricular and basal positions the anterior (11.4 +/- 9.2, 10.8 +/- 6.2, 9.3 +/- 6.3 mV) and lateral or posterior veins (10.1 +/- 10.7, 8.6 +/- 6.4, 7.7 +/- 4.3 mV) showed a trend favouring the apex without statistical significance. Pacing impedance, measured at the same sites and vein tributaries, ranged from 670 +/- 191 to 915 +/- 145 ohms. Pacing thresholds measured at apical and mid ventricular sites were significantly lower than at the base in the anterior vein 2.5 +/- 2.8 and 2.8 +/- 1.8 vs 5.6 +/- 2.7 V at 0.5 ms, P<0.001). Thresholds in the lateral/posterior veins showed a similar trend but did not reach statistical significance (3.0 +/- 1.7, 3.6 +/- 1.4 +/- 1.8 V at 0.5 ms). In patients, in whom thresholds were determined in more than one vein, the 'best' mean threshold was 1.6 +/- 0.7 V. CONCLUSION: The new 'over the wire' lead and guiding catheter system allows uncomplicated access to the coronary sinus and the depth of the coronary vein tributaries. Left ventricular sensing and pacing thresholds are acceptable for chronic use in implanted cardiac rhythm management systems.

[Experiences with a new transvenous electrode for left ventricular stimulation]. / Erfahrungen mit einer neuen transvenösen Sonde zur linksventrikulären Stimulation.

BACKGROUND: Left ventricular and biventricular pacing has recently been introduced as a new therapy for chronic heart failure in selected patients. We report our initial experience with a new electrode for transvenous left epicardial pacing via tributaries of the coronary sinus. PATIENTS AND METHOD: Inclusion criteria were: chronic heart failure NYHA > or = II, QRS-duration > 120 ms, left ventricular ejection fraction < 35%. Dual chamber pacemakers (CPI Contak TR) or defibrillators (CPI Contak CD) designed for atrial triggered biventricular stimulation were implanted in conjunction with the CPI Easytrak-lead for left ventricular pacing in a coronary vein. Lead placement was achieved via a subclavian vein access and a preformed guiding catheter for coronary sinus insertion. RESULTS: In 13 of 16 patients (81%) the left ventricular lead was implanted successfully in a mid to distal posterior or anterolateral vein. Lead insertion could not be achieved in 2 patients with significant cardiomegaly and right atrial enlargement (12.5%), while 1 patient with a history of myocardial infarction and small anterior ventricular aneurysm had inacceptable high left ventricular pacing thresholds intraoperatively. The implantation was well tolerated by all patients without complications. There was no case of lead dysfunction (mean follow-up time: 142 +/- 126 days). Intraoperative electrode measurements and chronic parameters (> or = 3 months, n = 8) are given in Table 1. CONCLUSION: In the past left ventricular pacing has mainly been achieved by epicardially placed electrodes after thoracotomy with conventional electrodes. This new approach for chronic left ventricular pacing uses the familiar transvenous over-the-wire technique in combination with a newly developed guiding catheter and electrode for pacing in left epicardial veins. Lead placement was shown to be safe and success rate was higher than in previous reports with standard electrodes. We conclude that left epicardial lead placement with the over-the-wire technique and a preformed guiding catheter for coronary sinus access presents as a safe and maybe more efficient method for left ventricular pacing.

[Cardiac resynchronization therapy in terminal heart failure: current status and prospects]. / Die kardiale Resynchronisationstherapie bei terminaler Herzinsuffizienz. Aktueller Stand und Perspektiven.

BACKGROUND: With regard to epidemiological aspects, heart failure has been shown an increasing incidence in constrast to coronary artery disease which counts decreasingly due to secondary and primary prevention. The present data show an incidence for heart failure of 2% per year. 4-5 million people are newly affected by the disease. The prognosis is limited after diagnosis is confirmed. According to the US Framingham study, the median life expectancy is 3.2 and 5.4 after diagnosis for male and female, respectively. For patients in an advanced stage of the disease the mortality rate is 27% within 3 years. AV SEQUENTIAL PACING: The introduction of AV sequential pacing by the Austrian group of Margarete Hochleitner in 1992 showed an improved left ventricular systolic function, an improved clinical benefit in terms of NYHA classification, an enhanced left ventricular ejection fraction, an improved systolic and diastolic blood pressure, a reduction of the heart-chest relationship as well as a reduction of the resting hart rate and the echocardiographic dimension parameters. STUDIES: First experimental approach for biventricular stimulation, which means the simultaneous activation of the right and the left chamber, relied on the observation of a distorted ventricular activation due to the presence of a bundle branch block. The bundle branch block is a result of the dilatation of the myocardial fibers, death of myocardial cells which are replaced by fibrous tissue. Resynchronization of both ventricles was associated with an improved left ventricular function and improved diastolic relaxation. Clinical studies of patients with heart failure, severe left ventricular systolic dysfunction, and left bundle branch block have shown that systolic function can be improved by electrically stimulating the site of late activation. The magnitude of the improvement seems to be associated with the duration of the intrinsic surface QRS complex and whether the ventricle ipsilateral with the conduction defect is stimulated. The effect of ventricular resynchronization therapy was optimized by timing of atrioventricular activation associated with a decrease in both systolic and diastolic mitral regurgitation. CONCLUSION: The prognosis of patients with end-stage heart failure is limited by two determinants: myocardial pump failure and sudden (arrhythmogenic) cardiac death. Due to the fact that the incidence for sudden cardiac death is considerably high in patients with end-stage heart failure it is reasonable to include the implantation of cardioverters/defibrillaters (ICD) in the concept of biventricular stimulation.

Pretreatment with recombinant Flt3 ligand partially protects against progressive cutaneous leishmaniasis in susceptible BALB/c mice.

Dendritic cells are potent antigen-presenting cells that also produce interleukin-12 (IL-12) during innate and adaptive cellular immune responses and that thereby promote the differentiation of gamma interferon (IFN-gamma)-producing Th1-type CD4(+) T lymphocytes. We hypothesized that expanded dendritic-cell populations in mice pretreated with the hematopoietic cytokine Flt3L would protect against cutaneous Leishmania major infection. Pretreatment of disease-susceptible BALB/c mice with 10 microg of recombinant Flt3L (rFlt3L) for 9 to 10 days before infection increased lymph node IL-12 p40 productive capacity 20-fold compared to that of saline-injected controls. Furthermore, 9 of 22 (40.9%) rFlt3L-pretreated BALB/c mice resolved their cutaneous infections, whereas none of the 22 control BALB/c mice healed. Healed, rFlt3L-pretreated mice did not develop disease following reinfection. Flt3L pretreatment also reduced parasite numbers 1,000-fold in the cutaneous lesions at 2 weeks after infection relative to numbers in lesions of untreated controls. However, Flt3L pretreatment did not significantly alter L. major-induced IFN-gamma and IL-4 production in lymph node culture at 1, 2, and 4 weeks after infection. Despite the lack of Th immune deviation, Flt3L ligand-pretreated lymph nodes expressed up to 10-fold higher levels of IL-12 p40 and inducible (type 2) nitric oxide synthase mRNA at 7 days after infection. In contrast, treatment with rFlt3L after infection failed to protect against disease despite comparable expansions of dendritic cells and IL-12 p40 productive capacity in both infected and uninfected BALB/c mice treated with rFlt3L. We conclude that rFlt3L pretreatment before infection with L. major reduces parasite load and promotes healing of cutaneous lesions without stable cytokine deviation towards a dominant Th1 cytokine phenotype.

IFN-gamma is necessary but not sufficient for anti-CD40 antibody-mediated inhibition of the Th2 response to Schistosoma mansoni eggs.

The injection of Schistosoma mansoni eggs into the footpads of mice results in a localized Th2 cytokine response and tissue eosinophilia. We examined whether treatment with CD40-activating Abs would block the development of Th2 cytokine responses and eosinophilic tissue pathology in this model. Seven days after C57BL/6 mice were injected with eggs and the FGK45 anti-CD40 Ab, Ag-specific synthesis of IL-4, IL-5, and IL-13 in lymph node culture was reduced (>10-fold) relative to control mice treated with eggs and rat IgG. In contrast, IFN-gamma and IL-12 were increased in both culture supernatants and in the serum. Similar changes in lymph node cytokine mRNA were observed in vivo, and tissue eosinophilia was reduced nearly 20-fold. Th2 cytokine responses in anti-CD40-treated IFN-gamma-/- and IL-12 p40-/- C57BL/6 mice were unaffected, although anti-CD40 induced high levels of systemic and local IFN-gamma production in both wild-type and IL-12 p40-/- mice. We conclude that CD40-activating treatments strongly reverse the immune phenotype generated in response to a classic, Th2-biasing stimulus and stimulate IFN-gamma through a novel IL-12-independent pathway. This model for Th1-deviating immune therapy may have relevance to the treatment of Th2-dependent diseases in general.

Interleukin-4-independent acceleration of cutaneous leishmaniasis in susceptible BALB/c mice following treatment with anti-CTLA4 antibody.

BALB/c mice are susceptible to progressive infection with Leishmania major due to the preferential development of CD4(+) T cells that secrete Th2 cytokines. Although Th2 cell development and susceptibility are disrupted by blockade of CD86 function early in infection, CD28-deficient BALB/c mice remain susceptible to leishmaniasis. We therefore examined whether the alternative CD86 ligand, CTLA4, contributes to the expression of susceptibility. BALB/c mice treated for 2 weeks of infection with anti-CTLA4 monoclonal antibody developed more rapidly progressive disease than sham-treated mice, whereas normally resistant C57BL/6 mice were unaffected. The draining lymph node cells of anti-CTLA4-treated BALB/c mice produced up to sixfold more interleukin-4 (IL-4) and IL-13 than control mice in the first 2 weeks of infection, but IFN-gamma synthesis was reciprocally decreased. Anti-CTLA4 treatment of BALB/c mice pretreated with neutralizing anti-IL-4 antibody or genetically deficient in IL-4 also caused significant worsening of leishmaniasis. Exacerbation in IL-4 KO mice was associated with increased IL-13 and decreased gamma interferon (IFN-gamma) and inducible nitric oxide synthase (iNOS) mRNA expression in vivo. These data indicate that anti-CTLA4 antibody induced earlier and more-polarized Th2 responses in susceptible BALB/c mice infected with L. major. The mechanism of disease worsening was partially IL-4 independent, indicating that increased IL-13 and/or decreased IFN-gamma production may have disrupted nitric oxide-based microbicidal responses. We conclude that CTLA4 significantly modulates Th2 development in murine leishmaniasis and that the Th2-polarizing effects of anti-CTLA4 treatment result in IL-4-independent exacerbation of disease.

Cure of progressive murine leishmaniasis: interleukin 4 dominance is abolished by transient CD4(+) T cell depletion and T helper cell type 1-selective cytokine therapy.

Progressive infection with Leishmania major in susceptible BALB/c mice is mediated by interleukin (IL)-4-producing T helper cell type 2 (Th2) CD4(+) T cells that, once established, become resistant to Th1-deviating therapies with recombinant (r)IL-12 and/or neutralizing anti-IL-4 antibodies. We sought to restore protective immunity in advanced leishmaniasis by depletion of Th2-biased CD4(+) populations and by cytokine-directed reconstitution of Th1 cellular responses during lymphocyte recovery. Treatment with cytolytic GK1.5 anti-CD4 mAb alone did not reverse disease in 3 wk-infected BALB/c mice, but GK1.5 combined with anti-IL-4 antibody and intralesional rIL-12 cured cutaneous lesions in 80% of mice and established a Th1-polarized cytokine response to L. major antigen protective against reinfection. The curative effects of GK1.5 were not replaced by cytotoxic anti-CD8 monoclonal antibody 2.43 or nondepleting anti-CD4 mAb YTS177, confirming that depletion of CD4(+) cells was specific and essential for therapeutic effect. Finally, combined CD4(+) depletion and IL-4 neutralization were curative, indicating that neither increased parasite burden nor altered accessory cell function independently biased towards Th2 reconstitution in advanced leishmaniasis. Advanced leishmaniasis can be cured by T cell depletion and cytokine-directed recovery of Th1 cellular responses, suggesting novel interventions for other immune-mediated diseases and identifying distinct roles for CD4(+) T cell and non-T cell in the maintenance of Th2 and Th1 phenotypes.

Adjuvant-guided type-1 and type-2 immunity: infectious/noninfectious dichotomy defines the class of response.

Traditionally, protein Ags have been injected in CFA (oil with inactivated mycobacteria) to induce immunity and with IFA (oil alone) to induce tolerance. We report here that injection of hen eggwhite lysozyme, a prototypic Ag, in CFA-induced and IFA-induced pools of hen eggwhite lysozyme-specific memory T cells of comparable fine specificity, clonal size, and avidity spectrum, but with type-1 and type-2 cytokine signatures, respectively. This adjuvant-guided induction of virtually unipolar type-1 and type-2 immunity was observed with seven protein Ags and in a total of six mouse strains. Highly polarized type-1 and type-2 immunity are thus readily achievable through the choice of adjuvant, irrespective of the genetic bias of the host and of the nature of the protein Ag. This finding should have far-reaching implications for the development of vaccines against infectious and autoimmune diseases. Furthermore, our demonstration that Ag injected with IFA is as strongly immunogenic for T cells as it is with CFA shows that the presence of the mycobacteria determines not the priming of naive T cells through the second-signal link but the path of downstream differentiation toward CD4 memory cells that express either type-1 or type-2 cytokines.

Endotoxin fails to induce IFN-gamma in endotoxin-tolerant mice: deficiencies in both IL-12 heterodimer production and IL-12 responsiveness.

Mice exposed to sublethal endotoxemia develop short-term endotoxin tolerance, a state characterized by decreased monokine production and enhanced protection against endotoxic lethality. We confirmed that TNF-alpha production is markedly impaired in endotoxin-tolerant mice and additionally found 2- to 6-fold decreases in serum IFN-gamma in these animals following endotoxin challenge. The IFN-gamma deficiency of endotoxin tolerance correlated with 8-fold decreases in the bioactive p40/p35 heterodimeric form of IL-12. In contrast, total circulating IL-12 p40 was reduced by only 30-50%. Endotoxin-tolerant mice were less responsive to IL-12 than control mice, as evidenced by 3-fold lower levels of IFN-gamma inducible in vivo when rIL-12 was administered at the time of endotoxin challenge. Similarly, spleen cell cultures of endotoxin-tolerant mice produced 3-fold less IFN-gamma in the presence of optimal concentrations of both IL-12 and IL-18. Finally, levels of IL-12R beta 2 subunit mRNA and the percent composition of NK lymphocytes in the spleen were both decreased in endotoxin-tolerant mice relative to controls. We conclude that endotoxin-tolerant mice are profoundly impaired in their ability to produce IFN-gamma in response to endotoxin and that this is associated with acquired defects in both the production of circulating IL-12 heterodimer response and the response to IL-12 by NK cells.