A patient with portal hypertension and blindness after transjugular intrahepatic portosystemic shunt.

We report on a case of recurrent variceal bleeding from gastric varices, which was treated with transjugular intrahepatic portosystemic shunt (TIPS) and Histoacryl injection into the gastric varices. Furthermore, the patient had a small patent foramen ovale without a right-to-left shunt. After the intervention, the patient developed acute neurological disorders as a result of a cerebral paradoxical embolism. In the following, we describe the potential risk of histoacryl in paradoxical embolization when used for the injection of variceal collaterals during TIPS placement in patients with portal hypertension. The present case report shows a very rare but important complication after TIPS implantation. To avoid this complication it is recommended to perform echocardiography before all TIPS placements.

L-carnitine and creatine in Friedreich's ataxia. A randomized, placebo-controlled crossover trial.

Impaired oxidative phosphorylation is a crucial factor in the pathogenesis of Friedreich's ataxia (FA). L-carnitine and creatine are natural compounds that can enhance cellular energy transduction. We performed a placebo-controlled triple-phase crossover trial of L-carnitine (3 g/d) and creatine (6.75 g/d) in 16 patients with genetically confirmed FA. Primary outcome measures were mitochondrial ATP production measured as phosphocreatine recovery by 31Phosphorus magnetic resonance spectroscopy, neurological deficits assessed by the international co-operative ataxia rating scale and cardiac hypertrophy in echocardiography. After 4 months on L-carnitine phosphocreatine recovery was improved compared to baseline (p<0.03, t-test) but comparison to placebo and creatine effects did not reach significance (p=0.06, F-test). Ataxia rating scale and echocardiographic parameters remained unchanged. Creatine had no effect in FA patients. L-carnitine is a promising substance for the treatment of FA patients, and larger trials are warranted.

[Determination of the renal blood flow in macro- and microcirculation by means of pulse inversion imaging]. / Bestimmung des renalen Blutflusses in Makro- und Mikrozirkulation mittels Pulse Inversion Imaging.

PURPOSE: To evaluate whether real-time and intermittent pulse inversion technology (PI) allows the analysis of blood flow in renal macro- and microcirculation. MATERIALS AND METHODS: The experiments were performed in a kidney perfusion phantom as an experimental model for the assessment of contrast replenishment in vascular regions of high flow velocity (medulla) and low flow velocity (cortex). During continuous infusion (0.03 ml/min) of Optison, contrast replenishment kinetics were assessed with intermittent PI at high emission power (MI: 1.3, with increasing trigger intervals) and with real-time PI at low emission power (MI: 0.09) at variable renal arterial blood flow (15 - 65 ml/min), using an HDI-5000 ultrasound unit (Philips Medical Systems). Regions of interest were placed in the major arteries of the medulla and the renal cortex to obtain replenishment curves of the macro- and microcirculation. Non-linear curve fitting was performed using the mathematical model y = A (1-e (-beta t)) with A as the parameter describing blood volume and beta as the parameter describing the speed of contrast replenishment. RESULTS: Replenishment curves could be obtained in all analyzed renal segments. For intermittent and real-time PI a strong linear correlation was found between renal arterial blood flow and A*beta (intermittent PI: cortex: R = 0.97; medulla: R = 0.98; real-time PI: cortex: R = 0.99; medulla: R = 0.96). The differences between the slopes of the regression lines (cortex: high power vs. low power, p = 0.844; medulla: high power vs. low power, p = 0.444) were not significant. CONCLUSION: Intermittent and real-time PI allows the assessment of renal blood flow in different vessel compartments.

[Interventional catheter closure of persistent foramen ovale (PFO) in a patient with paradoxical embolism and Brugada's syndrome]. / Katheterinterventioneller PFO-Verschluss bei einem Patienten mit paradoxer Embolie und Brugada-Syndrom.

HISTORY AND ADMISSION FINDINGS: A 41-year-man was admitted because of acute bluish-grey skin discoloration in cold sensation in the right hand. His brother had suffered sudden cardiac death, aged 42 years. INVESTIGATIONS: Angiography demonstrated embolic occlusion of the digital artery of the right thumb. Transesophageal echocardiography showed a persistent foramen ovale (PFO) with an aneurysm of the atrial septum (ASA) with marked right-to-left shunt of contrast medium during a Valsalva maneuvre as well as two smaller septal fenestrations. There was no evidence of any other source of embolism. The resting electrocardiogram showed an incomplete right bundle branch block with ST elevations in V (1)-V (3), changes like those described in Brugada's syndrome. TREATMENT AND COURSE: Paradoxical embolism having been demonstrated, the PFO with ASA were closed with a percutaneously introduced Helex septum occluder. Later an implantable cardioverter-defibrillator (ICD) was introduced. CONCLUSIONS: A PFO, particularly if associated with an atrial aneurysm, is an important site of paradoxical embolism. In symptomatic patients percutaneous transcatheter septal occlusion should be considered preceding any ICD insertion thought necessary for concurrent Brugada's syndrome.

[Biventricular thrombi dissolution and antibody development with lepirudin therapy]. / Biventrikuläre Thrombenauflösung und Antikörper-Bildung unter Lepirudin-Therapie.

HISTORY AND ADMISSION FINDINGS: A 50-year-old patient presented with clinical symptoms of heart failure with orthopnoe and edema (NYHA IV). INVESTIGATIONS: Echocardiography revealed a dilated left ventricle with severely reduced left ventricular function and biventricular floating thrombi, due to dilatative cardiomyopathy. TREATMENT AND COURSE: With a heart failure medication clinical symptoms reduced and body weight decreased > 10 kg in 3 weeks. Due to the high-risk constellation, anticoagulation was performed with lepirudin and the biventricular thrombi were dissolved within 17 days. At this point in time, the patient suffered from petechial bleedings, hemoptysis and gross hematuria. Despite breaking anticoagulation and substitution of PPSB with not measurable fibrinogen, subarachnoid hemorrhage occurred leading to exitus letalis. CONCLUSION: Lepirudin is a highly effective anticoagulant, that can induce severe hemorrhagic side effects in individual cases. The present case report demonstrates an immunological reaction as a rare cause with activation of prothrombin and formation of fibrin.

Atrial fibrillation: relation between clinical risk factors and transoesophageal echocardiographic risk factors for thromboembolism.

OBJECTIVE: To correlate clinical risk factors for thromboembolism with transoesophageal echocardiography (TOE) markers of a thrombogenic milieu. DESIGN: Clinical risk factors for thromboembolism and TOE markers of a thrombogenic milieu were assessed in consecutive patients with non-rheumatic atrial fibrillation. The following TOE parameters were assessed: presence of spontaneous echo contrast, thrombi, and left atrial appendage blood flow velocities. A history of hypertension, diabetes mellitus, or thromboembolic events, patient age > 65 years, and chronic heart failure were considered to be clinical risk factors for thromboembolism. SETTING: Tertiary cardiac care centre. PATIENTS: 301 consecutive patients with non-rheumatic atrial fibrillation scheduled for TOE. RESULTS: 255 patients presented with clinical risk factors. 158 patients had reduced left atrial blood flow velocities, dense spontaneous echo contrast, or both. Logistic regression analysis showed that a reduced left ventricular ejection fraction and age > 65 years were the only independent predictors of a thrombogenic milieu (both p < 0.0001). The probability of having a thrombogenic milieu increased with the number of clinical risk factors present (p < 0.0001). 17.4% of the patients without clinical risk factors had a thrombogenic milieu whereas 41.2% of the patients presenting one or more clinical risk factors had none. CONCLUSION: There is a close relation between clinical risk factors and TOE markers of a thrombogenic milieu. In addition, TOE examination allows for the identification of patients with a thrombogenic milieu without clinical risk factors.

The impact of emission power on the destruction of echo contrast agents and on the origin of tissue harmonic signals using power pulse-inversion imaging.

The purpose of this study was to determine the impact of emission power on ultrasound (US)-induced destruction of echocontrast microbubbles during real-time power pulse inversion imaging (PPI) in myocardial contrast echocardiography (MCE) and to evaluate the magnitude of noncontrast PPI signals arising from myocardial tissue at variable emission power to define the cut-off emission power for optimal MCE using low power technologies. In vitro studies were performed in a flow phantom using Optison, Definity and AFO 150. PPI signal intensity during real-time imaging at 27 Hz was compared with intermittent imaging at 0.1 Hz to evaluate bubble destruction at variable emission power (MI: 0.09 to 1.3). In healthy volunteers, PPI signal intensities during constant infusion of Optison(R) was studied in real-time PPI 22 HZ and during intermittent imaging triggered end-systolic frames every, every 3rd and every 5th cardiac cycle. In addition, the impact of emission power on nonlinear PPI signals from myocardial structures was studied. In vitro, there was a 40% decrease of real-time PPI signal intensity for Optison and AFO 150 at lowest emission power (0.09), whereas no signal loss was observed for Definity. Increase of emission power resulted in a faster decay for Optison(R) and AFO 150 as compared to Definity. In vivo, real-time PPI during continuous infusion of Optison(R) resulted in a 40% decrease of myocardial signal intensity as compared to intermittent imaging every 5th cardiac cycle, even at lowest possible emission power (mechanical index = 0.09). There was a strong positive relationship between MI and noncontrast myocardial PPI signals in all myocardial segments. PPI signal intensity was found to be lower than 1 dB only for extremely low emission power (MI < 0.2). Destruction of microbubbles during real-time imaging by use of PPI at low emission power varies considerably for different echo contrast agents. However, bubble destruction and the onset of tissue harmonic signals focus the use of real-time perfusion imaging to very low emission power.

Continuous-infusion contrast-enhanced US: in vitro studies of infusion techniques with different contrast agents.

PURPOSE: To evaluate the infusion properties of three ultrasonographic (US) contrast agents and to compare different infusion techniques for achieving constant signals during harmonic power Doppler US. MATERIALS AND METHODS: In vitro studies were performed in a flow phantom. SH U 508A, NC100100, or FS069 was continuously infused at clinically usable doses and infusion rates. To assess agent-specific physical properties, these agents were administered by using a vertically fixed infusion pump and varying infusion start times. The contrast agents were administered by also using a horizontally oriented infusion pump that was either fixed or continuously rotated to homogenize the agent in the syringe. RESULTS: With SH U 508A and NC100100, constant signals were achieved, regardless of the infusion modality used. Compared with conventional infusion, the continuous homogenization of SH U 508A, although not necessary for signal constancy, increased the agent's usefulness (P <.05). With FS069, only continuous homogenization yielded constant signals (P <.001). CONCLUSION: Continuous infusion of SH U 508A or NC100100 provided constant harmonic power Doppler US signals, regardless of the infusion modality used. Because of the special physical properties of FS069, only homogenization produced constant harmonic power Doppler US signals during continuous infusion of this agent.

Feasibility of the flash-replenishment concept in renal tissue: which parameters affect the assessment of the contrast replenishment?

The purpose of the study was to evaluate whether power pulse inversion (PPI) and pulse inversion (PI) techniques allow the measurement of indices of microcirculatory flow in real-time at low emission power using contrast microbubbles. PPI and PI imaging were performed in a kidney perfusion model during continuous infusion of Definity (0.12 mL/min). At steady state of tissue enhancement, contrast was destroyed by emission of echo bursts at high emission power (MI = 1.3). Consecutively, contrast replenishment was assessed at low emission power (MI = 0.09) in real-time imaging modes (PPI: 12 Hz; PI: 25 Hz). Regions-of-interest (ROI) of variable sizes were placed in the renal cortex and bigger arteries to compare replenishment of macro- and microcirculation. Nonlinear curve fitting was performed using the mathematical model y=s+A(1-e(-betat)), with A as the parameter describing blood volume and beta as a parameter describing the speed of microbubble contrast replenishment. Replenishment curves could be visually appreciated and quantitatively analyzed in all renal segments. A was significantly higher in bigger arteries compared to renal cortex (p < 0.001). beta was found to be significantly higher in the arteries as compared to the cortex (p < 0.001). The SD of beta diminishes with increasing size of the ROI. The acquisition of replenishment curves following ultrasound (US)-induced destruction of contrast microbubbles is feasible at low power using PPI and PI. Assessment of replenishment kinetics allows the differentiation between macro- and microcirculation. Size and position of the ROI have an important impact on the generation of replenishment curves in both imaging modalities, which has to be taken into account.

CD95-induced JNK activation signals are transmitted by the death-inducing signaling complex (DISC), but not by Daxx.

Here we investigated CD95-mediated JNK activation pathways and their physiological relevance by employing a variety of cell lines with deficiencies in individual signal transmitting proteins. JNK activation was completely dependent on the activation of caspases in type I and type II cells, as revealed by the inhibitory effects of the caspase inhibitors zVAD-fmk or the cowpoxvirus-encoded CrmA protein. Jurkat cells deficient in caspase-8 or expressing a dominant negative (DN) form of FADD were unable to induce JNK in response to CD95 ligation, indicating that these death-inducing signaling complex (DISC) proteins are required for signal transmission. Activation of caspases, JNK and apoptosis occurred with a markedly slower kinetics in cells expressing a DN version of ASK1, revealing an important contribution of ASK1 for these processes. A C-terminally truncated version of Daxx impaired CD95-mediated apoptosis without affecting the JNK signal. DN forms of FADD, MKK4 and MKK7 completely inhibited CD95-mediated JNK activation but remained without impact on cell killing, indicating that JNK activation is not required for the execution process of CD95-mediated cell killing.

Protein kinase C theta cooperates with Vav1 to induce JNK activity in T-cells.

Here we show that in human T-cell leukemia cells Vav1 and protein kinase C theta (PKCtheta) synergize for the activation of c-Jun N-terminal kinase (JNK) but not p38 MAP kinase. Vav1 and PKCtheta also cooperated to induce transcription of reporter genes controlled either by AP-1 binding sites or the CD28RE/AP composite element contained in the IL-2 promoter by stimulating the binding of transcription factors to these two elements. Dominant negative versions of Vav1 and PKCtheta inhibited CD3/CD28-induced activation of JNK, revealing their relative importance for this activation pathway. Gel filtration experiments revealed the existence of constitutively associated Vav1/PKCtheta heterodimers in extracts from unstimulated T-cells, whereas T-cell costimulation induced the recruitment of Vav1 into high molecular weight complexes. Several experimental approaches showed that Vav1 is located upstream from PKCtheta in the control of the pathway leading to synergistic JNK activation. Vav1-derived signals lead to the activation of JNK by at least two different pathways. The major contribution of Vav1 for the activation of JNK relies on the PKCtheta-mediated Ca(2+)-independent synergistic activation pathway, whereas JNK is also activated by a separate Ca(2+)-dependent signaling route.

Blood flow assessment by ultrasound-induced destruction of echocontrast agents using harmonic power Doppler imaging: which parameters determine contrast replenishment curves?

OBJECTIVE: To evaluate the feasibility of flow determinations by contrast replenishment using harmonic power Doppler imaging (H-PDI). BACKGROUND: The application of indicator dilution principles on contrast echocardiography is limited by numerous methodical problems. Recently, a new method was introduced that relies on ultrasound-mediated microbubble destruction and evaluation of the contrast replenishment. METHODS: Definity, a perfluorocarbon-derived contrast agent under development, was continuously infused into a steady flow phantom and H-PDI registrations were performed within a silicone tube (d = 8 mm). Replenishment interval between destruction and imaging frame was varied from 0.04-2 seconds. Nonlinear curve fitting was performed using an exponential mathematical model. RESULTS: Strong linear correlation between contrast dose and maximum signal intensity as well as between flow and the slope variable beta of the replenishment curve was found for all settings (r > 0.96). Maximum signal intensity and contrast replenishment rate were found to be a function of emission power and were significantly influenced by depth and focus position. CONCLUSION: The feasibility of flow assessment using replenishment curves obtained by H-PDI was demonstrated. However, in experimental conditions, flow analysis was severely influenced by ultrasound system settings and imaging conditions such as emission power, sound field geometry, and investigation depth. For a clinical use of this promising approach, algorithms that take specific system settings and imaging conditions into account have to be found. Imaging modalities that enable a most homogeneous scan field are best suited for the assessment of contrast replenishment.

Subendocardial steal effect seen with real-time perfusion imaging at low emission power during adenosine stress: replenishment M-mode processing allows visualization of vertical steal.

We present a patient in whom power pulse inversion imaging clearly demonstrated a subendocardial myocardial perfusion defect during contrast vasodilator stress using adenosine. The defect was best appreciated with M-mode postprocessing of power pulse inversion imaging data.

Enhancement of T cell receptor signaling by a mild oxidative shift in the intracellular thiol pool.

Exposure of T cells to the macrophage products hydrogen peroxide (HP) or L-lactate (LAC) was previously shown to enhance IL-2 production and to modulate glutathione (GSH) status. We now found that 50 microM HP and 30 mM LAC enhanced strongly the transcription from the IL-2 promoter in Jurkat T cells after stimulation with anti-CD28 together with or without anti-CD3 but not with anti-CD3 Abs alone. Therefore, we used anti-CD3 plus anti-CD28-stimulated cells to investigate the effect of the GSH reductase inhibitor 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on the signal cascade. BCNU enhanced the transcription to a similar extent as HP or LAC. Lowering the intracellular GSH/GSH disulfide ratio by BCNU, HP, or NO resulted in all cases in the fulminant enhancement of Jun-N-terminal kinase and p38 mitogen-activated protein kinase but not extracellular signal-regulated kinase 1/2. Jun-N-terminal kinase and NF-kappaB activation was enhanced through pathways involving Rac, Vav1, PKCTheta, p56(lck), p59(fyn), and IkappaB kinases. In a cell-free system, the autophosphorylation of rFyn was stimulated by GSH disulfide but not by HP. These findings suggest that the oxidation of the cellular thiol pool may play a role as an amplifying mechanism for TCR/CD3 signals in immune responses.

Synergistic activation of NF-kappa B by functional cooperation between vav and PKCtheta in T lymphocytes.

Here we identified PKCtheta as an activator of transcription factor NF-kappaB in T cells. PKCtheta-induced NF-kappaB activation was synergistically augmented by Vav. Several experimental approaches revealed that PKCtheta is located downstream from Vav in the control of the pathway leading to synergistic NF-kappaB activation. In addition to the synergistic activation cascade, Vav also triggered NF-kappaB activity on a separate route. CD3/CD28-induced activation of NF-kappaB was inhibited by dominant negative forms of Vav or PKCtheta, revealing their essential role in this activation pathway. The Vav/PKCtheta-mediated signals preferentially activated IkappaB kinase beta. Vav and PKCtheta were found to be constitutively associated in unstimulated T cells. Only the ligation of the costimulatory CD28 receptor, but not of the T cell receptor, resulted in the transient dissociation of the Vav-PKCtheta complex. In contrast, T cell receptor/CD28 costimulation resulted in faster dissociation and slower reassociation kinetics.

Glutathione is a factor of resistance of Jurkat leukemia cells to nitric oxide-mediated apoptosis.

We have previously reported that nitric oxide (NO) stimulates apoptosis in different human neoplastic lymphoid cell lines through mitochondrial damage (including degradation of cardiolipin, a major mitochondrial lipid) followed by activation of caspases. Here we demonstrate that Jurkat human leukemia cells which survive after 24 h treatment with NO form subpopulations with higher and lower cardiolipin content (designated as NAO(high) and NAO(low), respectively). Sorted NAO(high) cells were found to survive in culture whereas sorted NAO(low) cells died. Moreover, NAO(high) cells acquired an increased resistance to the exposure to NO donors which remained unchanged during long-term culture. These cells showed a similar cardiolipin content and expressed the same level of anti-apoptotic proteins Bcl-2 and Bcl-x(L) as APO-S unsorted cells but contained significantly higher concentration of the antioxidant glutathione. Depletion of glutathione in these cells with buthionine-sulfoximine (BSO) correlated with a significant stimulation of NO-mediated apoptosis whereas the exposure of NO-sensitive APO-S cells to the glutathione precursor N-acetylcysteine (NAC) resulted in a substantial suppression of this effect. Our data suggest a complex mechanism of the resistence to NO-induced apoptosis in Jurkat human leukemia cells in which glutathione plays an important role.

The pro- or anti-apoptotic function of NF-kappaB is determined by the nature of the apoptotic stimulus.

To test whether the behaviour of transcription factor NF-kappaB as a promoter or antagonist of apoptosis depends on the apoptotic stimulus, we determined the influence of NF-kappaB on cell killing elicited by a variety of inducers within a given cell type. Inhibition of NF-kappaB by genetic and pharmacological approaches rendered HeLa cells more susceptible to TNF-alpha-induced cell killing, but protected them almost completely from H2O2- and pervanadate-induced apoptosis. TNF-alpha was unable to protect HeLa from H2O2- and pervanadate-induced apoptosis and further enhanced the cytotoxicity induced by these two adverse agents. Supernatants from HeLa cells stably overexpressing a transdominant negative form of IkappaB-alpha selectively increased the cytotoxicity of TNF-alpha for HeLa cells, suggesting that the enhanced susceptibility of these cells can be attributed to one or more secretable factors. Supershift experiments showed that the various apoptotic stimuli induced the same subset of DNA-binding subunits. Therefore, the nature of the signals elicited by the respective death inducers determines whether NF-kappaB induction leads to apoptosis or survival, suggesting that the manipulation of NF-kappaB activity may provide a new approach to adjuvant therapy in cancer treatment.

Tyrosine-phosphorylated Vav1 as a point of integration for T-cell receptor- and CD28-mediated activation of JNK, p38, and interleukin-2 transcription.

In this study we identified tyrosine-phosphorylated Vav1 as an early point of integration between the signaling routes triggered by the T-cell receptor and CD28 in human T-cell leukemia cells. Costimulation resulted in a prolonged and sustained phosphorylation and membrane localization of Vav1 in comparison to T-cell receptor activation alone. T-cell stimulation induced the recruitment of Vav1 to an inducible multiprotein T-cell activation signaling complex at the plasma membrane. Vav1 activated the mitogen-activated protein kinases JNK and p38. The Vav1-mediated activation of JNK employed a pathway involving Rac, HPK1, MLK3, and MKK7. The costimulation-induced activation of p38 was inhibited by dominant negative forms of Vav1, Rac, and MKK6. Here we show that Vav1 also induces transcription factors that bind to the CD28RE/AP element contained in the interleukin-2 promoter. A detailed mutational analysis of Vav1 revealed a series of constitutively active and nonfunctional forms of Vav1. Almost all inactive versions were mutated in their Dbl homology domain and behaved as dominant negative mutants that impaired costimulation-induced activation of JNK, p38, and CD28RE/AP-dependent transcription. In contrast to NF-AT-dependent transcription, Vav1-mediated transcriptional induction of the CD28RE/AP element in the interleukin-2 promoter could only partially be inhibited by cyclosporin A, suggesting a dual role of Vav1 for controlling Ca(2+)-dependent and -independent events.

Caspase-dependent cleavage and inactivation of the Vav1 proto-oncogene product during apoptosis prevents IL-2 transcription.

Here we identify the hematopoietic proto-oncogene Vav1 as a caspase substrate during apoptosis in lymphoid cells. Cleavage of Vav1 is prevented by the caspase inhibitors zDEVD and zVAD as well as by expression of CrmA. Vav1 is cleaved in vivo at the evolutionary conserved caspase consensus cleavage site DLYD161C, generating the carboxy-terminal cleavage product Vav1p76 of intermediate stability. In vitro caspase assays reveal cleavage of Vav1 at position 161 either by apoptotic cell lysates or by recombinant caspase-3. Mutation of Asp 161 to Ala leads to the usage of the adjacent alternative cleavage sequence DQID150D. Mutation of both cleavage sites at position 150 and 161 protects Vav1 from caspase-mediated proteolysis in vitro and in vivo. The cleavage product Vav1p76 is capable of activating JNK in T-cells, but fails to induce the phosphorylation of p38/HOG1. Vav1p76 displays a diminished capacity to activate the transcription factors NF-AT, AP-1 and NF-kappaB, and thus completely fails to activate IL-2 transcription. Since Vav1 is essential for IL-2 production and plays a central role for cytoskeletal reorganization, its proteolytic inactivation during apoptosis affects multiple downstream targets.

Mixed-lineage kinase 3 delivers CD3/CD28-derived signals into the IkappaB kinase complex.

The phosphorylation of IkappaB by the multiprotein IkappaB kinase complex (IKC) precedes the activation of transcription factor NF-kappaB, a key regulator of the inflammatory response. Here we identified the mixed-lineage group kinase 3 (MLK3) as an activator of NF-kappaB. Expression of the wild-type form of this mitogen-activated protein kinase kinase kinase (MAPKKK) induced nuclear immigration, DNA binding, and transcriptional activity of NF-kappaB. MLK3 directly phosphorylated and thus activated IkappaB kinase alpha (IKKalpha) and IKKbeta, revealing its function as an IkappaB kinase kinase (IKKK). MLK3 cooperated with the other two IKKKs, MEKK1 and NF-kappaB-inducing kinase, in the induction of IKK activity. MLK3 bound to components of the IKC in vivo. This protein-protein interaction was dependent on the central leucine zipper region of MLK3. A kinase-deficient version of MLK3 strongly impaired NF-kappaB-dependent transcription induced by T-cell costimulation but not in response to tumor necrosis factor alpha or interleukin-1. Accordingly, endogenous MLK3 was phosphorylated and activated by T-cell costimulation but not by treatment of cells with tumor necrosis factor alpha or interleukin-1. A dominant negative version of MLK3 inhibited NF-kappaB- and CD28RE/AP-dependent transcription elicited by the Rho family GTPases Rac and Cdc42, thereby providing a novel link between these GTPases and the IKC.