CD4-BFFI: A novel, bifunctional HIV-1 entry inhibitor with high and broad antiviral potency

Resistance to antiretroviral drugs is a common problem in the treatment of HIV-1-infected patients. To overcome resistance, we generated a novel, bifunctional HIV-1 entry inhibitor by combining the anti-CD4 monoclonal antibody (mAb) 6314 with a fusion inhibitor similar to T-651 (anti-CD4 mAb based BiFunctional Fusion Inhibitor, CD4-BFFI). CD4-BFFI has potent antiviral activity against a multitude of HIV-1 isolates independent of their co-receptor usage and genetic background. It has higher antiviral potency compared to the fusion inhibitor T-651 or the anti-CD4 mAb 6314 used independently. More importantly, every HIV-1 strain tested was fully inhibited by CD4-BFFI while many strains were only partially inhibited by 6314. CD4-BFFI also retained antiviral potency against virus strains resistant to two fusion inhibitors, a CCR5 antagonist and an anti-CCR5 mAb. Pre-incubation of cells with a saturating concentration of anti-CD4 mAbs reduced the antiviral potency of CD4-BFFI, suggesting that binding of CD4-BFFI to the cell surface via its CD4 mAb portion is required for the antiviral potency of its fusion inhibitor moiety. Collectively, we present a novel HIV-1 inhibitor with a dual mode of action and excellent antiviral potency against wildtype and entry-inhibitor resistant virus strains suggesting that CD4-BFFI may have a high barrier to resistance.     

Portal vein embolization and autologous CD133+ bone marrow stem cells for liver regeneration: initial experience

PURPOSE: To prospectively evaluate the effectiveness of portal vein embolization (PVE) and CD133(+) bone marrow stem cell (BMSC) administration to the liver, compared with PVE alone, to augment hepatic regeneration in patients with large hepatic malignancies. MATERIALS AND METHODS: The study was approved by the institutional ethics committee; informed consent was obtained. Thirteen patients underwent PVE of liver segments I and IV-VIII to stimulate hepatic regeneration prior to extended right hepatectomy. In six patients (three men, three women; mean age, 61 years; range, 46-72 years) with a future liver remnant volume (FLRV) below 25% and/or limited quality of hepatic parenchyma, PVE alone did not promise adequate proliferation. These patients underwent BMSC administration to segments II and III (group I). In seven patients (three men, four women; mean age, 69 years; range, 63-75 years) with an FLRV below 25%, PVE alone was performed (group II). Two radiologists blinded to patients' identity and each other's results measured liver and tumor volumes with helical computed tomography. Absolute, relative, and daily FLRV gains were compared by using the t test or the Wilcoxon test. RESULTS: The increase of the mean absolute FLRV in group I from 239.3 mL +/- 103.5 (standard deviation) to 417.1 mL +/- 150.4 was significantly higher than that from 286.3 mL +/- 77.1 to 395.9 mL +/- 94.1 in group II (P = .049). The relative gain of FLRV after PVE in group I (77.3% +/- 38.2) was significantly higher than that in group II (39.1% +/- 20.4) (P = .039). The daily hepatic growth rate in group I (9.5 mL/d +/- 4.3) was significantly superior to that in group II (4.1 mL/d +/- 1.9) (P = .03). Time to surgery was 27 days +/- 11 in group I and 45 days +/- 21 in group II (P = .057). CONCLUSION: In patients with malignant liver lesions, the combination of PVE with CD133(+) BMSC administration substantially increased hepatic regeneration compared with PVE alone.     

Functional protein expression of multiple sodium channel α- and β-subunit isoforms in neonatal cardiomyocytes

Voltage-gated sodium channels are composed of pore-forming α- and auxiliary β-subunits and are responsible for the rapid depolarization of cardiac action potentials. Recent evidence indicates that neuronal tetrodotoxin (TTX) sensitive sodium channel α-subunits are expressed in the heart in addition to the predominant cardiac TTX-resistant Na_v1.5 sodium channel α-subunit. These TTX-sensitive isoforms are preferentially localized in the transverse tubules of rodents. Since neonatal cardiomyocytes have yet to develop transverse tubules, we determined the complement of sodium channel subunits expressed in these cells. Neonatal rat ventricular cardiomyocytes were stained with antibodies specific for individual isoforms of sodium channel α- and β-subunits. α-actinin, a component of the z-line, was used as an intracellular marker of sarcomere boundaries. TTX-sensitive sodium channel α-subunit isoforms Na_v1.1, Na_v1.2, Na_v1.3, Na_v1.4 and Na_v1.6 were detected in neonatal rat heart but at levels reduced compared to the predominant cardiac α-subunit isoform, Na_v1.5. Each of the β-subunit isoforms (β1-β4) was also expressed in neonatal cardiac cells. In contrast to adult cardiomyocytes, the α-subunits are distributed in punctate clusters across the membrane surface of neonatal cardiomyocytes; no isoform-specific subcellular localization is observed. Voltage clamp recordings in the absence and presence of 20 nM TTX provided functional evidence for the presence of TTX-sensitive sodium current in neonatal ventricular myocardium which represents between 20 and 30% of the current, depending on membrane potential and experimental conditions. Thus, as in the adult heart, a range of sodium channel α-subunits are expressed in neonatal myocytes in addition to the predominant TTX-resistant Na_v1.5 α-subunit and they contribute to the total sodium current.     

Clinical evaluation of two bilateral hand allotransplantations at six and three years follow-up

We report a six and three years follow-up clinical evaluation of two bilateral hand allotransplantations from brain-dead multi-organs donors performed in two young adult traumatic hand amputees. Lifelong immunosuppressive treatment included tacrolimus, mycophenolate mofetil and prednisolone. In both patients, early complications were observed but were successfully treated. At follow-up, clinical results allow useful hand function in both patients, by far superior to what can be provided by current myoelectric prostheses. Although the long-term risks-benefits ratio of bilateral hand transplantation is still unknown, these two cases demonstrate that this new treatment is feasible. Further experimental and clinical research is needed to better delineate its role in the future of hand surgery.     

Excitability of pontine startle processing neurones is regulated by the two-pore-domain K+ channel TASK-3 coupled to 5-HT2C receptors

The mammalian startle reflex is a fast response to sudden intense sensory stimuli that can be increased by anxiety or decreased by reward. The cellular integration of sensory and modulatory information takes place in giant neurones of the caudal pontine reticular formation (PnC). The startle reflex is known to be enhanced by 5-hydroxytryptamine (5-HT); however, signalling mechanisms that change the excitability of the PnC giant neurones are poorly understood. Possible molecular candidates are two-pore-domain K⁺ (K₂P) channels that generate a variable K⁺ background conductance and control neuronal excitability upon activation of G-protein-coupled receptors. We demonstrate by in situ hybridization that the K₂P channel TASK-3 is substantially expressed in PnC giant neurones. Brain slice recordings revealed a corresponding background K⁺ current in these cells that forms about 30% of the outward current at −30 mV. Inactivation of TASK-3 at pH 6.4 and by ruthenium red depolarized the cells by about 7 mV and increased the action potential frequency as well as duration. Specific activation of Gα_q-coupled 5-HT₂ receptors with α-methyl 5-HT evoked a similar increase of neuronal excitability. Consistently, we measured afferent synaptic inputs from serotonergic raphe neurones and detected 5-HT_2C receptors in PnC giant neurones by immunohistochemistry. Thus, neuronal excitability of PnC giant neurones in vivo is most likely increased by serotonergic projections via the K₂P channel TASK-3.     

The acid-sensitive potassium channel TASK-1 in rat cardiac muscle

OBJECTIVE: The outward current flowing through the two-pore domain acid-sensitive potassium channel TASK-1 (I(TASK)) and its inhibition via alpha1-adrenergic receptors was studied in rat ventricular cardiomyocytes. METHODS: Quantitative RT-PCR experiments were carried out with mRNA from rat heart. Patch-clamp recordings were performed in isolated rat cardiomyocytes. TASK-1 and other K+ channels were expressed in Xenopus oocytes to study the pharmacological properties of a new TASK-1 channel blocker, A293. RESULTS: TASK-1 channels were found to be strongly expressed in rat heart. Analysis of the sensitivity of various K+ channels to A293 in Xenopus oocytes showed that at low concentrations A293 was a selective blocker of TASK-1 channels. I(TASK) in rat cardiomyocytes was dissected by application of A293 and by extracellular acidification to pH 6.0; it had an amplitude of approximately 0.30 pA/pF at +30 mV. Application of 200 nM A293 increased action potential duration (APD(50)) by 31+/-3% at a stimulation rate of 4 Hz. The plausibility of the effects of A293 on APD50 was checked with a mathematical action potential model. Application of the alpha1-adrenergic agonist methoxamine inhibited I(TASK) in Xenopus oocytes co-injected with cRNA for TASK-1 and alpha1A-receptors. In cardiomyocytes, methoxamine inhibited an outward current with characteristics similar to I(TASK). This effect was abolished in the presence of the alpha1A-antagonist 5-methyl-urapidil. CONCLUSIONS: Our results suggest that in rat cardiomyocytes I(TASK) makes a substantial contribution to the outward current flowing in the plateau range of potentials and that this current component can be inhibited via alpha1A-adrenergic receptors.     

Modelling and expression studies of two novel mutations causing factor V deficiency

Human coagulation factor V (FV), a non-enzymatic cofactor of the prothrombinase complex, is required for the rapid generation of thrombin. FV deficiency is a rare autosomal recessive bleeding disorder. We describe two novel mutations, Tyr91Asn and Asp2098Tyr, found in two probands with a residual FV activity of 51% and 4%, respectively. Modelling and structural analysis of these mutations were performed following short-duration molecular dynamics (MD) simulation. Asp2098Tyr lead to abolishment of the highly conserved salt bridge Asp2098-Arg2171 presumably required for structural integrity of the C2 domain. MD studies suggest that additional conformational changes resulting from this mutation involve local rearrangements at Tyr2063 and Tyr2064 and so affect the phospholipid-membrane binding. MD modelling of the Try91Asn mutant revealed a conformational change nearby the Cu(2+) binding site that could affect overall stabilization of the heavy and light chains. These findings suggest that both mutations influence the structural integrity of FV protein. Transient expression data of wild-type and mutant FV variants in 293T human embryonic kidney cells showed FV-specific activity reduced to 26% for Asp2098Tyr and 56% for Tyr91Asn compared to that of wild-type. Thus, both the data from the short duration molecular dynamic simulation and from expression analysis indicate alterations of the FV protein variants that explain the clinical phenotype.     

DNA probes for studying streptothricin resistance evolution in enteric bacteria

Probes for the detection of streptothricin resistance genes have been derived from recombinant plasmids. These include the streptothricin resistance gene probe sat 1/2 derived from Tn 1826 and specific for both the sat-1 determinant of Tn 1825 and the sat-2 determinant of Tn 1826, and the probe sat D derived from and specific for the sat-1 determinant of transposon Tn 1825. A third streptothricin resistance gene probe, sat 3, represents the streptothricin resistance determinant sat-3 of the IncQ R plasmid pIE639. Hybridization studies did not reveal any sequence homology between sat-3 and the transposon-localized sat-1 and sat-2 determinants. Moreover, non of the different sat-determinants isolated from plasmids of gram negative bacteria hybridized with the analogous resistance determinant of Streptomyces noursei, which had been cloned and named nat by Krügel et al. (Gene, 1988, 62, 209-214). The sat 1/2 probe in combination with the sat D probe proved to be suitable for the identification and the differentiation of sat-1 and sat-2 determinants in different genetic environments. Streptothricin resistance genes related to those present on transposons Tn 1825 and Tn 1826 have been detected by hybridization with the probe sat 1/2 on plasmids isolated a long time ago before the application of streptothricins. The sat-3 determinant appears to be exclusively associated with the IncQ plasmid pIE639.     

Über muskuläre Drosselvorrichtungen („Zellknospen”, „-Polster”) in den Arterien der Schilddrüse

Zusammenfassung An der Hand von Reihenschnitten und Plattenmodellen werden 2 verschiedene muskuläre Drosselvorrichtungen an den Schilddrüsenarterien nachgewiesen. Es sind dies einerseits Polsterarterien im gebräuchlichen Sinne des Wortes, andererseits lippenförmige oder sphincterartige Ringmuskelwülste an den Abgangsstellen kleinerer Arterienäste aus größeren Stämmen. Die lichtungeinengende Wirksamkeit dieser Muskelwülste sieht man ganz besonders deutlich bei Arterien, bei denen eine Kontraktion der Muskulatur durch künstliche Sympathicusreizung erzielt wurde. Niedrigen Muskelleisten könnte die Bedeutung von Stromrichtern zukommen. Biologische Folgewirkungen dieser Blutstromregelung müssen in Erwägung gezogen werden.Mit 4 Abbildungen im Text