In vitro and in vivo modulation of MDR1/P-glycoprotein in HIV-infected patients administered highly active antiretroviral therapy and liposomal doxorubicin

P-glycoprotein (P-gp) transports a wide range of structurally unrelated drugs, such as HIV protease inhibitors (PIs) and cytotoxic compounds such as anthracyclines. Because modification of P-gp phenotype and function is an important underlying mechanism of drug interactions, the current study was conducted in order to evaluate whether highly active antiretroviral therapy (HAART), HIV plasma viral load (VL), or cancer chemotherapy may induce in vivo changes of P-gp phenotype in peripheral blood mononuclear cells (PBMCs) from HIV-infected treatment-naive and -experienced subjects at different stages of HIV infection and/or disease, including patients with HIV-associated Kaposi sarcoma (KS). Our results show that neither HAART nor HIV VL, nor the stage of HIV infection and/or disease, significantly alter P-gp expression on PBMCs. In particular, surface P-gp expression is expressed at low levels by T-cell subsets, B cells, and NK cells, whereas almost all monocytes are double positive and these results are not modified by HIV PI-containing regimens. By contrast, a significant phenotype modification is detected in PBMCs from AIDS/KS patients after challenge with the liposomal formulation of the anthracycline doxorubicin (L-DOX) with the higher expression reached 24 hours after the end of the drug infusion. In addition, accumulation of L-DOX is unaffected by P-gp-mediated drug efflux as documented by in vitro experiments, in sharp contrast to the kinetic of free DOX, based on HIV PI blockade experiments. Finally, P-gp expression was found in KS spindle cells from HIV-infected treatment-naive AIDS/KS patients. We conclude that P-gp phenotype in PBMCs and specific subsets is not altered by HAART and/or HIV, whereas a significant increase is induced by specific anticancer drugs such as L-DOX. Moreover, HIV PIs possess an inhibitory effect on P-gp function that may improve DOX sensitivity in KS spindle cells.     

Characterization of the large-conductance Ca-activated K channel in myocytes of rat saphenous artery

We used the patch-clamp method to characterize the BK channel in freshly isolated myocytes from the saphenous branch of the rat femoral artery. Single-channel recordings revealed that the BK channel had a conductance of 187 pS in symmetrical 150 mM KCl, was blocked by external tetraethylammonium (TEA) with a KD(TEA) of approx. 300 microM at +40 mV, and by submicromolar charybdotoxin (CTX). The sensitivity of the BK channel to Ca was especially high (KD(ca) approx. 0.1 microM at +60 mV) compared to skeletal muscle and neuronal tissues. We also investigated the macroscopic K current, which under certain conditions is essentially sustained by BK channels. This conclusion is based on the findings that the macroscopic current activated upon depolarization follows a single exponential time course and is virtually fully blocked by 100 nM CTX and 5 mM external TEA. We made use of this occurrence to assess the voltage and Ca dependence of the macroscopic BK current. In intact myocytes, the BK channel showed a strong and voltage-dependent reduction of the outward current (62% at +40 mV), most likely due to block by intracellular Ba and polyamines. The results obtained from macroscopic and unitary current indicate that approx. 2.5% of the BK channels are active under physiological conditions, sustaining approx. 20 pA of outward current. Given the high input resistance of these cells, few BK channels are required to open in order to cause a significant membrane hyperpolarization, and thus function to limit the contraction resulting from acute increases in intravascular pressure, or in response to hypertensive pathologies.     

Changes in EEG activity and hypothalamic temperature as indices for non-REM sleep to REM sleep transitions

A shift of physiological regulations from a homeostatic to a non-homeostatic modality characterizes the passage from non-NREM sleep (NREMS) to REM sleep (REMS). In the rat, an EEG index which allows the automatic scoring of transitions from NREMS to REMS has been proposed: the NREMS to REMS transition indicator value, NIV [J.H. Benington et al., Sleep 17 (1994) 28-36]. However, such transitions are not always followed by a REMS episode, but are often followed by an awakening. In the present study, the relationship between changes in EEG activity and hypothalamic temperature (Thy), taken as an index of autonomic activity, was studied within a window consisting of the 60s which precedes a state change from a consolidated NREMS episode. Furthermore, the probability that a transition would lead to REMS or wake was analysed. The results showed that, within this time window, both a modified NIV (NIV(60)) and the difference between Thy at the limits of the window (Thy(D)) were related to the probability of REMS onset. Both the relationship between the indices and the probability of REMS onset was sigmoid, the latter of which saturated at a probability level around 50-60%. The efficacy for the prediction of successful transitions from NREMS to REMS found using Thy(D) as an index supports the view that such a transition is a dynamic process where the physiological risk to enter REMS is weighted at a central level.     

Early atherosclerotic lesions in infancy: role of parental cigarette smoking

Cigarette smoking is associated with an increased incidence of atherosclerotic diseases. The aim of this study was to examine the progression of the preatherosclerotic lesions previously observed by us in coronary arteries of fetuses of smoker mothers and in infants with smoker parents. We examined the coronary arteries of 34 infants, aged 1–36 months, and the histological and biological [c-fos, proliferating cell nuclear antigen (PCNA), and apoptosis] features of the early atherosclerotic lesions. In 17 infants (50%), at least one parent smoked, generally more than five cigarettes a day. In 18 cases (53%), we observed variable thickening of the coronary walls from preatherosclerotic lesions to juvenile atherosclerotic plaques, related to parental smoking habit. This morphological progression of the lesions was accompanied by a sequence of biological changes in the smooth muscle cells of the tunica media. We suggest that the oxidants present in the gas phase of the parental cigarette smoke pass through the endothelium and induce at first the c-fos gene activation and subsequently the PCNA positivity, that is, a proliferative process.     

Loss of lamin A/C expression revealed by immuno-electron microscopy in dilated cardiomyopathy with atrioventricular block caused by<Emphasis Type="Italic"> LMNA</Emphasis> gene defects

Mutations of the LMNA gene encoding the lamin A and C nuclear envelope proteins cause an autosomal dominant form of dilated cardiomyopathy (DCM) with atrioventricular block (AVB). The aim of this study was to investigate ultrastructural nuclear membrane changes by conventional electron microscopy and protein expression by immuno-electron microscopy in the heart of patients with DCM and AVB due to LMNA gene mutations. Four immunohistochemical techniques were used: pre-embedding and post-embedding in Epon-Araldite resin and London Resin White (LRW), with and without silver enhancement. Parallel light microscopy immunohistochemistry studies were performed. Conventional electron microscopy showed a loss of integrity of the myocyte nuclei with blebs of the nuclear membrane, herniations and delamination of the nuclear lamina and nuclear pore clustering. Post-embedding LRW was the most informative technique for morphology and immuno-labelling. Immuno-labelling was almost absent in the nuclear envelope of patients with LMNA gene mutations, but intensely present in controls. The loss of labelling selectively affected myocyte nuclei; the endothelial cell nuclei were immunostained in patients and controls. Light immunohistochemistry confirmed the results. These findings confirm the hypothesis that LMNA gene defects are associated with a loss of protein expression in the selective compartment of non-cycling myocyte nuclei.     

Sclerosteosis: report of a case in a black African man

Sclerosteosis is a rare genetic disorder of bone modelling, similar to, but distinct from, van Buchem disease; it has been described almost exclusively in Afrikaners of South Africa, a white population of Dutch ancestry. Isolated cases have been reported in a girl in Japan, a boy in Spain, and in multiracial families in Brazil and USA.
Here we report a case of sclerosteosis in a black man born in Senegal. He presented with the full features of the disease: tall stature; syndactyly; nail dysplasia; massive sclerosis of the long tubular bones, the ribs, the pelvis and the skull; multiple cranial nerve involvement: optic atrophy, facial palsy and trigeminal neuralgia. Radiologic examination, visual and brainstem auditory evoked potentials, computerized tomography and magnetic resonance imaging of the skull were performed. This seems to be the first case of the disease in a black African individual, with no known relationship with Dutch ancestry.