RESUMEN
Reversible cerebral vasoconstriction syndrome (RCVS) is a poorly understood but increasingly recognized entity, likely multifactorial in nature and characterized by diffuse cerebral vasospasm that presents as sudden, intense, and fluctuating headaches. Due to insufficient evidence, there is currently no consensus on RCVS treatment guidelines. However, nicardipine, an L-type calcium channel blocker, may prove effective in RCVS treatment because of its ability to penetrate the blood-brain barrier. We report the concomitant use of intrathecal (IT) nicardipine and continuous intraarterial (IA) nicardipine infusion via microcatheter placed in the intracranial circulation for the treatment of a 58-year-old female with severe refractory RCVS. On presentation, this patient was noted to have a non-traumatic non-aneurysmal subarachnoid hemorrhage secondary to RCVS. Initially managed with oral verapamil, she later developed refractory symptomatic vasoconstriction requiring multiple angiograms for spasmolysis via balloon angioplasty and IA nicardipine. Due to the refractory nature of her spasm despite the IA therapy, we decided to attempt intrathecal nicardipine, starting at 4 mg q12 h via an external ventricular drain. This dose was escalated to 4 mg q6 h. The patient stabilized for 24 h but again decompensated, requiring continuous IA spasmolysis via a microcatheter placed in the left middle cerebral artery and left for continuous IA nicardipine infusion (5 mg/h). The patient showed slow incremental improvement clinically and a decrease in vasospasms on imaging, ultimately suffering minimal stroke burden. This patient's hospital course demonstrates that nicardipine, administered intrathecally or intraarterially, could be beneficial in select patients with refractory RCVS as a means of minimizing repeat angiography/angioplasty. Further studies are needed to better define a treatment paradigm for these patients.
RESUMEN
We previously discovered a class of recombinant lectin conjugates, denoted lectin DLIs ('dual-acting lytic inhibitors') that bind to the HIV-1 envelope (Env) protein trimer and cause both lytic inactivation of HIV-1 virions and cytotoxicity of Env-expressing cells. To facilitate mechanistic investigation of DLI function, we derived the simplified prototype microvirin (MVN)-DLI, containing an MVN domain that binds high-mannose glycans in Env, connected to a DKWASLWNW sequence (denoted 'Trp3') derived from the membrane-associated region of gp41. The relatively much stronger affinity of the lectin component than Trp3 argues that the lectin functions to capture Env to enable Trp3 engagement and consequent Env membrane disruption and virolysis. The relatively simplified engagement pattern of MVN with Env opened up the opportunity, pursued here, to use recombinant glycan knockout gp120 variants to identify the precise Env binding site for MVN that drives DLI engagement and lysis. Using mutagenesis combined with a series of biophysical and virological experiments, we identified a restricted set of residues, N262, N332 and N448, all localized in a cluster on the outer domain of gp120, as the essential epitope for MVN binding. By generating these mutations in the corresponding HIV-1 virus, we established that the engagement of this glycan cluster with the lectin domain of MVN*-DLI is the trigger for DLI-derived virus and cell inactivation. Beyond defining the initial encounter step for lytic inactivation, this study provides a guide to further elucidate DLI mechanism, including the stoichiometry of Env trimer required for function, and downstream DLI optimization.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Lectinas/metabolismo , Calorimetría , Epítopos/genética , Productos del Gen env/genética , Productos del Gen env/metabolismo , Glicosilación , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/genética , Humanos , Unión Proteica , Resonancia por Plasmón de SuperficieRESUMEN
We recently reported the discovery of a recombinant chimera, denoted DAVEI (dual-acting virucidal entry inhibitor), which is able to selectively cause specific and potent lytic inactivation of both pseudotyped and fully infectious human immunodeficiency virus (HIV-1) virions. The chimera is composed of the lectin cyanovirin-N (CVN) fused to the 20-residue membrane-proximal external region (MPER) of HIV-1 gp41. Because the Env gp120-binding CVN domain on its own is not lytic, we sought here to determine how the MPER(DAVEI) domain is able to endow the chimera with virolytic activity. We used a protein engineering strategy to identify molecular determinants of MPER(DAVEI) that are important for function. Recombinant mutagenesis and truncation demonstrated that the MPER(DAVEI) domain could be significantly minimized without loss of function. The dependence of lysis on specific MPER sequences of DAVEI, determination of minimal linker length, and competition by a simplified MPER surrogate peptide suggested that the MPER domain of DAVEI interacts with the Env spike trimer, likely with the gp41 region. This conclusion was further supported by observations from binding of the biotinylated MPER surrogate peptide to Env protein expressed on cells, monoclonal antibody competition, a direct binding enzyme-linked immunosorbent assay on viruses with varying numbers of trimeric spikes on their surfaces, and comparison of maximal interdomain spacing in DAVEI to that in high-resolution structures of Env. The finding that MPER(DAVEI) in CVN-MPER linker sequences can be minimized without loss of virolytic function provides an improved experimental path for constructing size-minimized DAVEI chimeras and molecular tools for determining how simultaneous engagement of gp120 and gp41 by these chimeras can disrupt the metastable virus Env spike.