When to switch and what to switch to: strategic use of antiretroviral therapy

Clinical cohort studies suggest that as many as 60% of patients experience virologic failure of a first-line antiretroviral regimen. Second-line and rescue (or salvage) regimens have a poorer success record: Most studies presented to date show a short-term virologic response rate of only approximately 30% in treatment-experienced individuals. That rate will improve with better understanding of what causes initial virologic failure, continued development of new antiretroviral agents (including drugs with new mechanisms of action) and new treatment strategies (including dual-protease inhibitor regimens), and more widespread use of resistance testing. Further clinical research is needed to improve salvage options, and physicians should consider enrolling treatment-experienced patients in clinical trials.     

Receptor-like function of heparin in the binding and uptake of neutral lipids

Molecular mechanisms regulating the binding, amphipathic stabilization, and metabolism of the major neutral lipids (e.g., cholesteryl esters, triglycerides, and fatty acids) are well studied, but the details of their movement from a binding compartment to a metabolic compartment deserve further attention. Since all neutral lipids must cross hydrophilic segments of plasma membranes during such movement, we postulate that a critical receptor-like site exists on the plasma membrane to mediate a step between binding and metabolism and that membrane-associated heparin is a key part of this mediator. For example, intestinal brush border membranes containing heparin bind homogeneous human pancreatic 125I-labeled cholesterol esterase (100 kDa) and 125I-labeled triglyceride lipase (52 kDa). This interaction is enzyme concentration-dependent, specific, and saturable and is reversed upon addition of soluble heparin. Scatchard analysis demonstrates a single class of receptors with a Kd of 100 nM and a Bmax of approximately 50-60 pmol per mg of vesicle protein. In contrast, enzymes associated with the hydrolysis of hydrophilic compounds such as amylase, phospholipase A2, and deoxyribonuclease do not bind to intestinal membranes in this manner. Human pancreatic cholesterol esterase also binds specifically and saturably to cultured intestinal epithelial cells (CaCo-2), and soluble heparin significantly diminishes the cellular uptake of the resultant hydrophobic reaction products (cholesterol and free fatty acids). We conclude that a physiological role for intestinal heparin is that of a mediator to bind neutral lipolytic enzymes at the brush border and thus promote absorption of the subsequent hydrolyzed nutrients in the intestine. This mechanism may be a generalizable pathway for transport of neutral lipids into endothelial and other cells.     

Residual human immunodeficiency virus (HIV) Type 1 RNA and DNA in lymph nodes and HIV RNA in genital secretions and in cerebrospinal fluid after suppression of viremia for 2 years

Residual viral replication persists in a significant proportion of human immunodeficiency virus (HIV)-infected patients receiving potent antiretroviral therapy. To determine the source of this virus, levels of HIV RNA and DNA from lymphoid tissues and levels of viral RNA in serum, cerebrospinal fluid (CSF), and genital secretions in 28 patients treated for < or =2.5 years with indinavir, zidovudine, and lamivudine were examined. Both HIV RNA and DNA remained detectable in all lymph nodes. In contrast, HIV RNA was not detected in 20 of 23 genital secretions or in any of 13 CSF samples after 2 years of treatment. HIV envelope sequence data from plasma and lymph nodes from 4 patients demonstrated sequence divergence, which suggests varying degrees of residual viral replication in 3 and absence in 1 patient. In patients receiving potent antiretroviral therapy, the greatest virus burden may continue to be in lymphoid tissues rather than in central nervous system or genitourinary compartments.     

Aerobactin Mediates Virulence and Accounts for Increased Siderophore Production under Iron-Limiting Conditions by Hypervirulent (Hypermucoviscous) Klebsiella pneumoniae

Hypervirulent (hypermucoviscous) Klebsiella pneumoniae (hvKP) strains are an emerging variant of “classical” K. pneumoniae (cKP) that cause organ and life-threatening infection in healthy individuals. An understanding of hvKP-specific virulence mechanisms that enabled evolution from cKP is limited. Observations by our group and previously published molecular epidemiologic data led us to hypothesize that hvKP strains produced more siderophores than cKP strains and that this trait enhanced hvKP virulence. Quantitative analysis of 12 hvKP strains in iron-poor minimal medium or human ascites fluid showed a significant and distinguishing 6- to 10-fold increase in siderophore production compared to that for 14 cKP strains. Surprisingly, high-pressure liquid chromatography (HPLC)-mass spectrometry and characterization of the hvKP strains hvKP1, A1142, and A1365 and their isogenic aerobactin-deficient (ΔiucA) derivatives established that aerobactin accounted for the overwhelming majority of increased siderophore production and that this was not due to gene copy number. Further, aerobactin was the primary factor in conditioned medium that enhanced the growth/survival of hvKP1 in human ascites fluid. Importantly the ex vivo growth/survival of hvKP1 ΔiucA was significantly less than that of hvKP1 in human ascites fluid, and the survival of outbred CD1 mice challenged subcutaneously or intraperitoneally with hvKP1 was significantly less than that of mice challenged with hvKP1 ΔiucA. The lowest subcutaneous and intraperitoneal challenge inocula of 3 × 10² and 3.2 × 10¹ CFU, respectively, resulted in 100% mortality, demonstrating the virulence of hvKP1 and its ability to cause infection at a low dose. These data strongly support that aerobactin accounts for increased siderophore production in hvKP compared to cKP (a potential defining trait) and is an important virulence factor.     

Comparison of Electrical and Ultrasound Neurostimulation in Rat Motor Cortex

Ultrasound (US) is known to non-invasively stimulate and modulate brain function; however, the mechanism of action is poorly understood. This study tested US stimulation of rat motor cortex (100 W/cm², 200?kHz) in combination with epidural cortical stimulation. US directly evoked hindlimb movement. This response occurred even with short US bursts (3?ms) and had short latency (10?ms) and long refractory (3?s) periods. Unexpectedly, the epidural cortical stimulation hindlimb response was not altered during the 3-s refractory period of the US hindlimb response. This finding suggests that the US refractory period is not a general suppression of motor cortex, but rather the recovery time of a US-specific mechanism.     

Reengineering inducible cardiac-specific transgenesis with an attenuated myosin heavy chain promoter

Despite the advantages of reversibly altering cardiac transgene expression, the number of successful studies with inducible cardiac-specific transgene expression remains limited. The utility of the current system is hampered by the large number of lines needed before a nonleaky inducible line is isolated and by the use of a heterologous virus-based minimal promoter in the responder line. We developed an efficient, experimentally flexible system that enables us to reversibly affect both abundant and nonabundant cardiomyocyte proteins. The use of bacterial-codon-based transactivators led to aberrant splicing, whereas other more efficient transactivators, by themselves, caused disease when expressed in the heart. The redesign of the system focused on developing stable transactivator-expressing lines in which expression was driven by the mouse alpha-myosin heavy chain promoter. A minimal responder locus was derived from the same promoter, in which the GATA sites and thyroid responsive elements responsible for robust cardiac specific expression were ablated, leading to an attenuated promoter that could be inducibly controlled. In all cases, whether activated or not, expression mimicked that of the parental promoter. By use of this system, an inducible expression of an abundant contractile protein, the atrial isoform of essential myosin light chain 1, and a powerful biological effector, glycogen synthase kinase-3beta (GSK-3beta), were obtained. Subsequently, we tested the hypothesis that GSK-3beta expression could reverse a preexisting hypertrophy. Inducible expression of GSK-3beta could both attenuate a hypertrophic response and partially reverse a pressure-overload-induced hypertrophy. The system appears to be robust and can be used to temporally control high levels of cardiac-specific transgene expression.