Safety, pharmacokinetics and neutralization of the broadly neutralizing HIV-1 human monoclonal antibody VRC01 in healthy adults.

VRC-HIVMAB060-00-AB (VRC01) is a broadly neutralizing HIV-1 monoclonal antibody (mAb) isolated from the B cells of an HIV-infected patient. It is directed against the HIV-1 CD4 binding site and is capable of potently neutralizing the majority of diverse HIV-1 strains. This Phase I dose-escalation study in healthy adults was conducted at the National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA). Primary objectives were the safety, tolerability and pharmacokinetics (PK) of VRC01 intravenous (i.v.) infusion at 5, 20 or 40 mg/kg, given either once (20 mg/kg) or twice 28 days apart (all doses), and of subcutaneous (s.c.) delivery at 5 mg/kg compared to s.c. placebo given twice, 28 days apart. Cumulatively, 28 subjects received 43 VRC01 and nine received placebo administrations. There were no serious adverse events or dose-limiting toxicities. Mean 28-day serum trough concentrations after the first infusion were 35 and 57 µg/ml for groups infused with 20 mg/kg (n = 8) and 40 mg/kg (n = 5) doses, respectively. Mean 28-day trough concentrations after the second infusion were 56 and 89 µg/ml for the same two doses. Over the 5-40 mg/kg i.v. dose range (n = 18), the clearance was 0.016 l/h and terminal half-life was 15 days. After infusion VRC01 retained expected neutralizing activity in serum, and anti-VRC01 antibody responses were not detected. The human monoclonal antibody (mAb) VRC01 was well tolerated when delivered i.v. or s.c. The mAb demonstrated expected half-life and pharmacokinetics for a human immunoglobulin G. The safety and PK results support and inform VRC01 dosing schedules for planning HIV-1 prevention efficacy studies.

Modulation of hexokinase association with mitochondria analyzed with quantitative three-dimensional confocal microscopy.

Hexokinase isozyme I is proposed to be associated with mitochondria in vivo. Moreover, it has been suggested that this association is modulated in coordination with changes in cell metabolic state. To test these hypotheses, we analyzed the subcellular distribution of hexokinase relative to mitochondria in paraformaldehyde-fixed astrocytes using immunocytochemistry and quantitative three-dimensional confocal microscopy. Analysis of the extent of colocalization between hexokinase and mitochondria revealed that approximately 70% of cellular hexokinase is associated with mitochondria under basal metabolic conditions. In contrast to the immunocytochemical studies, between 15 to 40% of cellular hexokinase was found to be associated with mitochondria after fractionation of astrocyte cultures depending on the exact fractionation conditions. The discrepancy between fractionation studies and those based on imaging of distributions in fixed cells indicates the usefulness of using techniques that can evaluate the distributions of "cytosolic" enzymes in cells whose subcellular ultrastructure is not severely disrupted. To determine if hexokinase distribution is modulated in concert with changes in cell metabolism, the localization of hexokinase with mitochondria was evaluated after inhibition of glucose metabolism with 2-deoxyglucose. After incubation with 2-deoxyglucose there was an approximate 35% decrease in the amount of hexokinase associated with mitochondria. These findings support the hypothesis that hexokinase is bound to mitochondria in rat brain astrocytes in vivo, and that this association is sensitive to cell metabolic state.

Compartmentation of glycolytic and glycogenolytic metabolism in vascular smooth muscle.

Vascular smooth muscle is characterized by a high rate of aerobic lactate production, which may be altered independently of oxidative phosphorylation. This finding suggested a cytoplasmic compartmentation of metabolism. Exogenous glucose was found to be the sole precursor of aerobic glycolysis under unstimulated conditions. Although tissue depolarization with high K+ resulted in a substantial reduction of endogenous glycogen, exogenous glucose remained the sole precursor of aerobic lactate production. These data showed unequivocally that carbohydrate metabolism is compartmentalized in vascular smooth muscle.

Superresolution three-dimensional images of fluorescence in cells with minimal light exposure.

Fluorescent probes offer insight into the highly localized and rapid molecular events that underlie cell function. However, methods are required that can efficiently transform the limited signals from such probes into high-resolution images. An algorithm has now been developed that produces highly accurate images of fluorescent probe distribution inside cells with minimal light exposure and a conventional light microscope. This method provides resolution nearly four times greater than that currently available from any fluorescence microscope and was used to study several biological problems.

Compartmentation of carbohydrate metabolism in vascular smooth muscle: evidence for at least two functionally independent pools of glucose 6-phosphate.

Previous work has indicated that there are at least two functionally independent Embden-Meyerhof pathways within the vascular smooth muscle of porcine carotid artery. We tested this hypothesis by analyzing the isotopic equilibrium between medium glucose and intracellular glucose 6-phosphate under basal conditions and after 30 min of mechanical activation, during which time the rate of glycogenolysis has been found to be substantial. Under basal conditions, the specific activity of glucose 6-phosphate equilibrated to a level which was not in isotopic equilibrium with medium glucose suggesting that there is a significant pool of glucose 6-phosphate which is not readily accessible to medium glucose. After 15 min of mechanical activation, the specific activity of intracellular glucose 6-phosphate was found to decrease significantly from its apparent steady-state distribution, indicating that glycogen was likely to be a significant source for glucose 6-phosphate. Since the specific activity of lactate was unaltered from its equilibrium distribution under similar stimulus conditions, these findings substantiate the existence of at least two independent pools of glucose 6-phosphate.

Localization of glucokinase gene expression in the rat brain.

The brain contains a subpopulation of glucosensing neurons that alter their firing rate in response to elevated glucose concentrations. In pancreatic beta-cells, glucokinase (GK), the rate-limiting enzyme in glycolysis, mediates glucose-induced insulin release by regulating intracellular ATP production. A similar role for GK is proposed to underlie neuronal glucosensing. Via in situ hybridization, GK mRNA was localized to hypothalamic areas that are thought to contain relatively large populations of glucosensing neurons (the arcuate, ventromedial, dorsomedial, and paraventricular nuclei and the lateral area). GK also was found in brain areas without known glucosensing neurons (the lateral habenula, the bed nucleus stria terminalis, the inferior olive, the retrochiasmatic and medial preoptic areas, and the thalamic posterior paraventricular, interpeduncular, oculomotor, and anterior olfactory nuclei). Conversely, GK message was not found in the nucleus tractus solitarius, which contains glucosensing neurons, or in ependymal cells lining the third ventricle, where others have described its presence. In the arcuate nucleus, >75% of neuropeptide Y-positive neurons also expressed GK, and most GK+ neurons also expressed KIR6.2 (the pore-forming subunit of the ATP-sensitive K+ channel). The anatomic distribution of GK mRNA was confirmed in micropunch samples of hypothalamus via reverse transcription-polymerase chain reaction (RT-PCR). Nucleotide sequencing of the recovered PCR product indicated identity with nucleotides 1092-1411 (within exon 9 and 10) of hepatic and beta-cell GK. The specific anatomic localization of GK mRNA in hypothalamic areas known to contain glucosensing neurons and the coexpression of KIR6.2 and NPY in GK+ neurons support a role for GK as a primary determinant of glucosensing in neuropeptide neurons that integrate multiple signals relating to peripheral energy metabolism.

Selection of fluorescent ion indicators for simultaneous measurements of pH and Ca2+.

The advent of fluorescent ion sensitive indicators has improved our understanding of the mechanisms involved in regulating pHi and [Ca2+]i homeostasis in living cells. However, changes in [Ca2+]i can alter pHi regulatory mechanisms and vice versa, making assignment of either ion to a particular physiological response complex. A further complication is that all fluorescent Ca2+ indicators are sensitive to protons. Therefore, techniques to simultaneously measure these two ions have been developed. Although several combinations of pH and Ca2+ probes have been used, few systematic studies have been performed to assess the validity of such measurements. In vitro analysis (i.e. free acid forms of dyes) indicated that significant quenching effects occurred when using specific dye combinations. Fura-2/SNARF-1 and MagFura-2/SNARF-1 probe combinations were found to provide the most accurate pH and [Ca2+] measurements relative to Fluo-3/SNARF-1, Ca2+-Green-1/SNARF-1, or BCECF/SNARF-1. Similar conclusions were reached when probes were calibrated after loading into cells. The magnitude of interactions between pH and Ca2+ probes could be a factor which may limit the use of certain specific combinations. Loading of probes that exhibit interactions into distinct intracellular compartments (i.e. separated by a biological membrane) abolished the quenching effects. These data indicate that interactions between the probes used to simultaneously monitor pH and Ca2+ must be considered whenever probe combinations are used.

pH and drug resistance. I. Functional expression of plasmalemmal V-type H+-ATPase in drug-resistant human breast carcinoma cell lines.

A major obstacle for the effective treatment of cancer is the phenomenon of multidrug resistance (MDR) exhibited by many tumor cells. Many, but not all, MDR cells exhibit membrane-associated P-glycoprotein (P-gp), a drug efflux pump. However, most mechanisms of MDR are complex, employing P-gp in combination with other, ill-defined activities. Altered cytosolic pH (pHi) has been implicated to play a role in drug resistance. In the current study, we investigated mechanisms of pHi regulation in drug-sensitive (MCF-7/S) and drug-resistant human breast cancer cells. Of the drug-resistant lines, one contained P-gp (MCF-7/DOX; also referred to as MCF-7/D40) and one did not (MCF-7/MITOX). The resting steady-state pHi was similar in the three cell lines. In addition, in all the cell lines, HCO3- slightly acidified pHi and increased the rates of pHi recovery after an acid load, indicating the presence of anion exchanger (AE) activity. These data indicate that neither Na+/H+ exchange nor AE is differentially expressed in these cell lines. The presence of plasma membrane vacuolar-type H+-ATPase (pmV-ATPase) activity in these cell lines was then investigated. In the absence of Na+ and HCO3-, MCF-7/S cells did not recover from acid loads, whereas MCF-7/MITOX and MCF-7/DOX cells did. Furthermore, recovery of pHi was inhibited by bafilomycin A1 and NBD-Cl, potent V-ATPase inhibitors. Attempts to localize V-ATPase immunocytochemically at the plasma membranes of these cells were unsuccessful, indicating that V-ATPase is not statically resident at the plasma membrane. Consistent with this was the observation that release of endosomally trapped dextran was more rapid in the drug-resistant, compared with the drug-sensitive cells. Furthermore, the drug-resistant cells entrapped doxorubicin into intracellular vesicles whereas the drug-sensitive cells did not. Hence, it is hypothesized that the measured pmV-ATPase activity in the drug-resistant cells is a consequence of rapid endomembrane turnover. The potential impact of this behavior on drug resistance is examined in a companion manuscript.

Modeling of exposure to carpet-cleaning chemicals preceding irritant-induced asthma in one patient.

42-year-old woman experienced an acute asthma attack, seizures, and unconsciousness immediately after a carpet-cleaning and deodorizing job was conducted in her home. Exposure modeling estimates that she was exposed to approximately 3.4-17 mg/m(3) of sodium tripolyphosphate and more than 14 mg/m(3) volatile organic compounds immediately after the cleaning. I derived two separate exposure models for these estimates that evidenced good consistency of exposure estimates. Asthmatics and carpet-cleaning companies should be advised about safety during carpet-cleaning operations, including adequate warnings about excess risk for asthmatics, temporary removal from the home, reduced detergent levels within cleaners, and reduced overall levels of cleaning solutions used within the home. Further studies of carpet-cleaning exposures are indicated.

Frontiers in the measurement of cell and tissue pH.

Investigation into the causes and consequences of aberrant pH regulation is entirely dependent on the ability to measure this parameter with specificity and sensitivity. Since their beginnings in the 1950s, techniques for measuring cell and tissue pH have undergone a number of significant advances. Following each of these advances, new investigators have been brought into the field, and new discoveries have been made. From microelectrode and dye distribution studies, measurement of pH underwent a revolution with the advent of pH-sensitive dyes that could be loaded into the cytosol. A further significant advance came from the measurement of cell and tissue pH in whole organisms by magnetic resonance spectroscopy (MRS). Frontiers in pH measurement exist in the development of pH-sensitive proteins and pH-sensitive MR contrast agents. These are predicted to bring even more people into this fascinating field, and generate more important discoveries.

Neuroimaging in neuro-ophthalmology.

The neuroradiologist and the clinician are partners in the search for pathology; the clinician must help the radiologist tailor the study by indicating where pathology is likely to be. The orbit, the sellar and parasellar regions, and new imaging modalities and interventional techniques are discussed in this light.

Microbiological contamination of compressed air used in dentistry: an investigation.

The purpose of this preliminary investigation was twofold: 1) to examine the possibility of cross-contamination between a dental-evacuation system and the compressed air used in dental operatories and 2) to capture and identify the most common microflora in the compressed-air supply. The investigation used swab, water, and air sampling that was designed to track microorganisms from the evacuation system, through the air of the mechanical room, into the compressed-air system, and back to the patient. Samples taken in the vacuum system, the air space in the mechanical room, and the compressed-air storage tank had significantly higher total concentrations of bacteria than the outside air sampled. Samples of the compressed air returning to the operatory were found to match the outside air sample in total bacteria. It was concluded that the air dryer may have played a significant role in the elimination of microorganisms from the dental compressed-air supply.

Energy metabolism of renal cell lines, A6 and MDCK: regulation by Na-K-ATPase.

The energy metabolism of two continuous cell lines of renal origin, MDCK (Madin-Darby dog kidney) and A6 (toad kidney), was investigated by measuring the oxygen consumption (QO2) and lactate production (Jlac) by cells taken into suspension from monolayer cultures. Cells suspended from fully differentiated monolayers produce approximately 80% of their ATP requirements from oxidative metabolism. The interrelationship between ion transport and metabolism was determined by analyzing the ouabain sensitive components of intermediary metabolism under control conditions and after the stimulation of active Na-K transport with nystatin. In both cell lines, approximately 25% of the net rate of ATP production was inhibited by ouabain. Ouabain inhibited Jlac by 40% in MDCK and 45% in A6 cells, whereas QO2 was decreased by only 20% in both cell lines. In the presence of 0.05 mg nystatin/mg cell protein, ouabain sensitive Jlac increased by 75% in MDCK and was more than doubled in A6, whereas the ouabain-sensitive QO2 was not statistically different than control. This preferential stimulation of glycolysis with nystatin was not due to a limited capacity of mitochondrial oxidative phosphorylation since nystatin treatment of cells incubated without glucose (no glycolysis) significantly elevated the rate of QO2. These data demonstrate that aerobic glycolysis is more sensitive than is QO2 to changes in hydrolytic activity of the Na-K-adenosine triphosphatase (ATPase), in both cell lines.

Compartmentation of carbohydrate metabolism in vascular smooth muscle.

In vascular smooth muscle, oxidative phosphorylation and glycolysis are independently regulated. Previous studies indicated that the independent regulation of these pathways was related to a compartmentation of carbohydrate metabolism. To further study carbohydrate metabolism, glucose transport and the incorporation of radiolabel from glucose into glycogen and lactate were measured after the oxidative and glycolytic pathways were independently altered. Ouabain stimulated mechanical activity, oxygen consumption, and glycogenolysis, whereas lactate production was decreased. Although glycogenolysis was substantial, glucose was the only substrate for lactate, indicating that intermediates derived from glycogen do not mix with those from glucose uptake. Thus glycogenolysis and glycolysis are carried out by independent enzymatic pathways. Insulin-stimulated lactate production and glucose transport without affecting the other parameters. Again, lactate was produced only from glucose. Phenytoin decreased isometric tension and oxygen consumption, whereas stimulating lactate production and glycogenolysis. Glycogen was the primary substrate for the lactate produced. Our findings indicate that the compartmentation of substrate utilization is ascribable to the coordination of glycogenolysis with increases in oxygen consumption and the coupling of glycolysis to the Na-K-adenosine triphosphatase. The coupling of independent energy providing pathways to specific endergonic processes indicates a mechanism by which cellular energetic efficiency may be optimized.

Coupling of aerobic glycolysis and Na+-K+-ATPase in renal cell line MDCK.

The relation between the activity of the Na+-K+-ATPase and the metabolic source of ATP was investigated in suspensions of MDCK cells. The pump activity of Na+-K+-ATPase was estimated from the initial rate of ouabain-sensitive K+ uptake into K+-depleted cells. Uptake was initiated by the reintroduction of K+ to the medium in which the cells were suspended. The metabolic source of ATP was varied by changing the substrates supplied to the suspension. Cells respiring on glutamine produced ATP from oxidative metabolism alone, whereas cells incubated with glucose and glutamine produced ATP via glycolysis and oxidative phosphorylation. Over a wide range of extracellular K+ concentrations, the initial rate of K+ uptake was faster in cells incubated with glucose and glutamine when compared with cells incubated with glutamine alone. Kinetic analysis together with ouabain-binding data demonstrated that this increase in K+ uptake was due to an increase in maximal velocity (Vmax) at a constant number of Na+-K+-ATPase transport sites. In addition, steady-state studies revealed that the addition of glucose to K+-depleted cells respiring on glutamine alone resulted in a net ouabain-sensitive influx of K+. These data demonstrate that in MDCK cells the maximal capacity for transport via the Na+-K+-ATPase is greater when ATP is produced from both glycolysis and oxidative phosphorylation than when ATP is produced from oxidative phosphorylation alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Energy metabolism and transduction in smooth muscle.

Early investigations into the nature of the coupling between energy transduction and metabolism in smooth muscle, particularly from the laboratories of Bülbring and Lundholm, suggested that specific metabolic pathways could independently supply energy for ion transport and actin-myosin interactions. Subsequent work has solidified the concept that oxidative phosphorylation is specifically coupled to tension generation and maintenance, whereas, aerobic glycolysis is not only a vital characteristic of smooth muscle metabolism, but also is likely to be independently coupled to Na-K transport at the plasmalemma. The independence of oxidative and glycolytic metabolism is reflected as a compartmentation of carbohydrate metabolism in the porcine carotid artery. The coupling of these independent metabolic pathways with specific energy utilizing processes, indicates a means by which energy production and transduction can be closely and efficiently regulated. The coupling of glycogenolysis to mitochondrial respiration may have evolved as a direct response to the energetic needs of VSM. That is, the large glycogenolytic response in the initial minutes of stimulation may be necessary to maximize the cellular production of ATP during the presteady state. Likewise, the coupling between aerobic glycolysis and Na-K transport indicates a sensitive and efficient means of coordinating energy metabolism with ion transport at the membrane level. Additionally, the regulation of substrate supply, i.e. glucose transport, also may be closely coordinated with changes in ion transport. One may speculate that alterations in the microenvironment of each compartment can independently regulate intermediary metabolism and therefore allow the cell to quickly and efficiently respond to localized stimuli. Thus, stimulation of Na-K transport could effectively regulate energy production at the membrane level without mobilizing or competing with the energy transduction of other cellular processes. This compartmentation of energy utilization may be highly advantageous, since oxidative metabolism is closely coordinated with mechanical activity and therefore regulation of blood flow. Future investigations will attempt to elucidate which intracellular signals which are responsible for the regulation of these functionally independent compartments of energy metabolism and transduction in VSM. In more general terms, our findings provide a basis from which future questions concerning the regulation of cellular metabolism must be directed. The cellular cytoplasm can no longer be envisioned as a homogeneous compartment, but rather a complex array of functional subcompartments which may be individual