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1.  Ceftriaxone reverses ketamine-induced lasting EEG and astrocyte alterations in juvenile mice 
Drug and alcohol dependence  2015;156:14-20.
Ketamine, an N-Methyl-D-aspartate receptor antagonist, is used as a pediatric anesthetic because of its favorable safety profile. It is also being investigated as an antidepressant. Unfortunately, ketamine causes adverse reactions including hallucinations and is associated with a high prevalence of abuse among adolescents. Although chronic ketamine use has been shown to produce cognitive impairments even years following cessation, little is known about its long-term consequences on adolescents. The beta-lactam ceftriaxone has been shown to attenuate alcohol withdrawal, and alleviate early brain injury and memory impairments following subarachnoid hemorrhage. However, its ability to reverse the effects of adolescent ketamine exposure is not known. Previous data indicate that ketamine causes a reduction in the number of Excitatory Amino Acid Transporter Type 2 (EAAT2)-containing astrocytes. Additionally, the beta lactam antibiotic ceftriaxone increased expression of EAAT2. As EAAT2 is a principal mechanism of glutamate clearance from the synapse, the current study tests the hypothesis that ceftriaxone may reverse functional consequences of ketamine exposure.
We examined the effects of chronic ketamine in juvenile mice as well as reversal by ceftriaxone using electroencephalography (EEG). Subsequently, we assessed the effects of these treatments on markers of astrocyte proliferation, using Glial Fibrillary Acidic Protein (GFAP), and function, as evidenced by EAAT2.
Juvenile mice exposed to chronic ketamine showed lasting alterations in EEG measurements as well as markers of astrocyte number and function. These alterations were reversed by ceftriaxone.
Data suggest that ceftriaxone may be able to ameliorate ketamine-induced long-term disturbances in adolescent brains.
PMCID: PMC4633341  PMID: 26442907
ketamine; mouse; EEG; glia; EAAT2
2.  Delayed emergence of behavioral and electrophysiological effects following juvenile ketamine exposure in mice 
Translational Psychiatry  2015;5(9):e635-.
Frequent ketamine abuse in adulthood correlates with increased risk of psychosis, as well as cognitive deficits, including disruption of higher-order executive function and memory formation. Although the primary abusers of ketamine are adolescents and young adults, few studies have evaluated its effects on juvenile cognition. Therefore, the current study analyzes the effect of adolescent ketamine exposure on cognitive development. Juvenile mice (4 weeks of age) were exposed to chronic ketamine (20 mg kg−1, i.p. daily) for 14 days. Mice were tested immediately after exposure in the juvenile period (7 weeks of age) and again as adults (12 weeks of age). Measures included electroencephalography (EEG) in response to auditory stimulation, the social choice test, and a 6-arm radial water maze task. Outcome measures include low-frequency EEG responses, event-related potential (ERP) amplitudes, indices of social behavior and indices of spatial working memory. Juvenile exposure to ketamine was associated with electrophysiological abnormalities in adulthood, particularly in induced theta power and the P80 ERP. The social choice test revealed that ketamine-exposed mice failed to exhibit the same age-related decrease in social interaction time as controls. Ketamine-exposed mice outperformed control mice as juveniles on the radial water maze task, but did not show the same age-related improvement as adult controls. These data support the hypothesis that juvenile exposure to ketamine produces long-lasting changes in brain function that are characterized by a failure to progress along normal developmental trajectories.
PMCID: PMC5068812  PMID: 26371763
3.  Rapid assessment of nonlinear optical propagation effects in dielectrics 
Scientific Reports  2015;5:7650.
Ultrafast laser processing applications need fast approaches to assess the nonlinear propagation of the laser beam in order to predict the optimal range of processing parameters in a wide variety of cases. We develop here a method based on the simple monitoring of the nonlinear beam shaping against numerical prediction. The numerical code solves the nonlinear Schrödinger equation with nonlinear absorption under simplified conditions by employing a state-of-the art computationally efficient approach. By comparing with experimental results we can rapidly estimate the nonlinear refractive index and nonlinear absorption coefficients of the material. The validity of this approach has been tested in a variety of experiments where nonlinearities play a key role, like spatial soliton shaping or fs-laser waveguide writing. The approach provides excellent results for propagated power densities for which free carrier generation effects can be neglected. Above such a threshold, the peculiarities of the nonlinear propagation of elliptical beams enable acquiring an instantaneous picture of the deposition of energy inside the material realistic enough to estimate the effective nonlinear refractive index and nonlinear absorption coefficients that can be used for predicting the spatial distribution of energy deposition inside the material and controlling the beam in the writing process.
PMCID: PMC4288215  PMID: 25564243
4.  Juvenile Exposure to Ketamine Causes Delayed Emergence of EEG Abnormalities During Adulthood in mice 
Drug and alcohol dependence  2013;134:123-127.
Increased susceptibility to cognitive impairment or psychosis in adulthood is associated with adolescent drug abuse. Studies in adults have identified impairments in attention and memory, and changes in EEG, as common consequences of ketamine abuse. In contrast, the effects of ketamine on the juvenile brain have not been extensively tested. This is a significant omission, since abuse of ketamine is often observed within this age group.
Juvenile mice (4–6 weeks of age) were administered ketamine (20 mg/kg) for 14 days. EEG was assessed in response to auditory stimulation both at one week following ketamine exposure at 7 weeks of age (juvenile) and again at 12 weeks of age (adult). EEG was analyzed for baseline activity, event-related power and event-related potentials (ERPs).
While no effects of ketamine exposure were observed during the juvenile period, significant reductions in amplitude of the P20 ERP component and event-related gamma power were seen following ketamine when re-tested as adults. In contrast, reductions in event–related theta were seen in ketamine-exposed mice at both time points.
Age related deficits in electrophysiological components such as P20 or event-related gamma may be due to an interruption of normal neural maturation. Reduction of NMDAR signaling during adolescence leads to delayed-onset disruption of gamma oscillations and the P20 component of the ERP. Further, delayed onset of impairment following adolescent ketamine abuse suggests that methods could be developed to detect and treat the early effects of drug exposure prior to the onset of disability.
PMCID: PMC4009692  PMID: 24210161
NMDA; gamma; oscillations; schizophrenia; theta; drug-abuse
5.  The circuitry of abulia: Insights from functional connectivity MRI 
NeuroImage : Clinical  2014;6:320-326.
Functional imaging and lesion studies have associated willed behavior with the anterior cingulate cortex (ACC). Abulia is a syndrome characterized by apathy and deficiency of motivated behavior. Abulia is most frequently associated with ACC damage, but also occurs following damage to subcortical nuclei (striatum, globus pallidus, thalamic nuclei). We present resting state functional connectivity MRI (fcMRI) data from an individual who suffered a stroke leading to abulia. We hypothesized that, although structural imaging revealed no damage to the patient's ACC, fcMRI would uncover aberrant function in this region and in the relevant cortical networks.
Resting state correlations in the patient's gray matter were compared to those of age-matched controls. Using a novel method to identify abnormal patterns of functional connectivity in single subjects, we identified areas and networks with aberrant connectivity.
Networks associated with memory (default mode network) and executive function (cingulo-opercular network) were abnormal. The patient's anterior cingulate was among the areas showing aberrant functional connectivity. In a rescan 3 years later, deficits remained stable and fcMRI findings were replicated.
These findings suggest that the aberrant functional connectivity mapping approach described may be useful for linking stroke symptoms to disrupted network connectivity.
•A method for single subject functional connectivity analysis is proposed.•In a case study, resting state fcMRI identifies reproducible disruption that corresponds to clinical deficit.•Damage to the anterior thalamus results in disrupted functional connectivity in the cingulate cortex.•Abulia without frontal lesion shows disrupted functional connectivity in the cingulo-opercular and default mode networks.
PMCID: PMC4215525  PMID: 25379445
Abulia; Executive function; Anterior cingulate; Functional connectivity; fMRI; apathy
6.  The scent of salience — Is there olfactory-trigeminal conditioning in humans? 
NeuroImage  2013;77:93-104.
Pavlovian fear conditioning has been thoroughly studied in the visual, auditory and somatosensory domain, but evidence is scarce with regard to the chemosensory modality. Under the assumption that Pavlovian conditioning relies on the supra-modal mechanism of salience attribution, the present study was set out to attest the existence of chemosensory aversive conditioning in humans as a specific instance of salience attribution. fMRI was performed in 29 healthy subjects during a differential aversive conditioning paradigm. Two odors (rose, vanillin) served as conditioned stimuli (CS), one of which (CS+) was intermittently coupled with intranasally administered CO2. On the neural level, a robust differential response to the CS+ emerged in frontal, temporal, occipito-parietal and subcortical brain regions, including the amygdala. These changes were paralleled by the development of a CS+-specific connectivity profile of the anterior midcingulate cortex (aMCC), which is a key structure for processing salience information in order to guide adaptive response selection. Increased coupling could be found between key nodes of the salience network (anterior insula, neo-cerebellum) and sensorimotor areas, representing putative input and output structures of the aMCC for exerting adaptive motor control. In contrast, behavioral and skin conductance responses did not show significant effects of conditioning, which has been attributed to contingency unawareness. These findings imply substantial similarities of conditioning involving chemosensory and other sensory modalities, and suggest that salience attribution and adaptive control represent a general, modality-independent principle underlying Pavlovian conditioning.
PMCID: PMC4132879  PMID: 23558094
Chemosensation; Pavlovian conditioning; Salience; Functional connectivity; fMRI
7.  Fatal Outcomes in Family Transmission of Mycoplasma pneumoniae 
Mycoplasma pneumoniae continues to be a significant cause of community-acquired pneumonia and can manifest as fulminant disease leading to mortality in healthy individuals.
Background. Mycoplasma pneumoniae continues to be a significant cause of community-acquired pneumonia and, on rare occasions, manifests as fulminant disease that leads to mortality, even in healthy individuals.
Methods. We conducted a retrospective study on members of a family who were quarantined by the Centers for Disease Control and Prevention in 2002 for respiratory failure and death of a 15-year-old brother (sibling 1) and a 13-year-old sister (sibling 2). Collected airway, cerebrospinal fluid (CSF), and serum samples from both deceased siblings and serum samples from both parents and the remaining 3 ill siblings (sibling 3–5) were tested using a range of diagnostic assays. Autopsy lung tissue samples from sibling 2 were also assessed using immunohistochemical and immunoelectron microscopic methods.
Results. Autopsy evaluation of sibling 1 revealed cerebral edema consistent with hypoxic ischemic encepatholopathy and pulmonary findings of bronchiolitis obliterans with organizing pneumonia (BOOP). Postmortem lung examination of sibling 2 revealed lymphoplasmacytic bronchiolitis with intraluminal purulent exudate, BOOP, and pulmonary edema. Results of diagnostic assays implicated the household transmission of M. pneumoniae among all 5 siblings and both parents. Further analysis of lung tissue from sibling 2 demonstrated the presence of M. pneumoniae organisms and community-acquired respiratory distress syndrome toxin. M. pneumoniae was cultured directly from sibling 2 autopsy lung tissue.
Conclusion. Evidence is provided that M. pneumoniae was readily transmitted to all members of the household and that the resulting infections led to a spectrum of individual responses with variation in disease progression, including lymphoplasmacytic bronchiolitis, BOOP, and death.
PMCID: PMC3245726  PMID: 22052890
8.  Cerebrospinal fluid hypocretin (orexin) levels are elevated by play but are not raised by exercise and its associated heart rate, blood pressure, respiration or body temperature changes 
Archives italiennes de biologie  2011;149(4):492-498.
Hypocretin (Hcrt) has been implicated in the control of motor activity and in respiration and cardiovascular changes. Loss of Hcrt in narcolepsy is linked to sleepiness and to cataplexy, a sudden loss of muscle tone which is triggered by sudden strong emotions. In the current study, we have compared the effects of treadmill running to yard play on cerebrospinal fluid (CSF) Hcrt level in normal dogs. We find that treadmill locomotion, at a wide range of speeds, does not increase Hcrt level beyond baseline, whereas yard play produces a substantial increase in Hcrt, even though both activities produce comparable increases in heart rate, respiration and body temperature. We conclude that motor and cardiovascular changes are not sufficient to elevate CSF levels of Hcrt and we hypothesize that the emotional aspects of yard play account for the observed increase in Hcrt.
PMCID: PMC3535459  PMID: 22205595
REM sleep; Breathing canine
9.  Modulation of hypothalamus and amygdalar activation levels with stimulus valence 
NeuroImage  2010;51(1):324-328.
In spite of long-standing evidence showing that the hypothalamus is instrumental in generating behaviors associated with positive and negative emotions, little is known about the role of the hypothalamus in normal human emotional processing. Recent findings have suggested that the hypothalamus plays a role beyond mere control of HPA-axis function; this is also supported by the existence of rich anatomical connections between the hypothalamus and the amygdala, a region known for its important role in emotional processing. However, evidence of emotion-induced hypothalamic activity from neuroimaging studies has been inconsistent, possibly due to methodological limitations (e.g., low spatial resolution). Taking advantage of recent improvements in fMRI technology we set out to explore a possible valence-dependent modulation of hypothalamic activity. Using second order parametric analysis of high-resolution BOLD fMRI, we assessed hypothalamic activation patterns during passive viewing of visual stimuli of varying valence, and compared the results with the activity pattern in the amygdalae, i.e. nuclei with known valence-dependent activity profiles. We show that both hypothalamic and amygdalar activation is modulated by the second-order stimulus valence term, i.e., there is increased neural activity following the processing of both positive and negative stimuli. Our results suggest that the hypothalamus may serve a role in generating emotions broader than generally assumed.
PMCID: PMC3535464  PMID: 20156568
fMRI; Hypothalamus; Amygdala; Emotion
10.  GABAB-mediated rescue of altered excitatory–inhibitory balance, gamma synchrony and behavioral deficits following constitutive NMDAR-hypofunction 
Translational Psychiatry  2012;2(7):e142-.
Reduced N-methyl-D-aspartate-receptor (NMDAR) signaling has been associated with schizophrenia, autism and intellectual disability. NMDAR-hypofunction is thought to contribute to social, cognitive and gamma (30–80 Hz) oscillatory abnormalities, phenotypes common to these disorders. However, circuit-level mechanisms underlying such deficits remain unclear. This study investigated the relationship between gamma synchrony, excitatory–inhibitory (E/I) signaling, and behavioral phenotypes in NMDA-NR1neo−/− mice, which have constitutively reduced expression of the obligate NR1 subunit to model disrupted developmental NMDAR function. Constitutive NMDAR-hypofunction caused a loss of E/I balance, with an increase in intrinsic pyramidal cell excitability and a selective disruption of parvalbumin-expressing interneurons. Disrupted E/I coupling was associated with deficits in auditory-evoked gamma signal-to-noise ratio (SNR). Gamma-band abnormalities predicted deficits in spatial working memory and social preference, linking cellular changes in E/I signaling to target behaviors. The GABAB-receptor agonist baclofen improved E/I balance, gamma-SNR and broadly reversed behavioral deficits. These data demonstrate a clinically relevant, highly translatable neural-activity-based biomarker for preclinical screening and therapeutic development across a broad range of disorders that share common endophenotypes and disrupted NMDA-receptor signaling.
PMCID: PMC3410621  PMID: 22806213
animal model; GABAergic signaling; gamma oscillation; neuropsychiatric disease; NMDA-receptor
Neuroscience  2006;144(1):239-246.
Currently available antipsychotic medications work primarily by antagonizing D2 dopamine receptors, thus raising intracellular cAMP levels. We hypothesized that intracellular stimulation of cAMP levels in the CNS would have similar effects to treatment with antipsychotic medication. To test this hypothesis, we studied the effect of an acute treatment of rolipram, an inhibitor of type 4 phosphodiesterases that degrade cAMP, on acoustic startle and prepulse inhibition (PPI) of the acoustic startle response in C57BL/6J mice known to exhibit poor PPI. PPI is disrupted in schizophrenia patients, and the ability of a drug to increase PPI in mice is predictive of antipsychotic efficacy. We show here that acute treatment with rolipram significantly increases PPI at doses that do not alter the acoustic startle response (lowest effective dose 0.66 mg/kg). In addition, rolipram (0.66 mg/kg) blocks the disruptive effects of amphetamine (10 mg/kg) on PPI. At a slightly higher dose (1.0 mg/kg), rolipram also induces catalepsy. Thus, phosphodiesterase-4 (PDE4) inhibition has many of the same behavioral effects as traditional antipsychotic medications. In contrast to traditional antipsychotics, these effects are achieved through alteration of an intracellular second messenger system rather than antagonism of neurotransmitter receptors. Given previous reports showing rolipram improves cognition, we conclude that PDE4 represents an important novel target for further antipsychotic drug development.
PMCID: PMC3313447  PMID: 17081698
prepulse inhibition; schizophrenia; animal model; psychopharmacology; novel targets; phosphodiesterase inhibitor
Neuroscience  2006;141(3):1257-1264.
People with schizophrenia display sensory encoding deficits across a broad range of electrophysiological and behavioral measures, suggesting fundamental impairments in the ability to transduce the external environment into coherent neural representations. This inability to create basic components of complex stimuli interferes with a high fidelity representation of the world and likely contributes to cognitive deficits. The current study evaluates the effects of constitutive forebrain activation of the Gsα G-protein subunit on auditory threshold and gain using acoustic brainstem responses and cortically generated N40 event-related potentials to assess the role of cyclic AMP signaling in sensory encoding. Additionally, we examine the ability of pharmacological treatments that mimic (amphetamine) or ameliorate (haloperidol) positive symptoms of schizophrenia to test the hypothesis that the encoding deficits observed in Gsα transgenic mice can be normalized with treatment. We find that Gsα transgenic mice have decreased amplitude of cortically generated N40 but normal acoustic brainstem response amplitude, consistent with forebrain transgene expression and a schizophrenia endophenotype. Transgenic mice also display decreased stimulus intensity response (gain) in both acoustic brainstem response and N40, indicating corticofugal influence on regions that lack transgene expression. N40 deficits in transgenic animals were ameliorated with low dose haloperidol and reversed with higher dose, suggesting dopamine D2 receptor-linked Gi activity contributes to the impairment. Consistent with this hypothesis, we recreated the Gsα transgenic deficit in wild type animals using the indirect dopamine agonist amphetamine. This transgenic model of sensory encoding deficits provides a foundation for identifying biochemical contributions to sensory processing impairments associated with schizophrenia.
PMCID: PMC3311921  PMID: 16750890
Gsα; N40; schizophrenia; cAMP; haloperidol; event-related potentials
13.  Chronic ketamine impairs fear conditioning and produces long-lasting reductions in auditory evoked potentials 
Neurobiology of disease  2009;35(2):311-317.
Ketamine is an NMDA receptor antagonist with a variety of uses, ranging from recreational drug to pediatric anesthetic and chronic pain reliever. Despite its value in the clinical setting, little is known about the immediate and long-lasting effects of repeated ketamine treatment. We assessed the effects of chronic administration of a subanesthetic dose of ketamine on contextual fear conditioning, detection of pitch deviants and auditory gating. After four, but not two, weeks of daily ketamine injections, mice exhibited decreased freezing in the fear conditioning paradigm. Gating of the P80 component of auditory evoked potentials was also significantly altered by treatment condition, as ketamine caused a significant decrease in S1 amplitude. Additionally, P20 latency was significantly increased as a result of ketamine treatment. Though no interactions were found involving test week, stimulus and treatment condition, these results suggest that repeated ketamine administration impairs fear memory and has lasting effects on encoding of sensory stimuli.
PMCID: PMC2726963  PMID: 19467327
ketamine; event related potential; auditory evoked potential; gating; fear conditioning; schizophrenia; drug abuse
14.  In vitro and in vivo demonstration of risperidone implants in mice 
Schizophrenia research  2007;98(1-3):66-78.
Nonadherence with medication is a critical limitation in current long-term treatment of schizophrenia and a primary factor in poor quality-of-life outcomes. However, few treatments have addressed this shortcoming using an implantable drug delivery approach. The goal of this study was to provide in vitro and in vivo proof of concept for a long-term implantable risperidone delivery system in mice.
Implantable formulations of risperidone were created using the biodegradable polymer Poly Lactic co Glycolic Acid (PLGA) combined with various drug loads. Implant bioactivity was tested using in vitro release and stability studies, as well as in vivo pharmacokinetic and behavioral studies in mice.
The pattern of risperidone release is influenced by various parameters, including polymer composition and drug load. In vitro measures demonstrate that risperidone is stable in implants under physiological conditions. Behavioral measures demonstrate the bioactivity of risperidone implants delivering 3mg/kg/day in mice, while pharmacokinetic analyses indicate that reversibility is maintained throughout the delivery interval.
The current report suggests that implantable formulations are a viable approach to providing long-term delivery of antipsychotic medications based on in vivo animal studies and pharmacokinetics. Implantable medications demonstrated here can last two months or longer while maintaining coherence and removability past full release, suggesting a potential paradigm shift in the long-term treatment of schizophrenia.
PMCID: PMC2561216  PMID: 17765477
Treatment adherence; schizophrenia; risperidone pharmacokinetics; drug delivery system; drug implant; in vitro/in vivo correlation
15.  Treatment trials in ankylosing spondylitis: current and future considerations 
Annals of the Rheumatic Diseases  2002;61(Suppl 3):iii24-iii32.
Emerging treatment options in ankylosing spondylitis (AS) are giving new hope to patients with this chronic and potentially disabling disease. Clinical development of new treatments requires that rigorous and well controlled trials be conducted to demonstrate safety and efficacy. A number of classification systems have been developed in recent years as a result of enhanced understanding of the pathogenesis of AS. Although new outcome measures have been developed and a consensus has been reached on the use of assessment instruments in clinical trials, there is still need for improvement and implementation.
The ASsessments in Ankylosing Spondylitis (ASAS) Working Group has addressed some of these dilemmas by establishing a core set of domains for the evaluation of AS and by selecting specific assessment methods for each domain. They have also published improvement criteria for assessing short term improvement with symptom modifying antirheumatic drugs and are presently in the process of developing response criteria for disease controlling antirheumatic treatment. Various experts are also currently examining discrepancies and inadequacies of classification systems for AS. Imaging studies, magnetic resonance imaging, in particular, may provide better classification criteria in the near future.
In addition to consensus on outcome assessment and classification of AS, lessons learnt from clinical trials in rheumatoid arthritis (RA) may serve as a template for AS. Guidance provided by the United States Food and Drug Administration (FDA) for clinical trials in RA may be of particular use. The FDA has defined the claims that sponsors can receive for RA products and the clinical trial data that would be expected to be submitted to support such claims.
PMCID: PMC1766730  PMID: 12381508
18.  Biliary tract diseases in the elderly: management and outcomes 
Gut  1997;41(4):433-435.
PMCID: PMC1891511  PMID: 9391238
19.  Monotremes and the evolution of rapid eye movement sleep. 
Early studies of the echidna led to the conclusion that this monotreme did not have rapid eye movement (REM) sleep. Because the monotremes had diverged from the placental and marsupial lines very early in mammalian evolution, this finding was used to support the hypothesis that REM sleep evolved after the start of the mammalian line. The current paper summarizes our recent work on sleep in the echidna and platypus and leads to a very different interpretation. By using neuronal recording from mesopontine regions in the echidna, we found that despite the presence of a high-voltage cortical electroencephalogram (EEG), brainstem units fire in irregular bursts intermediate in intensity between the regular non-REM sleep pattern and the highly irregular REM sleep pattern seen in placentals. Thus the echidna displays brainstem activation during sleep with high-voltage cortical EEG. This work encouraged us to do the first study of sleep, to our knowledge, in the platypus. In the platypus we saw sleep with vigorous rapid eye, bill and head twitching, identical in behaviour to that which defines REM sleep in placental mammals. Recording of the EEG in the platypus during natural sleep and waking states revealed that it had moderate and high-voltage cortical EEGs during this REM sleep state. The platypus not only has REM sleep, but it had more of it than any other animal. The lack of EEG voltage reduction during REM sleep in the platypus, and during the REM sleep-like state of the echidna, has some similarity to the sleep seen in neonatal sleep in placentals. The very high amounts of REM sleep seen in the platypus also fit with the increased REM sleep duration seen in altricial mammals. Our findings suggest that REM sleep originated earlier in mammalian evolution than had previously been thought and is consistent with the hypothesis that REM sleep, or a precursor state with aspects of REM sleep, may have had its origin in reptilian species.
PMCID: PMC1692309  PMID: 9720111
20.  Human Immunodeficiency Virus Type 1 cDNA Integration: New Aromatic Hydroxylated Inhibitors and Studies of the Inhibition Mechanism 
Integration of the human immunodeficiency virus type 1 (HIV-1) cDNA is a required step for viral replication. Integrase, the virus-encoded enzyme important for integration, has not yet been exploited as a target for clinically useful inhibitors. Here we report on the identification of new polyhydroxylated aromatic inhibitors of integrase including ellagic acid, purpurogallin, 4,8,12-trioxatricornan, and hypericin, the last of which is known to inhibit viral replication. These compounds and others were characterized in assays with subviral preintegration complexes (PICs) isolated from HIV-1-infected cells. Hypericin was found to inhibit PIC assays, while the other compounds tested were inactive. Counterscreening of these and other integrase inhibitors against additional DNA-modifying enzymes revealed that none of the polyhydroxylated aromatic compounds are active against enzymes that do not require metals (methylases, a pox virus topoisomerase). However, all were cross-reactive with metal-requiring enzymes (restriction enzymes, a reverse transcriptase), implicating metal atoms in the inhibitory mechanism. In mechanistic studies, we localized binding of some inhibitors to the catalytic domain of integrase by assaying competition of binding by labeled nucleotides. These findings help elucidate the mechanism of action of the polyhydroxylated aromatic inhibitors and provide practical guidance for further inhibitor development.
PMCID: PMC105796  PMID: 9736543
21.  Function and phenotype of immature CD4+ lymphocytes in healthy infants and early lymphocyte activation in uninfected infants of human immunodeficiency virus-infected mothers. 
The function and phenotypes of CD4+ lymphocytes in infants are different than in adults and are modulated by maturational changes and exposure to environmental antigens. Infants of non-human immunodeficiency virus (HIV)-infected mothers and uninfected infants of HIV-infected mothers, 0 to 6 months of age, were examined for CD4+ lymphocyte function by in vitro interleukin-2 (IL-2) production and for CD4+ phenotypes by three-color flow cytometry. A minority of these uninfected infants (28%) had functional responses similar to those of healthy adult women (IL-2 production in response to anti-CD3, alloantigen, and mitogen), while the remainder were capable of responding to alloantigen and mitogen but not to anti-CD3. We did demonstrate reduced phytohemagglutinin-stimulated IL-2 production in uninfected infants born to HIV-seropositive mothers compared to that in infants from seronegative mothers. The proportions of CD3+ CD4+, CD4+ HLA-DR- CD38+, and CD4+ CD45RA+ RO- (naive) lymphocytes were much higher in infants than in adults, and the proportions of CD4+ CD45RA- RO+ (memory) and CD4+ CD25+ (IL-2 receptor-bearing) lymphocytes were lower in infants than in adults. The proportions of activated (CD4+ HLA-DR+ CD38+) and memory (CD4+ CD45RA- RO+) lymphocytes were increased in uninfected infants of HIV-infected mothers compared to infants of uninfected mothers. Therefore, T-helper-cell function is immature in many infants, but the CD4+ lymphocytes of some HIV-exposed, uninfected infants have been stimulated by antigen at an early age.
PMCID: PMC170532  PMID: 9144377
22.  Comparison of the cellular fatty acid composition of a bacterium isolated from a human and alleged to be Bacillus sphaericus with that of Bacillus sphaericus isolated from a mosquito larvicide. 
The cellular fatty acid (CFA) composition of the cytoplasmic membrane of a bacillus isolated from a human lung and deposited in the National Collection of Type Cultures as Bacillus sphaericus NCTC 11025 was determined by gas-liquid chromatography. The CFA composition of B. sphaericus 2362, isolated from a microbial larvicide, and those of B. sphaericus reference strains obtained from public collections were also determined. Samples were grouped by hierarchical cluster analysis based on the unpaired-group method using arithmetic averages. Samples that linked at a Euclidean distance of < or = 2.0 U were considered to belong to the same strain. NCTC 11025 and the type strain of B. sphaericus, ATCC 14577, were mixed; all other isolates were monotypic. The predominant fatty acid in NCTC 11025 was 12-methyltetradecanoic acid, while the predominant fatty acid in the remaining isolates was 13-methyltetradecanoic acid. NCTC 11025 linked to the other isolates at a Euclidean distance of 83.8 U, and we concluded that it belongs to a different species that we could not identify. We could distinguish among six DNA homology groups of B. sphaericus by using fatty acids. Within DNA homology group IIA, strain 2362 could be distinguished from other strains belonging to serotype H5a, 5b. We concluded that CFA analysis is a useful technique to determine if future human isolates identified as B. sphaericus in fact belong to other species of bacteria or whether the isolates originated from commercial products.
PMCID: PMC168392  PMID: 9055417
23.  Laboratory findings in hypertensive disorders of pregnancy. 
This study sought to determine whether a normal platelet count is a reliable predictor of the absence of other coagulation abnormalities in patients with a hypertensive disorder of pregnancy. A retrospective review of laboratory data obtained from 80 patients with hypertensive disorders of pregnancy was carried out. Results of complete blood cell count, prothrombin time (PT), partial thromboplastin time (PTT), D-dimer, fibrin split products, and fibrinogen, bilirubin, and liver enzyme levels were reviewed. Minor abnormalities of PT, PTT, and fibrinogen level were frequent, even in the presence of a normal platelet count. These were found mostly in patients with severe pre-eclampsia. A baseline complete blood cell count including platelet count is probably sufficient in patients with a hypertensive disorder of pregnancy. Fibrinogen level and PT and PTT determinations are recommended in patients who have severe preeclampsia and for whom operative delivery or regional anesthesia is planned. This will detect minor abnormalities in a few patients despite a normal platelet count. This information may help prevent bleeding complications.
PMCID: PMC2608132  PMID: 8990805
24.  Evaluation of the immunofluorescence procedure for detection of Giardia cysts and Cryptosporidium oocysts in water. 
The accurate determination of the presence of Giardia cysts and Cryptosporidium oocysts in surface waters requires a reliable method for the detection and enumeration of these pathogenic organisms. Published methods have usually reported recovery efficiencies of less than 50% for both cysts and oocysts. Typically, the losses are greater for Cryptosporidium oocysts than they are for Giardia cysts. The purpose of this study was to examine procedures used for sample collection, elution, concentration, and clarification to determine when losses of cysts and oocysts occurred during processing. The results showed that major losses of cysts and oocysts occurred during centrifugation and clarification. Depending on the centrifugation force, oocyst losses of as high as 30% occurred for each centrifugation step. A 1.15-specific-gravity Percoll-sucrose gradient was needed to optimize recovery of oocysts from natural water samples. Minor improvements in the procedure could be accomplished by selecting a filter other than the recommended 1-micron-pore-size (nominal-porosity) polypropylene filter.
PMCID: PMC167330  PMID: 7574607
25.  Importance of factor Xa in determining the procoagulant activity of whole-blood clots. 
Journal of Clinical Investigation  1993;91(5):1877-1883.
The binding of thrombin to fibrin is thought to be an important mechanism by which thrombi exhibit procoagulant activity; however, the extent to which other procoagulants are associated with thrombi has not been previously defined. This study was designed to determine whether clotting factors other than thrombin are bound to whole-blood clots and can thereby contribute to significant procoagulant activity. Clots formed in vitro from human blood exhibited minimal thrombin activity when incubated in plasma depleted of vitamin K-dependent factors by barium-citrate adsorption, as indicated by increases in the concentration of fibrinopeptide A (FPA), a marker of fibrin formation, to 72 nM after 30 min. Incubation of clots in barium-absorbed plasma repleted with 0.9 microM human prothrombin under the same conditions resulted in marked increases in the concentration of FPA (> 1,000 nM) and clotting by 30 min. The increases in FPA were attributable to activation of the added prothrombin by clot-associated Factor Xa, judging from concomitant increases in the concentration of prothrombin fragment 1.2. Similar results were obtained with thrombi induced in the axillary arteries of dogs by vascular injury and incubated with plasma in vitro. Activation of prothrombin was inhibited in a dose-dependent manner by tick anticoagulant peptide, a direct inhibitor of Factor Xa, at concentrations of 0.5-5.0 microM. Clot-associated Factor Xa activity was resistant to inhibition by anti-thrombin III, judging from the lack of inhibition of prothrombin activation during incubation of clots in plasma containing heparin pentasaccharide, an anti-thrombin III-mediated inhibitor of Factor Xa. Thus, the activity of Factor Xa appears to be an important determinant of the procoagulant activity of whole-blood clots and arterial thrombi, and is resistant to inhibition by anti-thrombin III-dependent inhibitors.
PMCID: PMC288180  PMID: 8486759

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