Improved In Vitro Excystation Procedure for Giardia lamblia Cysts

Giardia lamblia cysts obtained from human symptomatic and asymptomatic donors were excysted in vitro. Excystation averaged 87% for cysts from symptomatic donors and 70% for cysts from asymptomatic donors.     

Characterization of Virus-Responsive Plasmacytoid Dendritic Cells in the Rhesus Macaque

Plasmacytoid dendritic cells (PDC) are potent producers of alpha interferon (IFN-α) in response to enveloped viruses and provide a critical link between the innate and adaptive immune responses. Although the loss of peripheral blood PDC function and numbers has been linked to human immunodeficiency virus (HIV) progression in humans, a suitable animal model is needed to study the effects of immunodeficiency virus infection on PDC function. The rhesus macaque SIV model closely mimics human HIV infection, and recent studies have identified macaque PDC, potentially making the macaque a good model to study PDC regulation. In this study, we demonstrate that peripheral blood PDC from healthy macaques are both phenotypically and functionally similar to human PDC and that reagents used for human studies can be used to study macaque PDC. Both human and macaque PBMC expressed IFN-α in response to herpes simplex virus (HSV), the prototypical activator of PDC, as measured by using an IFN bioassay and IFN-α-specific enzyme-linked immunospot assays. Similar to human PDC, macaque PDC were identified by using flow cytometry as CD123⁺ HLA-DR⁺ lineage⁻ cells. In addition, like human PDC, macaque PDC expressed intracellular IFN-α, tumor necrosis factor alpha, macrophage inflammatory protein 1β/CCL4, and IFN-inducible protein 10/CXCL10 upon stimulation with HSV, all as determined by intracellular flow cytometry. We found that IFN regulatory factor 7, which is required for the expression of IFN-α genes, was, similar to human PDC, expressed at high levels in macaque PDC compared to monocytes and CD8⁺ T cells. These findings establish the phenotypic and functional similarity of human and macaque PDC and confirm the utility of tools developed for studying human PDC in this animal model.     

Expression of the chemokine receptors CCR4, CCR5, and CXCR3 by human tissue-infiltrating lymphocytes.

Differential expression of adhesion molecules and chemokine receptors has been useful for identification of peripheral blood memory lymphocyte subsets with distinct tissue and microenvironmental tropisms. Expression of CCR4 by circulating memory CD4(+) lymphocytes is associated with cutaneous and other systemic populations while expression of CCR9 is associated with a small intestine-homing subset. CCR5 and CXCR3 are also expressed by discrete memory CD4(+) populations in blood, as well as by tissue-infiltrating lymphocytes from a number of sites. To characterize the similarities and differences among tissue-infiltrating lymphocytes, and to shed light on the specialization of lymphocyte subsets that mediate inflammation and immune surveillance in particular tissues, we have examined the expression of CCR4, CXCR3, and CCR5 on CD4(+) lymphocytes directly isolated from a wide variety of normal and inflamed tissues. Extra-lymphoid tissues contained only memory lymphocytes, many of which were activated (CD69(+)). As predicted by classical studies, skin lymphocytes were enriched in CLA expression whereas intestinal lymphocytes were enriched in alpha(4)beta(7) expression. CCR4 was expressed at high levels by skin-infiltrating lymphocytes, at lower levels by lung and synovial fluid lymphocytes, but never by intestinal lymphocytes. Only the high CCR4 levels characteristic of skin lymphocytes were associated with robust chemotactic and adhesive responses to TARC, consistent with a selective role for CCR4 in skin lymphocyte homing. In contrast, CXCR3 and CCR5 were present on the majority of lymphocytes from each non-lymphoid tissue examined, suggesting that these receptors are unlikely to determine tissue specificity, but rather, may play a wider role in tissue inflammation.     

Proportional Jerk: A New Measure of Motion as Applied to Eye Movements in Sleep and Waking

The third derivative of motion or the change in the rate of acceleration (also known as jerk) is examined in terms of its applicability to the study of psychophysiological function. An algorithm of this third derivative is presented to show that jerk can be derived from the arithmetic difference of two slopes which constitute the portion of motion being differentiated. By modifying the algorithm, a new parameter termed “proportional jerk’ or PJ is formulated whereby one slope is measured relative to the other slope; this PJ provides information about the smoothness of movement without being influenced by the velocity as is the traditional jerk measure. A practical application of the PJ to waking saccades and REMs in 11 human subjects revealed that REMs are significantly “jerkier’ or less smooth than waking calibration eye movements. Whereas the waking eye movements had a well-defined negative phase of the PJ, the REMs did not show such stereotypical behavior. This is in accord with previous work which showed that waking saccade velocity increases to a maximum and then decreases whereas REMs maintain their peak or near peak velocities for varying periods. It was suggested that PJ can be useful in detecting subtle temporal changes in the course of movements and may be used as a parameter of motion even when the absolute amplitude is unknown.     

Experimental Adenovirus Infection of the Mouse Adrenal Gland: II. Electron Microscopic Observations

A strain of mouse adenovirus, found to have a striking tropism for the weanling mouse adrenal gland, enabled electron microscopic examination of adrenals in various stages of infection. Nucleolar hypertrophy and the successive formation of three types of inclusion bodies in association with nucleoli preceded virion production. Angular crystals of virions formed in the affected nuclei. Virus was released by lysis of nuclear membranes; rapid degeneration of cytoplasmic organelles followed. Rupture of external cell membranes released virus into the extracellular spaces where virions crossed vascular basement membranes to enter endothelial cells. Virions were also phagocytized by inflammatory cells which reentered vascular sinusoids, and by adrenal parenchymal cells. Disruption of virus-laden phagocytic vacuoles in parenchymal cells released virions into the cytoplasm. Typical viral inclusion bodies also formed in vascular endothelial cells and in inflammatory cells, but virion replication was not detected. The possibility that virus directly entered parenchymal cells through the external cell membrane without phagocytosis is discussed.     

Mutation analysis of feline Niemann-Pick C1 disease

Niemann-Pick C (NPC) disease is an autosomal recessive neurovisceral lysosomal storage disorder that results in defective intracellular transport of cholesterol. The major form of human NPC (NPC1) has been mapped to chromosome 18, the NPC1 gene (NPC1) has been sequenced and several mutations have been identified in NPC1 patients. A feline model of NPC has been characterized and is phenotypically, morphologically, and biochemically similar to human NPC1. Complementation studies using cultured fibroblasts from NPC affected cats and NPC1 affected humans support that the gene responsible for the NPC phenotype in this colony of cats is orthologous to human NPC1. Using human-based PCR primers, initial fragments of the feline NPC cDNA were amplified and sequenced. From these sequences, feline-specific PCR primers were generated and designed to amplify six overlapping bands that span the entire feline NPC1 open reading frame. A single base substitution (2864G-C) was identified in NPC1 affected cats. Obligate carriers are heterozygous at the same allele and a PCR-based assay was developed to identify the geneotype of all cats in the colony. The mutation results in an amino acid change from cysteine to serine (C955S). Several of the mutations identified in people occur in the same region. Marked similarity exists between the human and feline NPC1 cDNA sequences, and is greater than that between the human and murine NPC1 sequences. The human cDNA sequence predicts a 1278aa protein with a lysosomal targeting sequence, several trans-membrane domains and extensive homology with other known mediators of cholesterol homeostasis.     

The feasibility,demand, and effect of integrating primary care services with HIV voluntary counseling and testing: evaluation of a 15-year experience in Haiti, 1985-2000

BACKGROUND: HIV voluntary counseling and testing (VCT) may be an effective strategy to prevent transmission of HIV in developing countries. Hypothesizing that primary care services and HIV VCT have synergistic benefits, we examine the feasibility, the demand, and the effect of integrating on-site primary care services into VCT at a stand-alone VCT center in Port au Prince, Haiti. METHODS: Through a retrospective review of patient records, we describe the integration of primary care services at the Groupe Haitien d'Etude du Sarcome de Kaposi et des Infections Opportunistes (GHESKIO) VCT center between1985 and 2000. RESULTS: Between 1985 and 1999, services for HIV care, tuberculosis care, treatment of sexually transmitted diseases, and reproductive health were sequentially integrated into HIV VCT at GHESKIO. The number of new people seeking voluntary counseling and testing at GHESKIO increased from 142 in 1985 to 8175 in 1999, with an increasing percentage of women, adolescents, symptom-free clients, and self-referred clients. Of new adults seeking VCT in 1999, the center was able to provide AIDS care to 17%, tuberculosis treatment to 6%, sexually transmitted infection management to 18%, and family planning to 19%. HIV transmission between discordant couples was 0 infections/100 follow-up years (95% CI, 0-3.2); vertical transmission from mother to child was 11 infections/100 live births (95% CI, 4.6-21.9); These rates are significantly lower than expected rates of transmission in Haiti. CONCLUSIONS: This report demonstrates the feasibility, demand, and effective synergy of integrating on-site primary care services into HIV VCT in Haiti. VCT is a good entry point for people in need of services for communicable diseases and reproductive health, and, reciprocally, services attract more people to VCT, including populations that are at high risk for HIV infection. This program is being duplicated elsewhere in Haiti and can serve as a model for other countries.     

Simultaneous inhibition of caudal medullary raphe and retrotrapezoid nucleus decreases breathing and the CO2 response in conscious rats

The medullary raphe (MR) and the retrotrapezoid nucleus (RTN) in the ventral medulla are two of many central chemoreceptor sites. We examine their combined function in conscious rats by focal inhibition using microdialysis. Inhibition of RTN neurons with the GABA_A receptor agonist muscimol, with simultaneous dialysis of artificial cerebrospinal fluid (ACSF) in or near to the caudal MR, causes hypoventilation (decrease in the ratio of minute ventilation to oxygen consumption,     ) and reduces the ventilatory response to 7% CO₂ by 24%. Inhibition of caudal MR serotonergic neurons with the 5-HT_1A receptor agonist ( R )-(+)-8-hydroxy-2(di- n -propylamino)tetralin (8-OH-DPAT), with simultaneous dialysis of ACSF in or near to the RTN, causes hypoventilation but has no significant effect on the CO₂ response. Inhibition of both the RTN and the caudal MR simultaneously produces enhanced hypoventilation and a 51% decrease in the CO₂ response. The effects of treatment on the CO₂ response are similar in wakefulness and in non-rapid eye movement sleep. Comparison of the effect of 8-OH-DPAT microdialysed into a more rostral portion of the MR, where the CO₂ response is reduced by 22%, demonstrates heterogeneity within the MR of the function of serotonergic neurons in breathing. We conclude that serotonergic neurons within the caudal MR provide a non-CO₂-dependent tonic drive to breathe and potentiate the effects of RTN neurons that contribute to a resting chemical 'drive to breathe' as well as the response to added CO₂. These effects of caudal MR serotonergic neurons could be at a chemoreceptor site, e.g. the RTN, or at 'downstream' sites involved in rhythm and pattern generation.     

Immunologic Mimicry Between Mouse Tissue and Enterobacterial Common Antigen

Organs of Swiss white albino and C57BL/6Ha mice were assessed for an antigen (CRA) which cross-reacts with common enterobacterial antigen (CA) of To this end, supernatant fluids (HKS) and ethanol-soluble fractions (ES) of heated homogenates of spleens, kidneys, and livers were examined for their capacities to react with CA hemagglutinins and to engender humoral and cellular events in the rabbit. The immunogenicity of CRA in the rabbit can not be predicted on the basis of CA hemagglutinin neutralization studies alone; although CRA was identified in the liver extracts of both mouse strains, according to this parameter, only the liver fraction of Swiss white albino mice elicited significant numbers of rosette-forming cells (RFC) in the spleens of rabbits. Also, kidney fractions, which primed the rabbits for booster with CA, were less effective in stimulating RFC in the spleens of the identical animals. Moreover, although extracts of mouse spleens failed to inhibit CA hemagglutination and did not prime rabbits for a CA hemagglutinin response, these same preparations clearly evoked RFC in rabbit spleens. Thus, the antigenicity and immunogenicity of CRA in target organs of mice reflect the mouse strain, extraction procedure, and testing method employed.     

Correlation of flunisolide plasma levels to eosinopenic response in humans

Plasma levels of flunisolide were measured in healthy male volunteers after the administration of single doses of the drug by the intravenous, oral, intranasal, and bronchial inhalation routes. The systemic availability of a 1-mg dose orally was only 21%. After a single dose of approximately 0.117 mg intranasally plasma levels ranged up to 1 ng/ml. When 1 mg was administered by bronchial inhalation, peak or near peak plasma levels were recorded at 2 min and remained near this level throughout the first hour before declining at a rate similar to that observed after flunisolide intravenously (plasma ). Gargling with an alcoholic mouthwash immediately after inhalation reduced plasma levels at 30 and 60 min but not earlier, suggesting rate-limiting dissolution of flunisolide in bronchial fluids or rate-limiting diffusion across the mucociliary blanket or pulmonary membrane. The systemic availabilities of the inhaled-mouthwash and inhaled-no mouthwash doses were 32% and 39%, respectively. Systemic potency of flunisolide, measured by eosinopenic response, was oral < inhaled < intravenous and correlated with the systemic availability of flunisolide after drug administration by these three routes. These pharmacokinetic properties of flunisolide are clinically advantageous in that relatively small doses are delivered topically to the target organs, i.e., the nasal mucosa and lungs, whereas a large portion of the dose is swallowed and subsequently extensively metabolized to relatively inactive metabolites.