Alterations in Notch signaling in neoplastic lesions of the human cervix.

The development of cancer is a cellular process that reflects and is partly driven by alterations in cell determination. Mutations in various molecules responsible for cell determination have been identified as being oncogenic, but little is known about the involvement of normal cell fate-determining mechanisms in the oncogenic process. The Notch pathway defines an evolutionarily conserved, general cell interaction mechanism that controls fundamental aspects of cell determination during vertebrate and invertebrate development. We have explored the involvement of the human Notch pathway in human cervical tissues, which define a cellular environment where cell fate changes take place and where neoplastic conditions have been well characterized. Our evidence suggests that Notch expression is associated with cell populations that are undergoing cell fate changes and that Notch activity can be used to monitor cell fate abnormalities in cervical as well as other epithelial neoplasias.

Persistent high frequency of human immunodeficiency virus-specific cytotoxic T cells in peripheral blood of infected donors.

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTLs) are thought to play a major role in the immune response to HIV infection. The HIV-specific CTL response is much stronger than previously documented in an infectious disease, yet estimates of CTL frequency derived from limiting-dilution analysis (LDA) are relatively low and comparable to other viral infections. Here we show that individual CTL clones specific for peptides from HIV gag and pol gene products are present at high levels in the peripheral blood of three infected patients and that individual CTL clones may represent between 0.2% and 1% of T cells. Previous LDA in one donor had shown a frequency of CTL precursors of 1/8000, suggesting that LDA may underestimate CTL effector frequency. In some donors individual CTL clones persisted in vivo for at least 5 years. In contrast, in one patient there was a switch in CTL usage suggesting that different populations of CTLs can be recruited during infection. These data imply strong stimulation of CTLs, potentially leading some clones to exhaustion.

Étude de la migration des populations de lymphocytes B du sang de patients infectés par le virus d’immunodéficience humaine (VIH)

La dérégulation du compartiment de cellules B est une conséquence importante de l’infection par le virus de l’immunodéficience humaine (VIH-1). On observe notamment une diminution des nombres de lymphocytes B sanguins ainsi qu’une variation des fréquences relatives des différentes populations de lymphocytes B chez les individus infectés par rapport aux contrôles sains. Notre laboratoire a précédemment démontré l’implication des cellules dendritiques dans la dérégulation des lymphocytes B via la roduction excessive de BLyS/BAFF, un stimulateur des cellules B. De plus, lors l’études menées chez la souris transgénique présentant une maladie semblable au SIDA, et chez la souris BLyS/BAFF transgénique, l’infection au VIH-1 fut associée à une expansion de la zone marginale (MZ) de la rate. De façon intéressante, nous observons chez les contrôleurs élites une diminution de la population B ‘mature’ de la MZ. Il s’agit du seul changement important chez les contrôleurs élites et reflète possiblement un recrutement de ces cellules vers la périphérie ainsi qu’une implication dans des mécanismes de contrôle de l’infection. Pour tenter d’expliquer et de mieux comprendre ces variations dans les fréquences des populations B, nous avons analysé les axes chimiotactiques CXCL13-CXCR5, CXCL12-CXCR4/CXCR7, CCL20-CCR6 et CCL25-CCR9. L’étude longitudinale de cohortes de patients avec différents types de progression clinique ou de contrôle de l’infection démontre une modulation des niveaux plasmatiques de la majorité des chimiokines analysées chez les progresseurs rapides et classiques. Au contraire, les contrôleurs élites conservent des niveaux normaux de chimiokines, démontrant leur capacité à maintenir l’homéostasie. La migration des populations de cellules B semble être modulée selon la progression ou le contrôle de l’infection. Les contrôleurs élites présentent une diminution de la population B ‘mature’ de la MZ et une augmentation de la fréquence d’expression du récepteur CXCR7 associé à la MZ chez la souris, suggérant un rôle important des cellules de la MZ dans le contrôle de l’infection au VIH-1. De façon générale, les résultats dans cette étude viennent enrichir nos connaissances du compartiment de cellules B dans le contexte de l’infection au VIH-1 et pourront contribuer à élaborer des stratégies préventives et thérapeutiques contre ce virus.

Assessment of vehicle safety problems for special driving populations. Final report.

National Highway Traffic Safety Administration, Washington, D.C.

Simple In Vitro Assay for Determining the Sensitivity of Plasmodium vivax Isolates from Fresh Human Blood to Antimalarials in Areas where P. vivax Is Endemic

The aim of this study was to develop a simple, field-practical, and effective in vitro method for determining the sensitivity of fresh erythrocytic Plasmodium vivax isolates to a range of antimalarials. The method used is a modification of the standard World Health Organization (WHO) microtest for determination of P.falciparum drug sensitivity. The WHO method was modified by removing leukocytes and using a growth medium supplemented with AB(+) serum. We successfully carried out 34 in vitro drug assays on 39 P. vivax isolates collected from the Mae Sod malaria clinic, Tak Province, Thailand. The mean percentage of parasites maturing to schizonts (six or more merozoites) in control wells was 66.5% +/- 5.9% (standard deviation). This level of growth in the control wells enabled rapid microscopic determination (5 min per isolate per drug) of the MICs of chloroquine, dihydroartemisinin, WR238605 (tafenoquine), and sulfadoxine. P. vivax was relatively sensitive to chloroquine (MIC = 160 ng/ml, 50% inhibitory concentration [IC50] = 49.8 ng/ml) and dihydroartemisinin (MIC = 0.5 ng/ml, IC50 = 0.47 ng/ml). The poor response of P. vivax to both tafenoquine (MIC = 14,000 ng/ml, IC50 = 9,739 ng/ml) and sulfadoxine (MIC = 500,000 ng/ml, IC50 = 249,000 ng/ml) was due to the slow action of these drugs and the innate resistance of P. vivax to sulfadoxine. The in vitro assay developed in our study should be useful both for assessing the antimalarial sensitivity of P. vivax populations and for screening new antimalarials in the absence of long-term P. vivax cultures.

T-cell clonality to Porphyromonas gingivalis and human heat shock protein 60s in patients with atherosclerosis and periodontitis

Individuals with periodontitis have been reported to have a significantly increased risk of developing coronary heart disease. Several studies have demonstrated that the immune response to heat shock protein 60 (HSP60) may be involved in the pathogenesis of both atherosclerosis and chronic periodontitis. To investigate this possible link between these diseases, cellular and humoral immune responses to HSP60 in atherosclerosis patients were compared with those in periodontitis patients and healthy subjects using human and Porphyromonas gingivalis HSP60 (GroEL) as antigens. Antibody levels to both human and P. gingivalis HSP60s were the highest in atherosclerosis patients, followed by periodontitis patients and healthy subjects. Clonal analysis of the T cells clearly demonstrated the presence of not only human HSP60- but also P. gingivalis GroEL-reactive T-cell populations in the peripheral circulation of atherosclerosis patients. Furthermore, these HSP60-reactive T cells seemed to be present in atherosclerotic lesions in some patients. These results suggest that T-cell clones with the same specificity may be involved in the pathogenesis of the different diseases.

Factors supporting the non-persistence of fruit fly populations in South Australia

For purposes of interstate and international fruit trade, it is necessary to demonstrate that in areas in which fruit fly species have not previously established permanent populations, but which are subject to introductions of fruit flies from outside the area, the introduced population once detected, has not become established. In this paper, we apply methodology suggested mainly by Carey (1991, 1995) to introductions of Mediterranean fruit fly (Medfly), Ceratitis capitata Weid., and Queensland fruit fly (QFF) Bactrocera tryoni Froggatt (Diptera: Tephritidae) to South Australia, a state in which these species do not occur naturally and in which introductions, once detected, are actively treated. By analysing historical data associated with fruit fly outbreaks in South Australia, we demonstrate that: (i) fruit flies occur seasonally, as would occur in established populations, except there is no evidence of the critical spring generation of either species; (ii) there is no evidence of increasing frequency of outbreaks, trapped flies or larval occurrences over 29 years; (iii) there is no evidence of decreasing time between catches of adult flies as the years progress; (iv) there is no decrease in the mean number of years between outbreaks in the same locations; (v) there is no statistically significant recurrence of outbreaks in the same locations in successive years; (vi) there is no evidence of spread of outbreaks outwards from a central location; (vii) the likelihood of outbreaks in a city or town is related to the size of the human population; (viii) introduction pathways by road from Western Australia (for Medfly) and eastern Australia (for QFF) are shown to exist and to illegally or accidentally carry considerable amounts of fruit into South Australia; and (ix) there was no association between the numbers of either Queensland fruit fly or Medfly and the spatial pattern of either loquat or cumquat trees as sources of larval food in spring. This analysis supports the hypothesis that most fruit fly outbreaks in South Australia have been the result of separate introductions of infested fruit by vehicular traffic and that most of the resultant fly outbreaks were detected and died out within a few weeks of the application of eradication procedures. An alternative hypothesis, that populations of fruit flies are established in South Australia at below detectable levels, is impossible to disprove with conventional technology, but the likelihood of it being true is minimised by our analysis. Both hypotheses could be tested soon with newly developed genetic techniques.

Virus-specific CD8+ T lymphocytes within the normal human liver

The frequency and phenotype of human antiviral memory CD8(+) T cells in blood are well studied, yet little is known about their distribution within tissues. Analysis of antiviral CD8(+) T cell populations derived from a unique set of normal liver and blood samples identified a consistent population of virus-specific cells within the liver. In comparison to the circulating T cells, the liver-derived T cells were present at frequencies which were variably enriched compared to that in the blood, and showed significant differences with regard to the expression of CD45RA, CD45RO, CD95, CCR7, CD27 and CD28. The differences in these cell surface markers are consistent with a mature 'effector memory' phenotype of antigen-specific CD8(+) T cells within the liver. An enrichment of an activated subset of NKT cells (Valpha24/Vbeta11) was also observed, a finding which may be relevant to the regulation of the antiviral population:.

An in vitro larval motility assay to determine anthelmintic sensitivity for human hookworm and Strongyloides species

With the implementation of programs to control lymphatic filariasis and soil-transmitted helminths using broad spectrum anthelmintics, including albendazole and ivermectin, there is a need to develop an in vitro assay for detection of drug resistance. This report describes an in vitro assay for measuring the effects of ivermectin and benzimidazoles on the motility of larvae of the hookworm species Ancylostoma ceylanicum, A. caninum, and Necator americanus, and Strongyloides species including Strongyloides stercoralis, and S. ratti. A dose-response relationship was demonstrated with each of the parasite species, with distinct differences observed between the various species. In pilot field testing of the assay with N. americanus larvae recovered from human fecal samples, a dose-response relationship was observed with ivermectin. While the assay has demonstrated the ability to determine drug responsiveness, its usefulness in resistance detection will require correlation with the clinical outcome among individuals infected with parasite strains showing different drug sensitivities.

Sunscreen penetration of human skin and related keratinocyte toxicity after topical application

Sunscreen skin penetration and safety assessment should be considered together in order to ensure that in vitro cytotoxicity studies examine relevant doses of these organic chemical UV filters to which viable epidermal cells are realistically exposed. In this study, we sought to determine whether sufficient topically applied sunscreens penetrated into human viable epidermis to put the local keratinocyte cell populations at risk of toxicity. The penetration and retention of five commonly used sunscreen agents ( avobenzone, octinoxate, octocrylene, oxybenzone and padimate O) in human skin was evaluated after application in mineral oil to isolated human epidermal membranes. Sunscreen concentration - human keratinocyte culture response curves were then defined using changes in cell morphology and proliferation ( DNA synthesis using radiolabelled thymidine uptake studies) as evidence of sunscreens causing toxicity. Following 24 h of human epidermal exposure to sunscreens, detectable amounts of all sunscreens were present in the stratum corneum and viable epidermis, with epidermal penetration most evident with oxybenzone. The concentrations of each sunscreen found in human viable epidermis after topical application, adjusting for skin partitioning and binding effects, were at least 5-fold lower, based on levels detected in viable epidermal cells, than those appearing to cause toxicity in cultured human keratinocytes. It is concluded that the human viable epidermal levels of sunscreens are too low to cause any significant toxicity to the underlying human keratinocytes. Copyright (C) 2005 S. Karger AG, Basel.

Moreton Bay, Queensland, Australia: An example of the co-existence of significant marine mammal populations and large-scale coastal development

Recent analyses assert that large marine vertebrates such as marine mammals are now 'functionally or entirely extinct in most coastal ecosystems'. Moreton Bay is a large diverse marine ecosystem bordering the fastest growing area in Australia. The human population is over 1.6 million and increasing yearly by between 10% and 13% with resultant impacts upon the adjoining marine environment. Nonetheless, significant populations of three species of marine mammals are resident within Moreton Bay and a further 14 species are seasonal or occasional visitors. This paper reviews the current and historical distributions and abundance of these species in the context of the current management regime and suggests initiatives to increase the resilience of marine mammal populations to the changes wrought by the burgeoning human population in coastal environments. (C) 2004 Elsevier Ltd. All rights reserved.

A golden age of human pigmentation genetics

The zebrafish golden mutation is characterized by the production of small and irregular-shaped melanin granules, resulting in a lightening of the pigmented lateral stripes of the animal. The recent positional cloning and localization of the golden gene, combined with genotype-phenotype correlations of alleles of its human orthologue (SLC24A5) in African-American and African-Caribbean populations, provide insights into the genetic and molecular basis of human skin colour. SLC24A5 promotes melanin deposition through maturation of the melanosome, highlighting the importance of ion-exchange in the function of this organelle.

Expression of human DEC-205 (CD205) multilectin receptor on leukocytes

DEC-205 (CD205) belongs to the macrophage mannose receptor family of C-type lectin endocytic receptors and behaves as an antigen uptake/processing receptor for dendritic cells (DC). To investigate DEC-205 tissue distribution in human leukocytes, we generated a series of anti-human DEC-205 monoclonal antibodies (MMRI-5, 6 and 7), which recognized epitopes within the C-type lectin-like domains 1 and 2, and the MMRI-7 immunoprecipitated a single similar to 200 kDa band, identified as DEC-205 by mass spectrometry. MMRI-7 and another DEC-205 mAb (MG38), which recognized the epitope within the DEC-205 cysteine-rich and fibronectin type II domain, were used to examine DEC-205 expression by human leukocytes. Unlike mouse DEC-205, which is reported to have predominant expression on DC, human DEC-205 was detected by flow cytometry at relatively high levels on myeloid blood DC and monocytes, at moderate levels on B lymphocytes and at low levels on NK cells, plasmacytoid blood DC and T lymphocytes. MMRI-7 F(ab')(2) also labeled monocytes, B lymphocytes and NK cells similarly excluding reactivity due to non-specific binding of the mAb to Fc gamma R. Tonsil mononuclear cells showed a similar distribution of DEC-205 staining on the leukocytes. DEC-205-specific semiquantitative immunoprecipitation/western blot and quantitative reverse transcriptase-PCR analysis established that these leukocyte populations expressed DEC-205 protein and the cognate mRNA. Thus, human DEC-205 is expressed on more leukocyte populations than that were previously assumed based on mouse DEC-205 tissue localization studies. The broader DEC-205 tissue expression in man is relevant to clinical DC targeting strategies and DEC-205 functional studies.

Imaging, anatomical, and molecular analysis of callosal formation in the developing human fetal brain

A complex set of axonal guidance mechanisms are utilized by axons to locate and innervate their targets. In the developing mouse forebrain, we previously described several midline glial populations as well as various guidance molecules that regulate the formation of the corpus callosum. Since agenesis of the corpus callosum is associated with over 50 different human congenital syndromes, we wanted to investigate whether these same mechanisms also operate during human callosal development. Here we analyze midline glial and commissural development in human fetal brains ranging from 13 to 20 weeks of gestation using both diffusion tensor magnetic resonance imaging and immunohistochemistry. Through our combined radiological and histological studies, we demonstrate the morphological development of multiple forebrain commissures/decussations, including the corpus callosum, anterior commissure, hippocampal commissure, and the optic chiasm. Histological analyses demonstrated that all the midline glial populations previously described in mouse, as well as structures analogous to the subcallosal sling and cingulate pioneering axons, that mediate callosal axon guidance in mouse, are also present during human brain development. Finally, by Northern blot analysis, we have identified that molecules involved in mouse callosal development, including Slit, Robo, Netrin1, DCC, Nfia, Emx1, and GAP-43, are all expressed in human fetal brain. These data suggest that similar mechanisms and molecules required for midline commissure formation operate during both mouse and human brain development. Thus, the mouse is an excellent model system for studying normal and pathological commissural formation in human brain development. (c) 2006 Wiley-Liss, Inc.

Alzheimer's disease: the relationship between the density of senile plaques, neurofibrillary tangles and A4 protein in human patients

The numerical density of senile plaques (SP) and neurofibrillary tangles (NFT) as revealed by the Glees silver method was compared with SP and NFT revealed by the Gallyas method and with amyloid (A4) deposits in immunostained sections in 6 elderly cases of Alzheimer's disease. The density of NFT was generally greater and A4 lower in tissue from hippocampus compared with the neocortex suggesting that A4 deposition was less important than the degree of paired helical filament (PHF) related damage in the hippocampus. The density of Glees SP was positively correlated Gallyas SP weakly correlated with A4 deposit number. A stepwise multiple regression analysis which included A4 deposit and Gallyas SP density and accounted for 54% of the variation in Glees SP density. Hence, different populations of SP were revealed by the different staining methods. The results suggested that the Glees method may stain a population of SP in a region of cortex where both amyloid deposition and neurofibrillary changes have occurred.