Enhanced photoemission from laser-excited plasmonic nano-objects in periodic arrays.

The process of photoelectron emission from gold surfaces covered with nano-objects that are organized in the form of a periodic array is addressed in the short laser pulse regime ([Formula: see text] fs) at moderate intensities [Formula: see text] W cm(-2) and for various laser wavelengths. The emission spectrum from a gold single crystal measured under the same conditions is used for reference. The comparison of the photo-emission yield and the energy of the ejected electrons with their counterparts from the (more simple) reference system shows that the periodic conditions imposed on the target surface drastically enhance both quantities. In addition to the standard mechanism of Coulomb explosion, a second mechanism comes into play, driven by surface plasmon excitation. This can be clearly demonstrated by varying the laser wavelength. This interpretation of the experimental data is supported by predictions from model calculations that account both for the primary quantum electron emission and for the subsequent surface-plasmon-driven acceleration in the vacuum. Despite the fact that the incident laser intensity is as low as [Formula: see text] W cm(-2), such a structured target permits generating electrons with energies as high as 300 eV. Experiments with two incident laser beams of different wavelengths with an adjustable delay, have also been carried out. The results show that there exist various channels for the decay of the photo-emission signal, depending on the target type. These observations are shedding light on the various relaxation mechanisms that take place on different timescales.

Investigation of laser-irradiated Ar cluster dynamics from K-shell x-ray emission measurements.

Intense (up to a few 10(17) W/ cm2) femtosecond (down to 40 fs) laser pulses are focused onto a partially clusterized argon gas jet. The target was previously characterized and optimized in order to get a homogeneous and dense jet of clusters with a well controlled size. The interaction leads to x-ray emission that is absolutely calibrated and spectrally resolved using a high resolution time-integrated spectrometer in the K-shell range (from 2.9 to 4.3 keV). X-ray spectra are investigated as a function of different laser temporal parameters such as the nanosecond prepulse contrast, the laser pulse duration, and the femtosecond delay between two different laser pulses. The cluster size ranges from 180 to 350 angstroms and irradiation by laser pulses with both linear and circular polarization is investigated. The experimental results are discussed in terms of the laser-cluster interaction dynamics. They are compared with the predictions of collision-dominated nanoplasma models. However, further interaction processes are required in order to explain the observed characteristic lines demonstrating highly charged ions up to Ar16+.

Allelic family-specific humoral responses to merozoite surface protein 2 (MSP2) in Gabonese residents with Plasmodium falciparum infections.

Merozoite surface protein 2 (MSP2) expressed by Plasmodium falciparum asexual blood stages has been identified as a promising vaccine candidate. In order to explore allelic family-specific humoral responses which may be responsible for parasite neutralization during natural infections, isolates from individuals with either asymptomatic infections or uncomplicated malaria and residing in a Central African area where Plasmodium transmission is high and perennial, were analysed using MSP2 as polymorphic marker. The family-specific antibody responses were assessed by ELISA using MSP2 synthetic peptides. We observed an age-dependence of P. falciparum infection complexity. The decrease of infection complexity around 15 years of age was observed simultaneously with an increase in the mean number of MSP2 variants recognized. No significant difference in the P. falciparum genetic diversity and infection complexity was found in isolates from asymptomatic subjects and patients with uncomplicated malaria. The longitudinal follow-up showed a rapid development of immune responses to various regions of MSP2 variants within one week. Comparing humoral responses obtained with the other major antigen on the merozoite surface, MSP1, our findings suggest that different pathways of responsiveness are involved in antibody production to merozoite surface antigens.

Three-flap anoplasty for imperforate anus: results for primary procedure or for redoes.

UNLABELLED: Three-flap anoplasty was first described in 1987 by Yazbeck for the treatment of rectal prolapse after pull-through operations for imperforate anus, and in 1992 in a case of anterior perineal approach. It is intended to reproduce the normal anatomy of a sensitive anal canal. The purpose of this study is to evaluate 14 children (9 boys and 5 girls) operated for imperforate anus. METHOD: Eleven children had an intermediate or high imperforate anus and 3 had a low imperforate anus. Nine were operated for the first time with a three-flap anoplasty (GrA), and 5 were reoperated with this technique because of fecal incontinence and/or rectal prolapse (GrB). Seven patients had associated malformations: one Hirschsprung's disease, one cloacal defect with renal insufficiency, three complex caudal malformations with tethered cord, one Down syndrome, and two psychological and psychomotor troubles. The patients (average age = 6 years) have been evaluated every year since 1997, with a questionnaire based on a clinical score validated with 30 healthy children, used as a control group. Ten anal manometries were carried out. RESULTS: In 1998, the GrA score was 16.1 (control = 22.5) and the GrB score was 11.5 (p = 0.25). In 1999, GrA and GrB score were approximately the same. The score of those without associated anomalies was 19.6 whereas the score of the children with other malformations or anomalies was 10 (p = 0.02). Anal manometry is almost normal in patients with low or intermediate imperforate anus (rectoanal relaxation reflex for 10 cm H2O, and basal resting pressure more than 40 cm H2O). Even though anal manometry is subnormal in patients with Down syndrome or psychomotor troubles, the clinical score remains low (score = 10). In cases of complex caudal malformations or high imperforate anus, the results of anal manometry and clinical score are bad (score 9.7). CONCLUSION: The three-flap anoplasty can be carried out in newborns without colostomy and often represents the only possible surgical approach to avoid a laparotomy. This plasty, proposed secondarily to correct a defect of continence, can be performed without colostomy, with satisfactory results.

Fixed, epitope-specific, cytophilic antibody response to the polymorphic block 2 domain of the Plasmodium falciparum merozoite surface antigen MSP-1 in humans living in a malaria-endemic area.

The MSP-1 merozoite surface antigen of the human malaria parasite Plasmodium falciparum is a major target of immune response. The domain called block 2 shows extensive allelic diversity, with more than 50 alleles identified, grouped into three allelic families. Presence of anti-block 2 antibodies has been associated with reduced risk for clinical malaria, but whether or not allele-specific antibodies are implicated remains unclear. To study the fine specificity of the human antibody response, we have used a series of 82 overlapping, N-biotinylated, 15-mer peptides scanning reference alleles and including numerous sequence variants. Peptide antigenicity was validated using sera from mice immunized with recombinant proteins. A cross-sectional survey conducted in a Senegalese village with intense malaria transmission indicated an overall 56 % seroprevalence. The response was specific for individuals and unrelated to the HLA type. Each responder reacted to a few peptides, unrelated to the infecting parasite genotype(s). Seroprevalence of each individual peptide was low, with no identifiable immunodominant epitope. Anti-block 2 antibodies were mostly of the IgG3 isotype, consistent with an involvement in cytophilic antibody-mediated merozoite clearance. The number of responders increased with age, but there was no accumulation of novel specificities with age and hence with exposure to an increasingly large number of alleles. A 15-month longitudinal follow up outlined a remarkably fixed response, with identical reactivity profiles, independent of the past or current parasite types, a pattern reminiscent of clonal imprinting. The implications of the characteristics of the anti-block 2 antibody response in parasite clearance are discussed.

Mimicking a conformational B cell epitope of the heat shock protein PfHsp70-1 antigen of Plasmodium falciparum using a multiple antigenic peptide.

The Pf72/Hsp70-1 antigen is a major target in the naturally acquired immunity against Plasmodium falciparum malaria. We carried out an extensive analysis of the responses to several epitopes on the least conserved C-terminal domain, according to the mode of sensitization: malaria infection or immunization with different immunogens. We found significant differences in the panel of B-cell epitopes recognized by animal models including primates, and by humans sensitized by natural infection. We focused the analysis on one epitope that is unique to Plasmodium species. It is specifically recognized by a monoclonal antibody that mediates the killing of infected hepatocytes in vitro. We produced a polymeric multiple antigenic peptide (MAP) form of this sequence, which enabled us to identify a new B-cell epitope not detected by ELISA with linear peptides. The polymer was strongly recognized by sera from monkeys or humans sensitized by natural infection, whereas the monomer was not. We modelled the three-dimensional structure of the Pf72/Hsp70-1 sequence, using known Escherischia coli DnaK structures as a template. This predicted that the corresponding region would form a loop in the native antigen. The results presented here suggest that the MAP strategy is also particularly useful as a means of obtaining suitable synthetic models for conformation-dependent epitopes.

[Determination of acceptability of 2 cutaneous estradiol gels, in a dose of 1.5 mg daily]. / Appréciation de l'acceptabilité de deux gels d'estradiol cutané, à la dose d'1,5 mg par jour.

OBJECTIVE: The aim was to compare acceptability of a percutaneous 0. 1% estradiol gel (Gel A, Estreva(R) Gel, Laboratoire Théramex, Monaco) to that of an 0.06% estradiol gel (Gel B, Oestrodose(R), Laboratoires Besins-Iscovesco) in its new formulation and packaging. MATERIAL AND METHODS: This randomized, crossed, simple-blind study was carried out in 48 volunteer healthy postmenopausal women. The volunteers applied on one forearm 1.5 mg/day of cutaneous estradiol in the form of either gel, according to randomized allocation, for four days without free period between the two therapeutic periods. The application and drying times of the two gels were measured during the first application; gel subjective women assessment was collected at the beginning and at the end of the study. RESULTS: Mean application and drying times with Gel A are significantly reduced, compared to Gel B (p=0.0259 and p=0.0001, respectively) with drying time 61% shorter; these data are confirmed by subjective women evaluation. The two gels are not significantly different regarding several criteria as consistency, ease of application and sensation of lasting stickiness. However, a significant difference is found in favour of Gel A on the following items: practicality of application (p=0.007), ease of penetration (p<0.001), quantity of gel to apply (p<0.001) after the first application. After four days of administration, a same significant difference is observed concerning practicality of the gel (p=0.0078), duration of use (p<0. 001), packaging, women opinion on the gel (p=0.022) and the product, gel and packaging (p<0.001). At the end of the study, gel A utilization is considered by women more practical (p=0.001) with an easier application (p<0.001) and less restricting while applying (p=0.001), compared to Gel B; 72.9% of women prefer the Gel A and 12. 5% of women prefer the Gel B. CONCLUSION: A better acceptability of the 0.1% estradiol gel and of its packaging compared to that of the 0.06% estradiol gel in this new formulation and packaging is observed in this study.

Accurate quantitation of Leishmania infection in cultured cells by flow cytometry.

BACKGROUND: Leishmaniases are major parasitic diseases caused by protozoans that are obligate intracellular parasites during the mammalian phase of their life cycle. Quantitation of experimental mammalian cell infections is usually performed by time-consuming microscopic examination. In this report a flow cytometry (FCM)-based assay suitable for studying in vitro infections by L.amazonensis is presented. METHODS: Intense fluorescence staining of the amastigote forms with a stage- and species-specific monoclonal antibody was obtained after permeabilization of both the host-cell cytoplasmic membrane and the parasitophorous vacuole membrane by saponin treatment. RESULTS: Upon flow cytometry (FCM) analysis, parasitized cells separated sharply from the auto-fluorescence of the mammalian host cells, giving the assay a high degree of sensitivity and specificity. Ninety to 98% of cells in the more fluorescent population harbored parasites visible by phase-contrast and UV-light microscopy, while no parasites were observed in more than 95% of the cells in the population with background fluorescence. Comparisons of the FCM results with those from microscope counting and analysis of various dilutions of parasitized cells confirmed the reliability of the method. CONCLUSIONS: The FCM assay provided rapid quantitation of Leishmania infection either in mouse macrophages, the natural host cell in murine leishmaniasis, or in Chinese hamster ovary (CHO) cells, a non-macrophage cell line proposed as an in vitro model for studying host-parasite interactions. The protocol described here should be adaptable to studies involving other parasites residing in nucleated cells.

Plasmodium falciparum: immune pressure in Saimiri sciureus monkeys can select for a parasite population inducing a protective immunity that is not controlled by antibody.

Protective immunity against a Plasmodium falciparum blood infection can be passively transferred by antibodies in humans and in the primate experimental malaria model Saimiri sciureus. We report here the emergence of a novel virulent parasite population after such passive transfer of hyperimmune serum in splenectomized monkeys. These FUP-2 parasites have been partially genotyped and phenotyped. Although no genotypic variation was detected for four polymorphic loci compared to the original FUP-1 parasite population, FUP-2-infected erythrocytes exhibit little or no detectable surface determinants, including those reacting with antibodies raised against FUP-1 surface antigens. In addition, FUP-2-infected erythrocytes exhibit no rosetting or autoagglutination. Interestingly, although Saimiri monkeys control efficiently FUP-2 parasites after repetitive infections, this protection cannot be passively transferred to naive recipients. Our results suggest that antibody-mediated and antibody-independent T-cell-mediated protective responses may cooperate in controlling P. falciparum infection in splenectomized Saimiri monkeys.

A method to recover, enumerate and identify lymphomyeloid cells present in an inflammatory dermal site: a study in laboratory mice.

We describe a new method to recover and study cells present in the dermis of mouse ear at homeostasis or after intradermal injection of disturbing agents (lipopolysaccharide or Listeria monocytogenes). The ears either left untreated or inoculated were handled and processed as culture explants of the dorsal and ventral leaflets, their dermal sides being spread on a buffered medium. Within this medium emigrate/sediment, with different kinetics: neutrophils, mononuclear phagocytes, dendritic leucocytes, T lymphocytes expressing either gamma delta or alpha beta TCRs, and other minor subsets, the identification of which deserves more relevant reagents: they are likely to be NK, mast cells, eosinophils and their local progenitors. All the major subsets were identified through a combination of immunocytochemical and flow cytometry labeling. Two examples illustrating the advantages and limitations of this new method are given: either 1 microgram of LPS or 10(4) Listeria monocytogenes were injected within the ear 48, 24, 12, 6, 3 h before ear explant culture. This ear explant culture has been further compared to the ear sheet treatment with collagenase/disease for three cell populations, the epidermal dendritic leucocytes, the gamma delta epidermal T cells as well as the alpha beta T cells recirculating within the steady state dermis. This method provides the first evidence of the existence of recirculating T CD4 lymphocytes in the mouse dermis.

Flow cytometry detection of surface antigens on fresh, unfixed red blood cells infected by Plasmodium falciparum.

The immunofluorescence detection of parasite-specific antigens on the surface of red blood cells infected by Plasmodium falciparum parasites is usually performed by visual detection under a fluorescence microscope. We describe here a technique permitting the analysis of surface immunofluorescence labelling by flow cytometry. Infected red blood cells are selected on the basis of their parasitic DNA and RNA content by Hoechst and Thiazole Orange vital dyes. Cytometric analysis of these labels, as well as general erythrocyte characteristics assessed by analysis of forward and side scatter allows the selection of viable intact infected erythrocytes from other blood cells. The integrity of these selected erythrocytes was confirmed by the absence of labelling with antibodies directed against internal components such as spectrin. This technique permits the detection of specific surface immunofluorescence staining on red blood cells infected with mature stages of P. falciparum by antibodies in sera from hyperimmune Saimiri monkeys. Using Thiazole Orange dye for detection of parasitised cells, this analysis was performed on a FACSscan apparatus equipped with a single laser.

Manipulating blood T cells and B cells from squirrel monkeys: some technical considerations.

The squirrel monkey Saimiri sciureus is an experimental host for a range of human pathogens, and for the assessment of vaccine candidate antigens and vaccine strategies. This experimental host is thus particularly suitable for the follow-up of humoral responses. To understand some of the mechanisms that underlie the defense against experimental pathogens, there is a need of basic knowledge on cellular immune effectors also. The authors report here their experience in characterizing squirrel monkey blood T and B lymphocytes, and in studying in vitro induced activation and proliferation of T and B cells. Particular emphasis is given to the in vitro differentiation of squirrel monkey B cells into immunoglobulin secreting cells, with respect to Plasmodium falciparum antigens.

Catecholaminergic neurons result from intracerebral implantation of embryonal carcinoma cells.

A replication-defective retrovirus was used to introduce the marker gene nlsLacZ into the murine embryonal carcinoma (EC) cell line PCC7-S-aza-R-1009. Undifferentiated EC cells were implanted into the central nervous system of adult rats. One month later, the grafted cells continued to express the nlsLacZ gene. Immunohistochemical analysis demonstrated the presence of EC-derived neurons. These neurons were capable of expressing tyrosine hydroxylase and extended neurites into the host parenchyma. EC-derived glial cells could not be detected. There was no evidence of tumorigenicity. These results demonstrate the utility of EC cells for introduction of exogenous gene products into the central nervous system in experimental models of gene therapy.

Expression of the alpha chain of human Fc epsilon RI in transfected rat basophilic leukemia cells: functional activation after sensitization with human mite-specific IgE.

The actual dilemma in studying the binding and triggering capacity of IgE from allergic patients is the lack of cultured basophils or mast cell analogs of human origin. Human IgE binds with exquisite species specificity to the high affinity IgE receptor (Fc epsilon RI) expressed on the surface of these cells. In rodents this receptor has been characterized as a tetrameric plasma membrane protein composed of an IgE-binding alpha chain, a beta chain and two disulfide-linked gamma chains. In order to establish a cell line expressing the alpha chain of human Fc epsilon RI which can be triggered with IgE from human patients and specific allergen, we transfected the cDNA coding for the human alpha subunit into rat basophilic leukemia cells. The resulting transfectants express the human alpha chain on the cell surface in the form of a hybrid complex associated with endogenous rat gamma chains. After sensitization with human IgE from mite-specific patients, the transfectant produces a calcium response upon incubation with allergen. The established cell line can be used as a model system to study the mechanism of mast cell triggering through IgE from allergic patients.

Plasmodium chabaudi p68 serine protease activity required for merozoite entry into mouse erythrocytes.

To define the role of malaria parasite enzymes during the process of erythrocyte invasion, we have developed an in vitro serum-free invasion assay of mouse erythrocytes by purified Plasmodium chabaudi merozoites. The sensitivity of a merozoite-specific serine protease (p68) to various inhibitors and the effect of these inhibitors on invasion indicate a crucial role for p68. The substrate specificity of the purified enzyme has been partially defined using fluorogenic peptides. Consistent with this, in vitro incubation of mouse erythrocytes with the merozoite enzyme led to the cleavage of band 3 protein. The possible implication of erythrocyte band 3 truncation for the successful entry of the merozoite into the erythrocyte is discussed.