Viscosimetric squeeze flow of suspensions.

The rheology of particle suspensions has been extensively explored in the case of a simple shear flow, but less in other flow configurations which are also important in practice. Here we investigate the behavior of a suspension in a squeeze flow, which we revisit using local pressure measurements to deduce the effective viscosity. The flow is generated by approaching a moving disk to a fixed wall at constant velocity in the low Reynolds number limit. We measure the evolution of the pressure field at the wall and deduce the effective viscosity from the radial pressure drop. After validation of our device using a Newtonian fluid, we measure the effective viscosity of a suspension for different squeezing speeds and volume fractions of particles. We find results in agreement with the Maron-Pierce law, an empirical expression for the viscosity of suspensions that was established for simple shear flows. We prove that this method to determine viscosity remains valid in the limit of large gap width. This makes it possible to study the rheology of suspensions within this limit and therefore suspensions composed of large particles, in contrast to Couette flow cells which require small gaps.

Droplets climbing a rotating helical fiber.

A liquid droplet is placed on a rotating helical fiber. We find that the droplet may slide down, attach or climb up the fiber. We inspect experimentally the domain of existence of these three behaviors as a function of the geometrical characteristics of the fiber, its angle relatively to the horizontal, the wetting properties of the fluid and the rotating speed of the helix. A theoretical model is proposed in order to capture the boundaries of the experimental phase diagram.

Successive reintrusions in a granular medium.

We measure and analyze the drag force experienced by a rigid rod that penetrates vertically into a granular medium and partially withdraws before sinking again. The drag during the successive reintrusions is observed to be significantly smaller than the force experienced in the first run. Two force regimes are evidenced depending on how the reintrusion depth compares with the withdrawal distance Δ. These two regimes are characterized by a force curve of positive and negative curvature and are separated by an inflection point, which is characterized experimentally. We approach the difference between the first intrusion and the following reintrusions by considering a modification in the stress field of the granular material after the partial extraction of the rod. A theoretical model for the stress modification is proposed and allows to rationalize all the experiments realized for different withdrawal distances Δ. This framework introduces a crossover length λ above which the stress modification in the granular medium is maintained and that is shown to depend linearly on Δ. Finally, the model provides a prediction for the location of the inflection points in reasonable agreement with observations.

Complete development of hepatic stages of Plasmodium falciparum in vitro.

An in vitro model was developed to study the hepatic phase of Plasmodium falciparum, the only malaria parasite lethal to man. Primary cultures of human hepatocytes were inoculated with sporozoites of Brazilian and African strains of P. falciparum. On days 1 through 7 after inoculation examination of fluorescence-labeled and Giemsa-stained preparations demonstrated the presence of many intracellular parasites. In three separate sets of experiments all cultures were found to be infected with as many as 650 liver schizonts measuring up to 40 micrometers. After the addition of red blood cells, intraerythrocytic forms of P. falciparum were detected on days 12 and 13 by an immunofluorescence assay, indicating that the hepatic cycle had been completed in vitro.

Effect of antibodies to recombinant and synthetic peptides on P. falciparum sporozoites in vitro.

Antibodies were raised in mice immunized with several recombinant and synthetic peptides of the circumsporozoite protein of Plasmodium falciparum. The antibodies were evaluated for protective activity in a human hepatocyte culture system. They exerted their protective effect against the parasite at three points: sporozoite attachment to the hepatocyte surface, entry, and subsequent intracellular development. Inhibition of attachment and entry were found to be related to the antibody titer against the authentic circumsporozoite protein on the sporozoite surface, especially when peptides were administered with alum or complete Freund's adjuvant. Even when invasion was not totally inhibited, the presence of abnormal trophozoites and a frequent inhibition of schizont development in long-term cultures suggested continued activity of antibodies at the intracellular level after sporozoite penetration had been completed.

Rotation of melting ice disks due to melt fluid flow.

We report experiments concerning the melting of ice disks (85 mm in diameter and 14 mm in height) at the surface of a thermalized water bath. During the melting, the ice disks undergo translational and rotational motions. In particular, the disks rotate. The rotation speed has been found to increase with the bath temperature. We investigated the flow under the bottom face of the ice disks by a particle image velocimetry technique. We find that the flow goes downwards and also rotates horizontally, so that a vertical vortex is generated under the ice disk. The proposed mechanism is the following. In the vicinity of the bottom face of the disk, the water eventually reaches the temperature of 4 °C for which the water density is maximum. The 4 °C water sinks and generates a downwards plume. The observed vertical vorticity results from the flow in the plume. Finally, by viscous entrainment, the horizontal rotation of the flow induces the solid rotation of the ice block. This mechanism seems generic: any vertical flow that generates a vortex will induce the rotation of a floating object.

T cell mapping of one epitope from thyroglobulin inducing experimental autoimmune thyroiditis (EAT).

These data collect the advance made in the last few years in our laboratory in defining one epitope from the thyroglobulin (Tg) molecule (660 KDa) inducing Experimental Autoimmune thyroiditis (EAT) in CBA/J mice. We achieved the characterization of one EAT-inducer Tg peptide by combining "in vitro" biochemical and immunological approaches and "in vivo" studies. Since T cells recognize degraded forms of the antigen and since endogenous antigens preferentially activate class I-restricted T cells, we hypothesized that one cytotoxic T cell hybridoma, named HTC2, which prevents further EAT induction in mice injected with Tg would be specific for one EAT inducer peptide. In order to identify one Tg epitope inducing EAT, enzymatic treatment of the protein by trypsin, HPLC purification and sequence analysis were performed. Simultaneously, tryptic digests were used to pulse CBA/J macrophages and tested for their ability to be recognized by HTC2 cells. Lastly, when digests were recognized by HTC2 cells their capacity to induce EAT in CBA/J mice was evaluated. To further assess the pathogenicity of the sequenced Tg peptide, one synthetic peptide was made and its capacity to induce EAT verified. By this procedure we identified for the first time one 40 amino-acid peptide from human thyroglobulin inducing EAT in CBA/J mice.

Polyarthritis in MRL-lpr/lpr mice: mouse type II collagen is antigenic but not arthritogenic.

In addition to a lupus-like syndrome and massive T cell proliferation, MRL-lpr/lpr(MRL/l) mice develop an arthritic process very similar serologically and histologically to human rheumatoid arthritis (RA). Recently, we have developed in DBA/1 mice an experimental model of autoimmune arthritis (EAA) which shares clinical features with RA, by injecting homologous type II collagen (CII). In order to investigate the possible relationship between the spontaneous polyarthritis of MRL/l mice and collagen induced EAA, we immunized MRL/l mice with mouse (M) CII. Our findings revealed that the injection of 100 micrograms M-CII in young or old MRL/l mice did not modify the articular pathology which spontaneously develops in non-injected mice. Circulating autoantibodies to native M-CII were found in the sera of immunized young mice but were not detected in non injected or immunized old mice. Conversely, denatured alpha 1 (II) chains or CB peptides derived from M-CII were recognized by most of the MRL/l sera whether mice had been immunized or not. The incidence of positive sera as well as the intensity of the response evaluated by Western blot analysis increased with the age of the mice. Taken together, our data suggest that, even if the injection of homologous CII in MRL/l mice may accelerate the onset of joint pathology, the spontaneous disease arises independently of an autoimmune response against native CII.

Characteristics of cytotoxic thyroglobulin-specific T cell hybridomas.

Clones of cytotoxic thyroid-specific T cell hybridomas were generated by fusion of thyroglobulin-primed, "in vitro"-boosted CBA lymph node cells with the AKR-derived lymphoma cell line BW 5147. One hundred and thirty one clones were obtained. Among them, 15 were able to induce the lysis of 51Cr-labeled syngeneic thyroid epithelial cells after 5 h of incubation at 37 degrees C. Two T cell clones, HTC1 and HTC2, were further studied. These clones, which exhibit cell surface characteristics of cytotoxic cells, were specific for only syngeneic thyroid cells (allogeneic thyroid cells or syngeneic epithelial cells were never lysed by these hybridomas). Moreover, by using Ag-pulsed syngeneic macrophages as targets, syngeneic cytotoxicity was shown to be specific for thyroglobulin and not for a nonrelated Ag. The lysis obtained with these autoreactive thyroid-specific T cell clones is restricted to class I major histocompatibility Ag. This property is assessed by both the blocking of syngeneic cytotoxicity toward thyroid epithelial cells or thyroglobulin-pulsed macrophages only by anti-class I mAb and by the detection of specific lysis of target cells exclusively when effector hybrid cells and target thyroid epithelial cells or thyroglobulin-pulsed macrophages shared at least class I major histocompatibility Ag.

Pattern of humoral reactivity to type II collagen in rheumatoid arthritis.

Humoral immunity directed against type II collagen (CII) is a common although not specific feature of rheumatoid arthritis (RA). We have shown that 10 to 15% of the sera either from RA patients (n = 88) or from healthy controls (n = 149) reacted with native human CII. Conversely, autoantibodies to the alpha-1 (II) chains were significantly more frequent in the RA group (26.1% versus 6.0%, P<0.001), suggestingthatdenaturedCII may bean autoantigenin RA. Thus, human CII was cleaved with cyanogen bromide (CB), and immunoblotting techniques were performed on 19 RA and 21 normal sera. Among the four major CB peptides, CB10 and CB11 were recognized by most of the sera tested without distinction between normal or RA sera. Inhibition experiments using an ELISA have shown that: (i) antibodies to the native CII molecule did not cross-react with those recognizing the CB peptides, and vice-versa; (ii) the binding of the sera to native CII was partially inhibited by pre-incubation with alpha-1 (II) chains, and vice-versa; (iii) pre-incubation of the sera with CB peptides partially blocked the binding to alpha-1 (II) chains, whereas pre-incubation of the sera with alpha-1 (II) chains totally inhibited the reactivity against CB peptides; and (iv) a substantial proportion of the epitopes recognized by anti-CII autoantibodies was neither species specific nor type specific. Taken together, these findings reveal the existence of several populations of anti-CII autoantibodies: some antibodies react exclusively with conformational determinants of the CII molecule, and others are directed towards linear structures of alpha-1 (II) chains.

Autoantibody specific for a thyroglobulin epitope inducing experimental autoimmune thyroiditis or its anti-idiotype correlates with the disease.

In order to elucidate the role of anti-thyroglobulin (Tg) autoantibodies (A-Ab) in experimental autoimmune thyroiditis (EAT), a kinetic study was conducted in EAT-susceptible (CBA/J) and non-susceptible (C57BL/6) strains of mice. From day 0 to 70 post-Tg immunization, titers of A-Ab to Tg and to the linear 5-10-kDa Tg tryptic fragment inducing EAT as well as anti-idiotypic A-Ab representing the internal image of the thyroidogeneic antigen were measured. EAT onset, development and recovery correlate with the presence of two subsets of A-Ab only in susceptible strains of mice. First, with the presence of anti-Tg A-Ab to one determinant borne by the linear 5-10-kDa Tg tryptic fragment, and second with the presence of anti-idiotypic A-Ab specific for the monoclonal anti-Tg A-Ab (3B8G9) paratope which binds to Tg determinant inducing EAT.

Protection from experimental autoimmune thyroiditis conferred by a monoclonal antibody to T cell receptor from a cytotoxic hybridoma specific for thyroglobulin.

A clonotypic mAb, AG7, has been prepared from splenocytes of CBA/J mice immunized with a cytotoxic T cell hybridoma, HTC2, specific for a pathogenic epitope of the thyroglobulin molecule in association with class I MHC Ag. AG7 binds to HTC2 cells but not to the other T cell hybridomas tested. Moreover, when used in functional studies, AG7 blocks the HTC2 capacity of specific target lysis. It also reacts with a determinant that comodulates with the CD3 Ag present on the surface of HTC2 cells. Immunoprecipitation of 125I-labeled solubilized HTC2 membranes demonstrated two bands located at 90 and 72 kDa under nonreducing conditions, which became a 46-kDa band under reducing conditions. Finally, when AG7 is injected into CBA/J mice, on day -1 before immunization with the pathogenic tryptic fragments of the thyroglobulin molecule, experimental autoimmune thyroiditis is abrogated. Thus, one of the multiple potential mechanisms of the protective immunity against EAT induced by HTC2 cells that we previously proposed, i.e., the generation of anti-clonotypic antibodies to HTC2 TCR, seems apparent.

Characterization and sequencing of a 40-amino-acid peptide from human thyroglobulin inducing experimental autoimmune thyroiditis.

We previously demonstrated that: a) a cytotoxic T cell hybridoma (HTC2) was able to induce lysis of syngeneic macrophages pulsed with either porcine thyroglobulin (pTg) or the tryptic fragments (TF) from pTg less than 10 kDa (M(r)) and that b) these low M(r) pTg TF included pathogenic epitopes because their injection into CBA/J mice induces thyroid lymphocytic infiltration typical of experimental autoimmune thyroiditis. Therefore the biochemical analysis of the TF preparation from pTg less than 10 kDa M(r) was undertaken and the characterized peptides were tested for their ability to be recognized or not by HTC2 cells. The sequencing of the selected peptides showed a 70% sequence homology with a portion of human thyroglobulin (hTg). The lack of a published sequence of pTg led us to synthesize a 40-amino acid peptide (F40D) similar to that portion of hTg. This F40D peptide was able to generate lymphocytic infiltrations in CBA/J mice thyroid glands, as was the native pTg molecule. Although the lymphocytic infiltrations were similar in the pTg or F40D-immunized mice, auto-antibodies to pTg or to hTg were only detectable in mice immunized with pTg. In contrast, autoantibodies levels to F40D peptide were significantly increased in serum from mice in which EAT had been induced by the F40D peptide. This highly hydrophobic peptide shows a M(r) of 4,492 kDa; it is located at the end of the second-third of the thyroglobulin molecule and up to now represents a unique sequence from the hTg molecule inducing experimental autoimmune thyroiditis.

[Obtaining hepatic stages of Plasmodium falciparum in vitro]. / Obtention in vitro des stades hépatiques de Plasmodium falciparum.

Sporozoites of Plasmodium falciparum, obtained by membrane feeding of Anopheles freeborni or A. stephensi with cultured gametocytes, were used to infect monolayers of human hepatocytes. Fluorescent labelling with an African serum as well as Giemsa staining performed from day one to day 7 of cultures, demonstrated the presence of numerous hepatic schizonts measuring up to 40 micron.