Search

Probiotics have been associated with prevention and improvement of symptoms in atopic diseases such as atopic dermatitis. However, few studies exist that document their efficacy for upper airways allergies such as allergic rhinitis.To investigate the effect of short-term oral administration of Lactobacillus paracasei ST11 on a nasal provocation test (NPT) with grass pollen.Thirty-one adult volunteers with allergic rhinitis were enrolled in a randomized, double-blind, placebo-controlled study, based on two 4-week cross-over periods of product consumption (ST11-fermented milk vs. placebo), separated by a wash-out period of 6-8 weeks. Objective and subjective clinical parameters of NPT as well as systemic and nasal immunological parameters were compared between the two treatment periods (registration number: NCT 011 50 253).Subjects that received ST11-fermented milk had lower nasal congestion than subjects under placebo (visual analogical scale; P0.05). Nasal pruritus followed the same trend. However, no significant change in combined nasal reaction threshold was observed between the two periods. IL-5 secretion by peripheral blood mononuclear cells and serum allergen-specific IgG4 were significantly lower in ST11-fermented milk group compared to placebo group. IL-8 and IL-10 secretion followed the same trend.Short-term treatment with ST11-fermented milk before NPT significantly improved a clinical marker of NPT (subjective nasal congestion) and down-regulated systemic immune markers (IL-5 from peripheral blood mononuclear cells and serum IgG4). These data strongly suggest that probiotics may down modulate key parameters of allergic rhinitis and warrant future evaluation in seasonal trials.

We report the first synthetic peptide vaccine eliciting strong CD8(+) and CD4(+) T lymphocyte responses in humans. The vaccine, representing the C-terminal region of the circumsporozoite protein of Plasmodium falciparum (amino acids 282-383) was well tolerated and strong sporozoite-specific antibodies were elicited. In addition, robust lymphocyte proliferation responses were equally elicited with concomitant in vitro production of IFN-gamma, crucial in the elimination of the parasite. Most importantly, we also observed the development of CD8(+) T lymphocyte responses decisive in the immunity to malaria. The latter finding opens new, possibly safer, avenues for vaccination strategies when a CD8(+) T cell response is needed.

Preclinical evaluation of synthetic peptides corresponding to the C-terminal regions of the circumsporozoite (CS) protein in various Plasmodia showed that these preparations were immunogenic and safe upon injection in various animal models. Additionally, the corresponding peptide from Plasmodium falciparum was widely recognized by sera and PBL obtained from semi-immune adults living in malaria endemic areas. Moreover, the CS C-terminal peptide derived from P. berghei conferred protection upon challenge with live sporozoites in mice. A GLP preparation of the synthetic peptide corresponding to residues 282-383 of the Pf CS, NF-54 strain is currently evaluated in a open, non-randomized, Phase I human trial. Data obtained after the second antigen injection show that the malaria vaccine Pf CS 282-383 is safe, well tolerated and gives rise to high antibody titre, CD4+ and CD8+ lymphocyte responses.

Tetanus toxoid (Ttxd) encapsulated in polyester microspheres (MS) for single injection immunization have so far given pulsatile in vitro release and strong immune response in animals, but no boosting effect. This has been ascribed to insufficient toxoid stability within the MS exposed to in vivo conditions over a prolonged time period. This study examined the effect of co-encapsulated putative stabilizing additives.Two different Ttxd were encapsulated in poly(D,L-lactic-co-glycolic acid) (PLGA 50:50) and poly(D,L-lactic acid) (PLA) MS by spray-drying. The influence of co-encapsulated additives on toxoid stability, loading in and release from the MS, was studied by fluorimetry and ELISA.Co-encapsulated albumin, trehalose and gamma-hydroxypropyl cyclodextrin all improved the toxoid encapsulation efficiency in PLGA 50:50 MS. Albumin increased the encapsulation efficiency of antigenic Ttxd by one to two orders of magnitude. Further, with albumin or a mixture of albumin and trehalose ELISA responsive Ttxd was released over 1-2 months following a pulsatile pattern.Optimized Ttxd containing MS may be valuable for a single-dose vaccine delivery system.

Antigenic proteins encapsulated in biodegradable polyester microspheres (MS) can slowly denature or aggregate, which results in decreased antigenicity. In this study, we have evaluated the ability of co-encapsulated additives to protect against the loss of tetanus toxoid (TT) antigenicity.Antibody responses were analyzed after immunization of mice with TT microencapsulated in the presence of additives (TT-MS-additive).Immunization with TT-MS-additives gave rise to higher responses than those obtained in the absence of additive. BSA, trehalose. Gamma-hydroxypropylcyclodextrin and calcium salts preserved the immunogenicity of the incorporated antigen with the highest efficacy. Sustained responses were obtained with mixtures of fast and slowly releasing TT-MS containing BSA plus trehalose or calcium salts.The selected additives may stabilize the antigen in MS during storage and rehydration in body fluids. Regulated antigen release from MS-based vaccines permits a reduction of the antigen dose and optimization of single-dose vaccine formulations.

The IgG subclass distribution was determined in six commercial and in four experimental human gamma-globulin preparations. The concentrations of IgG subclasses were measured in a modified radiommunoassay using subclass-specific antisera. In commercial gamma-globulins, the distribution of the subclasses corresponded roughly to the distribution in normal human serum. A considerable enrichment of the IgG 4 was found in experimental lots prepared either from the ethanol fraction III or from the rivanol-precipitable IgG.

Antibodies directed against viruses and bacteria are not equally distributed among the main classes of immunoglobulins, e.g. IgG, IgA and IgM. It has been found that IgM is mostly concerned with certain antibacterial activities (Salmonella, Escherichia coli and Pseudomonas) and IgA with high antibody titers for poliomyelitis virus I whereas antibody activities against many viruses such as influenza and measles virus occur preferentially in the IgG population. Furthermore, isolated immunoglobulin deficiency syndromes are actually well known. In the light of these findings, new concepts of immunotherapy have developed. Massive i.v. IgG-therapy is already widely used in congenital and acquired severe hypogammaglobulinemia. Preparations enriched in IgA and IgM are needed to complete the immunotherapeutical possibilities. Such a fraction called IgGAM has already been prepared in our Institute. Fraction III obtained during large scale fractionation is used as starting material and caprylic acid for the precipitation of most proteins other than the immunoglobulins present in fraction III. The immunoglobulin concentrate is finally obtained by ethanol precipitation of the caprylic acid supernatant. The present study is concerned with various modifications of the initial technique in order to obtain fractions more specially enriched in IgA or in IgM. In some cases the standard IgGAM fraction has been submitted to a further fractionation step, such as adsorption of IgG on DEAE-cellulose or precipitation of certain immunoglobulins achieved by Rivanol or by lowering the salt concentration. In other trials the fractionation procedure starting from fraction III has been modified. Rivanol has been used as a precipitating agent for the subfractionation of fraction III. It is well known that IgG is soluble in the presence of Rivanol. This technique was thus used in order to obtain preparations enriched mainly in IgM and IgA. The precipitate obtained after the addition of Rivanol was dissociated by NaCl and the solution further subfractionated by caprylic acid. In a similar way PEG was associated with the caprylic acid precipitation step. PEG precipitates proteins mainly in function of their molecular weight. However, the enrichment of IgM of the final fraction did not exceed 32% and much IgM was lost under the experimental conditions. It proved easiest to suspend fraction III in distilled water leaving IgM in the precipitate; it is dissolved and the solution submitted to a slightly modified caprylic acid precipitation step. This fraction contains 35-40% IgM, few (2-6%) IgA and about 50% IgG whereas an IgA (35%) enriched fraction is obtained when fraction III is solubilized with acetate at pH 6.2 and then submitted to precipitation by caprylic acid under slightly modified conditions as compared with our standard IgGAM. Thus, simple modifications of the standard procedure allow to prepare fractions enriched more specially in IgM or IgA. Fractions poor or almost devoid of IgG can also be obtained...

Resume Le systeme du complement est constitue par 9 composants ou 11 proteines distinctes, dont l'interaction sequentielle comporte des phenomenes enzymatiques et engendre diverses activites biologiques. Parmi celles-ci l'activite anaphylatoxique, ainsi que l'activite chimiotactique, sont supportees par des fragments resultant du clivage des composants C3 ou C5. Le systeme des kinines comporte des substrats (kininogenes), des enzymes (kininogenases), des polypeptides vaso-actifs (kinines) et des inactivateurs (kininases). Les deux systemes presentent des ressemblances et des relations dans leur mecanisme d'activation et leur inhibition ; ils presentent egalement des relations avec le systeme de la coagulation.

Resume L'activite anti-Gma, anti-Gmb et anti-Gmx, presente dans certains serums d'individus normaux, est de nature euglobulinique et en relation avec les fractions macroglobuliniques des γ-globulines (β2M). Cette activite parait etre supportee soit par la β2M seule, soit par l'association β2M-γ-globulines. Elle existe independamment du facteur rhumatoi'de. L'activite anti-Gm n'a pas ete rencontree dans des serums presentant une macroglobulinemie pathologique. Summary The anti-Gma, anti-Gmb and anti-Gmx activity present in the serum of certain normal individuals is a function of the euglobulin fraction and is related to the macroglobulin fraction of the γ-globulins (β2M). This activity appears to be exercised either by the β2M fraction alone or in association with the γ-globulins. It occurs independently of the rheumatoid arthritis factor. No anti-Gma activity was found in sera containing pathological macroglobulins. Zusammenfassung Die in den Seren gewisser gesunder Individuen gefundene Anti-Gma-, Anti-Gmb-und Anti-Gmx-Aktivitat ist an die Euglobulinfraktion gebunden und steht in Beziehung zur Makroglobulinfraktion der γ-Globuline (β2M). Diese Aktivitat scheint entweder an die s2M-Globuline oder an einen β2M-γ-Globulinkomplex gebunden zu sein. Sie existiert unabhangig vom Rheumafaktor. Seren mit pathologischer Makroglobulinamie zeigten keine Anti-Gm-Aktivitaten.

Resume Les auteurs presentent les resultats d'une etude du comportement electrophoretique du plasminogene obtenu a un haut degre de purete par chromatographie d'affinite. Le proenzyme se revele homogene en ultracentrifugation et en immuno-electrophorese alors qu'une microheterogeneite a ete observee en electrophorese sur gel d'amidon ou d'acrylamide, provoquee par l'influence de ces differents milieux. Cette heterogeneite a ete observee avec des preparations mettant en evidence un seul acide amine « N terminalalors qu'avec des preparations legerement modifiees, on peut observer, avec les memes gels, une bande diffuse. En electrophorese sur papier, sur acetate de cellulose et sur gel d'agarose, le plasminogene migre en tant que globuline γ1(β2) et en gelose, sa mobilite est α. La variabilite du comportement electrophoretique du plasminogene est discutee. Pour l'expliquer, on met en evidence l'importance de la technique de purification, des milieux de support et de la presence de l'acide E -aminocaproique. Il faut donc, quand on doit analyser les resultats de l'immunoelectrophorese, prendre en consideration le fait que le plasminogene migre differemment en agar et en agarose.

Resume L'inuline etant un activateur puissant du systeme de la properdine, il a ete recherche si cette substance pouvait declencher in vitro la lyse des erythrocytes d'un malade atteint d'hemoglobinurie paroxystique (HNP). Lorsque des erythrocytes HNP, des quantites variables d'inuline et du serum humain frais sont incubes a 37°C, il se produit une hemolyse. L'hemolyse maxima, soit 25,5 %, a ete obtenue apres addition de 2.000 μg d'inuline. La lyse est nulle ou negligeable en l'absence soit de serum soit d'inuline. Les constatations faites in vitro suggerent que des polysaccharides ou lipopolysaccharides pourraient declencher l'hemolyse intra-vasculaire, soit par la voie classique soit par la voie de la properdine. Il parait probable que le systeme de la properdine et les composants tardifs du complement puissent participer au mecanisme de l'hemolyse intravasculaire et peut-etre des episodes thrombotiques observes chez ces malades.

Two devices capable of automatically performing titrations are described. Both control an electromagnetically operated burette tap from the output of a suitable electrode. The first device, which uses an electronic valve amplifier, is adaptable to any titration the end point of which can be determined potentiometrically. The second, which makes use of a sensitive relay, is only suitable where the electrode used has a low internal resistance.

Resume Apres un rappel des travaux demontrant l'activite anticomplementaire des globulines γG agregees par chauffage, les methodes actuelles de mesure de cette activite soni exposees. Il resulte des travaux de nombreux auteurs que la globuline γG 7S serait depourvue d'activite anticomplementaire, alors que les formes polymerisees de globuline γG (9–10 S; 20–40 S) seraient douees d'activite anticomplementaire (aussi bien in vitro qu'in vivo) en raison d'une dimerisation du fragment Fc. L'inactivation du complement par la globuline γG polymerisee se produit selon un mecanisme identique a celui de la fixation du complement par un immun-complexe. Les resutats d'une etude effectuee sur des proteines isolees au Centre National de Transfusion Sanguine sont rapportes : aucune activite anti-complementaire n'a ete decelee, meme apres chauffage a 56° C ou 63° C, sur les proteines suivantes : transferrine, haptoglobine, orosomucoide, globuline β1C, globuline γA, globuline γM (de maladie de Waldenstrom) α-, γ- et β-lipoproteines (conservees a 4° C). Certaines preparations de ceruloplasmine presentent une activite anticomplementaire. Des preparations de γ-globulines injectables par voie intraveineuse cnt ete obtenues au Centre National de Transfusion Sanguine sans alteration de la structure moleculaire. Depourvues d'activite anticomplementaire in vitro et in vivo (chez l'animal normal et l'homme normal), elles sont bien tolerees par les sujets hypogammaglobulinemiques. Le mode de preparation de la globuline γG peut influer sur l'apparition d'une activite anticomplementaire. Le trace d'ultracentrifugation ne concordant pas toujours avec l'existence d'une activite anticomplementaire, l'induction d'une activite anticomplementaire pourrait relever d'un phenomene plus subtil que la simple agregation moleculaire.

A technique is described for the precipitation of the bulk of the plasma proteins with caprylic (octanoic) acid without affecting IgG, ceruloplasmin, and part of the IgA. The contaminants can be removed from the supernatant solution by batch-adsorption on DEAE-cellulose and pure IgG is obtained in good yield. The technique has been used for human, horse, sheep, and rabbit plasma.

A homogeneous preparation of pure human ceruloplasmin was submitted to isoelectric focusing a pH range of 4–6. The properties of the separated subfractions as studied by electrophoresis, copper assay, adsorbance ratio A 610 nm / A 280 nm revealed more or less heavy molecular alteration produced during the isoelectric focusing process.

When purified homogenous α2M is submitted to SDS polyacrylamide electrophoresis splitting in half molecules of MW of 380.000 is observed. If increasing amounts of plasmin are added to a given amount of α2M, a new component of MW 450,000 increases whereas as the MW 380,000 component decreases. This new component consists of one half molecule of α2M and one plasmin molecule. In the presence of reducing agents and SDS the half molecules of α2M are further cleaved showing that the molecule is composed of 4 chains having a MW of 180,000. If α2M/plasmin complexes are submitted to the same treatment, a major component MW of 95,000 appears proportionally to the amount of added plasmin. As already shown by Harpel this component is a split product to the proteolytic action of plasmin. Other components of major interest correspond to the heavy chain of plasmin MW 50,000 and a component of MW 120,000 which represents the association of the MW 95,000 fragment of α2M and the light chain of plasmin (25,000). This assumption is materialized by the use of Unlabeled plasmin. Both the 450,000 component and the 120,000 component are revaled by auto-radiography. Thus a stable, eventually covalent linkage between α2M and plasmin can be assumed.

Inter-alpha-trypsin inhibitor was isolated from human plasma and submitted to proteolytic degradation by plasmin. A split product of low molecular weight (18 000 daltons) is obtained by gel filtration or solubilisation in perchloric acid. This fragment reacts with an anti-inter-alpha-trypsin inhibitor immune serum and migrates as beta1 globulins. Its specific activity against trypsin (after absorption of residual plasmin on sepharose lysine) was estimated to be 900 mU1/mg. Thus one molecule of fragment can inhibit one molecule of trypsin. As well with native protein as with its fragment, complexes formed with trypsin can be dissociated by urea or sodium dodecyl sulfate. This fragment is similar to the small molecular weight inhibitors obtained directly by solubilisation in perchloric acid from serum, urine and bronchial secretions.

Antibodies directed against viruses and bacteria are not equally distributed among the main classes of immunoglobulins, e.g. IgG, IgA and IgM. It has been found that IgM is mostly concerned with certain antibacterial activities (Salmonella, Escherichia coli and Pseudomonas) and IgA with high antibody titers for poliomyelitis virus I whereas antibody activities against many viruses such as influenza and measles virus occur preferentially in the IgG population. Furthermore, isolated immunoglobulin deficiency syndromes are actually well known. In the light of these findings, new concepts of immunotherapy have developed. Massive i.v. IgG-therapy is already widely used in congenital and acquired severe hypogammaglobulinemia. Preparations enriched in IgA and IgM are needed to complete the immunotherapeutical possibilities. Such a fraction called IgGAM has already been prepared in our Institute. Fraction III obtained during large scale fractionation is used as starting material and caprylic acid for the precipitation of most proteins other than the immunoglobulins present in fraction III. The immunoglobulin concentrate is finally obtained by ethanol precipitation of the caprylic acid supernatant. The present study is concerned with various modifications of the initial technique in order to obtain fractions more specially enriched in IgA or in IgM. In some cases the standard IgGAM fraction has been submitted to a further fractionation step, such as adsorption of IgG on DEAE-cellulose or precipitation of certain immunoglobulins achieved by Rivanol or by lowering the salt concentration. In other trials the fractionation procedure starting from fraction III has been modified. Rivanol has been used as a precipitating agent for the subfractionation of fraction III. It is well known that IgG is soluble in the presence of Rivanol. This technique was thus used in order to obtain preparations enriched mainly in IgM and IgA. The precipitate obtained after the addition of Rivanol was dissociated by NaCl and the solution further subfractionated by caprylic acid. In a similar way PEG was associated with the caprylic acid precipitation step. PEG precipitates proteins mainly in function of their molecular weight. However, the enrichment of IgM of the final fraction did not exceed 32% and much IgM was lost under the experimental conditions. It proved easiest to suspend fraction III in distilled water leaving IgM in the precipitate; it is dissolved and the solution submitted to a slightly modified caprylic acid precipitation step. This fraction contains 35-40% IgM, few (2-6%) IgA and about 50% IgG whereas an IgA (35%) enriched fraction is obtained when fraction III is solubilized with acetate at pH 6.2 and then submitted to precipitation by caprylic acid under slightly modified conditions as compared with our standard IgGAM. Thus, simple modifications of the standard procedure allow to prepare fractions enriched more specially in IgM or IgA. Fractions poor or almost devoid of IgG can also be obtained...

Proteolytic activities arise during the activation of several biological plasma systems. More than one proteinase is necessary for the whole reaction sequence of certain of these systems. Thus the clotting system (Fig. 1) shows four different proteolytic activities (factors XII, XI, X, and II) and at least 3 antiproteinases are involved in the inhibition of these enzymes. In the classical sequence of the complement system (Fig. 2) 4 different proteolytic activities have already been demonstrated: Clr, Cls, C3-convertase, and C3-dependent peptidase. Normal C3-convertase activity arises by the molecular association of C2a and C4b(C4, 2), whereas C3 dependent peptidase (C4, 2, 3) needs, in addition, the association of C3b. Only the first 2 enzymes are actually known to be specifically inhibited by a plasma inhibitor (Cl inactivator or C1 INA), whereas no plasma antiproteinase has been identified for the other enzymes. Indeed the protein known as C3b inactivator is itself a proteolytic enzyme cleaving C3b into two additional fragments (in the same way as does trypsin) C3c and C3d. Again no inhibiting antiproteinase is actually known. In the alternate pathway of complement activation two other proteolytic activities are found corresponding to C3 Proactivator (C3PA) convertase and the C3 activator (Fig. 2) but none of the known plasmatic antiproteinases seem to be involved although the absence of a plasmatic inhibitor of C 3 PA convertase has been described in certain pathological conditions.