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The study described in this paper was conducted in the framework of the European nPSize project (EMPIR program) with the main objective of proposing new reference certified nanomaterials for the market in order to improve the reliability and traceability of nanoparticle size measurements. For this purpose, bimodal populations as well as complexly shaped nanoparticles (bipyramids, cubes, and rods) were synthesized. An inter-laboratory comparison was organized for comparing the size measurements of the selected nanoparticle samples performed with electron microscopy (TEM, SEM, and TSEM), scanning probe microscopy (AFM), or small-angle X-ray scattering (SAXS). The results demonstrate good consistency of the measured size by the different techniques in cases where special care was taken for sample preparation, instrument calibration, and the clear definition of the measurand. For each characterization method, the calibration process is described and a semi-quantitative table grouping the main error sources is proposed for estimating the uncertainties associated with the measurements. Regarding microscopy-based techniques applied to complexly shaped nanoparticles, data dispersion can be observed when the size measurements are affected by the orientation of the nanoparticles on the substrate. For the most complex materials, hybrid approaches combining several complementary techniques were tested, with the outcome being that the reliability of the size results was improved.

The immunomodulator OM-89 (bacterial extract from E. coli), known to act on the immune system by modulating both humoral and cellular responses, significantly increases ACTH and glucocorticoids secretion in normal Wistar rats. A comparative administration of IL-1 displays a similar pattern of release. Moreover, OM-89-induced responses are blocked by IL-1ra, the soluble receptor antagonist of IL-1. The effect of OM-89 can thus be explained, at least in part, via IL-1 which directly enhances the secretion of both stress hormones. Finally, a comparative experiment between control and stressed rats (by immobilization) shows that the responses to OM-89 measured in this study (ACTH and corticosterone secretion) are stress-modulated.

The fate of orally administered total 14C-labelled Escherichia coli extract (TEC, OM-89) was compared to that of its major 14C-labeled high molecular weight fractions (HEC, > 30 kD) in mice. High molecular weight substances (> 30 kD) were observed in blood 1 h after oral administration of the products, TEC and HEC. This suggests absorption from the digestive tract of the total E. coli extract. Blood clearance after 24 h indicates that the product was rapidly eliminated or taken up by the tissues. Its highest organ distribution among the measured organs was found in the liver, followed by the spleen and the Peyer's patches. Low molecular weight fractions (LEC, < 30 kD) were also used as control. This pilot study provides a rationale for in vivo pharmacological investigations with oral administration of the E. coli immunomodulating extract OM-89.

The activity of increasing oral doses of the bacterial extract OM-85 was determined by using the plaque-forming cells assay in mice. The results showed that the oral administration of OM-85 significantly increased the number of splenic antibody-secreting cells against sheep red blood cells (antigen) compared with controls. A maximal response was reached between 400 and 800 mg/kg body weight, and was reproducible with the different batches of OM-85 used. This study indicates that the oral administration of the immunomodulator allows the activation of splenic B-lymphocytes, probably through the gut-associated lymphoid tissue.

The action of diacetoxyscirpenol (DAS)--a mycotoxin that belongs to the family of trichothecenes--on the immune system of the mouse was investigated. Two experimental models were used: 1) bacterial infection with Salmonella typhimurium and 2) the PFC (plaque-forming cells) test on the splenic lymphocytes of the mouse. The results obtained showed that these were dependent on the chronological order of the administration of DAS. When the toxin was administered after a bacterial infection or antigenic stimulation, it was observed an increase in the mortality rate and a very significant decrease in the antibody response. By contrast, when this mycotoxin was administered before the bacterial infection or antigenic stimulation, the results obtained were close to those from the controls. The importance of the administration of an immunomodulator of bacterial origin OM-89 before the immunodepression with DAS was shown.

Although substances known as immunodulators are usually used to stimulate nonspecific immunity, their mode of action is not well understood. In an effort to clarify this mechanism, we investigated the effect of a lyophilized bacterial extract (Broncho-Vaxom) on experimental infections, on normal or irradiated mice, and on antigen processing.

Morphometric study of mouse bronchus-associated lymphoid tissue (BALT) using an image analyser and histological sections at close and regular intervals was carried out in lungs of control and influenza infected mice two to three months old. The mean area of BALT did not reach 0,1 p. 100 of the whole mean area of examined lung sections in control mice; this percentage was significantly greater one month after primary exposure to influenza virus.

Infection of Mice with S. typhimurium, was conducted by respiratory, oral and peritoneal routes. The results obtained showed that infection occurred more quickly by i. p. than by other routes. However i. n. inoculation is the more efficient concerning Bacteria concentration per Mousse to obtain the DL 100. A study of Salmonella growth in vivo was conducted in lungs, spleen, liver and blood. The results are discussed in relation to the calf respiratory Salmonellosis actually observed.

Nonspecific protection against infectious aerosols of influenza A virus was obtained in Swiss mice after vaccination by aerosols of bacterial ribosomes together with membranal glycoproteins extracted from Klebsiella pneumoniae as the adjuvant. It was shown that repeated stimulant aerosols were necessary to obtain this protection. Routine estimation of serum interferon levels after administration of the association of ribosomes plus membranal glycoproteins to the animals by aerosol or intravenous route showed that there was no correlation between protection and the presence of serum interferon. It was shown that the serum interferon-inducer activity was due to ribosomes. No induction of serum interferon was obtained with membranal glycoproteins used separately. Local liberation of interferon in the mucous membrane of the upper respiratory tract was not investigated.

In a previous chapter, the importance of macrophages in inflammatory processes was emphasized upon. The authors now mention the outstanding inflammatory agents utilized for experimental purposes, especially as regards the pattern of their action. Finally, this paper restates the question of the pharmacology and therapy in inflammation.

A blood investigation was carried out on 3,030 volunteer blood donors and the results treated on a computer by multivariant analysis. The authors noted a depressor effect of the urban environment on the red and white cells, a rise in small lymphocytes and a reduction in monocytes in patients living in industrial areas. This stimulation of the lymphocyte system may be compared with the experimental results obtained by the same authors in animals.

Various immunosuppressive treatments were used in order to induce a Salmonella typhi infection in mouse by oral route. Neither low protein diets nor administration of mucin, iron, streptomycin or silica could modify animal's sensitiveness, but, cyclophosphamide injections enhanced lethal effect of bacteria, while silica increased the number of bacteria in the blood and the spleen.

The immune response to Vibrio cholerae was studied in baboons, rhesus monkeys and marmosets inoculated by the oral, intraintestinal or intravenous route with various strains of V. cholerae. Sera were collected from all animals on days 2, 6, 10, 17, 24, 32, 45 and 60 after infection. Serum protein, immunoglobulins (IgA, IgG, IgM), GOT, and alkaline phosphatases were determined. The results showed that baboons and rhesus monkeys were not susceptible to cholera infection under these experimental conditions. However, we were able to induce a lethal cholera infection in cotton-topped marmosets. In baboons and rhesus monkeys serum IgG levels decreased significantly following inoculation with V. cholerae; however, the ratio of GOT, alkaline phosphatase and antibodies against toxin were only slightly modified.

A new process in taxinomic analysis clearly confirmed the presence of three different types of cells (epithelial, hypertrophic and mesenchymal) in the reticular zone of the thymus. The injection of hydrocortisol as well as adrenalectomy induced specific changes in nuclear surfaces of three cell types, while hydrocortisol tented to increase the surface of epithelial and mesenchymal cells, adrenalectomy reduced it, while a modification was seen in the percentages relative of the cell types. In adrenalectomized animals, the effect result was counteracted by injections of hydrocortisol.

As part of a general study of the biological activity of atmospheric micro constituants, we have perfected the methods for the rapid determination of polycyclic aromatic hydrocarbons. The method involves the use of two selective collectors, a high output rotatory freeze-drier, two-dimensional thin-layer chromatography and gas chromatography. A pollution map has been prepared of the Lyons areas showing the atmospheric concentrations of 13 polycyclic aromatic hydrocarbons and their variation with weather and sampling point.

We studied the effect of neuraminidase injection in rat's tumor at different doses: 5,10,50,100, 500 U and we concluded that: There was no difference between the rats treated with 5,10,50 U and the controls. The y died 3 weeks after the injection. But the rats treated by 100 at 500 U of NA died quickley, in the week, of long metastases.

In prelminary studies, growth of diploid cells (fetal calf buccal cells and chick embryo fibroblasts on dextran microcarriers was studied and compared to growth in monolayer. Growth coefficients obtained were always greater (by 2 fold) when microcarriers were used. Moreover, this technique had the advantage of reducing the number of manipulations and hence the risk of contamination. In two series of experiments, buccal epithelial cells were infected with vescicular stomatitis virus. Good virus multiplication was obtained.

The variations in anti-Salmonella typhimurium antibody levels was measured in BALB/c mice which had been orally and nasally vaccinated with S. typhimurium live mutants of different lipopolysaccharide lengths. Together with the demonstration of immunological memory, the intensity of the secondary response was correlated with the length of the polysaccharide chain at intestinal and serum levels. Nasally administered antigen seemed to be at least as immunogenic as when orally administered.

The efficiency of intranasal subcutaneous, aerosol and digestive vaccinations with killed Klebsiella pneumoniae as vaccine was studied on the survival curves after experimental disease with pathogen K. pneumoniae I inoculated by the same routes. Results show that the local immunization is an important factor of protection and the intranasal route is very convenient.

One of the earlier effects of poliovirus was the maintaining of a high level of Hela cells 5'-nucleotidase activity (maximal efficiency for a virus/cell ratio = 500 ID 50/cell) (ID50:50% Infections Dose). The activity decreased when cells were suspended in the presence of EDTA(EDTA:Ethylene Diamine Tetracetic Acid) and was partially restored by adding 10 mM Mg++ interactions are discussed.

The concentration of ACTH, corticosterone, LH and testosterone in the plasma of rats have been measured in normal, thymectomized and sham-operated rats. A group of rats, thymectomized at birth, received two i.p. injections weekly of two thymic extracts, each prepared in a separate way. Neonatal thymectomy was followed by the appearance of a transitory wasting disease during which a hormonal unbalance was manifest. Plasma corticosterone and ACTH levels increased at first at 30 days after thymectomy and then decreased at 60 days. The levels of the different hormones returned to normal by 90 days. The injections of thymic extracts rectified all these perturbations. The plasma levels of testosterone and corticosterone of hypophysectomized rats were compared to those of hypophysectomized and thymectomized animals. Results suggested that the thymus has a direct action of the adrenal cortex and in indirect action on the testes. The indirect effect is probably mediated through the hypophysis.

Isolation of pure neuraminidase and hemagglutinin from influenza virus A/Hong Kong 1/68 (H3N2) can be achieved using the coupled action of a new carboxypolypeptidase from Streptomyces fradiae and sodium dodecyl sulfate (SDS). Biochemical, physical and physico-chemical studies showed that the two molecules are in pure form and of glycoproteic nature.

Biological effects of microwaves (2450 MHz) were studied on cellular immune response of Swiss mouse. The obtained results show an increase of MIF, PFC and Phagocytosis values after microwaves irradiation.

Hepatitis A virus (HAV), obtained from human feces collected 3 days after the onset of jaundice, was propagated in human hepatocarcinoma cells (PLC/PRF/5). In the course of 5 passages, it was released into the supernatant without cytopathic effect. Released HAV was propagated after inoculation into PLC/PRF/5 cells. Its biophysical properties (buoyant density in CsCl: 1, 33 g/cm3; sedimentation coefficient: 155 S) were similar to those of the infectious particle present in the cells or in the stools of patients.

Pregnant Mice were stimulated with two substances, an antibronchitic lysate (LAB) and diethyldithiocarbamate (DTC). PFC response was studied in mothers and their offspring for 6 weeks after birth. An increase in PFC response was noted in the immunostimulated adult Mice. In the newborn Mice, the number of PFC increased between the 1st and 6th week. In comparison with controls, PFC response appeared later in the newborns to LAB-stimulated mothers and earlier in those to DTC-stimulated mothers.

We describe in this report the vaccinating power of a polysaccharidic preparation from Klebsiella pneumoniae type I. The essential part played by this extract in the vaccinating capacities of the ribosomal preparations from the same bacterium is demonstrated.

Experimental studies on cholera in Baboons (Papio, P. anubis) and Rhesus Monkeys, have led to unfavourable results. However we have obtained a typical cholera disease in cotton-top Marmoset Saguines oedipeus.

Eleven calves, 6 months old, vaccinated or not, have been infected experimentally with 10(7) Salmonella typhi-murium, administered by oral route. The control calves had a serious illness, characterized by a severe diarrhoea, hepatic and renal symptoms and a heavy infestation of the main organs. The other five calves, which were orally and subcutaneously vaccinated with ribosomal extracts of S. typhi-murium and S. dublin showed only a moderate alteration of their health while biochemical disorders at the level of liver and kidneys disappeared. However, salmonella were found in mesenteric lymph nodes, but in much lower amounts than in controls.

The effect of a phlogogenic agent and of six sulphur compounds on peritoneal macrophages, spleen B lymphocytes, and von Kupffer cells of the liver has been observed in BALB/C mice. The phlogogenic agent (0.5% carrageenin + 10% peptone) was a potent stimulant of the macrophages (increasing their number, protein content, and acid phosphatase activity), the lymphocytes (increase of haemolytic plaque counts), and the von Kupffer cells (increase of the colloidal carbon clearance rate which measures phagocytic capacity). Of four acid sulphur compounds (benzenesulphonates)-ethamsylate, diethylammonium persilate, calcium dobesilate, and piperazine sultosilate--not previously screened for antiinflammatory activity, all except piperazine sultosilate inhibited the macrophages, all except calcium dobesilate stimulated the lymphocytes, and only calcium dobesilate stimulated the von Kupffer cells. Two non-acid sulphur compounds--levamisole and pyritinol hydrochloride--had no effect on the macrophages but were potent stimulants of the lymphocytes and the von Kupffer cells. Because of these immunostimulant properties, levamisole and pyritinol hydrochloride may prove useful in the treatment of diseases such as rheumatoid arthritis in which the patient's immune potential has to be restored.

Sodium hypochlorite was used to suppress diacetoxyscirpenol cutaneous toxicity; mice skin can be decontaminated with "Eau de Javel" (1 p. 100 free chlorine) if application is performed less than 15 min. after contact with toxin.

The authors reviewed the data about inflammatory diseases acquired in the last few years. They have studied clinical aspects of inflammatory reaction, the various mediators of inflammation and physiological anti-inflammatory molecules.

Sera and bronchial washings from normal mice and from mice previously infected with influenza virus were analyzed for their concentration of four immunoglobulin (Ig) classes by the method of single radial immunodiffusion and for their content of specific antibody of these Ig classes by the immunofluorescent method. IgA were not detected in bronchial washings from normal mice. The IgA/IgG ratio was not higher than 0.33 in infected mice. The IgA level increased briefly in infected bronchial washings, but the levels of IgG2 and particularly of IgG1 exhibited a longer and higher increase. The maximal increase of these three Ig classes occurred by the seventh day and might have derived from transsudated serum. IgM was not identified in normal bronchial washings, but immunofluorescence detected IgM in infected washings, which also showed specific antibody in each of the four Ig classes. IgG2 and IgM contained antibody in greatest, IgA in lower concentration. Our results indicate that the most part of bronchial antibody was produced locally, since the ratio of Ig concentration to antibody titre of each Ig class was consistently higher for bronchial washings than for sera. An anamnestic secretory antibody response could not be demonstrated in bronchial washings.

131I labelled tetanus anatoxin was placed in vivo, in the sublingual area in the rat. The radioactivity appearing in blood was insignificant, even after that the disappearance rate in reference animal had been taken into account. It is concluded that immunisation with aerosols is fundamentally carried out through the respiratory tract.

A study of peritoneal macrophage activation has been conducted by simultaneous investigation in eleven enzymes in Balb/c Mice stimulated with bacterial extracts, by oral and parenteral route. The results showed important changes in Macrophage number and activity. On the other hand this activation depended upon the route of administration, antigen nature, and time interval between stimulation and peritoneal harvesting.

The neutral and aminosugar composition has been determined by gas-liquid chromatography for two neuraminidases, either bacterial, from Streptococcus pneumoniae, or viral, from Myxovirus influenzae A/Hong Kong/1/68.

The of efficiency of spraying as compared to intranasal and digestive ways was tested on Balb/c mouse by using on the one hand an immunostimulant product and on the other various types of test : Myxovirus influenzae by aerosol, Streptococcus pyogenes intranasally, Klebsiella pneumoniae intraperitoneally. Results showed that in the case of influenza virus the immunostimulant inhaled as liquid aerosol or powder aerosol, or deposited on the nasal mucosa was very active. When the test germ was Streptococcus and the contamination intra-nasal, only the homologous way was efficient. When the test germ was Klebsiella pneumonia all inoculation forms except powder aerosol were effective. The comparative study of aerosols granulometry helps grasping this phenomenon.

Antigenicity and immunogenicity of three influenza virus strains A/PR/8/34 (H1N1). A/Hong Kong/1/68 (H3N2) and A/Port Chalmers/1/73 (H-3H2) were assayed comparatively with their corresponding neuraminidase isolated by proteolysis, and with the recombinant virus X-42 (Heq1 N2). It was concluded that intranasal immunization of mice with live virus induced heterologous immunity. Except in homologous neuraminidase-vaccinated mice, the subunit always was shown less effective and demonstrated a significantly lower antibody response than the corresponding whole virus.

The study of the vaccinating power of ribosomal vaccines against Klebsiella pneumoniae led us to define the chemical nature which supports this protective activity. We tried to separate this support and the ribosomes by sucrose gradient ultracentrifugation. We isolated high protective membrane vesicles by this technique applied to salt-washed ribosomal preparations. When the ribosomal preparations were exposed to SDS, the protective activity was conserved all along the gradient, with no correlation with the ribosome concentration. The addition of bovine serum albumin to the ribosomal preparation focused the protective activity on the ribosomal peak. No correlation was observed between the response to capsular polysaccharide and the vaccinating power of the fractions.

The conditioned, passive hemagglutination method, which is simple, quick, economical and very sensitive, is suited for large-scale studies in which it is not possible to carry out the in vivo seroneutralization. However, our experience has confirmed that it has certain drawbacks and restrictions: (1) the technique must be applied very carefully and good laboratory training is required. The results obtained by this method vary according to several factors, especially the degree to which the antigen has been purified and its blood carriers, therefore these results are not always consistent; (2) the antibodies found were not a good indication of the degree of protection except at high titers. A study of correlations between HA titers and neutralizing (mice) titers carried out on the basis of 509 double titrations has demonstrated that, whereas they are satisfactory in clearly immune subjects, they show only mediocre results in subjects who are receptive or who are displaying a primary response: a wide range of variation has been observed as well as an optimalization of HA titers, perhaps because of IgM's. Therefore, the HA method, despite its usefulness, cannot provide precise evaluations for: --the amount of protection provided by an anti-tetanus vaccine, --the proportion of protected subjects in a certain group of people, --an injured person's anti-tetanus immunity. We should work to develop in vitro tests which are both sensitive and reliable in terms of the anti-tetanus protection threshold.

Ribosomes, ribosomal RNA (r-RNA), capsular polysaccharide (PS-K) and lipopolysaccharide (LPS) were isolated and purified from Klebsiella pneumoniae, type I. The protective capacity of these different fractions was investigated in function of their analytical composition. The results show that ribosomes, and in particular, ribosomal RNA have the greatest protective activity at the lowest concentrations. The role of PS-K in the specific immune response to ribosomes was also investigated. Even at very high levels PS-K failed to afford any significant protection. LPS gave no protection. A stable r-RNA/PS-K complex was isolated by means of affinity chromatography. This complex was uncontaminated by LPS and its existence indicated strong bonding between surface polysaccharide antigens and r-RNA. The r-RNA/PS-K complex was not antigenic in itself but conferred good protection when combined with an adjuvant.

A statistical method using the chi 2 distribution of variable was studied for survival data about experimental disease on S. typhimurium C5S infected mice via oral administration, and about vaccinal protection with S. typhimurium mutants. Our results showed that Ra Rc and Re S. typhimurium mutants were good experimental vaccines.

The trace elements are surrounding factors which are able to act on the yield of benzo(a)pyrene hydroxylation reaction of the hepatic tissue. This action was studied for each element and for various concentrations. The action of these elements may occurred to arylhydrocarbon hydroxylase by activating or inhibiting it, to epoxyde hydrase or to glutathione S epoxydase, favouring or inhibiting the way epoxyde dihydrodiol, and finally to arylhydrocarbon hydroxylase induction. Our results, obtained in vitro on hepatic tissue with many metallic salts, showed that some trace elements may have a cocarcinogenic action.