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In this study, we have searched for an effective mucosal delivery system for a purified E. coli antigen which elicits anticolonization and anti-toxic immunity. E. coli colonization factor antigen (CFA/I) and heat-labile enterotoxin (LT) were encapsulated in liposomes. To determine the efficacies of soluble and liposome-encapsulated E. coli antigens young rabbits were mucosally treated with three oral doses of E. coli antigens given 7 days apart. Ten days after the last booster, rabbits were orally challenged with 5 x 10(9) bacterial cells (O78:H11 serotype). The experimental results allow of making some remarks which can be correlated with the protection obtained in vaccinated animals: (a) immunization with E. coli antigens entrapped in liposomes ensured protection against ETEC strains; (b) lower protection against homologous and heterologous CFA/I +(LT- ST+) strains were noticed; (c) adhesion of labelled -3H-leucine-bacteria to the intestinal mucosa revealed a maximum distribution in duodenum-jejunum and minimum in the colonic mucosa; (d) it contributed to the release of inoculated virulent bacteria from intestinal tract; (e) humoral, cellular and histopathological findings confirm the afore mentioned observation. Summing up, these results suggest that liposomes are very good carriers for E. coli antigens and these findings highlight the potential use of LT and CFA/I antigens entrapped in liposomes as mucosal and humoral induction of immune response and make them a candidate for future use in prophylaxis of diarrhoea in man.

In this study, we investigated the colonizing ability as well as the association of Yersinia enterocolitica serotype 0:9 to epithelial cells of the intestinal tract, Peyer's patches, mesenteric lymph nodes, liver, spleen and lungs in Alloxan-induced diabetes mellitus in mice and controls. The results showed that: (a) in diabetic mice the Y. enterocolitica colonizing values were in range of 10(6.5)-10(8.25) CFU/g of feces; (b) maximum colonizing values were found in distal ileum and Peyer's patches and lower in colon; (c) the infection was progressive with dissemination of bacteria in the liver, spleen and lung; (d) in control (non-diabetic) mice, the colonizing values were 10-100 times lower than those found in the diabetic batch; (e) the main histopathological changes noticed, namely ileitis, mesenteric lymphadenitis and septicemia, were presumably induced by high bacterial load in the liver, spleen and lung leading to a septic course of infection as well as toxic effects of heat-stable enterotoxins of Y. enterocolitica (Yst). The results were confirmed by electron microscopy observations. Summing up, these results demonstrate that diabetic mice were more susceptible to Y. enterocolitica cells than normal mice.

Photodynamic therapy (PDT) is a promising local treatment modality based on the selective accumulation of a photosensitizer in malignant tissues and the subsequent irradiation with laser light. Photodynamic therapy of malignant tumors includes biological, photochemical and photophysical processes. These processes involve: (a) absorption of photosensitizing agent; (b) selective retention of the photosensitizer in tumors and (c) irradiation of sensitized tumor by laser radiation. This report provides a review of photosensitizers, photochemistry, subcellular targets, side effects and laser involved in photodynamic therapy. In addition, gradual increase in knowledge related to in vitro and in vivo mechanisms of action of PDT, as well as some clinical applications of photodynamic therapy are presented.

Human larynx carcinoma cells (HEp2) were sensitized with different concentrations of Hematoporphyrin and irradiated with a He-Ne laser at different fluences. The degree of PDT-effects were estimated by two parameters: a) macromolecular synthesis and b) observations using electron microscopy. All experiments were evaluated after 68 hr at 37 degrees C. The results showed that PDT exposure of HEp2 cells is characterized by: 1) inhibition of macromolecular synthesis and 2) different cellular and subcellular lesions. Summing up, these studies indicate the existence of a strong correlation between different PDT exposures and the degree of biochemical and ultrastructural changes in human larynx carcinoma cells in vitro.

Two immunotoxins containing ricin A-chain, staphylococcal protein A and mouse anti-Thy 1.2 antibodies of IgG(2a,b) subclass, were prepared. The two multivalent immunotoxins of 750 and 370 kDa and molar ratio A-chain: IgG of 1:2, were used for the treatment of mice bearing ascitic EL4 lymphoma. The immunotoxin-treatment, performed intraperitoneally, was combined or not with homologous or heterologous interleukin 2. The antitumor effects expressed by increase of mice survival time (p < 0.001 as compared with nontreated animals) corresponded to a proportion of 88-90% lymphoma cell-kill by immunotoxin and 93-95% by combination treatment (immunotoxin + interleukin 2), as calculated from the relationship between the survival time of nontreated mice and the number of tumor cells inoculated.

Two multivalent immunotoxins (ITs) with cytotoxic potential against Thy 1.2-expressing tumor cells were used in association with mouse interleukin 2 (IL2) for treatment of mice bearing ascitic EL4 lymphomas. The combined treatment, ITs + IL2, induced an enhanced antitumor effect revealed by a significant prolongation of the survival time of mice as compared to the simple treatment with ITs or IL2 alone. According to the survival of mice treated by combined therapy, the proportion of killed tumor cells rose up to 94% as resulted from the dose-dependent curve of the survival of nontreated mice versus the number of tumor cells inoculated.

Effects of pulsed near-ultraviolet laser beam on structural characteristics and macromolecular synthesis of carcinoma HEp2 cells were investigated. Laser irradiation damage induced in these eukaryotic cells could be characterized by two development stages: a) a reversible stage with minor morphological damages (1.5 kJ/m2) and 2) an irreversible one, at higher fluences, characterized by cellular membrane damage, necrobiosis and cells detachment from the substrate (4.5 kJ/m2). A. Studies performed referring to macromolecular syntheses of low laser fluences (1.5 kJ/m2)--irradiated HEp2 cells showed the following aspects: a) syntheses inhibiton phase in the first cycles of cellular replication and b) syntheses stimulation phases in the following cycle with total repair of laser-induced molecular lesions. B. At high laser fluences (3-4.5 kJ/m2), metabolic lesions repair was partially or totally blocked after prolonged culturing at 37 degrees C. Ths paper suggests some mechanisms of laser action on macromolecular synthesis and correlates them with morphological changes induced by laser exposure of carcinoma cells.

CFA/I antigen was isolated and purified from E. coli, mutant 279 B-1-14, serotype 0128:H12, and had the following biochemical and biological features: a) amino-acid content was similar to that of purified antigen prepared from strain H10407; b) latex particles sensitization with purified CFA/I antigen produced bovine and human erythrocytes group A/II hemagglutination in carbohydrates presence; c) purified anti-CFA/I specific antibodies agglutinated CFA/I-positive enterotoxigenic E. coli strains; d) 3H-leucine-labelled CFA/I antigen adhered to rabbits intestinal mucosa at significant values; e) intestinal mucosa pretreating with purified CFA/I antigen, followed by 3H-leucine labelled enterotoxigenic bacteria infection, had a least 3 local effects: 1) intestinal mucosa protection against parental enterotoxigenic bacteria; 2) inhibition of CFA/I-positive bacteria adherence to intestinal mucosa; 3) release of approximately 96% intraluminally inoculated bacteria.

The interactions between HeLa S3 tumoral cells and human fibroblasts after nitrogen-laser irradiation (337.1 nm) have been studied by using an in vitro cell invasion model. For the quantitative and morphological evaluation of nitrogen-laser radiation action upon tumoral adhesion to the fibroblast monostrate, we used: a) 3H-thymidine labelling of HeLa S3 tumoral cells; b) morphological modifications studies by phase contrast and scanning electron microscopy. The results emphasized the following aspects: 1. In non-irradiated cell cultures we noticed three interaction stages: adhesion, tumoral spreading and displacement with fibroblasts destruction; on the other side, we found a reduced adhesion to non-irradiated human fibroblasts of laser irradiated tumoral cells. 2. Significant percent increasing of non-irradiated tumoral cells adhesion to fibroblast monostrate, irradiated with various laser fluences (e.g. 0.2 kJ/m2--48.1%; 0.8 kJ/m2--63.8% and for 1.6 kJ/m2--79.5%). This phenomenon evidenced the close interrelation between irradiation fluences and tumoral adhesion rates. 3. The importance of numerical ratio between tumoral cells and fibroblasts in tumoral adhesion and invasion processes (e.g. ratio 1:10 tumoral adhesion reached 8.1%; in 1:5--25.9%; in 1:1--59.4% and for 2:1--83.9%). 4. Marked cytotoxic effects for both cell types after exposure to high and very high laser fluences (1.6--6.4 kJ/m2). Our results emphasize near UV-laser irradiation effects upon some of tumoral adhesion and invasion mechanisms and demonstrate the interrelations between cell populations manifesting a different vital potential.

HeLa S3 tumoral cells were used as an experimental model for studying the association of photodynamic therapy (PDT) and antitumoral agents. Tumoral monolayer cultures were incubated 18 hours at 37 degrees C with Photofrin II, trypsinized and suspended in Eagle medium supplemented with 10% FCS and then treated with antitumoral agents 90 minutes before He-Ne laser exposure. The tumoral cells were exposed to antitumoral agents in the following concentrations (equivalent to ED70): adriamycin (0.0297 micrograms); mitomycin C (0.0199 micrograms); 5-FU (0.4937 micrograms) and vinblastine (0.0109 micrograms) per 10(5) cells. Macromolecular syntheses (DNA, RNA and proteins) were investigated by use of radioactive precursors: 3H-thymidine, 3H-uridine and 3H-leucine, as expressed in percent referring to Photofrin II-pretreated controls; they were exposed to He-Ne laser but not treated with antitumoral agents. All experiments were followed for 72 hours incubation at 37 degrees C. The conclusions of the results of PDT associated with antitumoral agents sustain the following aspects: a) the antitumoral agents activity (adriamycin, mitomycin C, 5-FU, vinblastine) was more noticeable when applied 90 minutes before He-Ne laser irradiation; b) inhibition of radioactive precursors uptake in DNA, RNA and proteins was accompanied by suppression of in vitro tumoral cells development and c) PDT association with antitumoral agents could manifest at least three positive effects upon animals; 1) PDT potentiating effects with antitumoral agents; 2) suppressing effects on tumoral macromolecular synthesis; 3) antitumoral agents cytotoxic elimination (due to the low doses used).

In our case, where the difference between donor WHT/Ht(H--2d) and recipient C57B1/6 (H--2b) was at the H--2 locus of histocompatibility, the FSg rejection occurred between the 6th and the 10th day, while the SSg rejection was two days earlier. The morphological study emphasized that the cellular infiltrate in the FSg is predominantly lymphocytic, while in the SSg it is predominantly granulocytic. The vascularization settles on the second day postgrafting in the FSg as well as in the SSg, but is scarce and quickly destroyed in the SSg, thus being explained the scanty supply of lymphocytes in the latter graft type. The histochemical reactions demonstrated an increase of the NMPS in skin allograft from the moment of settling till the end of the rejection and a decrease of AMPS, till the complete disappearance, in the rejected graft. Little morphological differences were found between colateral draining nodes and contralateral ones during the reaction to the graft. An important feature described is the presence at the bed graft level of some cells morphologically identical to the IB encountered in great number in the TDA of lymph nodes in the allograft-bearing mice.

Normal macrophages and Salmonella typhi ribosomal antigen-activated (immune) ones were studied by electron microscopy. Examination of fine sections from samples incubated between 30 and 120 minutes at 37 degrees C, emphasized phagocytic ability of activated macrophages (29-69% activated macrophages phagocytized 3.7-5.6 bacteria/cell, in contrast to 23-43% with 2.6-4.4 bacteria/phagocytized by non-immune cells). The ultrastructural studies of activated macrophages, performed by electron microscopy, showed the following aspects: a) macrophages with pseudopodiform prolongations, presenting the tendency of bacterial sequestration: b) phagosomes with ingested virulent germs; c). bacteria presenting various degrees of wall and cytoplasm alterations; d). mitochondria with multiple cristae; e). cells with well developed Golgi apparatus and the presence of lysosomes in great numbers; f). existence of a space between the phagosome membrane and the bacterial wall. Experimental results demonstrated: I. an increased phagocytic activity of Salmonella typhi ribosomal antigen-activated macrophages and II. activation expressed as ultrastructural modifications at the level of immune macrophages and also of the bacteria phagocytized by them.

Experimental results of the present study evidenced the following aspects: a) the antigen, prepared from type I Escherichia coli purified fimbriae (H. 2946 strain), induced immunity at urinary tract level; b) the immunoprotection induced by oral vaccination with multiple doses of fimbriated antigen produced a significant decrease of acute pyelonephritis in newborn guinea pigs and at the same time, a local protection of the urinary tract; c) the immunoprophylaxis by vaccine prepared from fimbriae represents a preferential solution for urinary tract infections prevention in general and especially in children; d) the frequency distribution differences between "protected" and "non-protected" animals were evaluated by chi-square--test with YATES correction and proved to be statistically significant at probability levels.

The antitumor effect of two purified preparations of protein A obtained from Staphylococcus aureus strain Cowan 1 or the mutant strain A676 was investigated. This study using different doses of SpA (5 or 10 mg/animal) i.p. administered was performed in lymphosarcoma bearing rats at certain intervals of time after tumor transplantation. The treatment with SpA deriving from A676 strain which was started at the same time with the tumor transplantation led to a significant increase in the latency period of tumor growth and the prolongation of survival time of tumor bearing rats. Attempts to use a higher dose of SpA and also a smaller tumor inoculum did not improve the results with regard to inhibition of lymphosarcoma growth in inbred R rats.

A syndrome similar to GVHR is described in mice of C57B1/6 strain during the WHT/Ht skin allografts rejection period. In all the cases the described thymus involution was associated with the hypertrophy of lymph nodes. Their volume increase is due to the high number of blastic pyroninophilic and plasma cells concomitant with small lymphocytes depletion in the cortical area, and with a very pronounced hypertrophy of medullary cords by presence of a high number of plasmocytes and blastic cells. These changes have been noticed only in some animals sacrified during the first days after grafting and never later one. In agreement with the scarce data of the literature, we think that the immunocompetent passenger lymphocyte comprised in the skin grafts constitute an immunologic organ able to induce a GVHR at the beginning of the period of graft survival on the host. This GVHR, generally mild and without clinical and microscopic signs, becomes obvious only in animals which, for unknown reasons, present a low immunologic defense capacity. In these animals the described process seems to be reversible.

The monovalent V-1 fragment of protein A (fSpA) with a mol. wt of 13,000 obtained from an u.v. mutant of Staphylococcus aureus Cowan I strain was proved to be able to modulate significantly some of the effector functions of IgG, such as complement fixation, catabolism, attachment to Fc receptors and antibody-dependent cell-mediated cytotoxicity. Moreover fSpA-like protein A obtained from the A676 strain is mitogenic and enhances NK activity of human peripheral lymphocytes. The efficiency of fSpA was found to be lower than that of protein A with regard to its ability to inhibit complement fixation, EA rosette formation and antibody-dependent cell-mediated cytotoxicity. Both protein A and fSpA had the same efficiency in activation of the complement system after reaction with human or guinea pig IgG, and in increasing the IgG catabolism. Unlike fSpA the monovalent B fragment of protein A (with mol. wt of 7000) was not able to inhibit EA rosette formation and antibody-dependent cell-mediated cytotoxicity. The results recommend fSpA, substituting for protein A, as a molecular probe for the investigation of IgG antibody and lymphocyte effector functions.

Radiolabeled protein A from Staphylococcus aureus (SpA) injected i.v. into mice and rabbits forms a soluble [(IgG)2-(SpA)1]2 complex (Mr = 684 000) which is identical in composition to that formed by SpA in vitro with an equivalent amount or an excess of IgG. A soluble rabbit IgG-SpA complex injected into a mice or rabbits dissociates completely in vivo and a new complex is formed with the IgG of the recipient animal. The half-life of SpA administered to a mouse or a rabbit is therefore the half-life of the IgG-SpA complex formed in vivo. In mice and rabbits the half-life of the complexes formed is 9 and 30 h, respectively, whereas the half-life of rabbit IgG in these animals is 106 and 153 h, respectively. Fragment B of SpA (fSpA) reacts with IgG of mouse and rabbit and forms an (IgG)1-(fSpA)1 complex. Complexes of identical composition are formed if fSpA is injected i.v. into mice and rabbits. The half-life of the complexes in mice and rabbits are much shorter than those of the corresponding free IgG in these animals (up to 15 times). This result suggests that the binding of fSpA to the CH2 and the CH3 domains of IgG alters the function of the site, which controls the catabolism of IgG and is located in the CH2 domain. By contrast, fSpA does not change the Fc receptor-binding site of IgG, indicating that the Fc receptor site and the catabolic site are unrelated to each other.