Use of a diploid cell line for detecting the toxic components in medical immunobiological preparations

Besides specific antigens medical immunobiological agents (MIBA) contain chemical compounds (formaldehyde, aluminium hydroxide and mercury salt, merthiolate) in permissible concentrations. Therefore, the investigation of MIBA and their components should involve methods studying the effect of chemical compounds on cells and their structural components. For this purpose WHO recommends to use cell cultures. The results obtained show that cell cultures (constant and diploid lines) allow the differentiation in the degree of toxicity of chemical compounds constituting MIBA. Merthiolate had the strongest irreversible lethal effect. The technique can prove useful for more accurate evaluation of toxicity in inactivated bacterial and viral vaccines as well as in serum preparations. Cell culture can be successfully used for the detection of toxic components in vaccines and serum drugs, with the final safety tested by their injection to animals.     

Argasid tick lysozyme action on HEp-2 cells

The cytotoxic effect on Argasidae lysozyme was shown on a model of cell line HEp-2 in comparison to egg lysozyme. The lysozyme was obtained from homogenates of Ornithodoros papillipes of subfamily Ikodoidea. The lysozyme in concentrations of 300 and 500 gamma/ml had a cytotoxic effect, while in doses of 50 and 100 gamma/ml it has no such activity. The cells of line FL were not sensitive to the above concentrations of the lysozyme. In concentrations of 500 gamma/ml and higher the egg lysozyme had an analogous cytotoxic effect on the cells of line Hep-2. The comparative study of the cariogrammes of the cell monolayer treated with the Argasidae and egg lysozymes, as well as the study of the level of 3H-thimidine incorporation into the cell DNA showed the absence of the preparations effect of the mitotic activity of the cells and DNA synthesis by them.     

In vitro cytotoxicity of lipopolysaccharides for chinese hamster ovary cells

Abstract From our survey of various lipopolysaccharide (LPS) preparations, we demonstrated that three out of five commercial LPS preparations of Salmonella typhimurium were not cytotoxic for Chinese hamster ovary (CHO) cell monolayers at a concentration of 1000 μg/ml. One commercial LPS preparation produced cellular damage at a concentration of 1000 μg/ml and another at 400 μg/ml. Two S. typhimurium LPS preparations made in our laboratory were also cytotoxic at a concentration of 1000 μg/ml but not at lower concentrations. Cell-free sonic lysates of S. typhimurium TML R66 were cytotoxic when tested undiluted and up to a dilution of 1:20. Based on the 2-Keto-3-deoxyoctonate (KDO) content of all preparations, sonic lysateas were cytotoxic at KDO concentrations of 0.42 μg/ml while the KDO content of the most cytotoxic LPS preparation was 15.2 μg/ml. There was no apparent correlations between KDO content of the LPS preparations and cell detachment, leading to the conclusion that cell detachment activity of Salmonella cell lysates cannot be attributed to their LPS content.     

Substantiation of the composition, technology and standardization of the original medicinal forms of interferon and bactisporin

New medicinal forms of immunobiological preparations--human lekocytic interferon and bactisporin--have been developed for local application in dentistry, surgery, gynecology. As the basis for these preparations, the pharmacopeial collagen solution has been used. The results obtained in this investigation indicate that the preparations are safe and possess wound-healing properties.     

The chromosome-damaging action of 2 chemical vaccine contaminants on mice

The cytogenetic effects of two chemical agents, hydroxylamine used for the destruction of bacterial cells and thimerosal added to many immunobiological preparations as preservative, were studied in vivo by their action on the marrow cells of C57BL/6J mice. The preparations under study, when injected intraperitoneally in a wide range of doses, including subtoxic ones, induced no chromosomal aberrations. At the same time cyclophosphamide, an antitumor cytostatic agent used for positive control, produced a pronounced damaging effect on chromosomes.     

The current trends in the development of new immunobiological preparations

In this article information on the main immunobiological preparations, including vaccines and toxoids, phages, eubiotics, preparations obtained on the basis of antibodies, immunomodulators, new adaptogens of different origin, is presented. The problem of the development of new diagnostic preparations and biosensors whose action is based on specific immunological reactions is discussed. The expected use of more effective, more natural, as well as free from many drawbacks, modern immunobiological preparations in the medical practice of the XXI century is emphasized.     

The protective effect of preparations made from plant seedlings in experimental Pseudomonas aeruginosa infection

The influence of preparations obtained from oat and wheat seedlings (immunostimulating factors IF-1 and IF-2, respectively) on the natural resistance of mice to P. aeruginosa infection was studied. IF-1 and IF-2 were introduced intraperitoneally in a single injection in doses of 100 micrograms and 1000 micrograms per mouse 2 and 7 days prior to the inoculation of P. aeruginosa strain 8 in doses of 1 and 10 LD50. The presence of substances capable of stimulating the immunobiological reserves of the body in actively growing plants (seedlings) was shown.     

An experimental study of the safety of a chemical monovalent tableted cholera vaccine in enteral administration]

The safety of experimental chemical cholera monovalent vaccine in tablets, produced by the institute "Microbe" (Saratov, USSR), has been studied. The study has shown that the vaccine, administered to adult rabbits and germ-free suckling rabbits by the enteral route, retains residual toxicity, mainly due to the presence of O-antigen. One or two administrations of 1-2 human doses of this preparation to adult rabbits induce minimal structural changes admissible from the viewpoint of safety. After immunization made in two administrations immunobiological transformation develops more rapidly and is more pronounced than after immunization in a single administration.     

Peptide-like substances as antimicrobial barriers to Corynebacterium sp. adhesion to silicone catheters

AIMS: To show medical application of antimicrobial peptides such as Pep5 and epidermin in inhibiting adhesion of Corynebacterium spp. to silicone catheters. METHODS AND RESULTS: The inhibitory activity of crude preparations of Pep5 and epidermin was tested on Corynebacterium spp. isolated from catheter-related infections. The addition of these substances at 640 AU ml(-1) to a cell suspension of Corynebacterium sp. 633544 resulted in a decrease of 3 log cycles in the number of viable cells over a period of 12 h. When Pep5 and epidermin were added to in vitro catheter colonization experiments, there was a decrease of 1 log unit (P < 0.01) in the cell number of Corynebacterium spp. adhered to silicone catheters. Scanning electron microscopy revealed that antimicrobial-treated catheters presented zones with absence of adhered cells, and some parts of the catheter presented aggregates suggesting damaged cells. CONCLUSIONS: The crude preparations of Pep5 and epidermin were able to inhibit Corynebacterium sp. 633544 isolated from catheter-related infection. The capability of Pep5 and epidermin to inhibit catheter colonization may indicate their usefulness as a barrier to block or to reduce the bacteremia by Corynebacterium spp. SIGNIFICANCE AND IMPACT OF THE STUDY: Peptide-like antimicrobial substances used to reduce bacterial attachment to medical devices may represent a novel strategy to control catheter-related infections.     

Synergistic activation by recombinant mouse interferon-gamma and muramyl dipeptide of tumoricidal properties in mouse macrophages

Mouse peritoneal macrophages were activated to become cytotoxic against B16-BL6 melanoma cells by the combination of subthreshold amounts of murine interferon-gamma (IFN-gamma; 0.1 to 10 U/ml) and N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP; 0.001 to 10 micrograms/ml), but not by the combination of pH 2-treated IFN-gamma and MDP, heat-treated IFN-gamma and MDP, or IFN-gamma and the inactive stereoisomer of MDP, N-acetyl-muramyl-D-alanyl-D-isoglutamine (MDP-D). The encapsulation of intact IFN-gamma and MDP within the same liposome preparation was synergistic for macrophage activation. In contrast, the presentation of identical concentrations of IFN-gamma and MDP in separate liposome preparations did not activate macrophages. These data allow us to conclude that the encapsulation of genetically engineered IFN-gamma and synthetically produced MDP within the same liposome is highly efficient in producing synergistic activation of tumoricidal properties in mouse macrophages.     

Beeinflussung der Zytokinsekretion von Maus-Thymom-Zellen (EL-4) durch Ochratoxin A

The murine thymoma cell line EL-4 was used as an in vitro T-cell model to assess the immunomodulating effect of pure Ochratoxin A (OTA) and an Aspergillus ochraceus raw toxin preparation. Cytokine production (IL-2, IL-4, IL-5, IL-6) and cell viability of PMA-stimulated EL-4 cells were investigated in the presence of OTA. The cytotoxic effect of the raw toxin could be observed at lower concentrations than pure OTA. The IC50 values were 3 µg/ml and 11 µg/ml, respectively. Increasing concentrations of both OTA preparations caused an inhibition of cytokine production, but the inhibition effect of the raw toxin was stronger than of pure OTA. This is supposed to be the effect of further up to now not characterized substances in the raw toxin. Differences in the susceptibility of the mechanisms of production and regulation of each cytokine are indicated by the different concentrations for inhibition effects. Both toxin preparations showed also stimulating effects on some cytokines (IL-2 and IL-6) while others (IL-4 and IL-5) were depressed at these OTA concentrations. This indicates the immunomodulating properties of the toxin.     

Cytotoxic, genotoxic, and mutagenic effects of diphenyl diselenide in Chinese hamster lung fibroblasts

Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organic selenium compounds, and may increase the risk of human exposure to this chemical at the workplace. In a previous study, we demonstrated the pro-oxidant action and the mutagenic properties of this compound on bacteria and yeast. In the present study, we evaluated the putative cytotoxic, pro-oxidant, genotoxic, and mutagenic properties of this molecule in V79 Chinese lung fibroblast cells. When cells were treated with increasing concentrations of DPDS, its cytotoxic activity, as determined using four cell viability endpoints, occurs in doses up to 50 microM. The MTT reduction was stimulated, which may indicate reactive oxygen species (ROS) generation. Accordingly, the treatment of cells for 3h with cytotoxic doses of DPDS increased TBARS levels, and sensitized cells to oxidative challenge, indicating a pro-oxidant effect. The measurement of total, reduced, and oxidized glutathione showed that DPDS can lead to lower intracellular glutathione depletion, with no increase in the oxidation rate in a dose- and time-dependent manner. At the higher doses, DPDS generates DNA strand breaks, as observed using the comet assay. The treatment also induced an increase in the number of binucleated cells in the micronucleus test, showing mutagenic risk by this molecule at high concentrations. Finally, pre-incubation with N-acetylcysteine, which restored GSH to normal levels, annulled DPDS pro-oxidant and genotoxic effects. These findings show that DPDS-induced oxidative stress and toxicity are closely related to intracellular level of reduced glutathione. Moreover, at lower doses, this molecule has antioxidant properties, protecting the cell against oxidative damage induced by hydrogen peroxide.     

In vitro reproductive toxicity of polychlorinated biphenyls: effects on oocyte maturation and developmental competence in cattle

Polychlorinated biphenyls (PCBs) are one of the most persistent and widespread group of endocrine disrupting compounds in the ecosystem. High concentrations of these substances are known to be present in sewage sludge from industrial, agricultural, and domestic origin that is spread in increasing amounts on arable land and pasture as fertilizer and is found in water, representing an increasing risk for the reproductive health of farm animals. Objective of this study was to determine the impact of PCBs on maturation and developmental competence of cattle oocytes. Since PCBs are a family of 209 molecules present in the environment as a mixture, Aroclor-1254, a pool of more than 60 congeners, was used in these experiments as its composition is considered to be environmentally relevant. Cumulus-oocytes complexes were exposed during IVM to serial concentrations of Aroclor-1254 (between 1 microg/ml and 0.0001 microg/ml) and compared with control groups. Aroclor decreased the percentage of oocytes that reached metaphase II stage after 24 hr, at doses as low as 0.01 microg/ml. Groups treated with 0.001 microg/ml or above, showed an impaired fertilization rate and a dramatic increase of polyspermy. Moreover, exposure during maturation resulted in a reduced proportion of oocytes that cleaved and developed until blastocyst stage although no differences in embryo cell numbers were observed. The present study indicates that very low PCBs concentrations are sufficient to disrupt bovine oocyte maturation, its fertilization, and developmental competence. These results also provide a set of reference data for the assessment of the risk posed by these substances to animal reproductive health, though further work will be necessary to equate in vitro doses to in vivo exposures.     

Leptin promotes the growth of Colon 38 cancer cells and interferes with the cytotoxic effect of fluorouracil in vitro

INTRODUCTION: Epidemiological studies underline the fact that obesity represents a significant risk factor for the development of several cancers, one of which is cancer of the colon. Moreover, multiple recent data indicate that some adipose tissue-derived hormones may influence the growth of malignant cells. Leptin, the product of the ob gene, is one of these. However, the evidence from research is still contradictory regarding the role of leptin in colon cancer. The aim of our study was to examine the direct effect of leptin at various concentrations (from 10(-5) to 10(-12) M) when applied alone or jointly with fluorouracil (the classical cytotoxic drug for colon cancer) at two concentrations (0.25 mg/ml and 2.5 mg/ml) on the growth of murine Colon 38 cancer cells in vitro. MATERIAL AND METHODS: Colon 38 cancer cells were preincubated in RPMI 1640 medium supplemented with foetal calf serum for 24 hours. The cells were then cultured for a further 72 hours in the presence of various concentrations of the substances under examination, applied either alone or jointly. The growth of the Colon 38 cell line was assessed by a colorimetric kit based on the modified Mosmann method. RESULTS: We found that leptin increased the growth of murine Colon 38 cancer at concentrations of 10(-6), 10(-7) M and 10(-10), 10(-11), 10(-12) M. Its stimulatory effect was fairly slight, with an increase in cancer growth of 5% to 15% as compared to controls. As we expected, fluorouracil at both the concentrations examined inhibited the growth of Colon 38 cancer maximally up to 28% (2.5 mg/ml) and 34% (0.25 mg/ml) of controls, with a stronger effect obtained from higher doses. Leptin did not modulate the cytotoxic effect of fluorouracil applied at the higher concentration (2.5 mg/ml) but, unexpectedly, at concentrations of 1(-9) and 10(-10) M it heightened the cytotoxic effect of fluorouracil given at a lower concentration (0.25 mg/ml). CONCLUSIONS: These data indicate that leptin is involved in the regulation of colon cancer growth and it may even heighten the cytotoxic effect of fluorouracil.     

Immune phagocytosis inhibition by commercial immunoglobulins

Sixteen commercially available immunoglobulins (Ig) and 5 anti-Rho (D) hyperimmune globulins were investigated for immune phagocytosis inhibition (IPI) factors as well as for T, B lymphocytotoxic and monocytotoxic antibodies. All Ig contained IPI factors with lowest inhibitory IgG concentrations ranging from 0.08 to 50 mg/ml. Pepsin-digested Ig was noninhibitory. IPI factors in anti-D preparations were uniformly high (inhibitory IgG concentrations 0.6-2.5 mg/ml). Cytotoxic antibodies against T, B lymphocytes and monocytes were found in 2,2 and 7 products, respectively. Since we have recently shown that IPI is caused by antibodies against major histocompatibility complex antigens, most likely HLA, the hypothesis is put forward that IPI factors in Ig are HLA-related, cytotoxic as well as noncytotoxic antibodies which act via Fc receptor blockade of human monocytes.     

Evaluation of contents of A and B group substances in biological preparations]

The study was aimed at determination of blood A and B group substances in biological preparations used in Poland. Twenty three series were investigated, namely: Di-Te-Per, Ty-Te, Ty, Te, against cholera, vaccine according to Delbet and Panodin. Also were tested: 65 series of imported preparations of immunoglobulin g (i.v.) such as Endobulin, Sandoglobulin, Gamma-Venin, Veinoglobuline and 5 local series such as Bioglobulin, as well as 9 series of preparation LNI (i.m.) Human Gamma Globulin. Presence of substance A was detected in tetanus and botulinum horse antitoxins in amount from 3.75 micrograms/ml to 30 micrograms/ml. Group substances A and B contained 6 series of LNI preparations-Veinoglobuline. Amount of substance A was detected as 3.75 micrograms/ml-7.5 micrograms/ml and of substance B as 2,5 micrograms/ml-5 micrograms/ml. Group substances A and B were not present in vaccines used according to vaccination calendar.     

A new rabies vaccine in public health practice in the USSR

A new antirabies vaccine prepared on the basis of virus grown in the ovine brain, purified from 85-90% of brain-tissue ballast substances and inactivated with beta-propilactone has been developed at the Moscow Research Institute of Viral preparations (USSR Acad. Med. Sci.). The preparation produces no neuro-allergenic effect in tests on guinea pigs. When injected to humans, the vaccine shows much lower reactogenicity than Fermi vaccine. High antigenic and immunogenic activity of the new vaccine has made it possible to work out a less intensive immunization schedule in comparison with that used for immunization with Fermi vaccine and nonconcentrated tissue-culture vaccine, viz. doses of 3 ml for 12 days or doses of 3 ml for 20 days with two booster immunizations. The preparation has been introduced into medical practice.     

Effect of carbohydrates and polyols on amide content and protein fragmentation in a lactoglobulin preparation

Certain carbohydrates and polyols are used at various stages of the production of immunobiological preparations as stabilizers of biological activity, particularly in the production of lactoglobulin (against opportunistic pathogens) using membrane ultrafiltration. This study concerns the effect of these substances on changes in the amide content in proteins of this lactoglobulin. Lactoglobulin was incubated in near-physiological (0.9% NaCl, pH 5.5) 10% solutions of glucose, fructose, and sorbitol at 4 and 35 degrees C for 7, 14, and 28 days. A lactoglobulin solution in 0.9% NaCl, pH 5.5, was used as the control. All substances studied suppressed the reduction of the amide group content of asparagine and, in contrast, increased the rate of amide group removal from glutamine residues in proteins of lactoglobulin preparations.     

The improvement of immunobiological preparations against leptospirosis. An experimental study of a new concentrated purified vaccine against icterohemorrhagic leptospirosis for human immunization

The findings of the study of immunological structure of the population in regions endemic for leptospirosis indicate that the immune status of humans makes it impossible to obtain titrated blood sera for the preparation of antileptospirosis immunoglobulin. The data obtained in the study of the immunobiological properties of a new concentrated vaccine against icterohemorrhagic leptospirosis show the possibility of using this vaccine for the immunization of donors with the aim of obtaining blood sera to be used as raw material for the production of immunobiological preparations.     

Modulation of latent protein phosphatase activity from vascular smooth muscle by histone-H1 and polylysine

An apparently latent phosphatase which migrated as a protein of Mr 130,000 during sucrose density centrifugation, and a spontaneously active phosphatase (Mr 68,000) were isolated from bovine aortic smooth muscle. Basal phosphorylase phosphatase activity of the latent preparations was stimulated 12 fold by low concentrations of lysine-rich histone-H1 (30 micrograms/ml) and 6 fold by polylysine (Mr 17,000; 12 micrograms/ml), whereas the spontaneously active enzyme was only slightly affected. The enzymatic activity of the spontaneously active preparation was completely destroyed by beta-mercaptoethanol. In contrast, the apparently latent enzyme was converted to a more active form of lower molecular weight (Mr 86,000) following treatment with beta-mercaptoethanol and this form of the enzyme was still stimulateable by histone-H1. These findings show that the aortic spontaneous and apparently latent phosphatase actives are ascribable to separate enzymes and they suggest that the activity of latent phosphatase in living cells may be modulated by cationic proteins such as histones or similar effector molecules.