Immunogenetic analysis of microsomal and non-microsomal lipoproteins from normal and malignannt mouse tissues for histocompatibility-2 (H-2) antigens

The H-2 antigenic properties of lipoprotein fractions from malignant (L-5178Y leukemia) and normal (spleen, thymus, liver, kidney) mouse tissues have been studied by serological and immunological tests, and the results compared to the previously described activities of these fractions in homograft-sensitization tests. Although, in general, the relative activities in the different assays parallel each other some notable exceptions were found. The non-microsomal lipoproteins from leukemic tissue, inactive in homograft-sensitization tests, did elicit H-2 antibody. Also, the liver microsomal lipoproteins, which are inactive in homograft-sensitization tests in amounts 400 × the minimal effective doses of spleen preparations, exhibited, in in vitro agglutinin-inhibition tests, approximately one-fourth the H-2 activity of the latter. Other findings of note include the high antibody-eliciting potency of the spleen and leukemia microsomal lipoproteins (15 μg protein was sufficient to initiate primary immunization and 1 μg protein to cause an anamnestic response); and the quantitive identity of H-2 antigen activity of the microsomal lipoproteins from spleen and thymus.     

Effects of γ-ray treatment onCannabis saliva pollen viability

The viability and thein vitro germination capability of hemp pollen (cv. Carmagnola) were studied. Viability tests were based on the microscopic observation of the fluorescence of loaded fluorescein diacetate (FDA), while, for germinability tests, five different media were tested. The effects of irradiation with γ-rays on pollen viability and germination and on seed set were also studied, at three different irradiation doses (20, 60 and 100krad). The results show that in one of the media tested, about 85–90% of the pollen grains are viable and able to germinate in control samples, and that while viability measured by FDA test is not affected by increased γ-ray doses, the pollenin vitro germinability drops to about one-half of the controls at the maximum γ-ray dose employed, 100krad. Seed set of hemp plants pollinated with the irradiated pollen dropped to less than 1% of that of plants pollinated by untreated pollen for the higher dose used. The different media suitable forin vitro germination of hemp pollen, and the observed lack of correspondence between viability and germination capacity tests are discussed.     

Studies on the biological effects of deuteriated organic compounds

Investigations on the antimycotic properties of perdeuteriated fatty acids were carried out on Microsporum cains infections in vivo. The study was performed on experimental microsporie in guinea pigs using four different methods, all based on the ability of M. canis to cause alopecia. Perdeuteriated n-hendecanoic acid showed in vivo a statistically significant enhanced antimycotic effect compared to its unlabelled analogue. This is in accordance with our previous observations in vitro conditions. The remaining perdeuteriated fatty acids (C₁₂-C₁₈) showed no statistically significant growth retarding effect on M. canis infections in guinea pigs when compared with their unlabelled analogues.The present study attempts to ascertain if some perdeuteriated fatty acids have any antifungal activity in vivo conditions.Our previous papers (3, 4) concerning the evaluation of the antifungal activity of some perdeuteriated fatty acids on dermatophytes in vitro, demonstrated that the perdeuteriation of n-hendecanoic acid, lead to a pronounced antimycotic effect on common dermatophytes as e.g. E. floccosum, T. rubrum, M. canis and T. mentagrophytes. As our previous results indicate a decreasing of fungistasis with an increasing carbon-chain length of the perdeuteriated fatty acids, we have attempted to verify this observation in vivo conditions. A special interest was, of course, focused on the most promising compound, the perdeuteriated n-hendecanoic acid.     

An Archived Lot of Coccidioidin Induces Specific Coccidioidal Delayed-type Hypersensitivity and Correlates with in vitro Assays of Coccidioidal Cellular Immune Response

No test for assessing cellular immune response in coccidioidomycosis is currently available in the United States. In the present study, we tested 49 healthy subjects living in the coccidioidal endemic region with a 1:55.8 dilution of a single lot of coccidioidin archived since the 1970s. In this group, 23 evaluable subjects demonstrated ≥5 mm of induration at 24, 48 or 72 h, with a mean±SEM maximum induration of 18.4±4.0 mm. The induration results among 14 subjects reactive at 24 h were compared to those from 179 individuals in an historical cohort studied in the 1980s using a reference lot of coccidioidin. Results were within 5% and not significantly different (P=0.924). The maximum induration response of all evaluable subjects correlated significantly with the results of in vitro tests of coccidioidal cellular immunity using supernatant interferon-gamma concentration and CD69 expression on T cells (Spearman rank correlation coefficients 0.69 and 0.68, respectively; P<0.01 for both). These data suggest that archived coccidioidin retains its potency and specificity and that in vitro test of coccidioidal immunity may have utility in the measurement of coccidioidal cellular immunity.     

<Emphasis Type="Italic">Acmella radicans</Emphasis> var. <Emphasis Type="Italic">radicans: In vitro</Emphasis> culture establishment and alkamide content

Summary Acmella radicans var. radicans propagation was established in vitro. This plant belongs to the Asteraceae from which some species are known for their insecticide, fungicide and antibacterial activity. The complete Murashige and Skoog (MS) medium was the best in assisting seed germination. In order to obtain shoots in vitro, a complete MS medium and half-strength MS medium were assayed with explants from leaves, nodes, and internodes. The best medium for shoot production was the half-strength MS medium with no addition of plant growth regulators, and the highest shoot propagation was from single-node explants. Regeneration of roots on shoot explants in the medium was obtained without the addition of growth regulators. Of the plantlets that were acclimated, 90% of them were obtained from rooted shoots with completely expanded leaves. The alkamide content was evaluated for each tissue and the higher concentration was observed in flower heads. The main alkamides present in the leaves and the flower heads were N-(2-phenylethyl)-2Z,4E-octadienamide and 3-phenyl-N-(2-phenylethyl)-2-propenamide. This study describes the methodology for the establishment and propagation of Acmella radicans in vitro and the evaluation of different tissue alkamide contents in vitro and in the field.     

Modulation of lymphocyte functions by group A streptococcal membrane

Protoplast membranes isolated from group A streptococci suppress functions of mouse B cells in vivo and in vitro. Intraperitoneal injection 24 or 72 hr (but not 12 hr) before collection of lymphoid cells results in a selective decrease in the mitogenic response of bone marrow cells to dextran sulfate (DS). The response of bone marrow cells to lipopolysaccharide (LPS), and spleen cells to both DS and LPS, is unaltered. In vitro exposure of lymphocytes to membranes concomitantly with mitogen reduces the response to both DS and LPS, however, the DS response is more susceptible to low doses of membrane. Suppression of the response to DS in vitro is not mediated by cells bearing Thy 1.2 antigen. Neither the phytohemagglutinin (PHA)-responsive cells nor the adherent cells participate in suppression of the LPS response in vitro. In contrast to the suppression of B-cell functions neither the PHA nor concanavalin A (Con A) response of mouse bone marrow, spleen, or thymus cells is altered by streptococcal protoplast membranes injected 24 hr before collection of cells. In vitro exposure of spleen cells to a limited range of concentrations of membrane results in an enhanced proliferative response of spleen cells stimulated by suboptimal doses of PHA. This synergism is not mediated by the adherent cells. Addition of membranes to spleen cell cultures in vitro has no effect upon the response of spleen cells to suboptimal doses of Con A or to optimal doses of either Con A or PHA. Higher concentrations of membranes reduce the proliferative response of both control and mitogen-stimulated cells. This nonselective suppression by high doses of membranes is not due to toxicity. Delayed hypersensitivity to sheep erythrocytes is potentiated by injection of membranes. These studies suggest that streptococcal membranes preferentially suppress the immature B cells and enhance certain T-cell functions.     

Dissolution Testing of Sublingual Tablets: A Novel <Emphasis Type="Italic">In Vitro</Emphasis> Method

In the sublingual (SL) cavity, compared with the gastrointestinal tract, tablets are subjected to minimal physiological agitation, and a limited volume of saliva is available to facilitate disintegration and dissolution. None of the official compendial dissolution apparatuses and methods simulate these SL conditions. In this study, a custom-made dissolution apparatus was constructed, and a novel in vitro method that simulates SL conditions was evaluated. Several epinephrine 40 mg SL tablet formulations under development and two commercial SL tablets, isosorbide dinitrate 5 mg and nitroglycerin 0.6 mg, were studied. The dissolution medium was 2 mL of distilled water at 25°C. Dissolution was measured at 60 and 120 s. The novel in vitro method was validated for accuracy, reproducibility, and discrimination capability, and was compared with the official US Pharmacopeia (USP) dissolution method using apparatus 2 (Paddle). The data obtained following the novel in vitro method were accurate and reproducible. This method was capable of detecting minor changes in SL formulations that could not be detected using other in vitro tests. Results from the official USP dissolution method and our novel in vitro method were significantly different (p < 0.05). Results reflecting the dissolution of rapidly disintegrating tablets using simulated SL conditions were obtained using the novel in vitro dissolution method.     

Pseudomonas aeruginosa acid phosphatase and cholinesterase induced by choline and its metabolic derivatives may contain a similar anionic peripheral site

Summary Different compounds derived from choline, and obtained by demethylation or by oxidation of the primary alcohol group with subsequent N-demethylation, were tested as inducer agents of acid phosphatase and cholinesterase in Ps. aeruginosa. It was found that betaine and dimethylglycine were the most effective inducers of both enzyme activities. These metabolites including choline itself, were not inducers of acid phosphatase and cholinesterase in other Gram-negative bacteria such as: Escherichia coli, Salmonella typhimurium, Shigella flexneri, Enterobacter liquefacciens and Proteus mirabilis. The acid phosphatase activities found in these bacteria were not inhibited in vitro by choline, betaine and phosphorylcholine. From these results it may be concluded that the acid phosphatase activity from Ps. aeruginosa is different from the same activity observed in the other bacteria. In addition, it is also shown that Ps. aeruginosa acid phosphatase and cholinesterase were inhibited by a number of compounds containing a positively charged amino group, with methyl or ethyl groups bound to it. These results seem to confirm that Ps. aeruginosa acid phosphatase and cholinesterase may contain a similar anionic site.     

New semicarbazones as gorge-spanning ligands of acetylcholinesterase and potential new drugs against Alzheimer’s disease: Synthesis,molecular modeling,NMR, and biological evaluation

Two new compounds (E)-2-(5,7-dibromo-3,3-dimethyl-3,4-dihydroacridin-1(2H)-ylidene)hydrazinecarbothiomide (3) and (E)-2-(5,7-dibromo-3,3-dimethyl-3,4-dhihydroacridin-1(2H)-ylidene)hydrazinecarboxamide (4) were synthesized and evaluated for their anticholinesterase activities. In vitro tests performed by NMR and Ellman’s tests, pointed to a mixed kinetic mechanism for the inhibition of acetylcholinesterase (AChE). This result was corroborated through further docking and molecular dynamics studies, suggesting that the new compounds can work as gorge-spanning ligands by interacting with two different binding sites inside AChE. Also, in silico toxicity evaluation suggested that these new compounds can be less toxic than tacrine.     

Study of <Emphasis Type="Italic">In Vitro</Emphasis> Drug Release and Percutaneous Absorption of Fluconazole from Topical Dosage Forms

The present study aimed to evaluate different dosage forms, emulsions, emulgels, lipogels, and thickened microemulsion-based hydrogel, as fluconazole topical delivery systems with the purpose of determining a formulation with the capacity to deliver the whole active compound and maintain it within the skin so as to be considered a useful formulation either for topical mycosis treatment or as adjuvant in a combined therapy for Cutaneous Leishmaniasis. Propylene glycol and diethyleneglycol monoethyl ether were used for each dosage form as solvent for the drug and also as penetration enhancers. In vitro drug release after application of a clinically relevant dose of each formulation was evaluated and then microemulsions and lipogels were selected for the in vitro penetration and permeation study. Membranes of mixed cellulose esters and full-thickness pig ear skin were used for the in vitro studies. Candida albicans was used to test antifungal activity. A microemulsion containing diethyleneglycol monoethyl ether was found to be the optimum formulation as it was able to deliver the whole contained dose and enhance its skin penetration. Also this microemulsion showed the best performance in the antifungal activity test compared with the one containing propylene glycol. These results are according to previous reports of the advantages of microemulsions for topical administration and they are very promising for further clinical evaluation.     

The immunosuppressive activities of newly synthesized azaphenothiazines in human and mouse models

In this study, we evaluated the activities of new types of azaphenothiazines in the following immunological assays: the proliferative response of human peripheral blood mononuclear cells induced by phytohemagglutin A or anti-CD3 antibodies; lipopolysaccharide-induced cytokine production by human PBMC; the secondary, humoral immune response in mice to sheep erythrocytes (in vitro); and delayed-type hypersensitivity in mice to ovalbumin (in vivo). In some tests, chlorpromazine served as a reference drug. The compounds exhibited differential inhibitory activities in the proliferation tests, with 10H-2,7-diazaphenothiazine (compound 1) and 6-(3-dimethylaminopropyl)diquinothiazine (compound 8) being most suppressive. Compound 1 was selected for further studies, and was found to be strongly suppressive in the humoral immune response even at low concentrations (1 μg/ml). Compound 1 also inhibited the delayed-type hypersensitivity lipopolysaccharide-induced production of tumor necrosis factor and interleukin-6 in cultures of human blood cells. As there were only two subjects in this study, the effects of these compounds on human blood cells need to be confirmed. In this paper, we also discuss the structure-activity relationships of selected compounds.     

Effect of Fluorination on Skin Sensitization Potential and Fragrant Properties of Cinnamyl Compounds

A series of three α‐ and three β‐fluorinated representatives of the family of cinnamate‐derived odorants (cinnamaldehyde ( 1 ), cinnamyl alcohol ( 2 ), and ethyl cinnamate ( 3 )) as used as fragrance ingredients is described. Olfactive evaluation shows that the fluorinated compounds exhibit a similar odor profile to their parent compounds, but the olfactive detection thresholds are clearly higher. In vitro evaluation of the skin sensitizing properties with three different assays indicates that α‐fluorination of Michael acceptor systems 1 and 3 slightly improves the skin sensitization profile. α‐Fluorocinnamyl alcohol 2b is a weaker skin sensitizer than cinnamyl alcohol 2a by in vitro tests and the fluorinated product drops below the sensitization threshold of the KeratinoSens^® assay. On the other hand, β‐fluorination of compounds 1  –  3 results in highly reactive products which display a worsened in vitro skin sensitization profile.     

Different individual immune responses elicited by in vitro immunization

We have previously demonstrated that the addition of muramyl dipeptide (MDP), interleukin-2 (IL-2) and IL-4 effectively raises antibody production from L-Leucyl-L-leucine methyl ester (LLME)-treated human peripheral blood lymphocytes (PBLs) against specific soluble antigen when immunized in vitro. However, PBLs from individual donors were separate optimal conditions regarding concentrations for IL-2 and IL-4, which in turn required us to optimize each individual PBLs to effectively produce antigen specific human antibody by in vitro immunization. These individual differences in the requirement for IL-2 and IL-4 reflects the differences in individual immune responses against a specific soluble antigen, which can be elicited by in vitro immunization. In the present study, we investigated these individual differences in the requirement for IL-2 and IL-4 to induce antibody productionin vitro in the PBLs of 12 volunteers (9 healthy donors and 3 allergenic patients). IL-2 requirements for antibody production varied dependent upon each donor, while higher amounts of IL-4 inhibited IgM and IgG production in all of the healthy donors. However, some of the characteristic features for PBLs donated from allergenic included lowered IgM production compared to PBLs derived from healthy donors, and very high IgE production in the absence of cytokines and allergen. These results demonstrate that the sensitivity of PBLs against antigen sensitization differs between healthy donors and atopic patients, which suggests that the frequency of antigen sensitization might be reflected in differing activation states and/or differing subpopulations of lymphocytes in vivo. This revised version was published online in August 2006 with corrections to the Cover Date.     

The role of Ia antigens and Fc receptor-bearing T-cells in the inhibition of macrophage receptors for cytophilic antibody induced by soluble immune complexes

Soluble antigen-antibody complexes composed of 3 M KCl-extracted L1210 antigens and alloantibody to L1210 given to C3H mice caused immunosuppression in the mice. This was reflected in part by the inhibition of cytophilic antibody receptors on macrophages which could be used as a measure of the suppression. Thymocytes or splenic T cells from mice treated with immune complexes could adoptively transfer the suppression to normal syngeneic mice. These cells, which we have termed suppressor inducers, were found to be Ia positive: specifically, I-A⁺, I-J^?. Thus, treatment of the inducers with anti-la or anti-I-A antibodies and complement in vitro abrogated their ability to transfer the suppression to normal mice. In contrast treatment with anti-I-J serum and complement had no effect. Through a similar approach, the cooperating (acceptor) T cells were found to be I-A⁺, I-J^?. Pretreatment of mice with anti-Ia or anti-I-A serum before the administration of antigen-antibody complexes prevented the inhibition of macrophages. This was due at least in part to steric hindrance of adjacent Fc receptors on the FcR⁺ T cells with which the complexes interacted. Early interaction of immune complexes with FcR⁺ T cells was in fact demonstrated directly by the inability of the complexes to induce suppression when FcR⁺ T cells were depleted. The thymocytes or splenic T cells from anti-Ia-pretreated mice failed to transfer the suppression to recipient mice. In contrast, treatment with either anti-Ia or anti-I-A after the immune complexes did not abrogate the generation of suppressor inducers. Treatment of normal recipient mice with anti-Ia serum in vivo before they received the suppressor inducer cells did not prevent cooperation between the two types of cells. By the same token, blocking of Ia antigens of the inducers in vitro with anti-Ia serum (without complement) also did not impair the cooperative interaction. These results indicate that antigen-antibody complexes generate I-A-positive, I-J-negative T-suppressor inducer cells from FcR⁺ naive T cells. These in turn interact with Ia-positive (I-A⁺ and I-J^?) normal thymocytes or spleen T cells. This interaction most likely generates the ultimate suppressor T cells that suppress cytophilic antibody receptors on macrophages in vivo. However, the I-region determined antigens did not appear to be directly involved in the T-T interaction of suppressor inducer and acceptor cells.     

Double resistance by citrus green mould Penicillium digitatum to the fungicides guazatine and benomyl

In vitro dosage response data with different isolates of Penicillium digitatum and the fungicide guazatine indicated an approximate 10-fold shift in tolerance when compared with wild type strains. ED₅₀ values for resistant strains were approximately 0.5 μg/ml compared to approximately 0.05, μg/ml for the wild type strains. Colony growth of guazatine resistant isolates on selective media containing carbendazim showed that they were also resistant to the benzimidazole group of fungicides. In vivo tests in inoculated oranges with strains previously characterised by in vitro tests confirmed resistance to guazatine and benomyl. A combined treatment of these fungicides at 400 /μ/ml and 500 μg/ml respectively, which normally gives protection against decay, also failed to provide adequate mould control. Growth and pathogenicity of the resistant strains in these tests in oranges were indistinguishable from that of wild type strains.     

Alcohol-inducible gene expression in transgenic Populus

We tested the efficiency and optimized the conditions for controlled alcohol-inducible transgene expression in Populus using gus as a reporter gene. Specificity of induction, efficiency in different organs, effect of three chemical inducers, and induction methods were tested using up to 10 independent transgenic events generated in two different Populus genotypes. The optimal inducer concentration and the duration of induction period were determined in dose–response and in time–course experiments. Under in vitro conditions, β-glucuronidase (GUS) induction was efficient both in the aerial parts and in the roots of regenerated plantlets. Among the chemical inducers tested, ethanol was the most effective activator with no apparent phytotoxicity when concentrations were at or below 2%. After 5 days of treatment, fluorometrically-determined the GUS activity could be detected when inducing with ethanol at concentrations as low as 0.5%. Prolonged induction by ethanol vapors significantly increased the GUS activity in leaves from both the tissue culture plants and greenhouse-grown plants.Electronic Supplementary Material Supplementary material is available for this article at     

Interaction between Anti-Sarcoma 180 Serum and Various Lines of Tumor Cells

When mice were immunized with adequate doses (1.0~5.0 mg) of tumor cells attenuated with acetone-ether, complete resistance to the graft of Sarcoma 180 could be induced. The serum taken from mice immunized with repeated challenges was found to display immune adherence reactivity and the antibody titer of anti-Sarcoma 180 serum was higher than that of anti-sarcoma 37 or anti-Ehrlich serum prepared from respective tumor resistant mice.

The interaction between anti-Sarcoma 180 serum and various lines of tumor cells was investigated by the tests of immune adherence absorption and cytotoxicity.

Sarcoma 37 cells exhibited the same reactivity as Sarcoma 180 cells in both tests. Ehrlich cells showed lower reactivity than Sarcoma 180 or Sarcoma 37. Neither MH 134 cells nor myeloma cells exhibited a detectable reactivity in the test of cytotoxicity in vitro. On the other hand, in the test of cytotoxicity in vivo, MH 134 was slightly inhibited and myeloma was promoted in tumor growth.

These results suggest that anti-Sarcoma 180 serum prepared in this experimental system might be useful for the classification of tumor cells and in the study of tumor surface.     

Failure of Mouse Peritoneal Macrophage Activation by the Purified Excreted/Secreted Antigens of Toxoplasma gondii

This study was carried out to evaluate the effect of excreted/secreted antigens on macrophages infected by Toxoplasma gondii. Six proteins 28, 30, 45, 58, 63 and 145 kDa were separated by different chromatographic techniques. Mouse peritoneal macrophages were treated in vitro and in vivo with these purified fractions. Penetration and proliferation assays of T. gondii in the macrophages were performed in vitro. The different antigens used did not change the rate of penetration and proliferation of the parasites. Therefore, the secreted products, which are capable of provoking an immune response, could not directly activate the macrophages. Furthermore, the secreted products were not cytotoxic and neither did they possess a visible phospholipasic activity which would have increased penetration.     

Increased T‐cell stimulating activity by mutated SEC2 correlates with its improved antitumour potency

Aims: To investigate the improved antitumour activity of SAM‐3 compared with recombinant staphylococcal enterotoxins C2 (rSEC2). Methods and Results: Methylthiazol tetrazolium and flow cytometry assays showed that the antitumour activity of SAM‐3 in vivo was improved because of enhanced T‐cell stimulating potency, resulting in massive activation of T cells, particularly CD4 ⁺ and CD8 ⁺ T cells, and subsequent cytokine release. Quantitative real‐time PCR assay showed that despite similar Vβ specificities induced by rSEC2 and SAM‐3, the quantities of activated T cells bearing specific Vβin vitro were different. Conclusions: The results strongly suggested that the increased SAM‐3–T‐cell receptor (TCR) binding affinity contributed to massive T‐cell activation and cytokine release, substantially amplifying antitumour immune response in vivo. Significance and Impact of the Study: This study provided evidence for the mechanism of SAM‐3 antitumour activity improvement compared with rSEC2. Results indicated that SAM‐3 could be used as a potent powerful candidate agent for tumour treatment in clinics.     

Differentiation of the immunosuppressive and antiviral effects of interferon.

Virus-induced (virus-type) interferon suppression of the in vitro antibody response of mouse (C57B1/6) spleen cells to sheep red blood cells was blocked by 5 × 10^?5M 2-mercaptoethanol (2-ME). The blockade was not due to a direct effect on interferon since 2-ME was capable of blocking the suppression when added to cultures up to 48 hr after interferon. 2-ME blockade of virus-type interferon immunosuppression was not due to the immunoenhancing property of 2-ME. Similar protective effects of 2-ME were observed during immunosuppression by virus-type interferon inducers, but not T-cell mitogen inducers of interferon (immune interferon). The data suggest that the immunosuppressive properties of virus-type and immune interferon preparations involve different mechanisms. Virus-type interferon inhibited DNA synthesis in unstimulated spleen cell cultures and in 2-ME stimulated cultures, and the degree of inhibition of DNA synthesis appeared to be related to the immunosuppressive property of interferon in the absence or presence of 2-ME. 2-ME did not affect the antiviral properties of either virus-type or immune interferon in nonlymphoid cells. Further, the induction of virustype interferon in spleen cells was neither inhibited nor enhanced by 2-ME, while the induction of immune interferon was enhanced. This enhancement is consistent with 2-ME enhancement of the immunosuppressive effects of immune interferon inducers.There are two possibilities for 2-ME blockade of the immunosuppressive effect of virus-type interferon, while not affecting the antiviral property. Firstly, the immunosuppressive and antiviral properties of virus-type interferon may involve different mechanisms at the subcellular level. Secondly, the selectivity of the blockade by 2-ME could be due to the fact that spleen cells are the target cells in immunosuppression, while L cells are the target cells in inhibition of virus replication. Thus, virus-type interferon may suppress the immune response at the level of the macrophage and 2-ME may reverse this effect by replacing a blocked macrophage function.