Characteristics of the immunocyte cooperation in 2 strains of mice opposite in their sensitivity to dermaphytoses during the immune response to antigens of the causative agent

C57BL/6 and BALB/c mice, opposite in their sensitivity to dermatophytic infections, show similar activity of phagocytes as regards their capacity for the destruction of injected conidia of dermatophytes, but differ in the changes of this activity after immunization with dermatophytic antigenic complexes (DAC). Shortly after the injection of the antigens, the lymphocyte-mediated suppression of the fungicidal activity of macrophages, caused by the interaction of DAC with intact T-lymphocytes, was detected in the animals of both strains. Later in C57BL/6 mice resistant to mycosis the formation of cell-mediated immunity to DAC occurs, with the simultaneous production of factor stimulating the fungicidal activity of macrophages. In BALB/c mice sensitive to mycosis the injection of DAC induces active antibody production, but not the formation of delayed hypersensitivity with the resulting stimulation of the fungicidal activity of phagocytes. The injection of DAC into mice of the above-mentioned strains induces changes, peculiar to each strain, in the mitogen-induced proliferation of spleen cells and in the character of immune response to sheep red blood cells. Differences in the influence of DAC on the induction of immune response in C57BL/6 and BALB/c mice are realized by cells belonging to the population of T-lymphocytes.     

Antifungal potential of binary and mixed-ligand complexes of N,2'-diphenyl acetohydroxamic acid.

Chelating potential of N,2'-DPAHA with 3d metal ions such as Cu(II), Ni(II), Zn(II), and Cd(II) in the presence of Gly and Phen has been investigated. These experiments were designed to study the role of the stability of mixed-ligand complexes in the modulation of its fungicidal potential. The mixed-ligand complexes were found to be more stable than binary complexes. Enhanced stability of mixed-ligand complexes of Ni(II), Co(II), Zn(II), and Cd(II) is presumably due to pi-bonding effects. In the stabilization of the Cu(II) mixed-ligand complex system, the Jahn-Tellar effect may play a vital role, in addition to pi-bonding effects. Fungicidal activity of N,2'-DPAHA and its binary complexes with Cu(II), Ni(II), and Co(II) was examined against Fusarium oxysporum using the inhibition zone technique. Binary complexes of Zn(II) and Cd(II) with N,2'-DPAHA and mixed-ligand complexes M(II)-Gly or Phen-N,2'-DPAHA, where M(II) = Cu(II), Ni(II), Zn(II), Co(II), and Cd(II) were screened against Alternaria alternata by slide germination technique. All mixed-ligand complexes exhibited fungicidal activity but did not improve significantly compared to binary complexes. Synergistic action of primary and secondary ligands has increased the stability of the mixed-ligand complex compared to the binary complex (1:1) of the secondary ligand (N,2'-DPAHA), and the fungicidal potential of the mixed-ligand complex involving N,2'-DPAHA as secondary ligand was not increased.     

Design of novel peptide analogs with potent fungicidal activity,based on PMAP-23 antimicrobial peptide isolated from porcine myeloid

PMAP-23 is a 23-mer peptide derived from porcine myeloid. To develop novel antifungal peptides useful as therapeutic drugs, it would require a strong fungicidal activity against pathogenic fungal cells. To this goal, several analogs, with amino acid substitutions, were designed to increase the net hydrophobicity by Trp (W)-substitution at positions 10, 13, or 14 at the hydrophilic face of PMAP-23 without changing the hydrophobic helical face. The Trp (W)-substitution (P6) showed an enhanced fungicidal and antitumor activities, with the fungicidal activity inhibited by salts and the respiratory inhibitor, NaN(3). The results suggested that the increase of hydrophobicity of the peptides correlated with fungicidal activity. The fungicidal effects of analog peptides were further investigated using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a membrane probe. In Candida albicans, the analog peptide (P6) exerted its fungicidal effect on the blastoconidia in 20% fetal bovine serum by disrupting the mycelial forms. Furthermore, P6 caused significant morphological changes, and these facts suggested that the fungicidal function of the novel analog peptide (P6) was by damaging the fungal cell membranes. Thus, this peptide may provide a useful template for designing novel antifungal peptides useful for the treatment of infectious diseases.     

HP (2-20) derived from the amino terminal region of helicobacterpylori ribosomal protein L1 exerts its antifungal effects by damaging the plasma membranes of Candida albicans.

The fungicidal effects of the peptide HP (2-20). derived from the N-terminal sequence of Helicobacter pylori ribosomal protein L1 (RPL1). have been investigated. HP (2-20) displays a strong fungicidal activity against various fungi, without haemolytic activity against human erythrocyte cells, and the fungicidal activity is inhibited by Ca2+ and Mg2+ ions. In order to investigate the fungicidal mechanism(s) of HP (2-20). the amount of intracellular trehalose was measured in C. albicans. It was found that the amounts of intracellular trehalose were decreased when HP (2-20) was used. The action of the peptide against fungal cell membranes was further examined by the potassium-release test; HP (2-20) was found to increase the amount of K+ released from the cells. Furthermore, HP (2-20) caused significant morphological changes, as shown by scanning electron microscopy, and by testing the membrane disrupting activity using liposomes (phosphatidyl choline/cholesterol; 10: 1, w/w). Our results suggest that HP (2-20) may exert its antifungal activity by disrupting the structure of cell membranes, via pore formation or direct interaction with the lipid bilayers.     

Induction of allogeneic cytolytic T lymphocytes by partially purified membrane glycoproteins

It has been previously shown that P815 (H-2^d) purified plasma membranes can induce cytolytic activity from primed C57BL/6 (H-2^b) spleen cells. The secondary cytolytic T lymphocyte (CTL) inducing activity is retained when these P815 plasma membranes are solubilized in deoxycholate. Evidence is now presented that the cell surface antigens responsible for CTL induction can be partially purified in active form and these antigens can be incorporated into reconstituted membranes and phospholipid vesicles. The active antigens have the properties expected for H-2 molecules on lentil lectin chromatography and gel filtration.     

Structure-activity relationship studies on niphimycin, a guanidylpolyol macrolide antibiotic. Part 1: The role of the N-methyl-N'-alkylguanidinium moiety

Several N-methyl-N'-alkylguanidinium derivatives were synthesized and used as simplified analogues of niphimycin (NM), a guanidylpolyol macrolide, in structure-activity relationship studies. The C16-alkylated derivative exerted fungicidal activity by directly damaging the fungal plasma membrane and inducing oxidative stress in a manner similar to niphimycin. These results indicate that the N-methyl-N'-alkylguanidinium moiety is required for antifungal activity by NM.     

类球红细菌的免疫活性评价

目的 :评价类球红细菌的免疫活性。方法 :巨噬细胞吞噬试验、淋巴细胞转化试验、IL- 2的诱生和检测试验。结果 :类球红细菌 1号株具有调节和增强巨噬细胞吞噬功能 ,表现在免疫抑制组小鼠用光合细胞灌胃数周后 ,腹腔巨噬细胞吞噬百分率提高 6 1% ,吞噬指数提高 5 9% (P<0 .0 1)。在体外实验中 ,光合细菌三种不同抗原 (Ag1 、Ag2 、Ag3 )在一定程度上都有刺激脾淋巴细胞转化功能 ,特别是 Ag3 作用更为明显 ;在正常组 ,刺激指数提高了 6 5 % ;免疫抑制组 ,提高了 38% (P<0 .0 5 )。不管在正常组或免疫抑制组 ,Ag1 、Ag2 、Ag3 都具有诱生脾淋巴细胞产生 IL- 2的活性 ,其中 Ag2 的活性较明显 .结论 :类球红细菌 1号株对机体有一定的免疫活性。     

Structure-activity relationships of some hederagenin diglycosides: haemolysis, cytotoxicity and apoptosis induction

Hederagenin saponins are largely represented in nature and possess many biological activities such as haemolytic, antiviral, fungicidal, molluscicidal or cytotoxic, partially due to their interaction with the cell membrane. The lysis of erythrocytes (haemolysis) is a simple test to evaluate this adsorption, and this activity has been linked to the structure of the aglycone and also depends on the sugar moiety of the saponin. To further complete our study of the structure-activity relationships of triterpenoid saponins, alpha-hederin and related hederagenin diglycosides were synthesized to better understand the influence of the second sugar (alpha-L-rhamnose, beta-D-xylose or beta-D-glucose) and the substitution of this sugar on alpha-L-arabinose (position 2, 3 or 4). Haemolysis and cytotoxic activity on KB cells were tested. These compounds probably interact with membrane cholesterol and produce destabilization of the membrane inducing haemolysis. Cytotoxicity could involve the same mechanism, although some saponins induce an apoptotic process. The nuclear structure of the KB cell was thus investigated by confocal microscopy. The cytotoxic activity of a second group of hederagenin glucoside saponins was also evaluated. Our results showed that cytotoxicity was a result of both the sugar part and the structure of genin (carboxylic acid or methyl ester).     

Use of tetrameric antibody complexes to stain cells for flow cytometry

Bifunctional tetrameric complexes of monoclonal antibodies were used to stain cells for flow cytometry. These complexes consist of two different mouse monoclonal IgG1 antibodies (one with specificity for a cell surface antigen, the other with specificity for a fluorochrome) cross-linked by two molecules of a monoclonal rat anti-mouse IgG1. The use of this immunological approach to cross-link fluorochromes to cell surface antigens was studied with tetrameric complexes containing Leu-3a or Leu-2a antibodies and monoclonal antibodies specific for the fluorochromes B- and R-phycoerythrin. The ability of such cyclic immune complexes to stain T-cell subset antigens on human peripheral blood lymphocytes was demonstrated in single and double-staining experiments. The results demonstrate that tetrameric antibody complexes provide a simple and efficient alternative to covalently labeled antibodies for the flow cytofluorimetric analysis of cell-surface antigens.     

Structure–activity relationships of some hederagenin diglycosides: Haemolysis,cytotoxicity and apoptosis induction

Hederagenin saponins are largely represented in nature and possess many biological activities such as haemolytic, antiviral, fungicidal, molluscicidal or cytotoxic, partially due to their interaction with the cell membrane. The lysis of erythrocytes (haemolysis) is a simple test to evaluate this adsorption, and this activity has been linked to the structure of the aglycone and also depends on the sugar moiety of the saponin. To further complete our study of the structure–activity relationships of triterpenoid saponins, α-hederin and related hederagenin diglycosides were synthesized to better understand the influence of the second sugar (α-l-rhamnose, β-d-xylose or β-d-glucose) and the substitution of this sugar on α-l-arabinose (position 2, 3 or 4). Haemolysis and cytotoxic activity on KB cells were tested. These compounds probably interact with membrane cholesterol and produce destabilization of the membrane inducing haemolysis. Cytotoxicity could involve the same mechanism, although some saponins induce an apoptotic process. The nuclear structure of the KB cell was thus investigated by confocal microscopy. The cytotoxic activity of a second group of hederagenin glucoside saponins was also evaluated. Our results showed that cytotoxicity was a result of both the sugar part and the structure of genin (carboxylic acid or methyl ester).     

Occurrence of Conjugated Dienoic Fatty Acids in the Cellular Lipids of Pediococcus homari

Ninety-two thiolcarbamates with various substituents at the nitrogen and sulfur atoms, and their related compounds were synthesized, and their fungicidal activity against rice blast, Piricularia oryzae, and herbicidal activity against barnyardgrass, Echinochloa crus-galli, were determined in laboratory tests. The thiolcarbamate structure was necessary for the high fungicidal and herbicidal activities. The hydrophobicity of the substituents at the nitrogen atom was shown by the adaptive least-squares (ALS) method to be favorable to the fungicidal activity. The bulkier the substituents at the nitrogen atom, the less was the fungicidal activity. However, bulkiness of the substituents at the nitrogen and sulfur atoms was unfavorable to the herbicidal activity. The existence of a hydrogen atom at the nitrogen atom was favorable to fungicidal activity, but not to herbicidal activity. Correlation analyses were made to find compounds with both fungicidal and herbicidal activities against rice pests.     

Role of TNF-alpha in the induction of fungicidal activity of mouse peritoneal exudate cells against Cryptococcus neoformans by IL-12 and IL-18

We have recently demonstrated that two IFN-gamma-inducing cytokines, interleukin (IL)-12 and IL-18, synergistically induced the fungicidal activity of mouse peritoneal exudate cells (PEC) against Cryptococcus neoformans through NK cell production of interferon (IFN)-gamma and nitric oxide (NO) synthesis. In the present study, we further dissected these effects by examining the involvement of tumor necrosis factor (TNF)-alpha in the induction of IL-12/IL-18-stimulated PEC fungicidal activity. The addition of neutralizing anti-TNF-alpha mAb significantly suppressed IL-12/IL-18-stimulated PEC anticryptococcal activity. This effect was ascribed to the inhibition of macrophage NO synthesis, but not of IFN-gamma production by NK cells, because the same treatment inhibited the former response, but not the latter one. On the other hand, combined treatment with IL-12 and IL-18 synergistically induced the production of TNF-alpha by PEC and this effect was almost completely abrogated by neutralizing anti-IFN-gamma mAb. The cell type producing TNF-alpha among PEC was mostly macrophage. TNF-alpha significantly promoted macrophage NO production and anticryptococcal activity induced by IFN-gamma, and furthermore anti-TNF-alpha mAb partially inhibited these responses. Considered together, our results indicated that TNF-alpha contributed to the potentiation of IL-12/IL-18-induced PEC fungicidal activity against C. neoformans through enhancement of IFN-gamma-induced production of NO by macrophages, but not through increased production of IFN-gamma by NK cells.     

Reactive oxygen species modulate itraconazole-induced apoptosis via mitochondrial disruption in Candida albicans

Itraconazole (ITC), a well-known fungistatic agent, has potent fungicidal activity against Candida albicans. However, its mechanism of fungicidal activity has not been elucidated yet, and we aimed to identify the mechanism of ITC against C. albicans. ITC caused cell shrinkage via potassium leakage through the ion channel. Since shrunken cells could indicate apoptosis, we investigated apoptotic features. Annexin V-FITC and TUNEL assays indicated that fungicidal activity of ITC was involved in apoptosis. Subsequently, we confirmed an intracellular factor that could cause apoptosis. ITC treatment caused reactive oxygen species (ROS) accumulation. To confirm whether ROS is related with ITC-triggered cell death, cell viability was examined using the ROS scavenger N-acetylcysteine (NAC). NAC pretreatment recovered ITC-induced cell death, indicating that antifungal activity of ITC is associated with ROS, which is also confirmed by impaired glutathione-related antioxidant system and oxidized intracellular lipids. Moreover, ITC-induced mitochondrial dysfunction, in turn, triggered cytochrome c release and metacaspase activation, leading to apoptosis. Unlike the only ITC-treatment group, cells with NAC pretreatment did not show significant damage to mitochondria, and attenuated apoptotic features. Therefore, our results suggest that ITC induces apoptosis as fungicidal mechanism, and intracellular ROS is major factor to trigger the apoptosis by ITC in C. albicans.     

Interleukin-15 increases Paracoccidioides brasiliensis killing by human neutrophils

Interleukin-15 is a cytokine produced by a wide range of different cell types, including macrophages, in response to lipopolysaccharide or microbial infection. This cytokine may play a crucial role in the activation of phagocytic cells against pathogens, especially during innate immune response. The effects of IL-15 on human polymorphonuclear leukocyte fungicidal activity against a highly virulent Paracoccidioides brasiliensis strain were investigated. Pretreatment of human neutrophils from healthy individuals with IL-15 for 18 hours increased cell fungicidal activity in a dose-dependent manner. In addition, the exposure to IL-15 induced an increase in neutrophil oxidative burst as evaluated by superoxide anion and H(2)O(2) release. Catalase inhibited fungicidal activity supporting a role for H(2)O(2) in fungus killing. In contrast, IL-8 and TNF-alpha levels were not affected by IL-15 suggesting that its effects were not mediated by these cytokines. Together, these results show that IL-15 is a potent stimulant of antifungal activities in human neutrophils, at least in part by a mechanism dependent on oxidative metabolism.     

Neutron activation increases activity of ruthenium-based complexes and induces cell death in glioma cells independent of p53 tumor suppressor gene

Novel metal complexes have received great attention in the last decades due to their potential anticancer activity. Notably, ruthenium-based complexes have emerged as good alternative to the currently used platinum-based drugs for cancer therapy, providing less toxicity and side effects to patients. Glioblastoma is an aggressive and invasive type of brain tumor and despite of advances is the field of neurooncology there is no effective treatment until now. Therefore, we sought to investigate the potential antiproliferative activity of phosphine-ruthenium-based complexes on human glioblastoma cell lines. Due to its octahedral structure as opposed to the square-planar geometry of platinum(II) compounds, ruthenium(II) complexes exhibit different structure–function relationship probably acting through a different mechanism from that of cisplatin beyond their ability to bind DNA. To better improve the pharmacological activity of metal complexes we hypothesized that neutron activation of ruthenium in the complexes would allow to decrease the effective concentration of the compound needed to kill tumor cells. Herein we report on the effect of unmodified and neutron activated phosphine ruthenium II complexes on glioblastoma cell lines carrying wild-type and mutated p53 tumor suppressor gene. Induction of apoptosis/authophagy as well as generation of reactive oxygen species were determined. The phosphine ruthenium II complexes tested were highly active against glioblastoma cell lines inducing cell death both through apoptosis and autophagy in a p53 independent fashion. Neutron activation of ruthenium compounds rendered them more active than their original counterparts suggesting a new strategy to improve the antitumor activity of these compounds.     

Effect of cytokines on the in vitro fungicidal activity of monocytes from paracoccidioidomycosis patients

Peripheral blood monocytes obtained from paracoccidioidomycosis patients and healthy individuals were preactivated with recombinant gamma interferon (IFN-gamma) in different concentrations (250, 500 and 1000 U/ml) and evaluated for fungicidal activity against Paracoccidiodes brasiliensis strain 18 (Pb 18, high-virulence strain) and strain 265 (Pb 265, low-virulence strain) by plating of cocultures and counting of colony-forming units, after 10 d. Monocytes from healthy individuals failed to present fungicidal activity against P. brasiliensis even after IFN-gamma activation at the three concentrations. However, patient monocytes activated with IFN-gamma (1000 U/ml) showed a significant fungicidal activity when compared to that obtained with non-activated or activated cells with other IFN-gamma concentrations (250 and 500 U/ml). Moreover, patient monocytes presented higher fungicidal activity than the control, even before the activation process. These results may be explained by the activation state of patients' cells as a function of the in vivo contact with the fungus, which was confirmed by their higher capacity to release H(2)O(2) in vitro. Unlike the results obtained with Pb 18, patient and control cells presented a significant fungicidal activity against Pb 265, after priming with IFN- gamma. These results are explained by the higher levels of TNF-alpha in supernatants of cultures challenged with Pb 265. Moreover, higher levels of the cytokine were obtained in patient cell supernatants. Taken together, our results suggest that for effective killing of P. brasiliensis by monocytes, an initial activation signal induced by IFN-gamma is necessary to stimulate the cells to produce TNF-alpha. This cytokine may be involved, through an autocrine pathway, in the final phase activation process. The effectiveness of this process seems to depend on the virulence of the fungal strain and the activation state of the challenged cells.     

Platinum complexes with anticancer potential and their evaluation by a colorimetric lambda prophage induction assay

A simple biochemical phage induction assay (BIA) showed significant activity with 90% of the antitumor platinum compounds tested and lack of activity for all Pd(II) compounds and Pt(II) cationic complexes, compounds that are expected to be inactive. Structure-activity relationships for a large number of chemicals can be studied simultaneously by this simple, rapid, inexpensive and quantitative biochemical assay. Fifty-three platinum complexes were tested, including a number of ethylenediamines synthesized for this work. The magnitude of inducing activity varied over a 25-fold range; differences among analogs reflected structural differences in a chemically consistent manner. Seven platinum complexes showed greater activity than that of cis-diamminedichloroplatinum(II) (cisplatin, cis-DDP), while other compounds appeared to be substantially less toxic. The assay was predictive for most compounds with very high or very low activity in vivo against L1210. For compounds with intermediate levels of activity, no correlation between inducing and antitumor activity was observed.     

Induction of alpha interferon by membrane interaction between viral surface and peripheral blood mononuclear cells

Cells infected with viruses and fixed when viral antigens appeared at the cell membrane induced much higher alpha interferon (IFN-alpha) levels in human peripheral blood mononuclear cells (PBMC) than free virions. Relatively few inducer cells were sufficient for triggering IFN production. Optimal IFN yields depended on inducer/producer cell ratio. The response was peculiar to PBMC as it was not found in other cells in which IFN can normally be induced by free virions. IFN inducing activity was also exerted by live virus-infected PBMC, showing that this type of induction may have physiological importance. These findings confirm that viral induction of IFN-alpha is activated by some interaction between viral components presented at the cell surface and PBMC membrane. Thus induction of IFN by circulating cells infected by viruses and presenting viral antigens at the surface may be an efficient host defense mechanism. Since IFN yields close to 10(6) international units per milliliter are obtained, this system has potential for large scale production of native IFN-alpha.     

Antifungal activity of alkanols: inhibition of growth of spoilage yeasts

Primary aliphatic alkanols from C₆ to C₁₃ were tested for their antifungal activity against Saccharomyces cerevisiae using a broth dilution method. Undecanol (C₁₁) was found to be the most potent fungicide against this yeast with the minimum fungicidal concentration (MFC) of 25 μg/ml (0.14 mM), followed by decanol (C₁₀) with the minimum inhibitory concentration (MIC) of 50 μg/ml (0.31 mM). The time-kill curve study showed that undecanol was fungicidal against S. cerevisiae at any growth stages. This fungicidal activity was not influenced by pH values. Dodecanol (C₁₂) was the most effective fungistatic but did not show any fungicidal activity up to 1600 μg/mL. Fungistatic dodecanol quickly reduced cell viability, but the cell viability recovered shortly after and then finally became no longer different from the control indicating that the effect of dodecanol on S. cerevisiae was classified as a sublethal damage. However, fungistatic dodecanol combined with sublethal amount of anethole showed a fungicidal activity against this yeast. Anethole completely restricted the recovery of cell viability. Therefore expression of the synergistic effect was probably due to the blockade of the recovering process from dodecanol induced-stress. The alkanols tested inhibited glucose-induced acidification by inhibiting the plasma membrane H⁺-ATPase. Octanol (C₈) increased plasma membrane fluidity in the spheroplast cells of S. cerevisiae. The same series of aliphatic primary alkanols was also tested against a food spoilage fungus Zygosaccharomyces bailii and compared with their effects against S. cerevisiae. Decanol was found to be the most potent fungicide against Z. bailii with an MFC of 50 μg/ml (0.31 mM), whereas undecanol was found to be the most potent fungistatic with an MIC of 25 μg/ml (0.14 mM). The time-kill curve study showed that decanol was fungicidal against Z. bailii at any growth stage. This antifungal activity was slightly enhanced in combination with anethole. The primary antifungal action of medium-chain (C₉–C₁₂) alkanols comes from their ability as nonionic surfactants to disrupt the native membrane-associated function of the integral proteins. Hence, the antifungal activity of alkanols is mediated by biophysical process, and the maximum activity can be obtained when balance between hydrophilic and hydrophobic portions becomes the most appropriate.     

Characteristics of the reaction of healthy human blood serum with the epidermis

Indirect immunofluorescence has shown that the blood serum of normal subjects reacts with cytoplasm antigens of epidermis differentiated cells in 100% of the cases. The level of antibodies and the immunomorphological picture of the reaction are marked by high constancy and intense fluorescence of the cytoplasm of epidermis differentiated cells, while the reaction with basal layer cell antigens is observed comparatively seldom and little pronounced. The authors discuss possible participation of antibodies contained by the normal blood serum and of their complexes with tissue antigens in the regulation of vital activity of the cells and immune response to the host antigens.