The importance of different murine cell types in the immunopotentiation produced by amphotericin methyl ester.

Amphotericin R (AmB) or its methyl ester (AME) are potent stimulants of humoral and cell-mediated immunity in mice. When AME is injected simultaneously with antigen, it augments humoral immunity by expansion of the primary as well as the memory B cell pool. The most consistent and dramatic effect of amphotericin on humoral immunity is the enhancement of secondary IgG responses. This effect on the IgM-IgG switch mechanism depends on T cells and is reduced following thymectomy on adult mice. The route and timing of amphotericin administration also have dramatic quantitative, effects on immunopotentiation. Neither polyclonal B cell activation nor stimulation of antigen uptake in vitro by peritoneal macrophages was observed following intraperitoneal injection of AME. A tentative hypothesis is that antigen-stimulated T cells are important sites of the immunostimulant effects of AME on murine immune responses.     

The relationship between adjuvant and mitogenic effects of amphotericin methyl ester

The antifungal polyene amphotericin B (AmB) and its methyl ester derivative (AME) both show potent murine immunostimulant as well as B-cell activating effects. Under certain experimental conditions, AME is a much more potent polyclonal B-cell activator (PBA) than AmB. Notable features of the murine B-cell stimulation induced by AME include: (i) High concentrations of AME (50-100 microgram/ml) are required and even at this level exhibit little or no spleen cell toxicity. (ii) Several lines of evidence suggest that the B-cell activating properties of AME are not involved in the cellular mechanism of adjuvant activity in vivo. (iii) There is a strong correlation between the magnitude of the in vitro PBA effects and the in vivo adjuvant effects of AME in a survey of different mouse strains. This evidence suggests that there is genetic control of the murine lymphoid cell-stimulatory effects of AME and that a small number of genes determines the responsive phenotype.     

Minireview Murine models for Acquired Immune Deficiency Syndrome

The mouse has been suggested as a host for comparative studies of several aspects of Human Acquired Immune Deficiency Syndrome (AIDS). Models include studies where part or all of the genome of Human Immunodeficiency Virus (HIV) has been incorporated into murine DNA in living mice. However, the most promising oppurtunities for study of immunological changes, vaccine development, cofactor involvement in disease, and anti-retroviral and immunostimulatory drug testing involve infection with murine retroviruses which cause many functional changes similar to AIDS. The viruses' effects on immune systems are reviewed with special emphasis on LP-BM5 murine leukemia virus which infects T and B cells, and macrophages. LP-BM5 infection suppresses cell functions while causing polyclonal lymphocyte activation. Murine immunological characterization, availability of inbred mouse strains, economy of using mice versus primates or humans models, and similarity of immune change caused by murine retroviruses compared to those seen in AIDS caused by HIV encourage rapid development of the LP-BM5 murine leukemia model.     

Murine models for acquired immune deficiency syndrome

The mouse has been suggested as a host for comparative studies of several aspects of Human Acquired Immune Deficiency Syndrome (AIDS). Models include studies where part or all of the genome of Human Immunodeficiency Virus (HIV) has been incorporated into murine DNA in living mice. However, the most promising opportunities for study of immunological changes, vaccine development, cofactor involvement in disease, and anti-retroviral and immunostimulatory drug testing involve infection with murine retroviruses which cause many functional changes similar to AIDS. The viruses' effects on immune systems are reviewed with special emphasis. LP-BM5 murine leukemia virus which infects T and B cells, and macrophages. LP-BM5 infection suppresses cell functions while causing polyclonal lymphocyte activation. Murine immunological characterization, availability of inbred mouse strains, economy of using mice versus primates or humans models, and similarity of immune change caused by murine retroviruses compared to those seen in AIDS caused by HIV encourage rapid development of the LP-BM5 murine leukemia model.     

Co-administration of immunomodulator tuftsin and liposomised nystatin can combat less susceptible Candida albicans infection in temporarily neutropenic mice

In order to develop a prospective chemotherapeutic agent against opportunistic infections, it is important to know that host factors such as degree of immunological debility as well as recovery of immune functions to normality may contribute significantly to a successful elimination of the pathogens. We demonstrated previously that concomitant delivery of antimicrobial agents and immunomodulators to the pathogen harbouring-host contributes to the complete elimination of the deep-seated fungal infections (aspergillosis and candidiasis) in animals with normal immune status. Considering that neutropenic hosts are the main targets of such infections, it can be argued about the potential of the immunomodulator-based therapy in subjects with non-functional immune system. To resolve the hypothesis, we studied the role of immunomodulator tuftsin against experimental murine candidiasis in temporarily neutropenic Balb/c mice. The neutropenic mice were challenged with an isolate of Candida albicans that was showing less susceptibility to both free and liposomised-amphotericin B. The co-administration of tuftsin increased the efficiency of liposomised-polyene antibiotics (nystatin and amphotericin B) against experimental murine candidiasis in immunocompromised Balb/c mice. Pretreatment with liposomised tuftsin prior to C. albicans infection clearly enhanced protection against candidiasis, suggesting a prophylactic role of tuftsin in normal and temporarily neutropenic animals.     

Effects of amphotericin B on membrane permeability--kinetics of spin probe reduction

The effect of the polyene antibiotic amphotericin B on the permeability of both unilamellar and multilamellar model membranes is investigated. The method measures the loss of the electron paramagnetic resonance signal of a spin probe, trapped in the aqueous compartment of a lipid dispersion, upon addition of ascorbate ions to the bulk aqueous phase. Amphotericin B causes large increases in the permeability of cholesterol-containing egg phosphatidylcholine membranes, whereas the effects are small in the absence of sterol and do not depend on surface charge. The effect of amphotericin depends upon the antibiotic:sterol mole ratio. The antibiotic appears to be unable to cross the membrane, acting only on the outermost bilayer of a multibilayer dispersion. When a phospholipid in the gel phase is used, amphotericin B causes large increases in permeability, independently of the presence or absence of sterol. It is suggested that the mechanism of action of amphotericin B is different for lipids in the liquid crystalline or gel states.     

紫菜多糖对免疫细胞及肿瘤细胞生长的影响（英文）

采用细胞培养技术测定从条斑紫菜中得到的多糖PY3对小鼠免疫细胞及人肿瘤细胞K562生长的影响。结果表明,PY3对小鼠骨髓细胞和脾脏淋巴细胞的增殖以及对混合淋巴细胞反应均有一定的促进作用。PY3对血癌细胞K562的生长有一定的抑制作用,研究表明多糖PY3不仅能够提高小鼠免疫细胞的功能,而且有一定的抗肿瘤作用。     

The antifungal drug amphotericin B promotes inflammatory cytokine release by a Toll-like receptor- and CD14-dependent mechanism

Amphotericin B is the most effective drug for treating many life-threatening fungal infections. Amphotericin B administration is limited by infusion-related toxicity, including fever and chills, an effect postulated to result from proinflammatory cytokine production by innate immune cells. Because amphotericin B is a microbial product, we hypothesized that it stimulates immune cells via Toll-like receptors (TLRs) and CD14. We show here that amphotericin B induces signal transduction and inflammatory cytokine release from cells expressing TLR2 and CD14. Primary murine macrophages and human cell lines expressing TLR2, CD14, and the adapter protein MyD88 responded to amphotericin B with NF-kappaB-dependent reporter activity and cytokine release, whereas cells deficient in any of these failed to respond. Cells mutated in TLR4 were less responsive to amphotericin B stimulation than cells expressing normal TLR4. These data demonstrate that TLR2 and CD14 are required for amphotericin B-dependent inflammatory stimulation of innate immune cells and that TLR4 may also provide stimulation of these cells. Our results provide a putative molecular basis for inflammatory responses elicited by amphotericin B and suggest strategies to eliminate the acute toxicity of this drug.     

A leukemia virus-related protein in the murine pancreas

A protein that has been detected in the granules of islet cells in the murine pancreas is similar but not identical to the endogenous murine leukemia virus envelope protein gp70. The pancreatic protein was detected by several immunological methods using both polyclonal and monoclonal anti-murine gp70. On purification by affinity chromatography, it was shown to be different from murine gp70 in its subcellular location and its molecular size and the size of its precursor and by the effect of various reagents on its immunological activity as determined by the ELISA assay.     

Interaction between Candida agglutinins and antifungal agents

Antifungal agents alter the function and morphology of Candida cell membranes and cell walls. We observed that brief (30 minute) exposure to either amphotericin B or clotrimazole inhibited the agglutination of Candida blastoconidia by murine bronchoalveolar lavage fluid. This inhibition required continuous drug presence. Neither amphotericin nor clotrimazole inhibited Candida agglutination by concanavalin A or pooled human serum. These results demonstrate that antifungal drugs can produce rapid changes in the surface characteristics of some fungi.     

Ecotropic murine leukemia virus-induced fusion of murine cells.

Extensive fusion occurs upon cocultivation of murine fibroblasts producing ecotropic murine leukemia viruses (MuLVs) with a large variety of murine cell lines in the presence of the polyene antibiotic amphotericin B, the active component of the antifungal agent Fungizone. The resulting polykaryocytes contain nuclei from both infected and uninfected cells, as evidenced by autoradiographic labeling experiments in which one or the other parent cell type was separately labeled with [3H]thymidine and fused with an unlabeled parent. This cell fusion specifically requires the presence of an ecotropic MuLV-producing parent and is not observed for cells producing xenotropic, amphotropic, or dualtropic viruses. Mouse cells infected with nonecotropic viruses retain their sensitivity toward fusion, whereas infection with ecotropic viruses abrogates the fusion of these cells upon cocultivation with other ecotropic MuLV-producing cells. Nonmurine cells lacking the ecotropic gp70 receptor are not fused under similar conditions. Fusion is effectively inhibited by monospecific antisera to gp70, but not by antisera to p15(E), and studies with monoclonal antibodies identify distinct amino- and carboxy-terminal gp70 regions which play a role in the fusion reaction. The enhanced fusion which occurs in the presence of amphotericin B provides a rapid and sensitive assay for the expression of ecotropic MuLVs and should facilitate further mechanistic studies of MuLV-induced fusion of murine cells.     

Voriconazole, Combined with Amphotericin B, in the Treatment for Pulmonary Cryptococcosis Caused by C. neoformans (Serotype A) in Mice with Severe Combined Immunodeficiency (SCID)

Cryptococcosis is a subacute or chronic systemic mycosis with a cosmopolitan nature, caused by yeast of the genus Cryptococcus neoformans. The model of systemic cryptococcosis in mice with severe combined immunodeficiency (SCID) is useful for immunological and therapeutic study of the disease in immunodeficient hosts. Amphotericin B, fluconazole and flucytosine are the drugs most commonly used to treat cryptococcosis. Voriconazole is a triazole with high bioavailability, large distribution volume, and excellent penetration of the central nervous system. The objective of this study was to evaluate treatment with amphotericin B (AMB), voriconazole (VRC), and AMB, used in combination with VRC, of experimental pulmonary cryptococcosis in a murine model (SCID). The animals were inoculated intravenously (iv) with a solution containing 3.0 × 10(5) viable cells of C. neoformans ATCC 90112, (serotype A). Treatments were performed with amphotericin B (1.5 mg/kg/day), voriconazole (40.0 mg/kg/day) and AMB (1.5 mg/kg/day) combined with VRC (40.0 mg/kg/day); began 1 day after the initial infection; were daily; and lasted 15 days. Evaluations were performed using analysis of the survival curve and isolation of yeast in the lung tissue. There was a significant increase in survival in groups treated with AMB combined with VRC, compared with the untreated group and groups receiving other treatments (P < 0.05). In the group treated only with VRC and AMB combined with VRC, there was a significant reduction (P < 0.05) in the isolation of C. neoformans in lung tissue. Amphotericin B combined with voriconazole may be an effective alternative to increasing survival and may reduce yeast in the lung tissue of mice with pulmonary cryptococcosis and SCID.     

Stimulation of murine lymphocyte proliferation by a phosphorothioate oligonucleotide with antisense activity for herpes simplex virus

To investigate further the immunological properties of nucleic acids, the mitogenicity of a phosphorothioate oligonucleotide (S-oligo 1082) with anti-sense activity for herpes simplex virus was tested. This compound stimulated proliferation and antibody production by murine lymphocytes in in vitro cultures. Proliferation was dose-dependent and unaffected by T cell depletion. Furthermore, inclusion of a non-mitogenic DNA in the medium did not block stimulation. Since 1082 does not have homology to a known gene involved in lymphocyte activation, these observations suggest that S-oligo antisense compounds may display non-specific activating effects, at least on murine B cells.     

Immunological memory and acquired immunodeficiency syndrome pathogenesis

Infection with the human immunodeficiency virus results in profound perturbations in immunological memory, ultimately resulting in increased susceptibility to opportunistic infections and acquired immunodeficiency syndrome (AIDS). We have used rhesus macaques infected with the simian immunodeficiency virus (SIV) as a model to understand better the effects of AIDS virus infection on immunological memory. Acute infection with SIV resulted in significant deficits in CD4+ helper responses to cytomegalovirus (CMV) as well as CMV-specific cytotoxic T-lymphocyte and neutralizing antibody responses. Reactivation of CMV was associated with high levels of SIV replication and suppression of both T-helper and cytotoxic responses to CMV. We have also studied the effects of SIV infection on T-cell turnover in non-human primates. T-cell turnover was evaluated using the nucleoside analogue bromodeoxyuridine (BrdU) in combination with five-colour flow cytometric analysis. T cells in normal animals turned over at relatively rapid rates, with memory cells turning over more quickly than naive cells. In SIV-infected animals, the labelling and elimination rates of both CD4+ and CD8+ BrdU-labelled cells were increased by two- to threefold compared with normal controls. Further analysis of immunological memory in non-human primates should offer the opportunity to extend immunological insights from murine models to the pathogenesis and prevention of AIDS.     

Effects of temperature and molecular interactions on the vibrational infrared spectra of phospholipid vesicles

Infrared spectra were obtained as a function of temperature for a variety of phospholipid/water bilayer assemblies (80% water by weight) in the 3000-950 cm^?1 region. Spectral band-maximum frequency parameters were defined for the 2900 cm^?1 hydrocarbon chain methylene symmetric and asymmetric stretching vibrations. Temperature shifts for these band-maximum frequencies provided convenient probes for monitoring the phase transition behavior of both multilamellar liposomes and small diameter single-shell vesiclesof dipalmitoyl phosphatidylcholine/water dispersions. As examples of the effects of bilayer lipid/cholesterol/water (3 : 1 mol ratio) and lipid/cholesterol/amphotericin B/water (3 : 1 : 0.1 mol ratios) vesicles were examined using the methylene stretching frequency indices. In comparison to the pure vesicle form, the transition width of the lipid/cholesterol system increased by nearly a factor of two (to 8°C) while the phase transition temperature remained approximately the same (41° C). For the lipid/cholesterol/amphotericin B system, the phase transition temperature increased by about 4.5° C (to 45.5°C) with the transition width increasing by nearly a factor of four ($\text{to}\text{≈ 15°}\text{C}$) above that of the pure vesicles. The lipid/cholesterol/amphotericin B data were interpreted as reflecting the formation below 38°C of a cholesterol/amphotericin B complex whose dissociation at higher temperature (38–60°C range) significantly broades the gel-liquid crystalline phase transition.     

Amine fluorescamine compounds inhibit oxidative phosphorylation in rat liver mitochondria

The reaction of fluorescamine with ammonia, benzylamine, o,p-dimethylbenzylamine, 2-phenylethylamine, p-aminobenzoic acid, and the mycosamine-containing macrolide antibiotic, amphotericin B, yield compounds which induce significant effects on mitochondrial activities. From their effects on energy-yielding processes which lead to transmembranous proton movements, the compounds may be divided into three classes. While all modifiers significantly inhibit proton movement induced by both ATP hydrolysis and electron transfer in mitochondria, their influence on the primary energy yielding steps are quite different. Class I modifiers, e.g., the compound made from amphotericin B, inhibit electron transfer but have no effect on the Pi release associated with ATP hydrolysis. Class II modifiers, e.g., the compound made from benzylamine, inhibit respiration but stimulate Pi release. Class III modifiers, e.g., the compound made from p-aminobenzoic acid, on the other hand, only slightly increase Pi release but have no effect on redox reactions. These and other effects of the modifiers are taken to mean that the proton movements and their associated energy-yielding processes are only linked indirectly. The effects of the modifiers on State 3 mitochondrial activities were also investigated. Although all the modifiers decrease the rates of both State 3 respiration and its coupled ATP synthesis, the efficiency of energy conversion measured by the P/O ratio remains unaltered.     

Canine leishmaniasis: evolution of the chemotherapeutic protocols

Dogs are the domestic reservoir for Leishmania infantum (syn.: L. chagasi), the parasite causing zoonotic visceral leishmaniasis (ZVL) in both the Old and New Worlds. In foci of canine leishmaniasis (CanL), symptomatic disease occurs in less than 50% of infected dogs, and is characterized by chronic evolution of viscero-cutaneous signs. Among strategies recommended to control ZVL, detection and drug treatment of infected dogs are usually employed in the endemic countries of southern Europe. However, the conventional antileishmanial drugs successfully used in human therapy, such as pentavalent antimonials, amphotericin B, pentamidine or miltefosine, have low efficacy in the treatment of CanL. In dogs, these drugs induce only temporary remission of clinical signs, do not prevent occurrence of relapses, and often cause severe side effects. Leishmaniotic dogs may be classified into 4 groups: 1) Asymptomatic resistant dogs ("contacted dogs"), 2) Asymptomatic dogs (preclinical), 3) Dogs with minimal signs of leishmaniasis (oligosymptomatic dogs? Chronic form of leishmaniasis?), 4) Dogs suffering from different forms of clinical leishmaniasis (symptomatic dogs). The dog's immunological status and the associated clinical signs may influence the efficacy of antileishmanial drugs. Subjects belonging to groups 2, 3 and 4 should be always treated, in order to reduce their parasite load. Parameters that must be considered before starting the antileishmanial treatment are hemogram, renal and hepatic functions, electrophoretic protein pattern, antileishmania antibody titres, and bone marrow and lymph node parasite load. The most common antileishmanial drugs currently used in Italy to treat CanL are pentavalent antimonials (meglumine antimoniate) and allopurinol, alone or in combination. Other used drugs are aminosidine (syn.: paromomycin), pentamidine, metronidazole and spyramicin. Each drug regimen has different duration, from a few weeks (aminosidine), to a few months (meglumine antimoniate) or several months (allopurinol). One of the most recent drug used in human VL is liposomal amphotericin B (AmBisome--L-AMB), a powerful antileishmanial drug in both experimental murine models and in VL patients. In Italy, L-AMB is now considered the drug of choice for the treatment of human cases. However, in HIV co-infected patients high doses of L-AMB are ineffective in obtaining a radical cure. In dogs, L-AMB treatment rapidly leads to clinical recovery but is uneffective to eliminate the parasites. Drugs containing amphotericin B should not be used in veterinary practice in order to avoid selection of parasites resistant to the drug, as it already occurred for the pentavalent antimonials. Currently, there is not a standard protocol for CanL treatment in Italy, as there is an extreme variability of proposed dosages. Clinical studies on immunotherapeutics and new antileishmanial drugs, such as miltefosine and its derivates, are in progress.     

Efficacy of interferon-gamma and amphotericin B for the treatment of systemic murine histoplasmosis

The number of cases of systemic histoplasmosis has increased substantially in recent years, and improved therapy is needed. We examined the efficacy of immunomodulation with interferon (IFN)-gamma alone or in combination with a suboptimal regimen of amphotericin B for the treatment of primary systemic murine histoplasmosis. In the first study, BALB/c mice were infected with Histoplasma capsulatum G217B and treated with 10(5) U of IFN given every other day either preinfection and postinfection or only postinfection, alone or in combination with amphotericin B. IFN alone given subcutaneously (s.c.) postinfection prolonged survival over untreated controls (P < 0.01), whereas intravenous (i.v.) administration was ineffective. All combination regimens and amphotericin B alone significantly prolonged survival (P < 0.0001). The combination regimens of amphotericin B and IFN i.v. (pre- and postinfection) or IFN s.c. (postinfection) reduced the fungal burden in the liver and spleen; the latter regimen had superior efficacy in the spleen (P < 0.05) to either amphotericin B or IFN alone. After infection with a low-challenge inoculum, IFN given s.c. (pre- and postinfection) alone caused a significant reduction in fungal burden in the spleen (P < 0.001). In an acutely lethal model, combination regimens of IFN s.c. or i.v. and amphotericin B again prolonged survival (P < 0.01-0.001), with amphotericin B plus IFN given s.c. (pre- and postinfection) superior to all regimens (P < 0.05-0.01). This regimen also showed enhanced efficacy in causing the reduction of fungal burden in the spleen (P < 0.05). These results indicate that IFN in combination with AmB shows enhanced efficacy in the treatment of systemic histoplasmosis and support the potential utility of IFN as an adjunctive therapy.     

Feline Leukaemia Viral Antigens and Antisera to the Group Specific Antigens of the Murine Leukaemia Viruses

GEERING et al.¹ reported that feline leukaemia viruses shared one of the group specific antigens of the murine leukaemia viruses, gs-3, as detected by immunoprecipitation in agar gels with broadly reactive rat antisera to the group specific antigens of the murine leukaemia viruses (MuLV). Subsequently, they found that this shared group specific antigen was also present in the hamster and rat C-type viruses². Work by Schafer³ and our own immunodiffusion⁴ and complement fixation studies have confirmed the immunological reactivity between the feline leukaemia viral antigens and broad-reacting murine leukaemia group specific antisera. We have now applied this interspecies immunological reaction between the murine and feline C-type viruses to quantitative studies of the feline leukaemia viruses. Broad-reactive murine leukaemia-sarcoma group specific antisera prepared in rats by the induction of murine sarcoma virus (MSV) tumours^{5, 6} were found to be as useful and nearly as sensitive as a feline leukaemia-sarcoma specific, group specific antiserum for the in vitro detection and assay of the noncytopathogenic feline leukaemia virus (FeLV).     

Effect of cyclosporin A on immunological response in lungs of guinea pigs infected with Trichinella spiralis

The effects of cyclosporin A (CsA), a potent immunosuppressive drug with antiparasitic activity, on the innate immunological response in guinea pig lungs during an early period (6th and 14th days) after T. spiralis infection were studied. CsA treatment of T. spiralis-infected guinea pigs caused a significant attenuation of immunological response in lungs by decreasing lymphocyte infiltration into pulmonary alveolar space, inhibiting alveolar macrophage superoxide anion production and lowering both the production of NO metabolites measured in bronchoalveolar lavage fluid and expression of the iNOS protein in lung homogenates, allowing us to speculate that the T. spiralis-dependent immunological response is dependent on lymphocyte T function. Interestingly, CsA itself had a pro-inflammatory effect, promoting leucocyte accumulation and macrophage superoxide production in guinea pig lungs. This observation may have a relevance to the situation in patients undergoing CsA therapy. Macrophage expression of the iNOS protein, evaluated by immunoblotting was not influenced by treatment of animals with CsA or anti-TGF-antibody, indicating different regulation of the guinea pig and murine enzymes.