The drug resistance mechanisms in Leishmania donovani are independent of immunosuppression.

The protozoan parasite L. donovani resides inside macrophages as amastigotes and inflicts a potentially lethal disease visceral leishmaniasis (VL). Due to absence of a vaccine, chemotherapy with antimonials, amphotericin B, miltefosine or paromomycin remains the only option for treating VL. Prolonged treatment with a single drug resulted in parasite strains resistant to each of these drugs. As immuno-suppression characterizes the disease, we examined whether eliciting immunosuppressive cytokines is a mechanism of manifestation of drug-resistance. We infected BALB/c mice with the clinical isolates of L. donovani- BHU1066 (sensitive), NS2 (antimony-resistant), BHU1064 (miltefosine-resistant), BHU919 (Amphotericin B-resistant) and BHU1020 (paromomycin-resistant)- from the respective drug-unresponsive patients and assessed splenic parasite load and production of pro-inflammatory and anti-inflammatory cytokines. Although the splenic parasite loads in the drug-resistant L. donovani-infected BALB/c mice were higher than that observed in the drug-sensitive parasites-infected mice, the cytokine profiles were not significantly different between these two sets of mice. The drug-resistance in L. donovani results from innate drug modulation but perhaps not from host immune-suppressive cytokines.

Th-1 biased immunomodulation and synergistic antileishmanial activity of stable cationic lipid-polymer hybrid nanoparticle: biodistribution and toxicity assessment of encapsulated amphotericin B.

To address issues related to Amphotericin B (AmpB) clinical applications, we developed macrophage targeted cationic stearylamine lipid-polymer hybrid nanoparticles (LPNPs) with complementary characteristics of both polymeric nanoparticles and liposomes, for enhancement of therapeutic efficacy and diminishing toxic effect of encapsulated AmpB. The LPNPs (size 198.3 ± 3.52 nm, PDI 0.135 ± 0.03, zeta potential +31.6 ± 1.91 mV) provide core-shell type structure which has the ability to encapsulate amphiphilic AmpB in higher amount (Encapsulation efficiency 96.1 ± 2.01%), sustain drug release and stabilize formulation tremendously. Attenuated erythrocytes and J774A.1 toxicity of LPNPs demonstrated safe applicability for parenteral administration. Elevated macrophage uptake of LPNPs, rapid plasma clearance and higher drug allocation in macrophage abundant liver and spleen illustrated admirable antileishmanial efficacy of AmpB-LPNPs in vitro (IC50, 0.16 ± 0.04 µg AmpB/ml) and in vivo (89.41 ± 3.58% parasite inhibition) against visceral leishmaniasis models. Augmentation in antileishmanial activity due to Th-1 biased immune-alteration mediated by drug-free LPNPs which elevated microbicidal mediators of macrophages. Moreover, minimal distribution to kidney tissues and low level of nephrotoxicity markers (creatinine and BUN) demonstrated the safety profile of AmpB-LPNPs. Conclusively, reliable safety and macrophage directed therapeutic performance of AmpB-LPNPs suggest it as promising alternative to commercial AmpB-formulations for the eradication of intra-macrophage diseases.

Early detection of systemic candidiasis in the whole blood of patients with hematologic malignancies.

Systemic candidiasis is a significant cause of morbidity and mortality in patients with hematologic disorders. The aim of this study was to determine the prevalence of systemic candidiasis and the efficiency of the polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELIZA) method for the early detection of Candida spp. in patients with hematologic malignancies. From 2004 to 2006, 194 patients with hematologic malignancies were evaluated for systemic candidiasis. Collected blood samples were assayed using the PCR-ELISA method for the presence of the bands on ethidium bromide stained gel, and for hybridization with Candida spp. as well. The female-to-male ratio was 61:133, the mean age was 33.7 years, and the mean hospitalization period was 21.2 days. Twenty-five patients (12.9%) had positive PCR-ELISA results for systemic candidiasis. The etiologic agents were Candida albicans (21 cases), C. tropicalis (3 cases), and C. krusei (1 case). The mean interval of PCR-ELISA positivity in blood samples before the manifestation of clinical signs was 12.6 days. Fungal PCR-ELISA assay became negative after 14 days when patients were treated successfully with amphotericin B, and the assay remained positive until death when the treatment failed. The PCR-ELISA method can potentially serve as a useful tool for the management of patients suffering from hematologic malignancies and at risk for systemic candidiasis.

Enhanced immune response by amphotericin B following NS1 protein prime-oral recombinant Salmonella vaccine boost vaccination protects mice from dengue virus challenge.

A recombinant vaccine strain SL3261/pLT105 of attenuated aroA Salmonella enterica serovar Typhimurium SL3261 strain expressing a secreted dengue virus type 2 non-structural NS1 and Yersinia pestis F1 (Caf1) fusion protein, rNS1:Caf1, was generated. Immunological evaluation was performed by prime-boost vaccine regimen. Oral immunization of mice with 1 x 10(9)cfu of SL3261/pLT105 only induced low levels of NS1-specific antibody response and protective immunity following dengue virus challenge. The parenteral NS1 protein priming-oral Salmonella boosting protocol enhanced both NS1-specific serum IgG response and protective efficacy as compared to mice immunized with each type vaccine alone. Addition of an antifungal antibiotic amphotericin B (AmB) to Salmonella vaccine further enhanced the synergic effects of prime-boost vaccine regimen on the elicited NS1-specific serum IgG response and the protective efficacy. Together, the results demonstrated that the rNS1:Caf1 producing Salmonella SL3261/pLT105 strain fails to provide effective protection as an oral vaccine alone despite co-administration of AmB as an adjuvant capable of enhancing the immune responses, and moreover, the protein priming-oral Salmonella vaccine boosting approach in combination with AmB as an immunization regimen may have the potential to be further explored as an alternative approach for dengue vaccine development.

Secretion of proinflammatory cytokines and chemokines during amphotericin B exposure is mediated by coactivation of toll-like receptors 1 and 2.

Amphotericin B (AmB) is a ligand of toll-like receptor 2 (TLR2). Here, we demonstrate the participation of TLR1 in AmB-induced cell activation that led to the secretion of tumor necrosis factor alpha, interleukin 6 (IL-6), and IL-8. Hence, TLR2-TLR1 coactivation serves as the underlying mechanism for the proinflammatory toxicities associated with AmB.

Amphotericin-induced stridor: a review of stridor, amphotericin preparations, and their immunoregulatory effects.

BACKGROUND: Various adverse effects have been reported with the use of amphotericin B. The respiratory adverse effects include dyspnea, tachypnea, bronchospasm, hemoptysis, and hypoxemia. Stridor has not been previously reported with the use of amphotericin B. OBJECTIVE: To review the mechanism of action and reports of respiratory adverse effects for amphotericin B, the liposomal preparations of amphotericin B, and the differential diagnosis of stridor. DATA SOURCES: A MEDLINE search from 1966 to 2002 was performed to review the current literature for possible mechanisms and immunoregulatory effects related to the infusion of amphotericin B. RESULTS: Amphotericin B has been shown to increase tumor necrosis factor alpha (TNF-alpha) concentrations in macrophages. In addition, it induces prostaglandin E2 synthesis and increases the production of interleukin 1beta (IL-1beta) in mononuclear cells. The immunoregulatory effects of amphotericin B include increases in apoptosis, production of monocyte chemoattractant protein 1, superoxide anion, nitric oxide, and intercellular adhesion molecule 1 expression. CONCLUSIONS: Amphotericin B induces the production of TNF-alpha, interferon-gamma, and IL-1beta, which may potentiate its toxic effects. Some liposomal preparations induced lower levels of TNF-alpha and nitric oxide and may be useful in patients unable to tolerate amphotericin B deoxycholate.

Amphotericin B enzyme-linked immunosorbent assay.

Our purpose was to develop and characterize an enzyme-linked immunosorbent assay (ELISA) which could measure the concentration of amphotericin B in serum. Amphotericin B was assayed by competition ELISA. Multiwell ELISA plates coated with amphotericin B (1.0 micrograms/ml) conjugated to bovine serum albumin were used to test replicates of serum samples spiked with amphotericin B. Purified rabbit polyclonal antibody against amphotericin B (1.4 micrograms/ml) was added subsequent to the instillation of samples spiked with unknown amounts of amphotericin B. Experiments were performed to test the sensitivity, specificity, precision, and accuracy of the assay. The ability to measure lipid-associated amphotericin B was also evaluated in preliminary studies. Analysis of reference samples containing amphotericin B yielded a traditional sigmoidal curve. The limits of detection were 0.15 to 156 micrograms/ml. The sensitivity of the assay was affected by light and temperature exposure. Assay specificity was altered only by the presence of nystatin, a polyene antifungal agent similar to amphotericin B. Intrarun (coefficient of variation = 3.0%) and interrun (coefficient of variation = 12.8%) coefficients of variation were calculated and were comparable to those in similar assays. The assay's correlation coefficient (r = 0.907) demonstrated a statistically significant correlation between the optical density of the sample and the concentration of drug in the sample. The amphotericin B ELISA's ease, precision, and overall accuracy suggest that this assay could be used for assessments of serum amphotericin B concentrations. Multiple research questions concerning the role of serum amphotericin B concentrations in toxicity and efficacy have gone unanswered because of the labor-intensive nature of the assays which have been available to date. The ability to easily and rapidly measure 40 duplicate samples containing amphotericin B should also prove to be a distinct advantage for clinical research or reference laboratories in addressing these questions.

Candida albicans isoladas de pacientes com SIDA: sensibilidade "in vitro" aos agentes antifúngicos / Candida albicans from AIDS patients: susceptibility \"in vitro\" to antifungal agents

Resumo: Por meio da determinaçäo da CIM(Concentraçäo Inibitória Mínima) e da CFM(Concentraçäo Fungicoda Mínima), os autores verificaram a sensibilidade de candida albicans isoladas de pacientes com SIDA, frente a Anfotericina B, Nistatina, Cetoconazol e miconazol.Os autores näo verificaram a ocorrência de amostras resistente e discutem o fenômeno da resistência de C.albicans aos antifúngicos poliênicos e imidazólicos.

[Antibody interaction with the amphotericin channel]. / Vzaimodeistvie antitel s amfoteritsinovym kanalom.

The monoclonal antibodies against asymmetric channel formed in the lipid bilayer of polyene antibiotic amphotericin B and cholesterol after addition of the antibiotic to the compartment from the cis side of the membrane were obtained. The effect of the antibodies on ion conductance of the channel depends on the distribution of cholesterol in the membrane. When cholesterol was present on both sides of the lipid bilayer, three antibody molecules bound to the channel from the trans side of the membrane, thus markedly increasing the lifetime of the open state of the channel. When cholesterol was present in the cis monolayer only, the antibodies, added to the trans compartment of the cell, reduced the membrane conduction.

Acute immune hemolysis induced by a degradation product of amphotericin B.

We report here on an eight-year-old boy who first developed acute intravascular hemolysis following therapy with amphotericin B (AmB) and subsequently a delayed hemolytic transfusion reaction due to alloantibodies. Although there is as yet no evidence for metabolism of AmB in vivo, the hemolysis appeared to be the result of sensitization against a degradation product of the drug. The patient's serum contained a hemagglutinating IgM antibody that reacted with all red blood cells (RBC) tested in the presence of plasma obtained from patients receiving AmB (ex vivo antigen), but not in the presence of their urine, AmB itself, or with AmB-pretreated RBC. These findings indicate that the antibody was directed against a degradation product of AmB, presumably a trace metabolite, that has not yet been identified.

Antibodies affect the ionic conductance of channels formed by amphotericin B in a lipid bilayer.

For the first time poly- and monoclonal antibodies (class IgM) against the polyene antibiotic amphotericin B were obtained affecting the properties of a channel formed by the antibiotic and cholesterol in a lipid bilayer when amphotericin B was added to the solution at one (cis) side of the membrane. In the case of the symmetric distribution of cholesterol in the lipid bilayer, three molecules of monoclonal antibodies bind firmly to the channel at the trans-side of the membrane, thus strongly increasing the mean lifetime of the channel in the open state, and not changing practically the ion conductance of its open state. The antibodies did not alter the properties of these channels when added at the cis-side of the membrane as well as of the channels formed in the lipid bilayer when amphotericin B was added at both membrane sides. The antibodies obtained did not affect the conductance of channels in which amphotericin B and cholesterol were replaced with their analogs levorin and 5 alpha-androstan-3 beta-one, which points to a high specificity of the immunoglobulins isolated. When cholesterol was present only in the cis-monolayer of the lipid bilayer and was absent in the trans-monolayer, the same monoclonal antibodies when added at the trans-side of the membrane blocked the conductance of the channel formed by adding the antibiotic to the solution at the cis-side of the bilayer. The obtained evidence is of interest in elucidating the general features of interaction of antibodies with the ionic channels of cellular and model membranes.

Induction of amphotericin B-specific antibodies for use in immunoassays.

High-titered antisera specific for amphotericin B (AmB) were induced by immunization with a protein conjugate of the D-lysyl AmB methyl ester. These polyclonal anti-AmB antibodies reacted preferentially with AmB or the AmB methyl ester and discriminated sharply between nystatin and AmB. A solid-phase radioimmunoassay was developed with radioiodinated immunoglobulin G fractions derived from the anti-AmB antisera. This assay was capable of detecting AmB in the sera in the same concentration range that is regularly achieved during AmB treatment of systemic fungal infections. This study demonstrated the feasibility of immunoassays in measuring the concentration of AmB in blood and tissue fluids.

[Increase in the specific activity of killed influenza vaccines by using polyene antibiotics]. / Povyshenie spetsificheskoi aktivnosti ubitykh grippoznykh vaktsin s pomoshch'iu polienovykh antibiotikov.

It was shown experimentally that polyenic antibiotics, i. e. amphotericin B and sodium levorin markedly increased the specific immunogenic properties and interferonogenic activity of inactivated influenza virus vaccine prepared with various methods from highly reproductive recombinants. The rate of pneumonia and death from influenza among the vaccinated mice treated with inactivated influenza virus vaccine and one of the polyenic antibiotics was lower than that among the animals treated with the vaccine alone (P less than 0.05). Correlation between the increase in the immunological response, the decrease in the virus reproduction rate in the lungs and addition of the antibiotics into the vaccine was also observed. It is recommended that inactivated influenza virus vaccine be used in conjunction with polyenic antibiotics.

[Effect of amphoglucamine on humoral immunity indices]. / Vliianie amfogliukamina na nekotorye pokazateli gumoral'nogo immuniteta.

It was shown that amphoglucamine, a new Soviet polyenic antibiotic had no significant effect on the antibody titers in the reactions of agglutination, complement binding and precipitation, when administered at various periods of immunogenesis of rabbits immunized with killed C. immitis vaccine. No signific and difference in the serum complement titers of the immunized animals was found. The study on the protective properties of the immunized animal sera demonstrated that the antibiotic administered during immunogenesis rather inhibited the preventive properties of the immune sera. Amphoglucamine administered during immunogenesis suppressed intracutaneous allergic reactions to the antigen. The data should be considered in the diagnostic practice, since some times the allergic reactions are the only diagnostic sign.