Genomic and Metabolomic Polymorphism among Experimentally Selected Paromomycin-Resistant Leishmania donovani Strains.

Understanding the mechanism(s) underpinning drug resistance could lead to novel treatments to reverse the increased tolerance of a pathogen. In this study, paromomycin (PMM) resistance (PMMr) was induced in three Nepalese clinical strains of Leishmania donovani with different inherent susceptibilities to antimony (Sb) drugs by stepwise exposure of promastigotes to PMM. Exposure to PMM resulted in the production of mixed populations of parasites, even though a single cloned population was used at the start of selection. PMM 50% inhibitory concentration (IC50) values for PMMr parasites varied between 104 and 481 µM at the promastigote stage and 32 and 195 µM at the intracellular amastigote stage. PMM resistance was associated with increased resistance to nitric oxide at the amastigote stage but not the promastigote stage (P < 0.05). This effect was most marked in the Sb-resistant (Sbr) PMMr clone, in which PMM resistance was associated with a significant upregulation of glutathione compared to that in its wild type (P < 0.05), although there was no change in the regulation of trypanothione (detected in its oxidized form). Interestingly, PMMr strains showed an increase in either the keto acid derivative of isoleucine (Sb intermediate PMMr) or the 2-hydroxy acids derived from arginine and tyrosine (Sb susceptible PMMr and Sbr PMMr). These results are consistent with the recent finding that the upregulation of the branched-chain amino acid aminotransferase and d-lactate dehydrogenase is linked to PMMr In addition, we found that PMMr is associated with a significant increase in aneuploidy during PMM selection in all the strains, which could allow the rapid selection of genetic changes that confer a survival advantage.

What happened at Vienna's Allgemeines Krankenhaus after Semmelweis's contract as Assistant in the First Maternity Division was terminated?

Ignác Fülöp Semmelweis is famous for dramatically reducing puerperal mortality while he was an Assistant in Vienna's largest hospital, the Allgemeines Krankenhaus; he did this, mainly, by requiring medical personnel to disinfect their hands by washing in a chlorine solution. But Semmelweis was soon removed from his post as assistant. The conventional view, which is suggested by Semmelweis's own account, is that his contemporaries were skeptical of his results, that he was marginalized and that once he was no longer directly responsible for caring for maternity patients, puerperal mortality returned to its former high levels. In fact, the situation appears to have been quite different. In this paper, we calculate and discuss the number of deaths at the Allgemeines maternity clinic after Semmelweis was removed from his position. As we will see, his successors maintained a relatively low mortality rate roughly consistent with the rate Semmelweis himself achieved. This suggests that the chlorine washings were probably still used conscientiously after he left and that the opposition he encountered had other sources than doubts about the effectiveness of the chlorine washings.

Nanocarriers Made from Non-Ionic Surfactants or Natural Polymers for Pulmonary Drug Delivery.

BACKGROUND: Treatment by the pulmonary route can be used for drugs that act locally in the lungs (e.g. lung cancer) or non-invasive administration of drugs that act systemically (e.g. diabetes). The potential of using drug delivery systems (DDS) formed from non-ionic surfactants or natural products for pulmonary drug delivery are reviewed. METHODS: The effectiveness of each DDS depends on it ability to not only entrap the relevant drug and alter its bio distribution, but also its ability to withstand the physical stresses during nebulization and for the nebuliser to produce aerosol particles with the size for deposition in the appropriate part of the lungs. Different methods must be used to prepare nanoparticles (NP) using non-ionic surfactants, or biocompatible polymers from natural proteins or sugars, and the aqueous solubility of the drug also influences the manufacture method. RESULTS: NP produced using non-ionic surfactants, proteins such as collagen, albumin or gluten, and polysaccharides such as chitosan, hyaluronate, cellulose, carrageenans, alginate or starch has successfully delivered different types of drugs given by the pulmonary route. Drug entrapment efficiency depends on the DDS constituents and the manufacture method used. Large scale manufacture of DDS from natural products is technically challenging but changing from batch manufacture to continuous manufacturing processes has addressed some of these issues, and inclusion of a spray drying step has been beneficial in some cases. CONCLUSION: DDS for lung delivery can be produced using natural products but identifying a cost effective manufacture method may be challenging and the impact of using different type of nebulisers on the physiochemical characteristics of the aerosolised formulation should be an essential part of formulation development. This would ensure that some of the development work e.g. stability studies do not have to be repeated as they will identify if a carrier to protect the DDS from the physical trauma caused by nebulisation.

In vitro selection of miltefosine resistance in promastigotes of Leishmania donovani from Nepal: genomic and metabolomic characterization.

In this study, we followed the genomic, lipidomic and metabolomic changes associated with the selection of miltefosine (MIL) resistance in two clinically derived Leishmania donovani strains with different inherent resistance to antimonial drugs (antimony sensitive strain Sb-S; and antimony resistant Sb-R). MIL-R was easily induced in both strains using the promastigote-stage, but a significant increase in MIL-R in the intracellular amastigote compared to the corresponding wild-type did not occur until promastigotes had adapted to 12.2 µM MIL. A variety of common and strain-specific genetic changes were discovered in MIL-adapted parasites, including deletions at the LdMT transporter gene, single-base mutations and changes in somy. The most obvious lipid changes in MIL-R promastigotes occurred to phosphatidylcholines and lysophosphatidylcholines and results indicate that the Kennedy pathway is involved in MIL resistance. The inherent Sb resistance of the parasite had an impact on the changes that occurred in MIL-R parasites, with more genetic changes occurring in Sb-R compared with Sb-S parasites. Initial interpretation of the changes identified in this study does not support synergies with Sb-R in the mechanisms of MIL resistance, though this requires an enhanced understanding of the parasite's biochemical pathways and how they are genetically regulated to be verified fully.

Drug delivery: lessons to be learnt from Leishmania studies.

BACKGROUND: Leishmaniasis is a disease caused by infection with the protozoan parasite Leishmania, which is responsible for three main types of disease: cutaneous leishmaniasis, visceral leishmaniasis and mucocutaneous leishmaniasis based to the site of infection for the particular species. This presents a major challenge to successful drug treatment, as a drug must not only reach antileishmanial concentrations in infected macrophages, the parasites' host cell, but also reach infected cells in locations specific to the type of disease. In this paper we discuss how studies using Leishmania have contributed to our knowledge on how drug delivery systems can be used to improve drug efficacy and delivery.

Evaluating the potential of polyester nanoparticles for per oral delivery of amphotericin B in treating visceral leishmaniasis.

Leishmaniasis is a protozoan disease, which is responsible for response for major epidemics in many parts of the World. Amphotericin B (AMB) is one of the drugs used to treat leishmaniasis but it must be given intravenously and serious side effects such as nephrotoxicity can limit its use. Development of a formulation of AMB, which can be given by a non-invasive route but is still as effective as the conventional formulation, whilst causing minimal adverse side effects, is required. The present study describes a method for scale up production of a per oral nanoparticle formulation of AMB (AMB-NP) and compared its efficacy both in vitro and in vivo against Leishmania donovavni. Prophylactic studies showed that the AMB-NP formulation was significantly more effective (p < 0.05) than the same dose of AMB solution at suppressing parasite numbers compared to controls in bone marrow derived macrophages infected with L. donovani. Per oral treatment with AMB-NP resulted in a significant reduction in liver parasite burdens (p < 0.05) compared to control values and the formulation had a similar antileishmanial activity against parasites with different inherent susceptibilities to sodium stibogluconate.

The efficacy of aerosol treatment with non-ionic surfactant vesicles containing amphotericin B in rodent models of leishmaniasis and pulmonary aspergillosis infection.

Amphotericin B (AMB) is used to treat both fungal and leishmanial infections, which are of major significance to human health. Clinical use of free AMB is limited by its nephrotoxicity, whereas liposomal AMB is costly and requires parenteral administration, thus development of novel formulations with enhanced efficacy, minimal toxicity and that can be applied via non-invasive routes is required. In this study we analysed the potential of non-ionic surfactant vesicles (NIV) given by nebulisation to deliver AMB to the lungs, liver and skin. Treatment with AMB-NIV resulted in significantly higher drug levels in the lungs and skin (p<0.05) compared to similar treatment with AMB solution but significantly lower plasma levels (p<0.05). Treatment with AMB-NIV resulted in a significant reduction in fungal lung burdens in a rat model of invasive pulmonary aspergillosis (p<0.05) compared to treatment with the carrier alone. Treatment with AMB-NIV but not AMB solution significantly suppressed Leishmania donovani liver parasite burdens (p<0.05) but could not inhibit the growth of cutaneous Leishmania major lesions. The results of this study indicate that aerosolised NIV enhanced pulmonary and hepatic delivery whilst minimising systemic exposure and toxicity.

Comparative assessment of a DNA and protein Leishmania donovani gamma glutamyl cysteine synthetase vaccine to cross-protect against murine cutaneous leishmaniasis caused by L. major or L. mexicana infection.

Leishmaniasis is a major health problem and it is estimated that 12 million people are currently infected. A vaccine which could cross-protect people against different Leishmania spp. would facilitate control of this disease as more than one species of Leishmania may be present. In this study the ability of a DNA vaccine, using the full gene sequence for L. donovani gamma glutamyl cysteine synthetase (γGCS) incorporated in the pVAX vector (pVAXγGCS), and a protein vaccine, using the corresponding recombinant L. donovani γGCS protein (LdγGCS), to protect against L. major or L. mexicana infection was evaluated. DNA vaccination gave transient protection against L. major and no protection against L. mexicana despite significantly enhancing specific antibody titres in vaccinated infected mice compared to infected controls. Vaccination with the LdγGCS protected against both species but only if the protein was incorporated into non-ionic surfactant vesicles for L. mexicana. The results of this study indicate that a L. donovani γGCS vaccine could be used to vaccinate against more than one Leishmania species but only if the recombinant protein is used.

Vaccination with recombinant Leishmania donovani gamma-glutamylcysteine synthetase fusion protein protects against L. donovani infection.

Visceral leishmaniasis presents a serious health threat in many parts of the world. There is, therefore, an urgent need for an approved vaccine for clinical use to protect against infection. In this study, the ability of recombinant Leishmania donovani gamma-glutamyl cysteine synthetase protein (LdγGCS) alone or incorporated into a non-ionic surfactant vesicle (NIV) delivery system to protect against L. donovani infection was evaluated in a BALB/c mouse model. Immunization with LdγGCS alone or LdγGCS-NIV induced specific IgG1 and IgG2a antibodies compared to controls, with LdγGCS-NIV inducing significantly higher titers of both antibody classes (P < 0.05). Both formulations induced similar increases in splenocyte IFN-γ production following ex vivo antigen stimulation with LdγGCS compared with cells from control mice (P < 0.05). Similar levels of protection against infection were induced by LdγGCS alone and LdγGCS-NIV, based on their ability to suppress liver parasite burdens compared to control values (P < 0.01), indicating that using a carrier system did not enhance the protective responses induced by the recombinant protein. The results of this study indicate that LdγGCS may be a useful component in a vaccine against L. donovani.

DNA vaccination against the parasite enzyme gamma-glutamylcysteine synthetase confers protection against Leishmania donovani infection.

In this study the potential of using Leishmania donovani gamma-glutamylcysteine synthetase (glutamate-cysteine ligase, gamma-GCS) as a rational target for vaccine development was determined. Mice, immunised with plasmid containing the full gene sequence for gamma-GCS (pVAXgammaGCS) or plasmid alone (pVAX control), were challenged with a high dose of L. donovani amastigotes to give a stringent test of the ability of the vaccine to protect against infection. Vaccination with pVAXgammaGCS resulted in the production of specific IgG1 and IgG2a antibodies and resulted in significantly lower liver parasite burdens compared to controls. Protection was also associated with a significant increase in cell-mediated immunity, demonstrated as an increase in nitrite production by ConA stimulated splenocytes, an increase in the percentage of splenic CD3+CD4+ cells, and enhanced granuloma maturation, compared to control values.

Translation of an experimental oral vaccine formulation into a commercial product.

An effective experimental vaccine may fail to become a therapeutic reality for a number of scientific, regulatory or commercial reasons. In this review, we share some of our personal experiences as University-based researchers and provide an account of some of the problems that we have encountered during preliminary scale-up and assessment of an oral influenza vaccine formulation. Many of the problems we have faced have been non-scientific and related to identifying project-funding sources, finding suitable contract manufacturing companies that are GMP compliant, and protecting intellectual property generated from the scientific studies. The review is intended as a practical guide that will allow other researchers to adopt effective strategies to permit the translation of an effective experimental formulation to a viable commercial product.

Resistance of Leishmania donovani to sodium stibogluconate is related to the expression of host and parasite gamma-glutamylcysteine synthetase.

Sequencing studies showed that the gamma-glutamylcysteine synthetase (gamma-GCS) heavy chain genes from sodium stibogluconate (SSG)-resistant (SSG-R) and SSG-susceptible (SSG-S) Leishmania donovani strains were identical, indicating that SSG resistance was related to quantitative differences in gamma-GCS expression rather than gene interstrain polymorphisms. In vitro infection of murine macrophages with the SSG-R strain, but not the SSG-S strain, down regulated expression of host gamma-GCS, which would result in a reduction in intramacrophage glutathione (GSH) levels and promote an oxidative intramacrophage environment. This would inhibit, or minimize, the reduction of SSG pentavalent antimony to its more toxic trivalent form. Macrophage studies showed that the SSG-R strain expressed higher levels of gamma-GCS compared to the SSG-S strain, which would result in higher GSH levels, giving increased protection against oxidative stress and facilitating SSG efflux. However a similar differential effect on host and parasite gamma-GCS expression was not obtained when using tissues from infected mice. In this case gamma-GCS expression was organ and strain dependent for both the host and the parasite, indicating that environmental conditions have a profound effect on gamma-GCS expression. Consistent with the proposed mechanism from in vitro studies, increasing tissue GSH levels in the presence of SSG by cotreatment of L. donovani-infected mice with SSG solution and GSH incorporated into nonionic surfactant vesicles was more effective in reducing liver, spleen, and bone marrow parasite burdens than monotherapy with SSG. Together, these results indicate that SSG resistance is associated with manipulation of both host and parasite GSH levels by L. donovani.

Sodium stibogluconate resistance in Leishmania donovani correlates with greater tolerance to macrophage antileishmanial responses and trivalent antimony therapy.

Co-treatment of mice infected with different strains of Leishmania donovani with a non-ionic surfactant vesicle formulation of buthionine sulfoximine (BSO-NIV), and sodium stibogluconate (SSG), did not alter indicators of Th1 or Th2 responses but did result in a significant strain-independent up-regulation of IL6 and nitrite levels by stimulated splenocytes from treated mice compared to controls. The efficacy of BSO-NIV/SSG treatment was dependent on the host being able to mount a respiratory burst indicating that macrophages are important in controlling the outcome of treatment. In vitro studies showed that SSG resistance was associated with a greater resistance to killing by activated macrophages, treatment with hydrogen peroxide or potassium antimony tartrate. Longitudinal studies showed that a SSG resistant (SSG-R) strain was more virulent than a SSG susceptible (SSG-S) strain, resulting in significantly higher parasite burdens by 4 months post-infection. These results indicate that SSG exposure may favour the emergence of more virulent strains.

Pharmacokinetics, toxicities, and efficacies of sodium stibogluconate formulations after intravenous administration in animals.

The pharmacokinetics and toxicities of free sodium stibogluconate (SSG) and two vesicular formulations of this drug (a nonionic surfactant vesicular formulation of SSG [SSG-NIV] and SSG-NIV-dextran) were determined after treatment with a single intravenous dose in healthy dogs and were related to their antileishmanial efficacies in mice. Analysis of the curves of the concentrations in plasma after intravenous administration of SSG and SSG-NIV in dogs showed that both formulations produced similar antimony (Sb) pharmacokinetics. In contrast, treatment with SSG-NIV-dextran significantly modified the pharmacokinetics of the drug. The elimination half-life was four times longer (280 min) than that observed after administration of SSG (71 min) (P = 0.01), and the volume of distribution at steady state (V(SS)) was also increased (V(SS) for SSG, 0.21 liters/kg; V(SS) for SSG-NIV-dextran, 0.34 liters/kg [P = 0.02]), thus indicating that drug encapsulation favors the distribution of Sb into organs and increases its residence time in tissues. This would explain the superior antileishmanial efficacy of this formulation compared to those of the free drug in mice. No signs of toxicity were found in dogs after SSG and SSG-NIV administration. However, SSG-NIV-dextran treatment was associated with short-term toxicity, demonstrated by the development of chills and diarrhea, which cleared by 24 h postdosing, and hepatic dysfunction at 24 h postdosing (P < 0.05). The levels of all the biochemical parameters had returned to normal at 1 month postdosing. No signs of toxicity were observed in mice treated with all three formulations.

The in vivo susceptibility of Leishmania donovani to sodium stibogluconate is drug specific and can be reversed by inhibiting glutathione biosynthesis.

Resistance to pentavalent antimonial (Sb(v)) agents such as sodium stibogluconate (SSG) is creating a major problem in the treatment of visceral leishmaniasis. In the present study the in vivo susceptibilities of Leishmania donovani strains, typed as SSG resistant (strain 200011) or SSG sensitive (strain 200016) on the basis of their responses to a single SSG dose of 300 mg of Sb(v)/kg of body weight, to other antileishmanial drugs were determined. In addition, the role of glutathione in SSG resistance was investigated by determining the influence on SSG treatment of concomitant treatment with a nonionic surfactant vesicle formulation of buthionine sulfoximine (BSO), a specific inhibitor of the enzyme gamma-glutamylcysteine synthetase which is involved in glutathione biosynthesis, and SSG, on the efficacy of SSG treatment. L. donovani strains that were SSG resistant (strain 200011) and SSG sensitive (strain 200016) were equally susceptible to in vivo treatment with miltefosine, paromomycin and amphotericin B (Fungizone and AmBisome) formulations. Combined treatment with SSG and vesicular BSO significantly increased the in vivo efficacy of SSG against both the 200011 and the 200016 L. donovani strains. However, joint treatment that included high SSG doses was unexpectedly associated with toxicity. Measurement of glutathione levels in the spleens and livers of treated mice showed that the ability of the combined therapy to inhibit glutathione levels was also dependent on the SSG dose used and that the combined treatment exhibited organ-dependent effects. The SSG resistance exhibited by the L. donovani strains was not associated with cross-resistance to other classes of compounds and could be reversed by treatment with an inhibitor of glutathione biosynthesis, indicating that clinical resistance to antimonial drugs should not affect the antileishmanial efficacies of alternative drugs. In addition, it should be possible to identify a treatment regimen that could reverse antimony resistance.

The effect of nitrostyrene on cell proliferation and macrophage immune responses.

The use of mood enhancing drugs such as amphetamine and ecstasy are now prevalent in society. These compounds are known to produce serious psychological and physiological problems in users, which can, in some circumstances result in death. While there has been much research into the effects of these drugs on the body, little if any research has investigated the effect of the side products and synthetic reaction by-products which are a consequence of there illegal production. In the study the effects of nitrostyrene, a reaction by-product in one of the routes to synthesis of amphetamine sulphate, on cell viability and macrophage function was determined. Treatment with nitrostyrene at doses >0.75 microg/mL had a significant suppressive effect on the proliferation of stomach cancer lines. Treatment of macrophages with doses as high as 10 microg/mL did not effect cell viability. Nitrostyrene treatment of macrophages, stimulated with IFN gamma and LPS, resulted in a dose dependent differential inhibition in IL12, IL6 and nitrite production, even using doses < 0.5 microg/mL. Thus ranking of the three, on the basis of the suppressive effect obtained, is IL12 > nitrite > IL6. Thus ingestion of nitrostyrene contaminated ecstasy is likely to have a adverse effect on the immune responses of the recreational user.

Edwin Klebs's Grundversuche.

In 1876, discussions of the role of microorganisms in disease causation focused on anthrax and wound infections, and even in respect to these diseases there was controversy. In a series of papers on the pathologicality of bacteria, Edwin Klebs identified four "Grundversuche" (fundamental tests) that provided a basis for his own research strategy. The Grundversuche can be read as the following hypotheses: first, all bacteria are pathological; second, bacteria never occur spontaneously; third, every disease is caused only by bacteria; and fourth, the bacteria that cause distinguishable disease are distinguishable. Although some of these hypotheses are literally false, together they constitute part of a rational basis for a bacterial theory of disease, and, as such, they mark a true revolution in etiological thought.

Efficacies of vesicular and free sodium stibogluconate formulations against clinical isolates of Leishmania donovani.

In this study, the in vitro and in vivo efficacies of free sodium stibogluconate (SSG) and a nonionic surfactant vesicular formulation of SSG (SSG-NIV) against a laboratory strain of Leishmania donovani (MHOM/ET/67:LV82) and different clinical isolates of L. donovani were determined. Treatment with SSG-NIV was more effective against intramacrophage amastigotes than treatment with SSG. In vivo murine studies showed that there was interstrain variability in the infectivity of the different L. donovani strains, with two of the strains (20001 and 20003) giving low parasite burdens. In addition, interstrain variability in the antileishmanial efficacy of SSG in a single dose containing 300 mg of Sb(V)/kg of body weight was observed. This dose of free drug either caused a >97% reduction in liver parasite burdens or had no significant effect on parasite burdens compared with the result with the respective control. In some instances, treatment with this free SSG dose also caused a significant reduction in spleen (strain 20006) or bone marrow (strains 20001 and 20009) parasite burdens. Treatment with SSG-NIV was more effective than that with SSG against all of the strains tested. In SSG-responsive strains, the reduction in liver parasite burdens by SSG-NIV treatment was similar to that caused by free SSG. In SSG-nonresponsive strains, SSG-NIV treatment caused at least a 95% reduction in liver parasite burdens. Overall, these results indicate that the use of a vesicular formulation of SSG is likely to increase its clinical efficacy against visceral leishmaniasis.

Endogenous IL-4 is necessary for effective drug therapy against visceral leishmaniasis.

It is well established that a fully competent immune response is required for the successful drug treatment of visceral leishmaniasis. However, recent studies have cast some doubt as to which elements of the immune response synergize with chemotherapeutic treatment. The role of the Th2 response and IL-4 in particular during visceral leishmaniasis awaits clarification. We, therefore, examined the effectiveness of sodium stibogluconate treatment on Leishmania donovani infection in BALB/c wild-type and IL-4-/- mice. Parasite burdens in L. donovani-infected IL-4+/- and IL-4-/-, as we have previously shown for B6/129 mice, were similar, despite an apparent type 1 antibody response in infected IL-4-/- mice, demonstrated by increased levels of parasite-specific IgG2a and decreased IgG1. Unexpectedly IL-4-/- mice responded poorly to sodium stibogluconate treatment with increased parasite burdens in all tissues examined. Furthermore, drug therapy of IL-4-/- but not IL-4+/+ mice resulted in significant reductions in splenocyte IFN-gamma mRNA transcripts and in serum IFN-gamma levels. These results demonstrate that IL-4 has an important role in effective anti-leishmanial chemotherapy which seems to be related to modulation of IFN-gamma production.