Tolerance to NADH/NAD+ imbalance anticipates aging and anti-aging interventions.

Redox couples coordinate cellular function, but the consequences of their imbalances are unclear. This is somewhat associated with the limitations of their experimental quantification. Here we circumvent these difficulties by presenting an approach that characterizes fitness-based tolerance profiles to redox couple imbalances using an in silico representation of metabolism. Focusing on the NADH/NAD+ redox couple in yeast, we demonstrate that reductive disequilibria generate metabolic syndromes comparable to those observed in cancer cells. The tolerance of yeast mutants to redox disequilibrium can also explain 30% of the variability in their experimentally measured chronological lifespan. Moreover, by predicting the significance of some metabolites to help stand imbalances, we correctly identify nutrients underlying mechanisms of pathology, lifespan-protecting molecules, or caloric restriction mimetics. Tolerance to redox imbalances becomes, in this way, a sound framework to recognize properties of the aging phenotype while providing a consistent biological rationale to assess anti-aging interventions.

Comparison of conventional and lipid emulsion formulations of amphotericin B: Pharmacokinetics and toxicokinetics in dogs.

The major limiting factor in the use of amphotericin B (AmB) is cumulative nephrotoxicity. In previous studies, AmB mixed with Intralipid® 20% (AmB-IL), a parenteral fat emulsion, reduces its toxicity, increases its efficacy and is less expensive than other commercial amphotericin B lipid formulations. The pharmacokinetics and toxicity of the conventional deoxycholate AmB formulation (Fungizone®) and AmB-IL were compared in dogs. The pharmacokinetic of AmB was significantly modified and renal toxicity and infusion-related side effects were reduced when the drug was prepared in fat emulsion. In addition, pharmacokinetics and toxicity were evaluated after the administration of multiple doses of AmB-IL with the purpose of determining an optimal treatment protocol in dogs. When using a consecutive day administration regime, there was a significant drug accumulation together with an increase in creatinine values after each dose. However, when using three doses per week administration regime, similar maximum and minimum plasma concentrations were maintained. During the four weeks of treatment a moderate increase in the creatinine values was observed but none of the treatments were ended prematurely. All these data suggest that Intralipid®, similar to that seen previously in humans, favors AmB distribution to the organs, decreasing drug toxicity and increasing its therapeutic index in the dogs. The dose protocol evaluated (25mg/m2/48h/three times per week) produces maintenance of AmB plasma levels that were close to that obtained by others authors after administration of liposomal formulations of AmB and that have been demonstrated to be clinically effective.

Cytokines and chemokines measured in dried SLA-stimulated whole blood spots for asymptomatic Leishmania infantum and Leishmania donovani infection.

Whole blood stimulation with soluble Leishmania antigen (SLA), followed by plasma cytokine and chemokine determination, provides means of detecting subjects with asymptomatic Leishmania infection. This work examines the potential of Protein Saver 903 cards for the storage and transport of SLA-stimulated dried plasma spot samples. Blood was collected from asymptomatic and negative control subjects living in a Leishmania infantum- (Spain) and Leishmania donovani-endemic area (Bangladesh). After SLA-stimulation, three types of sample were prepared: frozen liquid plasma (-20 °C), and plasma dropped onto Protein Saver cards kept at -20 °C (DPS-FZ), and at ambient temperature (DPS-AT). The concentrations of IFN-γ, IL-2, CXCL10, CXCL9, CCL2 and CXCL8 in the thawed liquid plasma (TLP), DPS-FZ and DPS-AT samples were then determined. Strong correlations were seen between the TLP and DPS-FZ/AT samples for all the studied cytokines/chemokines in both the L. infantum and L. donovani areas. Protein Saver 903 cards would therefore appear to allow for the transport of SLA-stimulated plasma samples by courier at ambient temperature. The CXCL10 and CXCL9 detectable in these plasma spots provided robust markers for identifying asymptomatic subjects from both endemic areas. This easy procedure opens up new possibilities for field studies in resource-limited settings, which could help in Leishmania control.

Interleukin-2 as a marker for detecting asymptomatic individuals in areas where Leishmania infantum is endemic.

No field method exists for identifying asymptomatic individuals in areas where Leishmania infantum is endemic. This work reports that, 24 h after stimulating whole blood with soluble Leishmania antigen (SLA), plasma interferon-γ (IFN-γ) and interleukin-2 (IL-2) become significantly elevated in samples from asymptomatic individuals (n=47) compared with those from negative controls (n=50), all of them recruited from a blood bank. When compared with the reference test SLA-lymphoproliferative assay, IL-2 appears as a new, 100% sensitive and specific marker for asymptomatic individuals with a positive cellular response (compared with 100% and 84.78%, respectively, for IFN-γ). Further studies in other transmission areas and in other cohorts of exposed people need to be performed to confirm these results. Once validated, IFN-γ and IL-2 levels in SLA-stimulated whole blood could be reliably used in the field to estimate the prevalence of those asymptomatic individuals with Leishmania-specific cellular immune responses.

Leishmaniasis impact and treatment access.

According to disease burden estimates, leishmaniasis ranks third in disease burden in disability-adjusted life years caused by neglected tropical diseases and is the second cause of parasite-related deaths after malaria; but for a variety of reasons, it is not receiving the attention that would be justified seeing its importance. This is especially apparent in the unnecessarily and unacceptably poor access to timely and appropriate treatment for patients. To our knowledge, this is the first publication that addresses the major issues associated with poor access to drugs for leishmaniasis and that outlines a number of feasible and practical solutions.

Leishmania /HIV co-infections: epidemiology in Europe

It presents situation about Leishmaniasis and leishmania/HIV co-infection in Europe and also brings epidemiological features. Document in PDF format, required Acrobat Reader.

HIV and the transmission on Leishmania

It presents informations about the HIV and the transmission of leishmania and the change of the epidemiology of visceral leishmaniasis. Document in PDF format, required Acrobat Reader.

Immunization with H1, HASPB1 and MML Leishmania proteins in a vaccine trial against experimental canine leishmaniasis.

The protective capabilities of three Leishmania recombinant proteins - histone 1 (H1) and hydrophilic acylated surface protein B1 (HASPB1) immunized singly, or together as a protein cocktail vaccine with Montanide, and the polyprotein MML immunized with MPL-SE adjuvant - were assessed in beagle dogs. Clinical examination of the dogs was carried out periodically under blinded conditions and the condition of the dogs defined as asymptomatic or symptomatic. At the end of the trial, we were able to confirm that following infection with L. infantum promastigotes, five out of eight dogs immunized with H1 Montanide, and four out of eight dogs immunized with either the combination of HASPB1 with Montanide or the combination of H1+HASPB1 with Montanidetrade mark, remained free of clinical signs, compared with two out of seven dogs immunized with the polyprotein MML and adjuvant MPL-SE, and two out of eight dogs in the control group. The results demonstrate that HASPB1 and H1 antigens in combination with Montanide were able to induce partial protection against canine leishmaniasis, even under extreme experimental challenge conditions.

Leishmania/HIV co-infections: epidemiology in Europe.

As the AIDS pandemic spreads to rural areas and human visceral leishmaniasis (VL) becomes more common in suburban areas, there is an ever greater degree of overlap between the geographical distributions of the two diseases and, in consequence, an increasing incidence of Leishmania/HIV co-infection. Cases of the co-infection have been reported from 35 countries around the world but most have been recorded in south-western Europe. There has been a total of 1911 cases detected in Spain, France, Italy and Portugal. The incidence of Leishmania/HIV co-infection is expected to continue increasing in eastern Africa but to fall in south-western Europe as increasing numbers of HIV-positives in the latter region are given the new, highly active, antiretroviral therapy (HAART). In 1998, a world-wide network of surveillance for the co-infection, which now includes 28 member institutions, was established by the World Health Organization (WHO) and the Joint United Nations Programme on HIV/AIDS (UNAIDS). In south-western Europe, the surveillance system is based on 16 institutions and is already well established. The systematic use of standardized and recently computerized case-report forms, a central international registry at the WHO's headquarters in Geneva, and the use of a geographical information system (GIS) for mapping and monitoring the co-infections have together improved the overall quality of the epidemiological data-gathering. All member institutions of the global network report to the WHO on an annual basis. The data collected are then analysed and periodically disseminated through international publications. The GIS allows the relevant epidemiological and demographic data-sets to be integrated and permits all detected cases of co-infection to be mapped down to locality level. The system also allows the spatial distribution of cases to be visualised and analysed and the geographical spread of the co-infection to be monitored over time. The risk posed by co-infected patients, as a source of Leishmania infection for the sandflies feeding on them, has recently been confirmed. The parasites and HIV may also be transmitted as the result of needle-sharing among intravenous-drug users.

HIV and the transmission of Leishmania.

In many countries, Leishmania/HIV co-infection is now changing the epidemiology of visceral leishmaniasis. The levels of transmission of the parasites causing such leishmaniasis were previously dependent on the conventional zoonotic cycle, in which sandflies transmitted the parasites from infected canids to other canids or humans. The co-infection, however, has led not only to marked increases in the sandfly transmission of the parasites from immunodepressed individuals directly to other humans but also, probably, to artificial transmission between immunodepressed intravenous-drug users, as the result of needle sharing.

Iso-enzymatic variability of Leishmania infantum in Spain.

In many areas of the Mediterranean basin, leishmaniasis can now be found in HIV-positive individuals. Such cases of Leishmania/HIV co-infection are relatively common in southern Europe, Spain being the country that has reported the greatest number. Since 1984, 359 Spanish isolates of Leishmania infantum have been characterized at the Instituto de Salud Carlos III in Madrid. Most (94.6%) of the isolates came from HIV-positive patients. The results of iso-enzymatic analysis indicated a high level of variability among the isolates, the visceralization in HIV-positive individuals of variants considered to be dermotropic in the immunocompetent, and the appearance of new zymodemes among the HIV-positive human population.

Lower trypanosomatids in HIV/AIDS patients.

Although the family Trypanosomatidae includes parasites of plants, insects and vertebrates, only two genera in the family, Leishmania and Trypanosoma, are usually found in humans. Since 1995, however, other monoxenous trypanosomatids have been isolated from several HIV-positive individuals, in whom the parasites cause either visceral or cutaneous lesions. These odd cases are reviewed here. It appears that immunocompromised patients may be vulnerable to infection with trypanosomatids (and other parasites) that either fail to survive or never cause detectable morbidity in the immunocompetent.

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.

A nested polymerase chain reaction (Ln-PCR) for diagnosing and monitoring Leishmania infantum infection in patients co-infected with human immunodeficiency virus.

We investigated a Leishmania-specific nested polymerase chain reaction (Ln-PCR) for the diagnosis and treatment monitoring of L. infantum infections in patients co-infected with human immunodeficiency virus (HIV). Peripheral blood and bone marrow samples from 89 HIV patients in Spain suspected of having leishmaniasis were examined by different diagnostic techniques (Ln-PCR, microscopy, NNN culture and indirect fluorescent antibody test). The sensitivity of Ln-PCR compared with microscopy and culture of bone marrow was 95.45% using blood and 100% when using bone marrow. 38 of these patients with confirmed leishmaniasis were entered in a chemotherapy trial (reported elsewhere), and samples from them were collected before treatment, one month after treatment ended and during follow-up (1-20 months), and examined similarly. Ln-PCR was shown to be a good method for testing efficacy of treatment and for predicting relapses after treatment (relapses were predicted on average 5 months earlier than when using classical diagnostic techniques). We suggest that Ln-PCR (especially using peripheral blood) should be the technique of choice for diagnosis, monitoring the success of treatment, and predicting relapses in patients with HIV and suspected or confirmed L. infantum infection.

Molecular epidemiology of Leishmania infantum on the island of Majorca: a comparison of phenotypic and genotypic tools.

In the Mediterranean basin, Leishmania infantum is the causative agent of both visceral and cutaneous leishmaniasis, and is an important opportunistic parasite in patients infected with human immunodeficiency virus (HIV). The commonest method used to study the variability of Leishmania spp. is isoenzyme analysis. In addition to this, we employed 3 assays based on the polymerase chain reaction (PCR): random amplified polymorphic deoxyribonucleic acid (RAPD), intergenic region typing (IRT), based on the amplification of ribosomal ribonucleic acid internal transcribed spacers and restriction fragment length polymorphism (PCR-RFLP). We used 54 L. infantum stocks isolated from HIV co-infected patients, 38 isolated from dogs, 3 isolated from immunocompetent patients and 3 isolated from 1826 sand files in the island of Majorca (Spain), a closed ecological niche. Zymodemes MON-1 (70%), MON-24 (11%) and MON-34 (18%) were found among the human isolates, and MON-1 (95%) and MON-108 (5%) among those from dogs. RAPD and IRT could not discriminate among the strains as they all gave the same pattern, even when different zymodemes were examined. In contrast, PCR-RFLP was able to distinguish the strains and, furthermore, a dendrogram (unweighted pair group method with arithmetic average [UPGMA]) was constructed from the genetic distances derived from RFLP data. The Leishmania isolates from HIV-infected subjects formed a single cluster, supporting the existence of an artificial anthroponotic cycle previously proposed by our group, in which syringes have been substituted for sand flies, and in which certain clones have been spread among intravenous drug users. This contrasts with the clusters representing a zoonotic cycle, involving dogs, sand flies and both immunocompetent and immunocompromised humans.

Leishmania in discarded syringes from intravenous drug users.

Needle sharing by intravenous drug users (IVDUs) has been proposed as providing an alternative, artificial, and anthroponotic cycle for leishmania transmission. We looked for parasites in syringes discarded by IVDUs using two different PCR techniques. Leishmania spp were detected in 65 (52%) of 125 syringes collected in southern Madrid, Spain, in 1998, and in 52 (34%) of 154 collected in southwestern Madrid in 2000-01. We found shared restriction fragment length polymorphisms in 12 of 65 positive samples tested, suggesting that syringe sharing can indeed promote the spread of leishmania clones among IVDUs.

Semi-quantitative analysis of multiple cytokines in canine peripheral blood mononuclear cells by [correction of zby] a single tube RT-PCR.

Cytokines play an important role in the regulation of the immune system, but low circulating levels in plasma make routine measurement a difficult task. A new methodology based on single tube RT-PCR has been developed to determine the expression of multiple canine cytokines (TNF-alpha, IL-2, IFN-gamma, IL-18, IL-4, IL-6 and IL-10) using primers and protocols designed allow specific amplification of the mRNAs. The technique is performed in one tube in two consecutive steps, a specific transcription of the mRNA of a given cytokine and amplification of the corresponding gene by PCR. The technique was used to analyse the mRNA cytokine profile of peripheral blood mononuclear cells (PBMCs) from healthy dogs using two approaches: (i) analysis of PBMC isolated ex vivo; (ii) analysis of PBMC after in vitro cultures with or without the mitogen ConA. The samples were separated in agarose gels and the intensity of ethidium bromide signals quantified using standard video imaging equipment. Results were interpreted as the ratio of cytokine to GAPDH expression. The results obtained show that the method is easy to use and reproducible. Therefore, this method of monitoring the mRNA cytokine expression might be an useful tool for understanding the immune response in dogs.

Genotypic characterization of Toxoplasma gondii strains associated with human toxoplasmosis in Spain: direct analysis from clinical samples.

Genetic analysis of the SAG2 locus was performed to determine the prevalence of the different genotypes of Toxoplasma gondii (strain types I, II, and III) associated with human toxoplasmosis in Spain. This determination was made directly from primary clinical samples, obviating the previous process of isolation in mice or cell culture. A total of 34 isolates of T. gondii, collected from immunocompromised patients and congenital infection cases, were analyzed. Restriction fragment length polymorphism in PCR-amplified SAG2 products was used to group strains into one of the three genotypes of T. gondii. Complete characterization of the SAG2 gene was successful in 76.5% of the cases, demonstrating the feasibility of direct genotype analysis from clinical samples of different origins. Strains of T. gondii type II were the most prevalent in immunocompromised patients, with 52% of cases, while strains of type I were present in 75% of the congenital infection cases. These data differ from previous reports that show type II strains to be mostly associated with all kinds of human toxoplasmosis. These differences might be an effect of selection in the process of culture and isolation of the samples performed by other researchers prior to strain characterization.

Molecular tracking of infections by Leishmania infantum.

Leishmania infantum is a major opportunistic parasite in patients with acquired immune deficiency syndrome and is very variable in these subjects. Isoenzyme characterization is not able to explain this variability, since half of the stocks isolated from patients co-infected with human immunodeficiency virus and Leishmania belong to zymodeme MON-1. Amplification of L. infantum minicircles by the polymerase chain reaction (PCR) and digestion of the amplified product to reveal restriction fragment length polymorphisms (RFLP) has proved very useful in distinguishing between relapses and reinfections in co-infected, treated patients. We have confirmed the existence of a leishmaniasis outbreak among intravenous drug users in north-east Spain, previously detected by isoenzymatic analysis. We have documented persistence of the same strain of Leishmania in 2 treated co-infected patients throughout several years, regardless of the theoretical rapid evolution ascribed to kinetoplast deoxyribonucleic acid minicircle sequences. We suggest using this PCR-RFLP technique to detect reinfections in treated co-infected subjects.