Drug susceptibility in Leishmania isolates following miltefosine treatment in cases of visceral leishmaniasis and post kala-azar dermal leishmaniasis

Background

With widespread resistance to antimonials in Visceral Leishmaniasis (VL) in the Indian subcontinent, Miltefosine (MIL) has been introduced as the first line therapy. Surveillance of MIL susceptibility in natural populations of Leishmania donovani is vital to preserve it and support the VL elimination program.

Methodology and Principal Findings

We measured in vitro susceptibility towards MIL and paromomycin (PMM) in L. donovani isolated from VL and PKDL, pre- and post-treatment cases, using an amastigote-macrophage model. MIL susceptibility of post-treatment isolates from cured VL cases (n = 13, mean IC₅₀±SD = 2.43±1.44 µM), was comparable (p>0.05) whereas that from relapses (n = 3, mean IC₅₀ = 4.72±1.99 µM) was significantly higher (p = 0.04) to that of the pre-treatment group (n = 6, mean IC₅₀ = 1.86±0.75 µM). In PKDL, post-treatment isolates (n = 3, mean IC₅₀ = 16.13±2.64 µM) exhibited significantly lower susceptibility (p = 0.03) than pre-treatment isolates (n = 5, mean IC₅₀ = 8.63±0.94 µM). Overall, PKDL isolates (n = 8, mean IC₅₀ = 11.45±4.19 µM) exhibited significantly higher tolerance (p<0.0001) to MIL than VL isolates (n = 22, mean IC₅₀ = 2.58±1.58 µM). Point mutations in the miltefosine transporter (LdMT) and its beta subunit (LdRos3) genes previously reported in parasites with experimentally induced MIL resistance were not present in the clinical isolates. Further, the mRNA expression profile of these genes was comparable in the pre- and post-treatment isolates. Parasite isolates from VL and PKDL cases were uniformly susceptible to PMM with respective mean IC₅₀ = 7.05±2.24 µM and 6.18±1.51 µM.

Conclusion

The in vitro susceptibility of VL isolates remained unchanged at the end of MIL treatment; however, isolates from relapsed VL and PKDL cases had lower susceptibility than the pre-treatment isolates. PKDL isolates were more tolerant towards MIL in comparison with VL isolates. All parasite isolates were uniformly susceptible to PMM. Mutations in the LdMT and LdRos3 genes as well as changes in the expression of these genes previously correlated with experimental resistance to MIL could not be verified for the field isolates.     

Recent evolution of sedimentary heavy metals in a coastal lagoon contaminated by industrial wastewaters (Pialassa Baiona,Ravenna, Italy)

From 2000 to 2002, sediment contamination by Cd, Cu, Hg, Pb and Zn was analysed in the Pialassa Baiona salt marsh, which receives petrochemical wastewaters from the industrial district of Ravenna (Italy). The recent contamination levels were compared with data of previous studies carried out in 1982, in order to assess whether environmental policies and remedial measures reduced sediment pollution. Sedimentary profiles of Cu and Pb were homogeneous along the uppermost 0–10 cm horizon, which corresponded to the sedimentation in the last 30 years. Concentrations of Zn attained a peak (up to 800 mg kg⁻¹ dry weight) in the 0–4 cm sediment horizon, which was assumed to correspond to the last 10–15 years. A wide-spread contamination by Hg was detected in the salt marsh as well as in the main channel with peaks up to 20–40 mg kg⁻¹ dry weight. Nonetheless, recent sediments resulted less contaminated, since Hg discharge from industrial plants ceased about 20 years ago. Contamination levels by Hg values were two orders of magnitude higher than the international sediment quality standards. Cadmium, which was analysed for the first time in 2000–2002, attained a peak in the surface layers (1–2.5 mg kg⁻¹ d.w.), with a progressive decline along the sediment column. Through comparison with pre-industrial values detected in the deeper sediment horizons (before 1920), Hg showed the highest enrichment factor, up to 300 times. Cd and Zn concentrations in recent sediments were from 2 to 10 times higher than background values. In terms of possible adverse effects, Hg posed the highest risk, and Cd and Zn were frequently above the recommended thresholds.     

Interplay between formation of photosynthetic complexes and expression of genes for iron–sulfur cluster assembly in Rhodobacter sphaeroides?

Photosynthesis Research - Formation of photosynthetic complexes leads to a higher demand for Fe–S clusters. We hypothesized that in the facultative phototrophic alpha-proteobacterium...     

The heat shock protein HSP70 promotes mouse NK cell activity against tumors that express inducible NKG2D ligands

The stress-inducible heat shock protein (HSP) 70 is known to function as an endogenous danger signal that can increase the immunogenicity of tumors and induce CTL responses. We show in this study that HSP70 also activates mouse NK cells that recognize stress-inducible NKG2D ligands on tumor cells. Tumor size and the rate of metastases derived from HSP70-overexpressing human melanoma cells were found to be reduced in T and B cell-deficient SCID mice, but not in SCID/beige mice that lack additionally functional NK cells. In the SCID mice with HSP70-overexpressing tumors, NK cells were activated so that they killed ex vivo tumor cells that expressed NKG2D ligands. In the tumors, the MHC class I chain-related (MIC) A and B molecules were found to be expressed. Interestingly, a counter selection was observed against the expression of MICA/B in HSP70-overexpressing tumors compared with control tumors in SCID, but not in SCID/beige mice, suggesting a functional relevance of MICA/B expression. The melanoma cells were found to release exosomes. HSP70-positive exosomes from the HSP70-overexpressing cells, in contrast to HSP70-negative exosomes from the control cells, were able to activate mouse NK cells in vitro to kill YAC-1 cells, which express NKG2D ligands constitutively, or the human melanoma cells, in which MICA/B expression was induced. Thus, HSP70 and inducible NKG2D ligands synergistically promote the activation of mouse NK cells resulting in a reduced tumor growth and suppression of metastatic disease.     

Genital damage in the orb-web spider Argiope bruennichi (Araneae: Araneidae) increases paternity success

The morphology of male genitalia often suggests functions besidessperm transfer that may have evolved under natural or sexualselection. In several species of sexually cannibalistic spiders,males damage their paired genitalia during mating, limitingthem to one copulation per pedipalp. Using a triple-mating experiment,we tested if genital damage in the orb-web spider Argiope bruennichiincreases male fitness either through facilitating his escapefrom an aggressive female or by obstructing the female's inseminationducts against future copulation attempts from other males. Wefound no survival advantage for males damaging their pedipalps;however, copulations into a previously used insemination ductwere significantly shorter when the previous male had left partsof his genitalia inside the insemination duct. Because copulationduration determines paternity in this species, our result suggeststhat male genital damage in A. bruennichi is sexually selected.By breaking off parts of their intromittent organs inside avirgin female, males can reduce sperm competition and therebyincrease their paternity success.     

Pseudomonas community structure and antagonistic potential in the rhizosphere: insights gained by combining phylogenetic and functional gene-based analyses

The Pseudomonas community structure and antagonistic potential in the rhizospheres of strawberry and oilseed rape (host plants of the fungal phytopathogen Verticillium dahliae) were assessed. The use of a new PCR-DGGE system, designed to target Pseudomonas-specific gacA gene fragments in environmental DNA, circumvented common biases of 16S rRNA gene-based DGGE analyses and proved to be a reliable tool to unravel the diversity of uncultured Pseudomonas in bulk and rhizosphere soils. Pseudomonas-specific gacA fingerprints of total-community (TC) rhizosphere DNA were surprisingly diverse, plant-specific and differed markedly from those of the corresponding bulk soils. By combining multiple culture-dependent and independent surveys, a group of Pseudomonas isolates antagonistic towards V. dahliae was shown to be genotypically conserved, to carry the phlD biosynthetic locus (involved in the biosynthesis of 2,4-diacetylphloroglucinol - 2,4-DAPG), and to correspond to a dominant and highly frequent Pseudomonas population in the rhizosphere of field-grown strawberries planted at three sites in Germany which have different land use histories. This population belongs to the Pseudomonas fluorescens phylogenetic lineage and showed closest relatedness to P. fluorescens strain F113 (97% gacA gene sequence identity in 492-bp sequences), a biocontrol agent and 2,4-DAPG producer. Partial gacA gene sequences derived from isolates, clones of the strawberry rhizosphere and DGGE bands retrieved in this study represent previously undescribed Pseudomonas gacA gene clusters as revealed by phylogenetic analysis.