Amphotericin B-loaded nanoparticles for local treatment of cutaneous leishmaniasis

Cutaneous leishmaniasis (CL) is an infectious, parasitic disease caused by the protozoan Leishmania. Amphotericin B (AMB) is a macrolide polyene antibiotic presenting potent antifungal and antileishmanial activity, but due to poor water solubility at physiological pH, side effects, and toxicity, its therapeutic efficiency is limited. In the present study, poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with AMB were generated to reduce drug toxicity and facilitate localized delivery over a prolonged time. AMB NPs were characterized for particle size, zeta potential, polydispersity index, and degree of aggregation. In vitro assessments demonstrated its sustained activity against Leishmania major promastigotes and parasite-infected macrophages. A single intralesional administration to infected BALB/c mice revealed that AMB NPs were more effective than AMB deoxycholate in terms of reducing lesion area. Taken together, these findings suggest thatAMB NPs improve AMB delivery and can be used for local treatment of CL.This research was funded by the GIP program of the Deutsche Forschungsgemeinschaft (DFG) German Research Foundation. EZ wish to acknowledge the financial support of the RBNI-The Russell Berrie Nanotechnology Institute at the Technion. CLJ holds the Michael and Penny Feiwel Chair of Dermatology

Group G Streptococcal Bacteremia in Jerusalem

Recurrent group G Steptococcus bacteremia, associated with lymphatic disorders and possibly emm stG840.0, is described

Identification of a Secreted Casein Kinase 1 in <i>Leishmania donovani</i>: Effect of Protein over Expression on Parasite Growth and Virulence

<div><p>Casein kinase 1 (CK1) plays an important role in eukaryotic signaling pathways, and their substrates include key regulatory proteins involved in cell differentiation, proliferation and chromosome segregation. The <i>Leishmania</i> genome encodes six potential CK1 isoforms, of which five have orthologs in other trypanosomatidae. <i>Leishmania donovani</i> CK1 isoform 4 (<i>Ldck1.4</i>, orthologous to LmjF27.1780) is unique to <i>Leishmania</i> and contains a putative secretion signal peptide. The full-length gene and three shorter constructs were cloned and expressed in <i>E. coli</i> as His-tag proteins. Only the full-length 62.3 kDa protein showed protein kinase activity indicating that the N-terminal and C-terminal domains are essential for protein activity. LdCK1.4-FLAG was stably over expressed in <i>L. donovani</i>, and shown by immunofluorescence to be localized primarily in the cytosol. Western blotting using anti-FLAG and anti-CK1.4 antibodies showed that this CK1 isoform is expressed and secreted by promastigotes. Over expression of LdCK1.4 had a significant effect on promastigote growth in culture with these parasites growing to higher cell densities than the control parasites (wild-type or Ld:luciferase, P<0.001). Analysis by flow cytometry showed a higher percentage, ∼4–5-fold, of virulent metacyclic promastigotes on day 3 among the LdCK1.4 parasites. Finally, parasites over expressing LdCK1.4 gave significantly higher infections of mouse peritoneal macrophages compared to wild-type parasites, 28.6% versus 6.3%, respectively (p = 0.0005). These results suggest that LdCK1.4 plays an important role in parasite survival and virulence. Further studies are needed to validate CK1.4 as a therapeutic target in <i>Leishmania</i>.</p></div

Expression of CK1.4-FLAG by mutant <i>L. donovani</i> promastigotes (Ld:CK1.4-FLAG).

<p>SDS-PAGE and Western blot analysis of total cellular lysates (10⁷ promastigotes/lane) from wild type (wt) and Ld:CK1.4-FLAG (CK1.4) promastigotes using Panel A: anti-FLAG M2 monoclonal antibody (1/1000 dilution, Sigma - Aldrich Chemical Co.) or Panel B: rabbit polyclonal anti-HSP83 (1/5000 dilution) or anti-CK1.4 (1/7500 dilution).</p

Multi-alignment of leishmanial CK1.4 protein sequences.

<p>Predicted protein sequence for CK1.4 orthologs from <i>Leishmania major</i> (Lmaj - Gene Bank Accession No. CBZ12245.1), <i>L. donovani</i> (Ldon - JN225463), <i>L. infantum</i> (Linf - CAM69136.1), <i>L. mexicana</i> (Lmex - CBZ28278.1) and <i>L. braziliensis</i> (Lbra - CAM45467.1). Identical amino acids are marked by dots, and the gaps by red dashes. The blue brackets delineate the conserved protein kinase domain.</p

Time course of CK1.4 release by <i>Leishmania donovani</i> promastigotes.

<p>Cell-free supernatants were prepared from Ld:wt and/or Ld:CK1.4-FLAG parasites at different times (min) post-induction with ionomycin (5 µM)/EGTA (1 mM), and examined by SDS-PAGE - Western blotting. Panel A: Membranes were incubated with anti-FLAG M2 monoclonal antibody (1/1000 dilution, Sigma –Aldrich Chemical Co); Lysate is a positive control prepared from 10⁷ Ld:CK1.4-FLAG promastigotes collected at t = 0. Panel B: Membranes probed simultaneously with rabbit polyclonal anti-CK1.4 (1/7500 dilution) and anti-KMP 11 (1/2000 dilution) antibodies. Lanes labeled Lysate were prepared from either 10⁷ Ld:wt or Ld:CK1.4 promastigotes collected at t = 0 min. Antibody binding was detected by incubation of the membrane with a chemiluminescent substrate and exposure to X-ray film.</p

Dendrogram showing the evolutionary relationship of CK1 isoforms from different organisms.

<p>The tree was constructed by neighbor-joining analysis of the conserved catalytic domain from 18 CK1 sequences. Both complete and pairwise deletion options were tested and the bootstrap consensus tree built using 1000 replicates. Analyses were conducted using Mega (Molecular evolutionary genetic analysis) software, version 5.2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079287#pone.0079287-Tamura1" target="_blank">[25]</a>. Sequences examined include: LmjF isoform 1 - <i>Leishmania major</i> (LmjF35.1000); LmjF isoform 2 - <i>L. major</i> (LmjF35.1010); LinJ isoform 3 - <i>L. infantum</i> (LinJ04.1230); <i>L. donovani</i> isoform 4 - (JN225463), LmjF isoform 4 - <i>L. major</i> (LmjF27.1780), LinJ isoform 4 - <i>L. infantum</i> (LinJ27.1680); LinJ isoform 5 - <i>L. infantum</i> (LinJ25.1640); LinJ isoform 6 - <i>L. infantum</i> (LinJ30.3530). Other organisms: <i>T. b. gambiense</i> CK1 (CBH15205.1), <i>Entamoeba dispar</i> δ isoform (XP_001735831.1), <i>Trichomonas vaginalis</i> (XP_001327672.1), <i>Plasmodium falciparum</i> (AAn35960), <i>Toxoplasma gondii α</i> isoform (XP_002366683), <i>T. gondii</i> β isoform (XP_00236367), <i>Schizosaccharomyces pombe</i> CKi2 (NP_595380), <i>Homo sapiens</i> α1 isoform (AAV_38633.1), <i>Mus Musculus</i> δ isoform (NP_082150.1), <i>Mus Musculus</i> ε isoform (NP_038795.3).</p

Expression of His-tag CK1.4 wild-type and deletion constructs.

<p>Panel A. Schematic description of recombinant full-length CK1.4 and truncated polypeptides. Conserved protein kinase catalytic domain – speckled box; predicted signal polypeptide – solid black box; (His)₆ epitope tag – his; Oligonucleotide primers used for PCR: forward primers -MFCK1 and PFCK1; reverse primers – MRCK1 and P2MRCK1. Predicted molecular mass (kDa) of each recombinant protein (A – D) and casein kinase activity (rCK1) is given. Panel B. Western blot analysis of full length CK1.4 and truncated polypeptides expressed in <i>E. coli.</i> Expression of recombinant proteins was induced by addition of L-arabinose and IPTG for 18 hrs at 22°C (Lanes +). Non-induced bacteria were used as negative controls (Lanes −). Bacterial lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes and probed with Nickel conjugated Horseradish peroxidase (1/10000 dilution, HisDetector Nickel-HRP, KPL Inc, USA). Binding was detected using chemiluminescent substrate. His tagged recombinant proteins in Lanes: A - LdCK1.4; B - LdCK1.4Δ^1–90; C - LdCK1.4Δ^411–566, and D - LdCK1.4Δ^{1–90,411–566}.</p

emm Typing of M Nontypeable Invasive Group A Streptococcal Isolates in Israel

We performed emm typing of M nontypeable invasive group A streptococcal (GAS) isolates collected in a prospective population-based study in Israel. One hundred twenty of 131 isolates (92%) had emm sequences compatible with GAS, consisting of 51 different emm types. Eleven isolates were found to be group G streptococcus. Of the 120 isolates, 55 (46%) belonged to 32 types for which there were no typing sera available in the Streptococcal Reference Laboratory in Israel. The other 65 (64%) isolates, consisting of 19 types, had sera available and therefore could have been serotyped. Forty-three isolates had T and emm types which were not correlated according to standard M-typing protocols and were therefore missed. The principal effect of emm typing was the addition of 32 types not previously identified in Israel and the discovery of new associations between emm and T types. emm typing did not significantly change the proportion of M types; the five most common types were 3, 28, 2, 62, and 41. Twenty different types comprised 80% of all isolates. No new emm sequences were discovered. emm typing emphasized the unusually low incidence of M1 strains causing severe disease in Israel. As serological typing of GAS becomes more problematic due to lack of sera and the appearance of new emm types, reference laboratories should replace M typing with emm sequence typing. Development of a GAS vaccine relies on the emm type distributions in different geographical locations. In our study, 7% of isolates (types 41 and 62) are not included in a 26-valent vaccine that is being studied