Indigenous human cutaneous leishmaniasis caused by Leishmania tropica in Kenya

Six Leishmania isolates from 3 indigenous Kenyans (2 isolates from one patient) and 2 Canadian visitors in Kenya were characterized by cellulose acetate electrophoresis. The isolates were compared among themselves and with reference strains of Leishmania donovani, L. aethiopica, L. major, L. tropica, and L. arabica using 9 enzymes: malate dehydrogenase (MDH), malic enzyme (ME), phosphogluconate dehydrogenase (6PGD), glucose-6-phosphate dehydrogenase (G6PD), aspartate aminotransferase (ASAT), adenylate kinase (AK), mannose phosphate isomerase (MPI), glucose phosphate isomerase (GPI), and phosphoglucomutase (PGM). Enzyme migration patterns of isolates from the 3 indigenous Kenyans were indistinguishable from those of 2 L. tropica reference strains. The isolates from the 2 Canadians yielded migration patterns of 7 enzymes that were indistinguishable from those of 2 L. tropica reference strains. However, migration patterns of 2 enzymes, PGM and ME, differed from all migration patterns of the 10 reference strains. Balb/c mice were inoculated with stationary phase promastigotes cultured from 3 stabilates from the lesions of 2 of the Kenyan patients. The mice developed no gross pathological lesions in 6 months time. All of the study patients developed cutaneous leishmaniasis while living in or visiting districts in Central and Rift Valley Provinces, Kenya. This is the first report of human cutaneous leishmaniasis caused by L. tropica indigenous to Africa south of the Sahara.     

Flow cytometry technique for analysing Leishmania promastigote phagocytosis by human polymorphonuclear leucocytes and monocytes

In this study, we developed a flow cytometry technique for studying Leishmania (L.) mexicana phagocytosis by human polymorphonuclear leucocytes (PMNs) and monocytes. Leishmania promastigotes are elongated in shape and flagellated. This influences the light scatter when phagocytosis is measured by flow cytometry. Accordingly, we developed an oxidative burst method for measuring the phagocytic process. As this is an indirect marker of phagocytosis, we used confocal, light and electron microscopy to verify that promastigotes were, indeed, internalized by the phagocytes. For both PMNs and monocytes, the optimal conditions for achieving high sensitivity in flow cytometry detection were 5% pooled human serum and 15 min. incubation time. Incubations at 35, 37 and 39°C were also equally efficient for both PMNs and monocytes. Optimal parasite ratios were 10 parasites per PMN and 20 parasites per monocyte. Under these conditions, Leishmania were readily phagocytosed by human PMNs and monocytes and the effects of other influences, such as treatment, would be readily detectable. This indicated that these cells may play a role in the immune response against Leishmania.     

Experimental acquisition,development, and transmission of Leishmania tropica by Phlebotomus duboscqi

We report experimental infection and transmission of Leishmania tropica (Wright), by the blood-feeding sand fly Phlebotomus duboscqi (Neveu-Lemaire). Groups of laboratory-reared female sand flies that fed “naturally” on L. tropica-infected hamsters, or artificially, via membrane feeding device, on a suspension of L. tropica amastigotes, were dissected at progressive time points post-feeding. Acquisition, retention and development of L. tropica through procyclic, nectomonad, and leptomonad stages to the infective metacyclic promastigote stage, and anterior progression of the parasites from abdominal midgut bloodmeal to the thoracic midgut were demonstrated in both groups. Membrane feeding on the concentrated amastigote suspension led to metacyclic promastigote infections in 60% of sand flies, whereas only 3% of P. duboscqi that fed naturally on an infected hamster developed metacyclics. Sand flies from both groups re-fed on naïve hamsters, but despite infections in 25–50% of membrane-fed and 2–3.5% of naturally fed flies, no skin lesions developed in the hamsters. After four months of observation these animals were euthanized and necropsied. Screening of the organs and tissue by polymerase chain reaction (PCR) that targeted the small subunit RNA gene, amplified generic Leishmania DNA from liver, spleen, bone marrow, and blood, but only from hamsters bitten by membrane-infected P. duboscqi. These results are notable in demonstrating the ability of P. duboscqi, originating from Kenya, to acquire, retain, develop, and transmit a Turkish strain of L. tropica originally isolated from a human case of cutaneous leishmaniasis. This marks the first demonstration of complete development and transmission of L. tropica by a member of the Phlebotomus subgenus of sand flies.     

Comparative electron microscope studies on the labelling Leishmania donovani, Leishmania tropica and Leishmania braziliensis with ferritin (author's transl)]

The labelling of negative charges on the cell surface of the developing promastigote forms in Leishmania donovani, L. tropica and L. braziliensis was determined using cationized ferritin and electron microscopy. Ferritin deposits were seen exclusively on the pellicle, since they cannot penetrate the cell membrane. The ferritin particles were distributed in regular rows covering the whole cell surface. The mean diameter of the particles was 8.76 nm and at a magnification of 104.000 an average of 9.8 particles was detected on 1 cm cell surface. Only in the membrane of the proximal part of the reservoir labelling was frequently absent. The possible explanations for this observation were discussed. There was no difference in the pattern size of the ferritin particles or in their number per cm cell surface among the various strains of L. donovani on the one hand or between L. donovani, L. tropica and L. braziliensis on the other.     

First report on natural Leishmania infection of Phlebotomus sergenti due Leishmania tropica by high resolution melting curve method in Southeastern Iran

Objective:To identify the Leishmania species in infected sand flies by Real-time PCR coupled with HRM analysis.Methods:Real-time PCR coupled with HRM analysis targeting the first internal transcribed spacer(ITS1)of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish Lelthmania species in sand flies specimens.Results:Three out of 115females of Phlebotomus sergenti(P.sergenti)(2.6%)were positive to Leishmania tropica(L.tropica).Conclusions:This is the first report on P.sergenti as the main and proven vector of anthroponitic cutaneous leishmaniasis in Dehbakri County using Real-time PCR coupled with HUM analysis.This method is rapid,sensitive and specific for diagnosing of parasites in infected Sand flies and ideal for large scale genotyping projects.     

A nucleotidase from Leishmania tropica Promastigotes: partial purification and properties

Nucleotidase activity which had been found in various subcellular fractions of Leishmania tropica has been partially purified from the supernatant of the Triton X-100 treated pellet of the 100 000 g centrifugation of the L. tropica homogenate by CM-cellulose column chromatography, Con A-Sepharose affinity chromatography, isoelectrofocusing and column chromatography on Sephacryl S-300. The enzyme showed the characteristics of a nucleotidase (EC 3.1.3.31) with a broad substrate specificity. It was inhibited by the products of the reaction and by alpha, beta-methylene adenosine 5'diphosphate and adenosine 5'-9-thiomonophosphate. Nucleotidase activity of intact Leishmania cells and the inhibitory effect of Concanavalin A on the activity of the enzyme suggest also surface-associated properties of the enzyme.     

Lattice defects induced by microtubule-stabilizing agents exert a long-range effect on microtubule growth by promoting catastrophes

Microtubules are dynamic cytoskeletal polymers that spontaneously switch between phases of growth and shrinkage. The probability of transitioning from growth to shrinkage, termed catastrophe, increases with microtubule age, but the underlying mechanisms are poorly understood. Here, we set out to test whether microtubule lattice defects formed during polymerization can affect growth at the plus end. To generate microtubules with lattice defects, we used microtubule-stabilizing agents that promote formation of polymers with different protofilament numbers. By employing different agents during nucleation of stable microtubule seeds and the subsequent polymerization phase, we could reproducibly induce switches in protofilament number and induce stable lattice defects. Such drug-induced defects led to frequent catastrophes, which were not observed when microtubules were grown in the same conditions but without a protofilament number mismatch. Microtubule severing at the site of the defect was sufficient to suppress catastrophes. We conclude that structural defects within the microtubule lattice can exert effects that can propagate over long distances and affect the dynamic state of the microtubule end.

Microtubules are cytoskeletal polymers that rapidly switch between phases of growth and shortening, and this behavior, termed dynamic instability, plays a crucial role in the formation, maintenance, and reorganization of microtubule arrays during cell division, migration, and differentiation (1, 2). The transition from growth to shrinkage, an event called catastrophe, is known to occur when the protective cap of guanosine triphosphate (GTP)–bound tubulin subunits is reduced or lost, but the underlying mechanisms are still the subject of investigation (3, 4). One interesting property of microtubules is that the frequency of catastrophes depends on microtubule age: Microtubules that are growing for a longer time have a higher chance to switch to depolymerization (5, 6). Changes occurring at the microtubule end, such as loss of individual protofilaments or end tapering, have been shown to promote catastrophe (7–9). In principle, it is also possible that the catastrophe frequency at the plus end is affected by structural features in the microtubule lattice farther away from the tip, but this possibility has so far remained untested.Structural studies have established that tubulin can form tubes with different protofilament numbers (10), dependent on the species, nucleation template, presence of different microtubule-associated proteins, and other properties of the polymerization reaction (e.g., ref. 11; reviewed in ref. 12). An important consequence of the structural plasticity of the microtubule lattice (13) is the formation of lattice defects, such as sites where a microtubule gains or loses one or more protofilaments (11, 14–17). A recent cryoelectron tomography analysis showed that in some cell types, such as Drosophila neurons, variations and transitions in protofilament number are readily detectable (18) and are thus likely to be physiologically relevant. Switches in protofilament number can be introduced during microtubule growth, and their presence may affect microtubule dynamics in different ways. For example, defects can be repaired through tubulin incorporation, and the resulting islands of GTP-tubulin can trigger microtubule rescue (19–22). On the other hand, the presence of defects could potentially also induce catastrophes (as proposed in ref. 14), since conformational properties of the microtubule lattice might propagate over some distances (23).To study the relationship between lattice defects and microtubule catastrophes, one should be able to directly correlate the presence of defects with the dynamics of microtubule ends. We recently found that fluorescent analogs of microtubule-stabilizing agents (MSAs) can be used to induce microtubule lattice defects that can be visualized by fluorescence microscopy. When present at low concentrations, MSAs preferentially bind to microtubule plus ends that enter a “precatastrophe” state (24), which is manifested by the gradual loss of the GTP cap and reduced recruitment of end-binding (EB) proteins that detect GTP-bound microtubule lattice (25–27). Strong accumulation of MSAs at precatastrophe microtubule ends leads to the formation of stabilized patches of microtubule lattice, where the tube is incomplete and keeps incorporating GTP-tubulin but is not fully repaired (24). When microtubules switch to depolymerization, such persistent lattice defects, which coincide with the hotspots of MSA binding, can induce repeated rescues and, therefore, they were termed “stable rescue sites” (24).Here, we used MSA-induced lattice defects to address two questions. First, what prevents complete repair of an MSA-induced persistent lattice defect? And second, does the presence of such a persistent defect affect the dynamics of the microtubule plus end? Since different MSAs are known to affect the number of protofilaments (15, 28–33), we hypothesized that persistent lattice defects could be associated with the changes in protofilament number and thus could not be fully repaired for geometrical reasons. We tested this idea by generating stable microtubule seeds with one MSA and then elongating them in the presence of another MSA, with the same or different preference for protofilament number. Use of fluorescent MSAs allowed us to directly follow drug binding. We found that precatastrophe microtubule ends accumulated MSAs in all conditions; however, the outcome of drug binding was different. When there was no mismatch in protofilament number between the seeds and the elongation conditions, drug accumulations were short in duration and length, and microtubule growth beyond such sites was processive. In contrast, when, based on the MSA properties, a mismatch in protofilament number could be expected, large and persistent drug accumulations were formed. The existence of such mismatches was confirmed by cryoelectron microscopy (cryo-EM) and by measuring microtubule growth rate, which became higher with increasing protofilament number. When microtubule ends extended beyond a mismatch-containing lattice defect, they displayed elevated catastrophe frequency. Laser-mediated severing of a microtubule at the site of the persistent defect reduced catastrophe frequency at the plus end. Our data demonstrate that local perturbations in microtubule structure can affect the state of the dynamic end at a distance of several micrometers.     

Predominance of Leishmania major and rare occurrence of Leishmania tropica with haplotype variability at the center of Iran

Background

Leishmania major is a causative agent of zoonotic cutaneous leishmaniasis in the center of Iran, Abarkouh district. Molecular characterization and precise incrimination of Leishmania species was carried out to perform controlling measurements and to design treatment programs for zoonotic cutaneous leishmaniasis.

Effect of leishmanial excreted factor on the activities of adenylate cyclase from hamster liver and Leishmania tropica

The carbohydrate segment of the excreted factor of culture cells from L. tropica and L. donovani was shown to interact with the adenylate cyclase from hamster liver. At a concentration of 1 mg/ml the excreted factor inhibited the stimulated activity of the mammalian enzyme by about 50%, independent of the activation by glucagon, 5'-guanylylimidodiphosphate and forskolin, respectively. In contrast to the adenylate cyclase system from hamster liver the enzyme from L. tropica-culture cells was not stimulated by addition of glucagon, 5'-guanylylimidodiphosphate and forskolin. In addition, the activity of the adenylate cyclase from L. tropica was not affected by addition of its endogenous excreted factor.     

Combined effect of the essential oil from Chenopodium ambrosioides and antileishmanial drugs on promastigotes of Leishmania amazonensis

To date, there are no vaccines against Leishmania, and chemotherapy remains the mainstay for the control of leishmaniasis. The drugs of choice used for leishmaniasis therapy are significantly toxic, expensive and with a growing frequency of refractory infections. Because of these limitations, a combination therapy is the better hope. This work demonstrates that the essential oil from Chenopodium ambrosioides shows a synergic activity after incubation in conjunction with pentamidine against promastigotes of Leishmania amazonensis. However, an indifferent effect has been found for combinations of meglumine antimoniate or amphotericin B and the essential oil.     

Rosiglitazone impacts negatively on bone by promoting osteoblast/osteocyte apoptosis

Thiazolidinediones (TZDs) increase peripheral tissue insulin sensitivity in patients with type 2 diabetes mellitus by activating the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma). In bone marrow stromal cell cultures and in vivo, activation of PPARgamma by high doses (20 mg/kg/day) of TZDs has been reported to alter stem cell differentiation by promoting commitment of progenitor cells to the adipocytic lineage while inhibiting osteoblastogenesis. Here, we have examined the in vivo effects of low-dose rosiglitazone (3 mg/kg/day) on bone, administered to mice by gavage for 90 days. Rosiglitazone-treated mice had increased weight when compared with controls, with no significant alterations in serum levels of glucose, calcium or parathyroid hormone (PTH). Bone mineral density (BMD) at the lumbar vertebrae (L1-L4), ilium/sacrum, and total body was diminished by rosiglitazone treatment. Histologically, bone was characterized by decreased trabecular bone volume and increased marrow space with no significant change in bone marrow adipocity. Decreased osteoblast number and activity due to increased apoptotic death of osteoblasts and osteocytes was apparent while osteoclast parameters and serum levels of osteocalcin, alkaline phosphatase activity, and leptin were unaltered by rosiglitazone treatment. Therefore, the imbalance in bone remodeling that follows rosiglitazone administration arises from increased apoptotic death of osteogenic cells and diminished bone formation leading to the observed decrease in trabecular bone volume and BMD. These novel in vivo effects of TZDs on bone are of clinical relevance as patients with type 2 diabetes mellitus and other insulin resistant states treated with these agents may potentially be at increased risk of osteoporosis.     

Identification of a 94-kilodalton antigen on Leishmania promastigote forms and its specific recognition in human and canine visceral leishmaniasis

We have analysed by immunoblotting sera from humans and dogs with visceral leishmaniasis, from the Old World as well as the New. When lysates of promastigotes are used as antigens, antibodies against a 94 kDa Leishmania component are detected, regardless of the age and geographical origin of the patient, the serum antibody titre as measured by indirect immunofluorescence, and the number of arcs in counterimmunoelectrophoresis. Low dilutions of sera from patients with Old and New World cutaneous leishmaniasis did not react with the 94-kDa antigen, whatever the species of Leishmania used as antigens. Sera from patients with other infections than leishmaniases, or without infection, are negative, even at low dilution. Anti-94 kDa antibodies were detected in the sera of Leishmania-infected dogs from both the Old and the New World. When lysates of Leishmania mexicana axenic amastigotes are used as antigens, the 94-kDa antigen was little or none identified by sera from humans and dogs with visceral leishmaniasis, and never recognized by control sera. Thus, the specific recognition of the 94-kDa promastigote antigen in human and canine visceral leishmaniasis suggests that this antigen could be a potential candidate in the differential immunodiagnosis of the disease.