In vitro 'time-to-kill' assay to assess the cidal activity dynamics of current reference drugs against Leishmania donovani and Leishmania infantum.

OBJECTIVES: Despite a continued search for novel antileishmanial drugs, treatment options remain restricted to a few standard drugs, e.g. antimonials, miltefosine, amphotericin B and paromomycin. Although these drugs have now been used for several decades, their mechanism of action still remains partly hypothetical and their dynamics of cidal action and time-to-kill are still poorly documented. METHODS: An in vitro time-to-kill assay on intracellular amastigotes of the laboratory reference strains Leishmania donovani (MHOM/ET/67/L82) and Leishmania infantum [MHOM/MA(BE)/67/ITMAP263] evaluated the cidal action dynamics of the listed reference drugs at three different concentrations: at IC50, 2 × IC50 and the near cytotoxic dose level (CC90: determined on MRC-5 cells). This assay focused on identifying the minimal exposure time needed to completely eliminate viable intracellular amastigotes, using the standard microscopic Giemsa assay and the promastigote back-transformation assay. RESULTS: While 100% reduction was microscopically apparent for most drugs, the promastigote back-transformation assay clearly demonstrated a concentration- and time-dependent cidal mechanism. The time-to-kill at 2 × IC50 was ≥240 h for pentavalent antimony (77 µg eq./mL), 96 h for trivalent antimony (44 µg eq./mL), 168 to >240 h for miltefosine (10 µM), 168 h for paromomycin (100 µM) and >240 h for amphotericin B (2 µM). No differences were noted between both Leishmania species. CONCLUSIONS: Evaluation of the concentration- and time-dependent cidal activity using the promastigote back-transformation assay revealed striking differences in efficacy of the different antileishmanial reference drugs. This assay should allow in-depth pharmacodynamic evaluation of novel drug leads in comparison with the existing antileishmanial drug repertoire.

Evidence of a drug-specific impact of experimentally selected paromomycin and miltefosine resistance on parasite fitness in Leishmania infantum.

OBJECTIVES: Although miltefosine and paromomycin were only recently introduced to treat visceral leishmaniasis, increasing numbers of miltefosine treatment failures and occasional primary resistance to both drugs have been reported. Understanding alterations in parasite behaviour linked to drug resistance is essential to assess the propensity for emergence and spread of resistant strains, particularly since a positive effect on fitness has been reported for antimony-resistant parasites. This laboratory study compared the fitness of a drug-susceptible parent WT clinical Leishmania infantum isolate (MHOM/FR/96/LEM3323) and derived miltefosine and paromomycin drug-resistant lines that were experimentally selected at the intracellular amastigote level. METHODS: Parasite fitness of WT, paromomycin-resistant and miltefosine-resistant strains, in vitro and in vivo parasite growth, metacyclogenesis, infectivity and macrophage stress responses were comparatively evaluated. RESULTS: No significant differences in promastigote fitness were noted between the WT and paromomycin-resistant strain, while clear benefits could be demonstrated for paromomycin-resistant amastigotes in terms of enhanced in vitro and in vivo growth potential and intracellular stress response. The miltefosine-resistant phenotype showed incomplete promastigote metacyclogenesis, decreased intracellular growth and weakened stress response, revealing a reduced fitness compared with WT parent parasites. CONCLUSIONS: The rapid selection and fitness advantages of paromomycin-resistant amastigotes endorse the current use of paromomycin in combination therapy. Although a reduced fitness of miltefosine-resistant strains may explain the difficulty of miltefosine resistance selection in vitro, the growing number of miltefosine treatment failures in the field still requires further exploratory research.

Lack of correlation between the promastigote back-transformation assay and miltefosine treatment outcome.

OBJECTIVES: Widespread antimony resistance in the Indian subcontinent has enforced a therapy shift in visceral leishmaniasis treatment primarily towards miltefosine and secondarily also towards paromomycin. In vitro selection of miltefosine resistance in Leishmania donovani turned out to be quite challenging. Although no increase in IC50 was detected in the standard intracellular amastigote susceptibility assay, promastigote back-transformation remained positive at high miltefosine concentrations, suggesting a more 'resistant' phenotype. This observation was explored in a large set of Nepalese clinical isolates from miltefosine cure and relapse patients to assess its predictive value for patient treatment outcome. METHODS: The predictive value of the promastigote back-transformation for treatment outcome of a set of Nepalese L. donovani field isolates (n = 17) derived from miltefosine cure and relapse patients was compared with the standard susceptibility assays on promastigotes and intracellular amastigotes. RESULTS: In-depth phenotypic analysis of the clinical isolates revealed no correlation between the different susceptibility assays, nor any clear link to the actual treatment outcome. In addition, the clinical isolates proved to be phenotypically heterogeneous, as reflected by the large variation in drug susceptibility among the established clones. CONCLUSIONS: This in vitro laboratory study shows that miltefosine treatment outcome is not necessarily exclusively linked with the susceptibility profile of pre-treatment isolates, as determined in standard susceptibility assays. The true nature of miltefosine treatment failures still remains ill defined.

In Vivo Selection of Paromomycin and Miltefosine Resistance in Leishmania donovani and L. infantum in a Syrian Hamster Model.

In 2002 and 2006, respectively, miltefosine (MIL) and paromomycin (PMM) were licensed in the Indian subcontinent for treatment of visceral leishmaniasis; however, their future routine use might become jeopardized by the development of drug resistance. Although experimental selection of resistant strains in vitro has repeatedly been reported using the less relevant promastigote vector stage, the outcome of resistance selection on intracellular amastigotes was reported to be protocol and species dependent. To corroborate these in vitro findings, selection of resistance in Leishmania donovani and Leishmania infantum was achieved by successive treatment/relapse cycles in infected Syrian golden hamsters. For PMM, resistant amastigotes were already obtained within 3 treatment/relapse cycles, while their promastigotes retained full susceptibility, thereby sharing the same phenotypic characteristics as in vitro-generated PMM-resistant strains. For MIL, even five treatment/relapse cycles failed to induce significant susceptibility changes in either species, which also corresponds with the in vitro observations where selection of an MIL-resistant phenotype proved to be quite challenging. In conclusion, these results argue for cautious use of PMM in the field to avoid rapid emergence of primary resistance and highlight the need for additional research on the mechanisms and dynamics of MIL resistance selection.

Intracellular amastigote replication may not be required for successful in vitro selection of miltefosine resistance in Leishmania infantum.

Although miltefosine (MIL) has only recently been positioned as a first-line therapeutic option for visceral leishmaniasis, field reports note an increasing trend in treatment failures. Study of laboratory selected MIL-resistant strains is needed in the absence of confirmed resistant clinical isolates. In contrast to promastigotes, experimental in vitro selection of MIL-resistance on intracellular amastigotes has not yet been documented. This study reports for the first time the selection of MIL-resistance in Leishmania infantum LEM3323, a strain which clearly shows active intracellular replication. Starting from the hypothesis that active multiplication may be essential in the resistance selection process; several other L. infantum strains were evaluated. Although strain LEM5269 showed only marginally lower intracellular multiplication, selection for resistance failed, as was also the case for several other strains showing poor or no intracellular replication. These results suggest that intracellular multiplication may not be an absolute prerequisite for the outcome of experimental in vitro MIL-resistance selection in clinical field isolates.

The opioid methadone induces a local anaesthetic-like inhibition of the cardiac Na⁺ channel, Na(v)1.5.

BACKGROUND AND PURPOSE: Treatment with methadone is associated with severe cardiac arrhythmias, a side effect that seems to result from an inhibition of cardiac hERG K⁺ channels. However, several other opioids are inhibitors of voltage-gated Na⁺ channels. Considering the common assumption that an inhibition of the cardiac Na⁺ channel Na(v)1.5, is the primary mechanism for local anaesthetic (LA)-induced cardiotoxicity, we hypothesized that methadone has LA-like properties leading to a modulation of Na(v)1.5 channels. EXPERIMENTAL APPROACH: The whole-cell patch clamp technique was applied to investigate the effects of methadone on wild-type and mutant human Na(v)1.5 channels expressed in HEK293 cells. A homology model of human Na(v)1.5 channels was used to perform automated ligand-docking studies. KEY RESULTS: Methadone inhibited Na(v)1.5 channels in a state-dependent manner, that is, tonic block was stronger with inactivated channels than with resting channels and a use-dependent block at 10 Hz. Methadone induced a concentration-dependent shift of the voltage dependency of both fast and slow inactivation towards more hyperpolarized potentials, and impaired recovery from fast and slow inactivation. The LA-insensitive mutants N406K and F1760A exhibited reduced tonic and use-dependent block by methadone, and docking predictions positioned methadone in a cavity that was delimited by the residue F1760. Dextromethadone and levomethadone induced discrete stereo-selective effects on Na(v)1.5 channels. CONCLUSIONS AND IMPLICATIONS: Methadone interacted with the LA-binding site to inhibit Na(v)1.5 channels. Our data suggest that these channels are a hitherto unrecognized molecular component contributing to cardiac arrhythmias induced by methadone.

Patient perceptions concerning pain management in the treatment of rheumatoid arthritis.

Previous qualitative studies have revealed discrepancies between patients' and physicians' perceptions of rheumatoid arthritis (RA) and its treatment. Questionnaires were administered to 2795 patients with RA (756 from Europe; 2039 from the USA) to measure patients' perceptions regarding pain management in RA. Although the majority of patients reported their RA as somewhat-to-completely controlled, 75% of European and 82% of US patients reported their pain as moderate-to-severe in the previous 2 months. The majority of European (60%) and US (65%) patients reported dissatisfaction with their arthritis pain. Patients' pain levels corresponded with their disease severity. A higher percentage of patients who reported severe pain were being treated for depression than those who had moderate or mild pain. Patients in the USA rated pain relief as the top required benefit from their RA medication. A comprehensive examination of patients' perspectives regarding pain could lead to better patient care and pain management strategies.

Induced micronucleus frequencies in peripheral lymphocytes as a screening test for carriers of a BRCA1 mutation in breast cancer families.

Enhanced sensitivity to the chromosome-damaging effects of ionizing radiation is a feature of many cancer-predisposing conditions. It has been suggested that women with breast cancer are deficient in the repair of radiation-induced DNA damage. We have now investigated whether mutagen sensitivity is related to mutations in the breast cancer gene BRCA1. We studied the induction and repair of DNA damage in lymphocytes of women from families with familial breast cancer and breast and ovarian cancer. The mutagens used were gamma-irradiation and hydrogen peroxide and the DNA effects were determined with the micronucleus test and the comet assay. Women with a BRCA1 mutation (n = 12) and relatives without the familial mutation (n = 10) were compared to controls (i.e., healthy women without family history of breast or ovarian cancer; n = 17). Our results indicate a close relationship between the presence of a BRCA1 mutation and sensitivity for the induction of micronuclei. Compared to a concurrent control, 10 of 11 women with a BRCA1 mutation showed elevated radiation sensitivity. Of the 10 related women without the familial mutation, only 2 had clearly enhanced micronucleus frequencies. In addition to the sensitivity toward gamma-irradiation, hypersensitivity toward hydrogen peroxide was also observed, indicating that the mutagen sensitivity is not solely due to a defect in the repair of DNA double strand breaks. In contrast to the results with the micronucleus assay, we found no significant difference between women with and without a BRCA1 mutation with respect to the induction and repair of DNA damage in the comet assay. This finding suggests a normal rate of damage removal and points to a disturbed fidelity of DNA repair as a direct or indirect consequence of a BRCA1 mutation. Our results support the usefulness of induced micronucleus frequencies as a biomarker for cancer predisposition and suggest its application as a screening test for carriers of a BRCA1 mutation in breast cancer families.

The demonstration of progesterone, but not of estrogen, receptors in the developing human placenta.

Since presence of steroid receptors in the human placenta has been the subject of dispute, we have investigated the existence of estrogen (ER) and progesterone (PR) receptors in trophoblasts across gestational age by a variety of different techniques. Fresh human placental tissue of trimesters 1 to 3 was paraffin-embedded or snap-frozen (-80 degrees C) and sliced (5 microns). Other tissue fragments from identical placentae were dispersed and incubated in monolayer cultures for up to 5 days. Immunocytochemistry (ICC) was performed for ER and PR in both trophoblast cells in culture and in whole tissue slices, using the sandwich antibody technique with subsequent horse-radish peroxidase reaction for colorization. In addition, long-term perifusion studies were conducted with explants of term placentae, using perifusion medium with estradiol (E, 2 ng/ml) and/or progesterone (P, 200 ng/ml). Perifused explants were then subjected to further ICC staining. Furthermore, RT-PCR for both ER and PR mRNA was performed for detection of the gene products in placentae of different gestational ages. Lastly, binding studies with iodine or tritium-labeled E and P were conducted on cytosol fractions. In placental sections and cultured trophoblasts, PR was clearly demonstrable in all placentae across different gestational ages. Abundant PR signal was found adjacent to the nuclei, and additionally in the dendrite-like pseudopods of syncytiotrophoblast cells. In contrast, no such staining signal was detected for the ER; this finding applied under all conditions investigated and at all gestational ages. Again, no staining for ER by ICC was detected in any tissue after perifusion with sex steroids. RT-PCR revealed no product for ER, but only for PR, in placentae across all gestational ages. Binding studies with labeled E and P showed no binding for either compound. Taken together, these observations suggest the presence of PR, but not of ER, in human placenta throughout gestation. Our failure to detect the ER does not entirely preclude the presence of this receptor in human trophoblasts, but might be attributed to a relatively low number and density of ER on these cells. Alternatively, estrogen's action on the placenta may be mediated by a different type of ER, such as by a non-classical membrane-bound receptor.

Contribution of a tyrosine side chain to ribonuclease A catalysis and stability.

An intricate architecture of covalent bonds and noncovalent interactions appear to position the side chain of Lys 41 properly within the active site of bovine pancreatic ribonuclease A (RNase A). One of these interactions arises from Tyr 97, which is conserved in all 41 RNase A homologues of known sequence. Tyr 97 has a solvent-inaccessible side chain that donates a hydrogen bond to the main-chain oxygen of Lys 41. Here, the role of Tyr 97 was examined by replacing Tyr 97 with a phenylalanine, alanine, or glycine residue. All three mutant proteins have diminished catalytic activity, with the value of Kcat being perturbed more significantly than that of Km. The free energies with which Y97F, Y97A, and Y97G RNase A bind to the rate-limiting transition state during the cleavage of poly(cytidylic acid) are diminished by 0.74, 3.3, and 3.8 kcal/mol, respectively. These results show that even though Tyr 97 is remote from the active site, its side chain contributes to catalysis. The role of Tyr 97 in the thermal stability of RNase A is large. The conformational free energies of native Y97F, Y97A, and Y97G RNase A are decreased by 3.54, 12.0, and 11.7 kcal/mol, respectively. The unusually large decrease in stability caused by the Tyr-->Phe mutation could result from a decrease in the barrier to isomerization of the Lys 41-Pro 42 peptide bond.

[Alfred Kirstein (1863-1922)--pioneer in direct laryngoscopy]. / Alfred Kirstein (1863-1922)--Pionier der direkten Laryngoskopie.

On April 23rd 1895, in Berlin, Alfred Kirstein performed the first direct examination of the interior of the larynx. 23 days after his first view of larynx, he gave a comprehensive demonstration of autoscopy to the Berlin Medical Association. Until then laryngologists had been content with the technique of indirect laryngoscopy using mirrors, a method popularised by Garcia, Türck and Czermak. Kirstein named the combination of the electroscope and the oesophagoscope "The autoscope" and direct examination of the larynx he termed "autoscopy". Despite the infancy of autoscopy and the autoscope, Kirstein already recognised the potential of his new discovery. He reported that the removal of foreign bodies from the trachea must be easier through an autoscope then by means of a tracheostomy; furthermore, catheterisation of the bronchi should now present no great difficulties. The similarity between the blades he used and those described in the 1940s by Macintosh and Miller is remarkable.

Inductive effects on the structure of proline residues.

4(S)-Hydroxyproline (Hyp) residues constitute about 10% of most forms of collagen, the most abundant protein in vertebrates. X-Ray diffraction analysis was used to ascertain how the structure of proline residues is affected by the inductive effect elicited by the hydroxyl group of Hyp residues. N-Acetylproline methylester (1), N-acetyl-4(S)-hydroxyproline methylester (2) and N-acetyl-4(S)- fluoroproline methylester (3) were synthesized, and their crystalline structures were determined at high resolution. The amide bond of crystalline 1 was in the cis conformation, which is the minor isomer in solution, and the pyrrolidine ring of 1 had C gamma-endo pucker. In crystalline 2 and 3 the amide bonds were in the trans conformation, and the pyrrolidine rings had C-exo pucker. The lengths of the bonds between sp3-hybridized carbon atoms in the pyrrolidine ring were significantly shorter in 2 and 3 than in 1, as was predicted by ab initio molecular orbital calculations at the RHF/3-21G level of theory. No significant change in bond length was observed in the other bonds of 1,2 or 3. The pyramidylization of the nitrogen atom increased dramatically in the order: 1 < 2 < 3. Together, these results indicate that electron-withdrawing substituents in the 4-position of proline residues can have a significant influence on the structure of these residues. In particular, the change in pyramidylization suggests that such substituents increase the sp3-character of the prolyl nitrogen atom and could thereby alter the rate of prolyl peptide bond isomerization.

Curt Schimmelbusch (1850-1895)--development of a mask for chloroform and ether anesthesia based on primary aseptic principles. / Curt Schimmelbusch (1860-1895)--Entwicklung einer Maske für Chloroform- und Athernarkosen aus primär aseptischen Uberlegungen.

The German surgeon Curt Schimmelbusch is not generally recognised as a pioneer in anaesthesia, although he is well known by his investigations concerning the development and scientific foundation of mechanical sterilisation methods which he performed under Ernst von Bergmann in Berlin at the end of the 19th century. His book "Anleitung zur aseptischen Wundheilung" is one of the classics in the history of asepsis. Curt Schimmelbusch was born on November 16th, 1860, as the son of a lord of a manor in Gross-Nogath/West-Prussia. Having graduated from high school in 1882, he studied medicine at Würzburg, Göttingen, Berlin and Halle. The physician who was awarded the doctor's degree in Halle in 1886 as a co-worker of the histologist and pathologist Eberth at first dealt with research in the field of thrombosis. His surgical training took place in Cologne where he worked in 1888 with the famous surgeon Bardenheuer at the Bürgerhospital. A year later he returned to Berlin to do research work at the institute at the Ziegelstrasse. Under the main aspects of hygiene he described a mask in 1890, which he used predominantly for ether anaesthesia, although he considered it to be suitable also for chloroform anaesthesia, in itself an exciting contribution he has made characterising the progress of anaesthesia in German-speaking countries up to the mid-50's of the 20th century.

NMR strategy for determining Xaa-Pro peptide bond configurations in proteins: mutants of staphylococcal nuclease with altered configuration at proline-117.

A general approach has been developed for configurational analysis (cis or trans) of Xaa-Pro peptide bonds in proteins. This approach, which entails selective 13C labeling of Xaa and Pro residues in the protein and isotope-edited NMR, has been applied to mutants of staphylococcal nuclease with suspected altered configurations of the Lys116-Pro117 peptide bond. The technique for monitoring proline configurations is based on differences in interproton distances between the H alpha of residue Xaa and the proline H delta or H alpha protons. Short (< 2.5 A) Xaa H alpha-Pro H delta interproton distances are diagnostic for the trans configuration, whereas short (< 2.5 A) Xaa H alpha-Pro H alpha interproton distances are diagnostic for the cis configuration. Biosynthetic incorporation of [alpha-13C]Xaa and [delta-13C]proline facilitates detection of trans Xaa-Pro peptide bonds, whereas incorporation of [alpha-13C]Xaa and [alpha-13C]proline facilitates detection of cis Xaa-Pro peptide bonds. Provided that the Xaa-Pro peptide bond is unique within the protein sequence, symmetric off-diagonal NOE cross peaks in the isotope-edited NOE spectrum allow for simultaneous chemical shift assignment and determination of the prolyl peptide bond geometry. We have used this technique to determine the predominant configuration of the Lys116-Pro117 peptide bond in recombinant V8 staphylococcal nuclease A (H124L) and two of its single amino acid mutants (D77A+H124L and G79S+H124L). The results are consistent with conclusions reached on the basis of indirect arguments concerning changes in the chemical shifts of histidine 1H epsilon 1 NMR signals.(ABSTRACT TRUNCATED AT 250 WORDS)

[Thoracic outlet syndrome and tumor embolism]. / Thoracic outlet Syndrom und Tumorembolie.

This is probably the first report of a rare tumor-embolization from a leiomyosarcoma into the brachial artery simultaneously linked with a thoracic outlet syndrome. After transaxillary rib resection and thromboembolectomy only histological examination of the resected specimen allowed the final diagnosis: leimyosarcomatous embolus.

Gain model for microchannel plates.

It is shown that microchannel plates (MCPs) tend to act as if they were discrete stage electron multipliers with a fixed number of stages or dynodes if a plausible assumption is made regarding the behavior of the secondary electrons emitted from the semiconducting sidewalls of tubular channel electron multipliers under the grazing incidence conditions predominantly encountered in these multipliers. The shape of the resultant predicted gain-voltage transfer characteristic for the MCP fits well with experimental data, confirming the assumption made and permitting the use of curve matching techniques to determine such important MCP parameters as the average number of active dynodes, the gain per stage, the crossover potential for the MCP wall material, the transit time through the multiplier, etc.

Image transfer properties of proximity focused image tubes.

Experimental measurements of the lateral (i.e., radial) spreading of photoelectrons from a multialkali photocathode in an unfocused image dissector tube have shown that (a) the distribution of the radial emission energies is approximately Maxwellian and (b) the radial emission energies are smaller than expected based on the difference between the input photon energies and the long wavelength threshold energy of the multialkali photocathode ( congruent with1.4 eV). Both results are favorable with respect to the predicted electron optical image transfer properties of proximity focused image tubes. The observed data indicate that at least a 40-lp/mm or a 1600-lp/40-mm image field diameter should be resolvable for white light input to commercially available tubes-and that this resolving power should increase to at least 60 lp/mm for input wavelengths longer than approximately 600 nm and decrease to 30 lp/mm or less for input wavelengths shorter than approximately 400 nm.