Efficacy of Off-Label Anti-Amoebic Agents to Suppress Trophozoite Formation of Acanthamoeba spp. on Non-Nutrient Agar Escherichia Coli Plates.

Acanthamoeba keratitis (AK) is a dangerous infectious disease, which is associated with a high risk of blindness for the infected patient, and for which no standard therapy exists thus far. Patients suffering from AK are thus treated, out of necessity, with an off-label therapy, using drugs designed and indicated for other diseases/purposes. Here, we tested the capability of the off-label anti-amoebic drugs chlorhexidine (CH; 0.1%), dibromopropamidine diisethionate (DD; 0.1%), hexamidine diisethionate (HD; 0.1%), miltefosine (MF; 0.0065%), natamycin (NM; 5%), polyhexamethylene biguanide (PHMB; 0.02%), povidone iodine (PVPI; 1%), and propamidine isethionate (PD; 0.1%) to suppress trophozoite formation of Acantamoeba castellanii and Acanthamoeba hatchetti cysts on non-nutrient agar Escherichia coli plates. Of the eight off-label anti-amoebic drugs tested, only PVPI allowed for a complete suppression of trophozoite formation by drug-challenged cysts for all four Acanthamoeba isolates in all five biological replicates. Drugs such as NM, PD, and PHMB repeatedly suppressed trophozoite formation with some, but not all, tested Acanthamoeba isolates, while other drugs such as CH, DD, and MF failed to exert a relevant effect on the excystation capacities of the tested Acanthamoeba isolates in most, if not all, of our repetitions. Our findings suggest that pre-testing of the AK isolate with the non-nutrient agar E. coli plate assay against the anti-amoebic drug intended for treatment should be performed to confirm that the selected drug is cysticidal for the Acanthamoeba isolate.

Phenotypic Diversity of Cryptococcus neoformans var. neoformans Clinical Isolates from Localized and Disseminated Infections.

Cryptococcus neoformans var. neoformans is the second most prevalent agent of cryptococcosis in central Europe. Infections mostly present with localized skin and disseminated infections. Previous studies did not find these presentations to be determined by the fungal genotype as detected by multilocus sequence typing (MLST). However, phenotypic fungal traits may impact clinical presentation. Here, we studied the growth and virulence factors of C. neoformans var. neoformans isolates from disseminated and localized infections and an environmental isolate. We used coincubation with Acanthamoeba castellanii and the Galleria mellonella infection model to identify phenotypic characteristics potentially associated with clinical presentation. Clinical isolates of C. neoformans var. neoformans present a substantial phenotypic variability. Median survival of G. mellonella varied between 6 and 14 days. C. neoformans var. neoformans isolates from disseminated infections showed stronger melanization and larger capsules. They demonstrated superior uptake into an amoeba and increased cytotoxicity for the amoeba. Differences of strains from localized and disseminated infections in coincubation with amoeba are in line with the importance of phagocytes in the pathogenesis of disseminated cryptococcosis. Phenotypic traits and non-vertebrate infection models may help understand the virulence potential of C. neoformans var. neoformans isolates.

Evaluation of vectorcardiography parameters matrixed synthesized

The form of analysis of the cardiac signal most used today is the electrocardiogram (ECG). However, in addition to this form of data visualization, there is the vectorcardiogram (VCG), that allows a visualization of the signal in 3 dimensions. This study aims to compare the different ECG to VCG transformation matrices Kors and Inverse Dower (iDower), by analyzing some known parameters taken from VCG's mathematically synthesized from 12-lead ECG's of amyloidosis patients. The idea is also to compare that similarity for patients with different types of amyloidosis. The study was done through the analysis of electrocardiograms taken from a sample of 12 humans who have amyloidosis, either mutant or wild-type. The results indicated that there is not much similarity between the signals, although the similarity was higher for patients with mutant amyloidosis than for those with wild-type amyloidosis.

Análise de parâmetros do VCG obtidos a partir de matrizes de transformação do ECG em pacientes com hipertrofia cardíaca / Analysis of VCG parameters obtained from ECG transformation matrices in patients with cardiac hypertrophy

INTRODUÇÃO: O eletrocardiograma (ECG) é uma ferramenta diagnóstica primordial em cardiologia. Através de técnicas de pós-processamento do ECG digital é possível extrair dados do clássico vetorcardiograma (VCG) e ampliar as possibilidades do método. OBJETIVO: Comparar os dados matriciais do ECG entre os pacientes portadores de hipertrofia ventricular patológica (Cardiomiopatia Hipertrófica e Amiloidose Cardíaca) e o grupo controle utilizando duas matrizes diferentes de transformação de ECG para VCG: a matriz inversa de Dower (iDower) e a matriz de Kors. MÉTODOS: Foram analisados VCGs sintetizados a partir das duas matrizes, iDower e Kors, e tais exames foram obtidos para o grupo hipertrófico, composto por 11 pacientes, e para o grupo controle, com cinco pacientes. O tempo de coleta do ECG foi de 10 minutos e, para cada paciente, foram analisados 10 VCGs, referentes a cada minuto da coleta. Foram analisados a amplitude cúbica (mV), por meio da análise da variação dos valores, e o ângulo do complexo QRS e da onda T, por meio da análise de dados circulares. RESULTADOS: Em relação aos ângulos, as médias dos ângulos de cada grupo tiveram maior proximidade no grupo controle e menor no grupo hipertrófico. A variação destes ângulos foi maior utilizando a matriz de Kors para o grupo controle (Figura.1); no entanto, no grupo hipertrófico a maior variação no observada na matriz iDower (Figura 2). Em relação à amplitude cúbica das ondas, a variação foi pequena para ambos os grupos, porém com proximidade maior na onda T, e não houve diferença notável em relação às matrizes. CONCLUSÃO: Analisando as variáveis angulares e de amplitude cúbica nas ondas QRS e T para o grupo controle e hipertrófico, não foi observada diferença significativa entre os grupos. Tal proximidade entre as variáveis também foi observada considerando as matrizes de transformação.

The Effect of Anti-Amoebic Agents and Ce6-PDT on Acanthamoeba castellanii Trophozoites and Cysts, In Vitro.

Purpose: The purpose of this study was to analyze the concentration-dependent effects of biguanides (polyhexamethylene biguanide [PHMB], chlorhexidine [CH]); diamidines (hexamidine-diisethionate [HD], propamidine-isethionate [PD], dibromopropamidine-diisethionate [DD]); natamycin (NM); miltefosine (MF); povidone iodine (PVPI), and chlorin e6 PDT on Acanthamoeba trophozoites and cysts, in vitro. Methods: Strain 1BU was cultured in peptone-yeast extract-glucose medium. Trophozoites or cysts were cultured in PYG medium containing each agent at 100%, 50%, and 25% of maximum concentration for 2 hours. The percentage of dead trophozoites was determined using a non-radioactive cytotoxicity assay and trypan blue staining. Treated cysts were also maintained on non-nutrient agar Escherichia coli (E.coli) plates and observed for 3 weeks. Results: All tested drugs displayed significant cytotoxic effects on 1BU cells based on the biochemical and staining-based viability assays tested. On non-nutrient agar E. coli plates, neither trophozoites nor freshly formed cysts were observed after PHMB, PD, NM, and PVPI treatment, respectively, within 3 weeks. However, CH-, HD-, DD-, and MF-treated cysts could excyst, multiply, and encyst again. Conclusions: The off-label drugs PHMB, PD, NM, and PVPI are under in vitro conditions more effective against strain 1BU than CH, HD, DD, and MF. Our findings also suggest that the non-nutrient agar E.coli plate assay should be considered as method of choice for the in vitro analysis of the treatment efficacy of anti-amoebic agents. Translational Relevance: Ophthalmologists may optimize the treatment regime against Acanthamoeba keratitis by pre-testing the in vitro susceptibilities of the Acanthamoeba strain against drugs of interest with the non-nutrient E.coli agar plate assay.

Comparison of in vitro assays to study the effectiveness of antiparasitics against Acanthamoeba castellani trophozoites and cysts.

We aimed to compare LDH release assay, trypan blue and fluorescent stainings, and non-nutrient Escherichia coli plate assay in determining treatment efficacy of antiamoebic agents against Acanthamoeba castellanii trophozoites/cysts, in vitro. 1BU trophozoites/cysts were challenged with 0.02% polyhexamethylene biguanid (PHMB), 0.1% propamidine isethionate (PD), and 0.0065% miltefosine (MF). Efficacies of the drugs were determined by LDH release and trypan blue assays, by Hoechst 33343, calcein-AM, and ethidium homodimer-1 fluorescent dyes, and by a non-nutrient agar E. coli plate assay. All three antiamoebic agents induced a significant LDH release from trophozoites, compared to controls (p < 0.0001). Fluorescent-dye staining in untreated 1BU trophozoites/cysts was negligible, but using antiamoebic agents, there was 59.3%-100% trypan blue, 100% Hoechst 33342, 0%-75.3% calcein-AM, and 100% ethidium homodimer-1 positivity. On E. coli plates, in controls and MF-treated 1BU trophozoites/cysts, new trophozoites appeared within 24 h, encystment occurred after 5 weeks. In PHMB- and PD-treated 1BU throphozoites/cysts, irregularly shaped, smaller trophozoites appeared after 72 h, which failed to form new cysts within 5 weeks. None of the enzymatic- and dye-based viability assays tested here generated survival rates for trophozoites/cysts that were comparable with those yielded with the non-nutrient agar E. coli plate assay, suggesting that the culture-based assay is the best method to study the treatment efficacy of drugs against Acanthamoeba.

Reducing morbidity with surgical adhesives following inguinal lymph node dissections for the treatment of malignant skin tumors.

BACKGROUND: Inguinal lymph node dissection (ILND) is associated with a high rate of morbidity. To evaluate the clinical benefit of surgical adhesives to reduce complications in patients undergoing ILND, we compared the use of TissuGlu(®) Surgical Adhesive and ARTISS(®) fibrin sealant with a control population. MATERIAL AND METHODS: We conducted a retrospective analysis of patients undergoing ILND for metastatic malignant skin tumors at one hospital, Fachklinik Hornheide (Münster, Germany), from January 2011 through September 2013, assessing 137 patients with a total of 142 procedures. RESULTS: Complications occurred in 22/60 procedures in the TissuGlu group (TG), in 8/17 in the ARTISS group (AG), and in 29/65 in the control group (CG). Prolonged drainage and seroma were recorded in 16 (26.7%), four (23.5%), and 26 (40%) respectively (non-significant). TG showed less extended drainage vs. CG (p=0.082). Mean daily drain volumes were significantly lower in AG vs. CG (p=0.000). With regard to wound infection, there was a 15% reduction in TG and 74% increase in AG group. Revision surgery was reduced by 36% in TG and increased by 54% in AG. Mean daily drain volumes were significantly lower in AG vs. CG (p=0.000). Mean total post-operative drain volume was lower in TG and AG vs. CG (p<0.001 among groups, CG vs. TG p<0.001, CG vs. AG p<0.001). The mean body mass index (BMI) was significantly higher in patients with complications, 29.4±5.8 vs. 25.3±4.1 (p=0.000). CONCLUSION: The use of TissuGlu in our ILND patients was associated with a reduction in post-operative wound related complications and the need for revision surgeries compared to the control group. Daily drainage was significantly lower within the first 7 post-operative days with the use of ARTISS, but the benefit was lost due to the higher occurrence of wound infection and revision surgery. BMI above 29 is a risk factor for complications following ILND. ( LEVEL OF EVIDENCE: level IV, retrospective case study).

Probing Charges on the Atomic Scale by Means of Atomic Force Microscopy.

Kelvin probe force spectroscopy was used to characterize the charge distribution of individual molecules with polar bonds. Whereas this technique represents the charge distribution with moderate resolution for large tip-molecule separations, it fails for short distances. Here, we introduce a novel local force spectroscopy technique which allows one to better disentangle electrostatic from other contributions in the force signal. It enables one to obtain charge-related maps at even closer tip-sample distances, where the lateral resolution is further enhanced. This enhanced resolution allows one to resolve contrast variations along individual polar bonds.

Influence of the respirator on volatile organic compounds: an animal study in rats over 24 hours.

Long-term animal studies are needed to accomplish measurements of volatile organic compounds (VOCs) for medical diagnostics. In order to analyze the time course of VOCs, it is necessary to ventilate these animals. Therefore, a total of 10 male Sprague-Dawley rats were anaesthetized and ventilated with synthetic air via tracheotomy for 24 h. An ion mobility spectrometry coupled to multi-capillary columns (MCC-IMS) was used to analyze the expired air. To identify background contaminations produced by the respirator itself, six comparative measurements were conducted with ventilators only. Overall, a number of 37 peaks could be detected within the positive mode. According to the ratio peak intensity rat/ peak intensity ventilator blank, 22 peaks with a ratio >1.5 were defined as expired VOCs, 12 peaks with a ratio between 0.5 and 1.5 as unaffected VOCs, and three peaks with a ratio <0.5 as resorbed VOCs. The peak intensity of 12 expired VOCs changed significantly during the 24 h measurement. These results represent the basis for future intervention studies. Notably, online VOC analysis with MCC-IMS is possible over 24 h in ventilated rats and allows different experimental approaches.

Resistance to intranasal infection with Balamuthia mandrillaris amebae is T-cell dependent.

T and B cell-deficient BALB/c SCID mice become severely ill and die of amebic encephalitis after intranasal infection with Balamuthia mandrillaris, while adult immunocompetent BALB/c wild-type (WT) mice are resistant. To further investigate the role of lymphocytes in protection from Balamuthia amebic encephalitis (BAE), SCID mice were reconstituted with and WT mice selectively depleted of lymphocytes before infection. Reconstitution of SCID mice with whole spleen cells from WT mice rendered the recipients as resistant to BAE as WT mice. SCID mice that had received spleen cells depleted of CD4(+) T cells remained susceptible. When adult WT mice were depleted of both CD4(+) and CD8(+) T cells or of CD4(+) T cells alone, these mice also became susceptible to BAE. Depletion of CD8(+) T cells alone increased susceptibility only marginally. All morbidity and mortality data were underpinned by histological analysis of the brain.

Fatal Balamuthia mandrillaris infection in a gorilla - first case of balamuthiasis in Germany.

BACKGROUND: A 12-year-old female western lowland gorilla died in a zoological garden in Germany after exhibiting general neurological signs. METHODS: Balamuthia mandrillaris was identified as causative agent by indirect immunofluorescent staining of brain sections and confirmed by PCR and respective sequencing. RESULTS: The animal suffered from a chronic progressive necrotizing amebic meningoencephalitis. CONCLUSION: This is the first case of Balamuthia amebic encephalitis in Germany.

German Randomized Acupuncture Trial for chronic shoulder pain (GRASP) - a pragmatic, controlled, patient-blinded, multi-centre trial in an outpatient care environment.

UNLABELLED: The German Randomized Acupuncture Trial for chronic shoulder pain (GRASP) comprised 424 outpatients with chronic shoulder pain (CSP) > or =6 weeks and an average pain score of VAS > or =50 mm, who were randomly assigned to receive Chinese acupuncture (verum), sham acupuncture (sham) or conventional conservative orthopaedic treatment (COT). The patients were blinded to the type of acupuncture and treated by 31 office-based orthopaedists trained in acupuncture; all received 15 treatments over 6 weeks. The 50% responder rate for pain was measured on a VAS 3 months after the end of treatment (primary endpoint) and directly after the end of the treatment (secondary endpoint). RESULTS: In the ITT (n=424) analysis, percentages of responders for the primary endpoint were verum 65% (95% CI 56-74%) (n=100), sham 24% (95% CI 9-39%) (n=32), and COT 37% (95% CI 24-50%) (n=50); secondary endpoint: verum 68% (95% CI 58-77%) (n=92), sham 40% (95% CI 27-53%) (n=53), and COT 28% (95% CI 14-42%) (n=38). The results are significant for verum over sham and verum over COT (p<0.01) for both the primary and secondary endpoints. The PPP analysis of the primary (n=308) and secondary endpoints (n=360) yields similar responder results for verum over sham and verum over COT (p<0.01). Descriptive statistics showed greater improvement of shoulder mobility (abduction and arm-above-head test) for the verum group versus the control group immediately after treatment and after 3 months. The trial indicates that Chinese acupuncture is an effective alternative to conventional orthopaedic treatment for CSP.

Delivery of amphotericin B nanosuspensions to the brain and determination of activity against Balamuthia mandrillaris amebas.

Amphotericin B was formulated as nanosuspensions to develop a nanoparticulate brain delivery system. Nanosuspensions were produced with different surfactant solutions by high-pressure homogenization and then characterized by laser diffractometry and photon correlation spectroscopy. Before in vitro and in vivo testing all nanosuspensions were investigated for protein adsorption by two-dimensional polyacrylamide gel electrophoresis to predict brain-targeting capacities. Selected nanosuspensions were tested for amebicidal activity against Balamuthia mandrillaris, an agent of lethal encephalitis. Our results indicate that nanosuspensions coated with polysorbate 80 and sodium cholate markedly increased drug brain delivery and inhibited the parasite in vitro, though less in vivo. From the clinical editor: The antifungal Amphotericin B was formulated as nanosuspensions to develop a nanoparticulate brain delivery system. The results indicate that nanosuspensions coated with polysorbate 80 and sodium cholate markedly increased drug brain delivery and inhibited the parasite in vitro, though less in vivo.

Balamuthia and Acanthamoeba-binding antibodies in West African human sera.

Little is known about the prevalence of Balamuthia mandrillaris amoebae and Balamuthia amoebic encephalitis in Africa. As an approach, relative concentrations of amoebae-binding serum antibodies (Ab) were assessed by flow cytometry using formaldehyde-fixed B. mandrillaris, Acanthamoeba lenticulata 72-2 and Acanthamoeba castellanii 1BU amoebae for specific Ab capture (B.m.-Ab, A.l.-Ab, A.c.-Ab). One hundred and ninety-two sera from West African (Côte d'Ivoire) donors aged 11-95years (mean 38 a; 51% males), and living in villages surrounded by rainforest near the Liberian border, were tested and related to reference sera from Berlin. While B.m.-Ab tended to increase with donor age, A.l.-Ab and A.c.-Ab did not. Accordingly, B.m.-Ab correlated only weakly with A.l.-Ab or A.c.-Ab. Of the nine individuals with the highest B.m.-Ab concentrations, most were elderly (mean 58 a), male (78%), and professed intensive outdoor activity (hunting, farming). Only three of these sera also showed elevated A.l.-Ab, and none elevated A.c.-Ab.

Assessment of Balamuthia mandrillaris-specific serum antibody concentrations by flow cytometry.

A flow cytometry (fluorescence-activated cell sorter)-based assay was adapted to detect and quantify antibodies to Balamuthia mandrillaris, a causative agent of fatal amoebic encephalitis (BAE), and to Acanthamoeba species. With sera from BAE patients for positive and a group of inconspicuous volunteers for negative reference, most of the 237 sera from random blood donors, patients with atypical encephalitis, atypical pneumonitis, visceral amoebiasis and toxoplasmosis and from subjects working with primates and other mammals were rated negative, 19% elevated and of these 2% high. In comparison, 23 of 25 West Africans living in rural areas revealed elevated, of these 15 high, and one very high B. mandrillaris-binding antibody titers, the latter well in the range of BAE patients. To date, none of the tested individuals have developed symptoms indicative of BAE. Criss-cross analysis with rabbit hyper immune sera against B. mandrillaris, Acanthamoeba comandoni (group 1), Acanthamoeba castellanii (group 2) and Acanthamoeba lenticulata (group 3) confirmed that cross-reactivity between B. mandrillaris and Acanthamoeba sp. is negligible while accentuating antigenic differences between the three morphological groups of Acanthamoeba.

3D blood flow characteristics in the carotid artery bifurcation assessed by flow-sensitive 4D MRI at 3T.

To determine three-dimensional (3D) blood flow patterns in the carotid bifurcation, 10 healthy volunteers and nine patients with internal carotid artery (ICA) stenosis > or =50% were examined by flow-sensitive 4D MRI at 3T. Absolute and mean blood velocities, pulsatility index (PI), and resistance index (RI) were measured in the common carotid arteries (CCAs) by duplex sonography (DS) and compared with flow-sensitive 4D MRI. Furthermore, 3D MRI blood flow patterns in the carotid bifurcation of volunteers and patients before and after recanalization were graded by two independent readers. Blood flow velocities measured by MRI were 31-39% lower than in DS. However, PI and RI differed by only 13-16%. Rating of 3D flow characteristics in the ICA revealed consistent patterns for filling and helical flow in volunteers. In patients with ICA stenosis, 3D blood flow visualization was successfully employed to detect markedly altered filling and helical flow patterns (forward-moving spiral flow) in the ICA bulb and to evaluate the effect of revascularization, which restored filling and helical flow. Our results demonstrate the feasibility of flow-sensitive 4D MRI for the quantification and 3D visualization of physiological and pathological flow patterns in the carotid artery bifurcation.

Detection of Balamuthia mandrillaris DNA by real-time PCR targeting the RNase P gene.

BACKGROUND: The free-living amoeba Balamuthia mandrillaris may cause fatal encephalitis both in immunocompromised and in - apparently - immunocompetent humans and other mammalian species. Rapid, specific, sensitive, and reliable detection requiring little pathogen-specific expertise is an absolute prerequisite for a successful therapy and a welcome tool for both experimental and epidemiological research. RESULTS: A real-time polymerase chain reaction assay using TaqMan probes (real-time PCR) was established specifically targeting the RNase P gene of B. mandrillaris amoebae. The assay detected at least 2 (down to 0.5) genomes of B. mandrillaris grown in axenic culture. It did not react with DNA from closely related Acanthamoeba (3 species), nor with DNA from Toxoplasma gondii, Leishmania major, Pneumocystis murina, Mycobacterium bovis (BCG), human brain, various mouse organs, or from human and murine cell lines. The assay efficiently detected B. mandrillaris DNA in spiked cell cultures, spiked murine organ homogenates, B. mandrillaris-infected mice, and CNS tissue-DNA preparations from 2 patients with proven cerebral balamuthiasis. This novel primer set was successfully combined with a published set that targets the B. mandrillaris 18S rRNA gene in a duplex real-time PCR assay to ensure maximum specificity and as a precaution against false negative results. CONCLUSION: A real-time PCR assay for B. mandrillaris amoebae is presented, that is highly specific, sensitive, and reliable and thus suited both for diagnosis and for research.

Chinese acupuncture for chronic low back pain: an international expert survey.

BACKGROUND: Chinese acupuncture is widely accepted in western countries, and a number of clinical trials are testing Chinese acupuncture, especially for chronic low back pain (cLBP). However, little is known about how practitioners perform acupuncture treatment in daily clinical work and whether treatment styles differ among practitioners and countries. OBJECTIVE: To investigate whether styles of Chinese acupuncture treatment for cLBP are heterogeneous among different practitioners and countries. METHODOLOGY: Literature review, a prospective survey of international experts using a questionnaire with open and closed questions, and personal interviews. RESULTS: Data were collected from 18 experts in 10 different countries. The typical treatment regimen for cLBP, based on a composite of experts' responses, consisted of 11 sessions lasting 25 minutes each, held twice weekly. The typical practitioner inserted 12 needles, would select individual points according to channels and syndromes, and would identify syndromes such as kidney deficiency (yin and yang), cold dampness, and qi and blood stagnation. Preferred points were BL 23, BL 40, GB 34, BL 54, BL 60, BL 54, and Huatuo, although there was a high variation in additional local and distal points. Generally speaking, no expert would administer fewer than 6 treatments, omit Ahshi points, restrict needle insertion to 1 cm or less, or adhere solely to fixed points for all patients. CONCLUSIONS: For the first time it has been shown that there is a broad consistency in fundamental aspects of Chinese acupuncture for cLBP across different kinds of practitioners and different countries. These data can be used as minimal standards for the design and funding of future clinical trials.

Intracellular parasite kill: flow cytometry and NO detection for rapid discrimination between anti-leishmanial activity and macrophage activation.

Transgenic Leishmania expressing fluorescent reporter proteins such as green fluorescent protein (GFP) have opened the way for a flow cytometry (FACS)-based method to assess the killing of Leishmania parasites inside their macrophage host. Compared with counting parasites in microscopic preparations, the assessment of anti-leishmanial effects by FACS analysis promises both strict objectivity and significant reduction of labour-per-sample while scanning thousands of cells within seconds. Compared with other semi-automated methods based on host cell lysis and biochemical quantification of released parasites, the procedure is more direct and simple, reducing handling artefacts. An assay system is described using highly pure murine bone marrow-derived macrophages infected in vitro as a suspension culture with GFP-transfected Leishmania major promastigotes. The cells were rested for 24 h, allowing intracellular promastigotes to transform into amastigotes, and then exposed to macrophage-activating agents (IFN-gamma, LPS) or standard anti-leishmanial therapeutics. Within 48 h, the GFP signal from parasitized macrophages became indiscernible by FACS analysis, both in activated host cells and in cultures treated with the anti-leishmanials. In cultures activated with rIFN-gamma+LPS this coincided with the release of nitric oxides, but this was not the case in cultures treated with anti-leishmanials. Furthermore, by adding propidium iodide immediately before FACS analysis, the effect of treatment on the viability of the host cell was assessed at the same time. The combination of FACS analysis, and PI and NO detection offers a rapid and objective means of testing for intracellular anti-leishmanial effects and general cytotoxicity and gives a first indication of whether the former is due to direct leishmanicidal activity or indirect functions via macrophage activation.

Stimulus (polyphenol, IFN-gamma, LPS)-dependent nitric oxide production and antileishmanial effects in RAW 264.7 macrophages.

The effects of interferon (IFN-gamma), lipopolysaccharide (LPS), and some polyphenols as individual stimuli, as well as in various combinations on NO production in non-infected and infected macrophage-like RAW 264.7 cells were investigated, with emphasis on the NO/parasite kill relationship. In non-infected and in Leishmania parasitized cells, gallic acid significantly inhibited the IFN-gamma and LPS-induced NO detected in the supernatant. This effect was less prominent in IFN-gamma- than in LPS-stimulated cells. Interestingly, and in contrast to non-infected cells, gallic acid inhibited NO production only when added within 3h after IFN-gamma+LPS. Addition of gallic acid following prolonged incubation with IFN-gamma+LPS periods (24 h) no longer inhibited, sometimes even enhanced NO release. Notably, an excellent NO/parasite kill relationship was evident from all the experiments. This study was extended to a series of polyphenols (3-O-shikimic acid, its 3,5-digalloylated analogue, catechin, EGCG, and a procyanidin hexamer) with proven immunostimulatory activities. Although these compounds themselves were found to be weak NO-inducers, the viability of intracellular Leishmania parasites was considerably reduced. Furthermore, their dose-dependent effects on macrophage NO release was determined in the presence of IFN-gamma and/or LPS. Again, non-infected and infected cells differed significantly in the NO response, while inhibition of IFN-gamma and/or LPS-induced NO production by the tested polyphenols strongly depended on the given time of exposure and the sequence of immunological stimuli. A strong inverse correlation between NO levels and intracellular survival rates of Leishmania parasites supported the assumption that the observed inhibition of NO was not simply due to interference with the Griess assay used for detection.