Nitrous oxide production in sputum from cystic fibrosis patients with chronic Pseudomonas aeruginosa lung infection.

Chronic lung infection by Pseudomonas aeruginosa is the major severe complication in cystic fibrosis (CF) patients, where P. aeruginosa persists and grows in biofilms in the endobronchial mucus under hypoxic conditions. Numerous polymorphonuclear leukocytes (PMNs) surround the biofilms and create local anoxia by consuming the majority of O2 for production of reactive oxygen species (ROS). We hypothesized that P. aeruginosa acquires energy for growth in anaerobic endobronchial mucus by denitrification, which can be demonstrated by production of nitrous oxide (N2O), an intermediate in the denitrification pathway. We measured N2O and O2 with electrochemical microsensors in 8 freshly expectorated sputum samples from 7 CF patients with chronic P. aeruginosa infection. The concentrations of NO3(-) and NO2(-) in sputum were estimated by the Griess reagent. We found a maximum median concentration of 41.8 µM N2O (range 1.4-157.9 µM N2O). The concentration of N2O in the sputum was higher below the oxygenated layers. In 4 samples the N2O concentration increased during the initial 6 h of measurements before decreasing for approximately 6 h. Concomitantly, the concentration of NO3(-) decreased in sputum during 24 hours of incubation. We demonstrate for the first time production of N2O in clinical material from infected human airways indicating pathogenic metabolism based on denitrification. Therefore, P. aeruginosa may acquire energy for growth by denitrification in anoxic endobronchial mucus in CF patients. Such ability for anaerobic growth may be a hitherto ignored key aspect of chronic P. aeruginosa infections that can inform new strategies for treatment and prevention.

A complex multilevel attack on Pseudomonas aeruginosa algT/U expression and algT/U activity results in the loss of alginate production.

Infection by the opportunistic pathogen Pseudomonas aeruginosa is a leading cause of morbidity and mortality seen in cystic fibrosis (CF) patients. This is mainly due to the genotypic and phenotypic changes of the bacteria that cause conversion from a typical nonmucoid to a mucoid form in the CF lung. Mucoid conversion is indicative of overproduction of a capsule-like polysaccharide called alginate. The alginate-overproducing (Alg(+)) mucoid phenotype seen in the CF isolates is extremely unstable. Low oxygen tension growth of mucoid variants readily selects for nonmucoid variants. The switching off mechanism has been mapped to the algT/U locus, and the molecular basis for this conversion was partially attributed to mutations in the algT/U gene itself. To further characterize molecular changes resulting in the unstable phenotype, an isogenic PAO1 derivative that is constitutively Alg(+) due to the replacement of the mucA with mucA22 (PDO300) was used. The mucA22 allele is common in mucoid CF isolates. Thirty-four spontaneous nonmucoid variants, or sap (suppressor of alginate production) mutants, of PDO300 were isolated under low oxygen tension. About 40% of the sap mutants were rescued by a plasmid carrying algT/U (Group A). The remaining sap mutants were not (Group B). The members of Group B fall into two subsets: one similar to PAO1, and another comparable to PDO300. Sequence analysis of the algT/U and mucA genes in Group A shows that mucA22 is intact, whereas algT/U contains mutations. Genetic complementation and sequencing of one Group B sap mutant, sap22, revealed that the nonmucoid phenotype was due to the presence of a mutation in PA3257. PA3257 encodes a putative periplasmic protease. Mutation of PA3257 resulted in decreased algT/U expression. Thus, inhibition of algT/U is a primary mechanism for alginate synthesis suppression.

Immunophenotypic pattern and cytokine profiles of dry type cutaneous leishmaniasis.

BACKGROUND: Dry type localized cutaneous leishmaniasis, one of the most prevalent cutaneous parasitic infections in Kerman Province, is presented as a polarized disease in which cytokine profiles secreted by immune cells play a major role in its presentation. In order to clarify the idea, immunohistochemical study of skin biopsies were performed to elucidate the cytokine release capabilities of immune cells. METHODS: Skin biopsies of acute, chronic nonlupoid, and chronic lupoid recidivans lesions of dry type localized cutaneous leishmaniasis were studied by immunohistochemical staining methods for immunophenotypic patterns (CD4, CD8, CD14, CD19, CD56, CD11a, CD18, CD1a, HLA-DR, CD54) and cytokines (INF-gamma, IL-12, IL-4, TNF-alpha) released by immune inflammatory cells. RESULTS: The descriptive analysis of data showed that the mean percentage of positive immunostained cells of CD4, CD8, and CD14; antigen-presenting cells (CD1a, HLA-DR); and markers of the extravasated positive memory T cells (CD11a, CD18, CD54) are more frequent in lupoid recidivans than in acute active and chronic nonlupoid lesions, in order of frequency. CONCLUSION: Based on the results, it seems that Th1-like response is predominant in acute active form and lupoid recidivans while Th2-like response is predominant in chronic nonlupoid lesions. It seems that lupoid recidivans is a type IV hypersensitivity reaction to the reactivation of hidden antigens.

Pathology of post-kala-azar dermal leishmaniasis: a light microscopical, immunohistochemical, and ultrastructural study of skin lesions and draining lymph nodes.

BACKGROUND: Whereas the clinical manifestations and treatment of post-kala-azar dermal leishmaniasis (PKDL) have been adequately described before, the pathology received little attention, particularly the African form of PKDL which shows some clinical differences from the disease in India. Therefore, our aim was to characterize the pathology and the immunohistopathology in PKDL lesions and correlate the histopathological findings with the clinical features of the disease. METHODS: Biopsies of skin lesions were examined for histopathological changes in formalin-fixed tissues and for cell phenotypes and adhesion molecules by immunohistochemistry. RESULTS: The epidermis showed various changes in different combinations. The dermis was infiltrated by lymphocytes and macrophages, but plasma cells were scanty or absent. The majority of cells were CD3 T cells, with a preponderance of CD4 over CD8 cells. Degenerating basal keratinocytes expressed HLA-DR, ICAM-1 and Leishmania antigen and closely interacted with CD4 T cells. Regional lymph nodes showed hyperplasia of the B- and T-cell zones. CONCLUSIONS: The inflammatory reaction in PKDL lesions is in response to Leishmania parasites and/or antigen. The majority of cells are CD4 T cells. Degeneration of the basal keratinocytes is probably due to the action of cytotoxic CD4 T cells interacting with leishmania-expressing epidermal cells. Ismail A, Gadir AFA, Theander TG, Kharazmi A, El Hassan AM. Pathology of post-kala-azar dermal leishmaniasis: a light microscopical, immunohistochemical, and ultrastructural study of skin lesions and draining lymph nodes.

Leishmania donovani: an in vitro study of antimony-resistant amphotericin B-sensitive isolates.

Drug sensitivity of clinically antimony-unresponsive Leishmania donovani isolates from Eastern Sudan was evaluated in an in vitro culture system against sodium stibogluconate (Pentostam) and Amphotericin B. Eight isolates, six from antimony-resistant and two from clinically responsive patients were included in the study. Parasites were tested as promastigotes and four of them were selected to be tested as amastigotes using a murine macrophage-like cell line. The results indicated that the conventional promastigotes and amastigotes-screening assays did not correlate with the clinical picture of patients. In vivo unresponsiveness does not necessarily mean primary parasite resistance. Amphotericin B could be a suitable second line drug in patients unresponsive to pentostam and without concomitant diseases, if close hospital monitoring is available. Promastigotes sensitivity testing concentrations are virtually incomparable with the in vivo clinically curable doses and the amastigotes/macrophage test concentrations.

Conformationally restricted anti-plasmodial chalcones.

Chalcones can exist as Z- or E-isomers and it is generally anticipated that both isomers are equipotent. In order to determine the active isomer of anti-plasmodial chalcones a series of analogues locked in the Z- or the E-form were prepared and evaluated for their anti-plasmodial activity. It was shown that the Z-locked analogue was nearly inactive, whereas the E-locked analogues were equipotent to the parent chalcones, indicating that the E-isomer is the active conformation.

[Ginseng modulates the immune response via its effect on cytokine production--secondary publication]. / Ginseng modulerer immunresponset via påvirkning af cytokinproduktionen--sekundaerpublikation.

Ginseng has been shown to modulate the immune system. In this study, ginseng"s effects against chronic P. aeruginosa lung infection in animal models and its cytokine-modulating effects on leukocytes were investigated. Leukocytes cultured with ginseng produced more interleukin-12. Animal models treated with ginseng s.c. showed a much faster bacterial clearance, milder lung pathology, activation of phagocytes and a Th1 immune response. These results suggest that ginseng treatment induced a Th1-like immune response, which improved the pathologic course in our animal models.

Synthesis and in vitro leishmanicidal activity of 2-(1-methyl-5-nitro-1H-imidazol-2-yl)-5-substituted-1,3,4-thiadiazole derivatives.

A series of 2-(1-methyl-5-nitroimidazol-2-yl)-5-(1-piperazinyl, 1-piperidinyl and 1-morpholinyl)-1,3,4-thiadiazoles (3a-g) were synthesized and evaluated for in vitro leishmanicidal activity against Leishmania major promastigotes. The leishmanicidal data revealed that compounds 3a-g had strong and much better leishmanicidal activity than the reference drug pentostam. Compound 3c (piperazine analog) was the most active compound (IC50=0.19 microM).

Serum concentrations of GM-CSF and G-CSF correlate with the Th1/Th2 cytokine response in cystic fibrosis patients with chronic Pseudomonas aeruginosa lung infection.

The inflammation in cystic fibrosis (CF) patients with chronic Pseudomonas aeruginosa lung infection is dominated by polymorphonuclear neutrophils (PMNs). There seems to be a relationship between the PMN-dominated inflammation, pronounced antibody production and a Th2-dominated response. Apart from mobilizing monocytes and PMNs from the bone marrow, GM-CSF, G-CSF and IL-3 select subsets of dendritic cells, which subsequently induce distinct Th responses. Therefore, the present study examines the correlation between the mobilizing cytokines in serum and the Th responses. The IFN-gamma and IL-4 production by peripheral blood mononuclear cells, and the concentrations of GM-CSF and G-CSF in serum as well as lung function, were determined in 37 CF patients with and 6 CF patients without chronic P. aeruginosa lung infection. The GM-CSF/G-CSF ratio correlated both with the IFN-gamma production and good lung function. In addition, an inverse correlation between IL-3 and IFN-gamma was observed. The results indicate involvement of endogenous GM-CSF, G-CSF and IL-3 in the skewed Th response in CF, and change to a Th1-dominated response might be achieved with GM-CSF treatment.

Synthesis and in vitro leishmanicidal activity of 2-(5-nitro-2-furyl) and 2-(5-nitro-2-thienyl)-5-substituted-1,3,4-thiadiazoles.

A series of 2-(5-nitro-2-furyl) and 2-(5-nitro-2-thienyl)-5-substituted-1,3,4-thiadiazoles (5a-d and 6a-j) were synthesized and evaluated against Leishmania major promastigotes using (3)H-thymidine incorporation. Most of the compounds showed activity better than the reference drug sodium stibogluconate (Pentostam). The most active compound was 6c (IC(50)=0.1 microM).

A quantitative study of epidermal Langerhans cells in cutaneous leishmaniasis caused by Leishmania tropica.

OBJECTIVE: The purpose of this study was to characterize the number and distribution of epidermal Langerhans cells in different clinical forms of dry-type cutaneous leishmaniasis (CL). METHODS: Sixteen cases of dry-type cutaneous leishmaniasis caused by Leishmania tropica were studied. These cases were classified clinically as five cases of acute leishmaniasis with indurated papules, nodules and plaques with central crust formation and duration < 2 years, six cases of lupoid leishmaniasis with characteristic papules around previous scars of cutaneous leishmaniasis with duration > 2 years, and five cases of chronic nonlupoid type with nonhealing lesions of duration > 2 years. Paraffin-embedded blocks were stained with hematoxylin and eosin (H&E) and stained immunohistochemically for CD1a. RESULTS: The number of Langerhans cells per millimeter length of epidermis was increased in acute cases compared to chronic and lupoid cases. CONCLUSIONS: Lesions of acute leishmaniasis contain the greatest amounts of antigen for presentation, so Langerhans cells increase in number and in trafficking to present antigens derived from Leishman bodies to the cellular immune system. In chronic leishmaniasis, the Langerhans cell population is reduced, perhaps because of exhaustion of the source of Langerhans cells, or because of reduced response to modified antigen.

Effect of smoking and abstention on oxidative burst and reactivity of neutrophils and monocytes.

BACKGROUND: Smoking is associated with surgical wound infections, impaired wound healing, and tissue-destructive disorders. The mechanisms are largely unknown, but changes in the function and activity of inflammatory cells may be involved. METHODS: Seventy healthy volunteers (54 smokers and 16 never smokers) were included. The smokers were studied while they smoked and after 20 days of abstinence. After the first 10 days of abstinence, they were randomized to double-blind treatment with transdermal nicotine patch 25 mg per day or placebo. Venous blood neutrophils and monocytes were sampled and isolated. In 22 randomly selected smokers and in all never smokers, the oxidative burst and chemotaxis were determined by a chemiluminescence response assay and a modified Boyden chamber technique, respectively. Stimulants were opsonized zymosan, formyl-Met-Leu-Phe, and zymosan-activated serum. RESULTS: The neutrophil and monocyte oxidative burst was 50% and 68% lower, respectively, in smokers compared to never smokers (P < .05). Neutrophil chemotaxis was 93% higher in smokers (P < .05). Monocyte chemotaxis was lower in smokers compared to never smokers (P < .05). After 20 days of abstinence, neutrophil oxidative burst increased to the level of never smokers (P < .05); monocyte oxidative burst increased by 50% (P < .05). Chemotaxis was only marginally affected. The changes induced by abstinence were less pronounced in the transdermal nicotine patch group compared to the placebo group. CONCLUSIONS: Smoking attenuates the oxidative burst of inflammatory cells and increases chemotaxis. Three weeks of abstinence normalize the oxidative burst, but affect chemotaxis only marginally.

Ginseng modulates the immune response by induction of interleukin-12 production.

In infections with intracellular microorganisms such as mycobacteria and Leishmania parasites as well as certain extracellular chronic infections such as Pseudomonas aeruginosa a Th1 response with activation of macrophages is desirable. Several studies indicate that such a response is associated with better recovery from infection, improved course of the chronic infection, and higher survival rate. In Th1 responses there is increased interferon-gamma (IFN-gamma) and interleukin-12 (IL-12) production, whereas that of interleukin-10 (IL-10) is decreased. The present study indicated that Ginseng modulation of stimulated peripheral blood mononuclear cells (PBMC) results in a higher IL-12 production. The enhanced IL-12 production could induce a stronger Th1 response, resulting in better protection against infection with a variety of pathogens.

Alginate production affects Pseudomonas aeruginosa biofilm development and architecture, but is not essential for biofilm formation.

Extracellular polymers can facilitate the non-specific attachment of bacteria to surfaces and hold together developing biofilms. This study was undertaken to qualitatively and quantitatively compare the architecture of biofilms produced by Pseudomonas aeruginosa strain PAO1 and its alginate-overproducing (mucA22) and alginate-defective (algD) variants in order to discern the role of alginate in biofilm formation. These strains, PAO1, Alg+ PAOmucA22 and Alg- PAOalgD, tagged with green fluorescent protein, were grown in a continuous flow cell system to characterize the developmental cycles of their biofilm formation using confocal laser scanning microscopy. Biofilm Image Processing (BIP) and Community Statistics (COMSTAT) software programs were used to provide quantitative measurements of the two-dimensional biofilm images. All three strains formed distinguishable biofilm architectures, indicating that the production of alginate is not critical for biofilm formation. Observation over a period of 5 days indicated a three-stage development pattern consisting of initiation, establishment and maturation. Furthermore, this study showed that phenotypically distinguishable biofilms can be quantitatively differentiated.

Pseudomonas aeruginosa alginate is refractory to Th1 immune response and impedes host immune clearance in a mouse model of acute lung infection.

Pseudomonas aeruginosa is an opportunistic respiratory pathogen that accounts for most of the morbidity and mortality in cystic fibrosis (CF) patients. In CF-affected lungs, the bacteria undergo conversion from a non-mucoid to a non-tractable mucoid phenotype, due to overproduction of alginate. The effect of alginate production on pathogenicity was investigated by using an acute lung infection mouse model that compared a non-mucoid P. aeruginosa strain, PAO1, to its constitutive alginate-overproducing derivative, Alg(+) PAOmucA22, and an alginate-defective strain, Alg(-) PAOalgD. Bacterial suspensions were instilled into the left bronchus and examined 24 and 48 h post-infection. The highest bacterial loads and the most severe lung pathology were observed with strain Alg(-) PAOalgD at 24 h post-infection, which may have been due to an increase in expression of bacterial elastase by the mutant. Significantly lower lung and spleen bacterial loads were found in the two non-mucoid (PAO1 and Alg(-) PAOalgD) groups, compared to the mucoid Alg(+) PAOmucA22 group, between 24 and 48 h post-infection. The positive correlation between lung bacteriology and lung macroscopic pathology in the Alg(+) PAOmucA22 group suggests that alginate production not only impedes pulmonary clearing, but also results in severe lung damage. Positive correlations between IL12 levels and lung macroscopic pathology, and between IL12 and IFN-gamma levels in the Alg(+) PAOmucA22 group, suggested a possible contribution of these pro-inflammatory cytokines to tissue damage. No significant differences were found between the three groups in lung cytokine responses at 24 or 48 h post-infection. However, on comparison within each group at 24 and 48 h post-infection, a significant increase in the pro-inflammatory cytokine IFN-gamma was observed. Higher ratios of IFN-gamma/IL4 and IFN-gamma/IL10, but lower IL10 levels, were also found in all three groups. These results indicate a Th1-predominated immune response in these animals. Such cytokine responses could have aided the clearance of non-mucoid P. aeruginosa, but were not sufficient to alleviate infection by the mucoid variants. Alginate production may promote survival and persistence of this pathogenic micro-organism in the lung.

An antiinflammatory galactolipid from rose hip (Rosa canina) that inhibits chemotaxis of human peripheral blood neutrophils in vitro.

The galactolipid (2S)-1,2-di-O-[(9Z,12Z,15Z)-octadeca-9,12,15-trienoyl]-3-O-beta-d-galactopyranosyl glycerol (1) isolated from dried and milled fruits of Rosa canina by bioassay-guided fractionation is an antiinflammatory agent with inhibitory effects on chemotaxis of human peripheral blood neutrophils in vitro. The inhibition of cell migration is not related to toxicity. The presence of 1 in rose hips may explain the clinically observed antiinflammatory properties of rose hip herbal remedies.

Cytokine modulating effect of ginseng treatment in a mouse model of Pseudomonas aeruginosa lung infection.

The major cause of morbidity and mortality in cystic fibrosis (CF) patients is chronic Pseudomonas aeruginosa lung infection. In a mouse model of P. aeruginosa lung infection mimicking that in CF patients, the effects of ginseng treatment on cytokine responses and the correlation between the changes in cytokine production and the lung pathology were studied. Mice were challenged with alginate beads containing P. aeruginosa (10(9) CFU/ml). A saline extract of ginseng was injected subcutaneously at a dosage of 250 mg/kg of body weight/day for 7 days. Saline was used as a placebo control. One week after challenge, a significantly lower mortality was found in the ginseng treated group (P < 0.005). The lung cells from the ginseng treated group produced more interferon-gamma (IFN-gamma) (P < 0.04) and tumor necrosis factor-alpha (TNF-alpha) (P < 0.03) but less interleukin-4 (IL-4) (P < 0 .02) with a higher ratio of IFN-gamma/IL-4 (P < 0.004) after 6 and/or 24 h of incubation with specific and non-specific antigens as compared to the control group. The ginseng treated splenocytes produced more TNF-alpha (P < 0.03) and IFN-gamma (P0.05) than the control spleen cells. Furthermore, a significantly milder lung pathology (P < 0.025) and a faster bacterial clearance (P < 0.038) from the lungs were also found in the ginseng treated group compared to the control group. These results indicate a Th1-like immune response in the mice with P. aeruginosa lung infection after 7 days of ginseng treatment, which is an important mechanism accounting for ginseng's favorable action. We therefore believe that Th1 response might benefit the host with P. aeruginosa lung infection and ginseng treatment might be a promising alternative measure for the treatment of chronic P. aeruginosa lung infection in CF patients.

Gerimax ginseng regulates both humoral and cellular immunity during chronic Pseudomonas aeruginosa lung infection.

BACKGROUND: Chronic lung infection among patients with cystic fibrosis (CF), diffused panbronchiolitis, and chronic obstructive bronchiecteisis is often because of Pseudomonas aeruginosa. High morbidity and mortality in patients with CF are because of P. aeuruginosa that undergoes genotypic and phenotypic changes during prolonged stay in the lung resulting in increased antibiotic resistance, necessitating a search for alternative or supplement drugs. OBJECTIVE: In this study we compared the therapeutical effect of Gerimax (Dansk Droge A/S, Ishøj, Denmark) ginseng with placebo control by using a rat model of chronic P. aeruginosa lung infection mimicking that in patients with CF. METHODS AND INTERVENTIONS: The animals were challenged intratracheally with the prototypic P. aeruginosa PAO1 in alginate beads (1 x 10(9) colony-forming units per milliliter [CFU/mL]) followed by subcutaneous injection of ginseng extract (150 mg/kg body weight once per day) and examined on days 7 and 21. RESULTS: The day 7 analyses show that ginseng treatment resulted in lowering serum immunoglobulin M (IgM) and lung interleukin-4 (IL-4) levels compared to the control group. On day 21, higher lung IgA, upregulated serum IgG2a, stronger lung responses of interferon-gamma, IL-4, and tumor necrosis factor-alpha with milder lung pathology and enhanced lung bacteriology were detected in the ginseng-treated group when compared to those of the control group. CONCLUSION: These results suggest that the Gerimax ginseng treatment can modulate the immune system in favor of clearing the infection with P. aeruginosa in the lungs of rats. Thus, ginseng might be a promising alternative supplement for the treatment of chronic P. aeruginosa lung infection in patients with CF.

Antiplasmodial compounds from Cochlospermum tinctorium.

Fractionation of an ethanol extract of roots of Cochlospermum tinctorium afforded five compounds: 3-O-E-p-coumaroylalphitolic acid (1), cochloxanthin (2), dihydrocochloxanthin (3), alphitolic acid (4), and 1-hydroxytetradecan-3-one (5). This is the first example of a 1-hydroxyalkan-3-one obtained from plant material after gentle workup. The antiplasmodial activities of the compounds were determined, and the IC(50) value of 3-O-E-p-coumaroylalphitolic acid was 2.3 microM.

Aurones interfere with Leishmania major mitochondrial fumarate reductase.

A series of aurones was analyzed for the ability to inhibit respiratory functions of mitochondria of Leishmania parasites. The aim of this study was to find a rational explanation for the activity of certain aurones and auronols as novel antiprotozoal compounds of plant origin. In a cell-free assay mitochondrial fumarate reductase from L. donovani was inhibited in a concentration-dependent manner. The most active compounds were 4',6-dihydroxyaurone and 6-methoxyaurone which inhibited parasite enzyme activity at 25 nM by over ninety percent.