Arsalan Kharazmi

Pseudomonas aeruginosa and the in vitro and in vivo biofilm mode of growth.

The biofilm mode of growth is the survival strategy of environmental bacteria like Pseudomonas aeruginosa. Such P. aeruginosa biofilms also occur in the lungs of chronically infected cystic fibrosis patients, where they protect the bacteria against antibiotics and the immune response. The lung tissue damage is due to immune complex mediated chronic inflammation dominated by polymorphonuclear leukocytes releasing proteases and oxygen radicals.

Licochalcone A, a new antimalarial agent, inhibits in vitro growth of the human malaria parasite Plasmodium falciparum and protects mice from P. yoelii infection.

Licochalcone A, isolated from Chinese licorice roots, inhibited the in vitro growth of both chloroquine-susceptible (3D7) and chloroquine-resistant (Dd2) Plasmodium falciparum strains in a [3H]hypoxanthine uptake assay. The growth inhibition of the chloroquine-resistant strain by licochalcone A was similar to that of the chloroquine-susceptible strain. To examine the activity of licochalcone A on the different asexual blood stages of the parasite, licochalcone A was added to highly synchronized cultures containing rings, trophozoites, and schizonts. The growth of the parasites at all stages was inhibited by licochalcone A. The in vivo activity of licochalcone A was tested in a mouse model of infection with P. yoelii. Licochalcone A administered either intraperitoneally or orally for 3 to 6 days protected the mice from the otherwise lethal P. yoelii infection. These results demonstrate that licochalcone A exhibits potent antimalarial activity and might be developed into a new antimalarial drug.

Inhibition of Fumarate Reductase in Leishmania major and L. donovani by Chalcones

ABSTRACT Our previous studies have shown that chalcones exhibit potent antileishmanial and antimalarial activities in vitro and in vivo. Preliminary studies showed that these compounds destroyed the ultrastructure of Leishmania parasite mitochondria and inhibited the respiration and the activity of mitochondrial dehydrogenases of Leishmania parasites. The present study was designed to further investigate the mechanism of action of chalcones, focusing on the parasite respiratory chain. The data show that licochalcone A inhibited the activity of fumarate reductase (FRD) in the permeabilized Leishmania major promastigote and in the parasite mitochondria, and it also inhibited solubilized FRD and a purified FRD from L. donovani. Two other chalcones, 2,4-dimethoxy-4′-allyloxychalcone (24m4ac) and 2,4-dimethoxy-4′-butoxychalcone (24mbc), also exhibited inhibitory effects on the activity of solubilized FRD in L. majorpromastigotes. Although licochalcone A inhibited the activities of succinate dehydrogenase (SDH), NADH dehydrogenase (NDH), and succinate- and NADH-cytochrome c reductases in the parasite mitochondria, the 50% inhibitory concentrations (IC50) of licochalcone A for these enzymes were at least 20 times higher than that for FRD. The IC50 of licochalcone A for SDH and NDH in human peripheral blood mononuclear cells were at least 70 times higher than that for FRD. These findings indicate that FRD, one of the enzymes of the parasite respiratory chain, might be the specific target for the chalcones tested. Since FRD exists in the Leishmaniaparasite and does not exist in mammalian cells, it could be an excellent target for antiprotozoal drugs.

The immune response to chronic Pseudomonas aeruginosa lung infection in cystic fibrosis patients is predominantly of the Th2 type

Most cystic fibrosis (CF) patients with chronic Pseudomonas aeruginosa lung infection have a persistent acute type lung inflammation dominated by polymorphonuclear neutrophils (PMN) and a pronounced antibody response against P. aeruginosa. We speculated whether this immune response in CF is of the Th2 type and whether a change to a Th1 type immune response could improve the prognosis. Therefore, we studied 14 CF patients with (CF +P) and 14 CF patients without (CF –P) chronic P. aeruginosa lung infection. The specific production of interferon‐gamma (IFN‐γ) and interleukin‐4 (IL‐4) by peripheral blood mononuclear cells was determined. Cells from CF +P patients had lower IFN‐γ (p<0.05) and higher IL‐4 (p<0.005) production as compared to cells from CF ‐P patients. Furthermore, a positive correlation between IFN‐γ production and lung function was found (FVC: Rho=0.637; p<0.03; FEV1: Rho=0.524; p<0.07). We conclude that a Th2 type immune response is most frequent in CF patients with chronic P. aeruginosa lung infection, and the patients with a Th1‐dominated immune response had the best lung function. The clinical implication is that a change to a Th1 type immune response might improve the prognosis in these patients.

Blood Monocyte and Neutrophil Functions in the Acquired Immune Deficiency Syndrome

The acquired immune deficiency syndrome (AIDS) is characterized by infections with microorganisms against which phagocytes (especially monocytes/macrophages) play an important role. Therefore, various functions of blood monocytes and neutrophils were tested in 16 patients with AIDS. Neutrophil chemotactic responses towards casein and N‐formyl‐methionyl‐L‐leucyl‐L‐phenylalanine were depressed in patients with a short duration of disease (n=9), whereas they were normal in those with a longer duration (n=5), P<0.05. Neutrophil superoxide anion release was normal. In contrast, we found no evidence of an altered monocyte activity in the patients, since chemotactic responsiveness, phagocytosis of opsonized Candida albicans, and superoxide anion release were all normal. These findings suggest that the depressed neutrophil chemotaxis may play an important role in the high incidence of opportunistic infections in AIDS. Furthermore, it appears that in AIDS the immune deficiency does not extend to peripheral blood monocytes, but it does not exclude the possibility that the function of tissue macrophages is abnormal.

Induction of beta-lactamase production in Pseudomonas aeruginosa biofilm.

Imipenem induced high levels of beta-lactamase production in Pseudomonas aeruginosa biofilms. Piperacillin also induced beta-lactamase production in these biofilms but to a lesser degree. The combination of beta-lactamase production with other protective properties of the biofilm mode of growth could be a major reason for the persistence of this sessile bacterium in chronic infections.

Mechanisms involved in the evasion of the host defence by Pseudomonas aeruginosa

Pseudomonas aeruginosa, an extracellular opportunistic pathogen, utilizes two major mechanisms to evade the host defence system. One of these mechanisms is the production of a large number of extracellular products, such as proteases, toxins, and lipases. The two proteases, alkaline protease and elastase, inhibit the function of the cells of the immune system (phagocytes, NK cells, T cells), inactivate several cytokines (IL-1, IL-2, IFN-r, TNF), cleave immunoglobulins and inactivate complement. Inhibition of the local immune response by bacterial proteases provides an environment for the colonization and establishment of chronic infection. The other mechanism by which P. aeruginosa evades the host defence system is the biofilm mode of growth of the bacteria in chronic infections. The biofilm-grown bacteria induce a low phagocyte response, and provide a barrier for the bacteria against antibodies, complement, and the cells of the immune system. Protection from the host defence system combined with increased antibiotic resistance of the bacteria in the biofilm are the major reasons for the persistence of P. aeruginosa in chronic infections.

Polymorphonuclear leucocytes consume oxygen in sputum from chronic Pseudomonas aeruginosa pneumonia in cystic fibrosis

Background: Chronic lung infection with Pseudomonas aeruginosa is the most severe complication for patients with cystic fibrosis (CF). This infection is characterised by endobronchial mucoid biofilms surrounded by numerous polymorphonuclear leucocytes (PMNs). The mucoid phenotype offers protection against the PMNs, which are in general assumed to mount an active respiratory burst leading to lung tissue deterioration. An ongoing respiratory burst by the PMNs has, however, not been demonstrated previously in endobronchial secretions from chronically infected patients with CF. Objective: Based on the accumulating evidence for depletion of molecular oxygen (O2) in the mucus in infected CF bronchi, it was hypothesised that the O2 depletion in the mucus in infected CF bronchi may be accelerated by the respiratory burst of the PMNs due to the reduction of O2 to the superoxide anion (O-2) by the phagocyte NADPH oxidase (Phox). Methods: Methods were established to isolate the O2 consumption by the respiratory burst from aerobic respiration in freshly expectorated sputum from chronically infected patients with CF. Results: Inhibition of the Phox with diphenylene iodonium (DPI) delayed O2 depletion, nearly abolished staining of O-2-producing PMNs with hydroethidine and inhibited the rapid luminol-enhanced chemiluminescence in sputum. Furthermore, the total O2 consumption was correlated to the concentration of PMNs in the sputum samples. Conclusion: The results demonstrate that CF sputum contains PMNs with an active consumption of O2 for O-2 production and suggest that the respiratory burst is ongoing and causes accelerated O2 depletion due to formation of O-2 in the lungs of chronically infected patients with CF.

Comparison of the Immune Profile of Nonhealing Cutaneous Leishmaniasis Patients with Those with Active Lesions and Those Who Have Recovered from Infection

ABSTRACT Th1-type cellular immune responses play a critical role in protection against infection with Leishmania parasites, whereas activation of Th2-type cells results in progressive disease. Cutaneous leishmaniasis caused by Leishmania major is often a self-healing disease; however, persistent nonhealing forms are also known. In the present study, we have described cell-mediated immune responses in nonhealing patients by measuring T-cell proliferation, cytokine production, and phenotypic characterization of these cells. The responses were compared with those of patients with active lesions, patients who had recovered from infection, and healthy controls. Peripheral blood mononuclear cells from patients with active lesions and recovered donors proliferated vigorously and produced Th1-type cytokine when stimulated with L. major antigens, whereas in nonhealing patients the proliferative responses were significantly lower and showed a Th2-type response to Leishmaniaantigens. Interleukin-10 (IL-10) production was not a feature ofL. major stimulation. Flow cytometric analysis revealed that L. major antigen induced proliferation of the CD4-positive population and that these cells were the major source of gamma interferon and IL-4. These results show a distinct dichotomy in the cytokine response to L. major infection.

Interleukin 6 primes human neutrophil and monocyte oxidative burst response

Interleukin 6 (IL-6), a 26-kDa inducible protein, is a cytokine with multiple biological activities. This paper reports on the regulatory role of rIL-6 on the function of human polymorphonuclear and mononuclear leukocytes, a property not described previously. rIL-6 by itself did not exhibit any chemotactic activity and it could not activate these cells for an oxidative burst response. Preincubation of both cell types with rIL-6 at concentrations of 5 and 50 ng/ml primed the cells for enhanced generation of oxygen radicals following stimulation with the chemotactic peptide f-Met-Leu-Phe or the phorbol ester PMA. The enhancement of the oxidative burst response occurred both at the level of superoxide anion generation, an early step in the activation pathway, and at the level of the hydrogen peroxide-myeloperoxidase mediated response, a later step in the oxidative burst pathway. The priming ability was abolished by heat treatment of rIL-6 at 100 degrees C but not at 70 degrees C. Stimulation of B cell growth and immunoglobulin production combined with enhancement of oxidative burst response of phagocytic cells by IL-6 provide an effective mechanism of fighting against invading micro-organisms.