A. Kharazmi

Mucoid conversion of Pseudomonas aeruginosa by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung

The leading cause of mortality in patients with cystic fibrosis (CF) is respiratory failure due in large part to chronic lung infection with Pseudomonas aeruginosa strains that undergo mucoid conversion, display a biofilm mode of growth in vivo and resist the infiltration of polymorphonuclear leukocytes (PMNs), which release free oxygen radicals such as H2O2. The mucoid phenotype among the strains infecting CF patients indicates overproduction of a linear polysaccharide called alginate. To mimic the inflammatory environment of the CF lung, P. aeruginosa PAO1, a typical non-mucoid strain, was grown in a biofilm. This was treated with low levels of H2O2, as if released by the PMNs, and the formation of mucoid variants was observed. These mucoid variants had mutations in mucA, which encodes an anti-sigma factor; this leads to the deregulation of an alternative sigma factor (sigma22, AlgT or AlgU) required for expression of the alginate biosynthetic operon. All of the mucoid variants tested showed the same mutation, the mucA22 allele, a common allele seen in CF isolates. The mucoid mucA22 variants, when compared to the smooth parent strain PA01, (i) produced 2-6-fold higher levels of alginate, (ii) exhibited no detectable differences in growth rate, (iii) showed an unaltered LPS profile, (iv) were approximately 72% reduced in the amount of inducible-beta-lactamase and (v) secreted little or no LasA protease and only showed 44% elastase activity. A characteristic approximately 54 kDa protein associated with alginate overproducing strains was identified as AlgE (Alg76) by N-terminal sequence analysis. Thus, the common phenotype of the mucoid variants, which included a genetically engineered mucA22 mutant, suggested that the only mutation incurred as a result of H2O2 treatment was in mucA. When a P. aeruginosa biofilm was repeatedly exposed to activated PMNs in vitro, mucoid variants were also observed, mimicking in vivo observations. Thus, PMNs and their oxygen by-products may cause P. aeruginosa to undergo the typical adaptation to the intractable mu- coid form in the CF lung. These findings indicate that gene activation in bacteria by toxic oxygen radicals, similar to that found in plants and mammalian cells, may serve as a defence mechanism for the bacteria. This suggests that mucoid conversion is a response to oxygen radical exposure and that this response is a mechanism of defence by the bacteria. This is the first report to show that PMNs and their oxygen radicals can cause this phenotypic and genotypic change which is so typical of the intractable form of P. aeruginosa in the CF lung. These findings may provide a basis for the development of anti-oxidant and anti-inflammatory therapy for the early stages of infection in CF patients.

High levels of plasma IL-10 and expression of IL-10 by keratinocytes during visceral leishmaniasis predict subsequent development of post-kala-azar dermal leishmaniasis

Some patients develop post‐kala‐azar dermal leishmaniasis (PKDL) after they have been treated for the systemic infection kala‐azar (visceral leishmaniasis). It has been an enigma why the parasites cause skin symptoms after the patients have been successfully treated for the systemic disease. We report here that PKDL development can be predicted before treatment of visceral leishmaniasis, and that IL‐10 is involved in the pathogenesis. Before treatment of visceral leishmaniasis, Leishmania parasites were present in skin which appeared normal on all patients. However, IL‐10 was detected in the keratinocytes and/or sweat glands of all patients who later developed PKDL (group 1) and not in any of the patients who did not develop PKDL (group 2). Furthermore, the levels of IL‐10 in plasma as well as in peripheral blood mononuclear cell culture supernatants were higher in group 1 than in group 2.

Improved outcome of chronic Pseudomonas aeruginosa lung infection is associated with induction of a Th1-dominated cytokine response

Repeated challenge with antigen is involved in the pathogenesis of a variety of pulmonary diseases. Patients with cystic fibrosis (CF) experience recurrent pulmonary colonization with Pseudomonas aeruginosa before establishment of chronic lung infection. To mimic recurrent lung infections in CF patients, the lungs of susceptible BALB/c mice were re‐infected with P. aeruginosa 14 days after the initial infection. Singly‐infected BALB/c mice, as well as non‐infected mice, were used as controls. Decreased mortality and milder lung inflammation in re‐infected BALB/c mice, as well as a tendency for improved clearance of bacteria, was observed when compared with singly‐infected mice. The improved outcome in re‐infected mice correlated with changes in CD4 cell numbers. Surface expression of LFA‐1 on pulmonary CD4 cells was increased in re‐infected compared with singly‐infected mice. Moreover, resistance to re‐infection was paralleled by a shift towards a Th1‐dominated response and increased IL‐12 production. No significant increase in serum IgG was observed in the re‐infected mice. In conclusion, these results indicate a protective role for a Th1‐dominated response, independent of antibody production, in chronic P. aeruginosa lung infection in CF.

Chalcones from Chinese liquorice inhibit proliferation of T cells and production of cytokines.

Licochalcone A (LicA), an oxygenated chalcone, has been shown to inhibit the growth of both parasites and bacteria. In this study, we investigated the effect of LicA and four synthetic analogues on the activity of human peripheral blood mononuclear cell proliferation and cytokine production. Four out of five chalcones tested inhibited the proliferation of lymphocytes measured by thymidine incorporation and by flow cytometry. The production of pro- and anti-inflammatory cytokines from monocytes and T cells was also inhibited by four of five chalcones. Furthermore, intracellular detection of cytokines revealed that the chalcones inhibited the production rather than the release of the cytokines. Taken together, these results indicate that LicA and some analogues may have immunomodulatory effects, and may thus be candidates not only as anti-microbial agents, but also for the treatment of other types of diseases.

Evaluation of the polymerase chain reaction in the diagnosis of cutaneous leishmaniasis due to Leishmania major: a comparison with direct microscopy of smears and sections from lesions

We have compared the sensitivity of the polymerase chain reaction (PCR) as a diagnostic tool against conventional microscopical diagnostic techniques in patients with cutaneous leishmaniasis from the Sudan. Twenty-eight patients were diagnosed according to clinical criteria followed by microscopical examination of histological sections and slit or impression smears. The PCR had a sensitivity of 86% when used alone, and 93% when combined with Southern blotting. In contrast, microscopy of histological sections had a sensitivity of 76% and slit and impression smears of only 55% and 48%, respectively. The PCR should be considered as a valuable and sensitive diagnostic tool in the diagnosis of cutaneous leishmaniasis; it has the added advantage of identification of the species of Leishmania causing the lesion.

Immunopathology of post kala-azar dermal leishmaniasis (PKDL): T-cell phenotypes and cytokine profile

In Sudan, post kala‐azar dermal leishmaniasis (PKDL) caused by Leishmania donovani develops in half of the patients treated for visceral leishmaniasis (kala‐azar). In most patients lesions heal spontaneously, but in others symptoms are severe and persist for years. This study examined the immunological response in lesions of PKDL patients by immunohistochemistry and compared the findings with results obtained using peripheral blood mononuclear cells (PBMCs). In all lesions, parasites or parasite antigen were present and provoked the formation of an inflammatory infiltrate consisting of a mixture of macrophages, lymphocytes, and plasma cells. In patients who had high interferon‐gamma (IFNγ) responses to Leishmania antigen in vitro, compact epithelioid granulomas were formed. The inflammatory cells were mainly CD3+ and interleukin‐10 (IL10) was the most prominent cytokine in the lesions. However, IFNγ was found in all and IL4 in most lesions, in varying amounts. PBMCs from all patients responded to Leishmania antigen by IFNγ production or proliferation. The results indicate that PKDL develops as a result of an influx of immunocompetent cells into skin, which harbours parasites. The inflammatory response to the parasites is complex. It involves several cell types and cytokines, of which some are antagonistic. It is conceivable that the balance between these cytokines determines the outcome of the disease. Copyright

The development of post-kala-azar dermal leishmaniasis (PKDL) is associated with acquisition of Leishmania reactivity by peripheral blood mononuclear cells (PBMC)

PKDL develops in about 50% of Sudanese patients treated for visceral leishmaniasis (kala‐azar). Patients with kala‐azar were entered into this study and followed for a period of up to 2u2003years. During follow up 12 patients developed PKDL and eight did not. Proliferative responses and cytokine production to Leishmania donovani and control antigens were measured inu2003vitro using PBMC isolated at the time of diagnosis of kala‐azar, after treatment of visceral leishmaniasis, during follow up, and at the time of diagnosis of PKDL. Proliferative responses and interferon‐gamma (IFN‐γ) production were low at diagnosis and increased after treatment of kala‐azar in both patients who developed (group 1) and those who did not develop PKDL later (group 2). In group 1, development of PKDL was always associated by an increased PBMC response to Leishmania antigen in proliferation and IFN‐γ production assays. There were no differences in Leishmania antigen‐induced production of IL‐4, IL‐5 and IL‐10 between or within the two groups. We have previously shown that Leishmania parasites spread to the skin during visceral leishmaniasis and proposed that PKDL was the result of an immunological attack on parasites, which have survived in the skin despite the drug treatment. The finding that PKDL develops after treatment of kala‐azar as Leishmania‐reactive T cells start to circulate in peripheral blood in sufficient numbers to be detected in inu2003vitro assays supports this hypothesis.

Faster activation of polymorphonuclear neutrophils in resistant mice during early innate response to Pseudomonas aeruginosa lung infection.

Polymorphonuclear neutrophils (PMNs) are crucial for the outcome of Pseudomonas aeruginosa lung infection in patients with cystic fibrosis. We compared PMNs and inflammatory cytokines in the lungs and blood from susceptible BALB/c and resistant C3H/HeN mice 1 and 2 days after intratracheal challenge with alginate embedded P. aeruginosa. These parameters were correlated with the quantitative bacteriology and histopathology of the lungs. After challenge, the content of granulocyte colony‐stimulating factor (G‐CSF) and macrophage inflammatory protein‐2 (MIP‐2) was increased in the lungs and the sera and the percentage of PMNs was increased in the blood. However, 2 days after challenge the concentration of G‐CSF and MIP‐2 was higher in the lungs and sera of BALB/c mice. CD11b expression was higher on the PMNs of the C3H/HeN mice. The expression of CD62L on PMNs of both strains of mice was decreased 1 day after bacterial challenge, whereas the expression was increased after 2 days of challenge on PMNs of C3H/HeN mice only. These changes were accompanied by a more severe lung inflammation in BALB/c mice and faster clearance of the bacteria in C3H/HeN mice. In conclusion, the rapid early bacterial clearance in the lungs of C3H/HeN mice could be explained by faster activation of the PMNs, as indicated by the higher up‐regulation of CD11b. The severe lung inflammation in BALB/c mice may be caused by the early higher content of G‐CSF in the sera mobilizing PMNs from the bone marrow and the persistent chemotactic gradient provided by MIP‐2 in the lungs.

Detection and characterization of Leishmania in tissues of patients with post kala-azar dermal leishmaniasis using a specific monoclonal antibody

Sections from skin lesions and draining lymph nodes of patients with post kala-azar dermal leishmaniasis were examined using an immunoperoxidase method and a monoclonal antibody directed against Leishmania donovani. Parasites were detected in 22 of 25 biopsies (88%). In parallel sections stained by haematoxylin and eosin, parasites were detected in only 5 of 25 of the biopsies (20%). The demonstration of whole parasites or parasite antigen is diagnostic.

Molecular characterization of a Leishmania donovanii cDNA clone with similarity to human 20S proteasome a-type subunit

Using plasma from patients infected or previously infected with Leishmania donovanii, we isolated a L. donovanii cDNA clone with similarity to the proteasome a-type subunit from humans and other eukaryotes. The cDNA clone, designated LePa, was DNA sequenced and Northern blot analysis of L. donovanii poly(A(+))mRNA indicated the isolation of a full length cDNA clone with a transcript size of 1.9 kb. The expressed recombinant LePa fusion protein induced proliferation of peripheral blood mononuclear cells in one out of seven patients who had suffered from visceral leishmaniasis. Plasma from 16 out of 25 patients with visceral leishmaniasis and four out of 18 patients with cutaneous leishmaniasis contained IgG antibodies which reacted with the purified LePa fusion protein as evaluated in an ELISA. The LePa DNA sequence was inserted into an eukaryotic expression vector and Balb/c mice were vaccinated. DNA vaccination of Balb/c mice with LePa generated an initial significant reduction in lesion size after challenge.