Omid Rasool

Experimental neonatal group B streptococcal pneumonia : effect of a modified porcine surfactant on bacterial proliferation in ventilated near-term rabbits

ABSTRACT: We studied bacterial proliferation in relation to surfactant treatment in a model of neonatal group B streptococcal (GBS) pneumonia. Surfactant (Curosurf) was isolated from pig lungs with a method preserving only polar lipids and hydrophobic proteins. Near-term rabbit fetuses were ventilated in a body plethysmograph system. At 15 min, a suspension of GBS strain 090 la LD (5 mL/kg, concentration ~109/mL) was instilled intratracheally. At 30 min, surfactant (n = 12) or sterile saline (n = 13) was administered via the airways (2.5 mL/kg). A control group (n = 12) received the same volumes of saline. After 5 h the animals were killed, and samples for blood cultures and blood gases were taken from the heart. The left lung was aseptically removed, weighed, homogenized, serially diluted, and cultured on blood agar plates. The results were expressed as mean log10 colony forming units/g lung ± SD. Compared with animals (n = 12) killed immediately after GBS instillation (8.13 ± 0.54), there was a significant increase in bacterial numbers in both groups ventilated for 5 h, but values for surfactant-treated animals (8.96 ± 0.38) were lower than those for animals receiving saline (9.46 ± 0.50; p < 0.05). After 5 h, 96% of GBS-infected animals had positive blood cultures. Light microscopic examination of the right lung of GBS-infected animals revealed inflammatory changes that tended to be less prominent in surfactant-treated rabbits. We conclude that intratracheal inoculation of near-term rabbits with GBS resulted in a significant bacterial proliferation during 5 h of ventilation and that bacterial growth was mitigated by treatment with surfactant.

Higher pH level, corresponding to that on the skin of patients with atopic eczema, stimulates the release of Malassezia sympodialis allergens

Background:  The opportunistic yeast Malassezia is a trigger factor in atopic eczema (AE). Around 30–80% of patients with AE have an IgE and/or T‐cell reactivity to the yeast. Several IgE‐binding components have been identified in Malassezia extracts and 11 allergens have been cloned and sequenced. The pH of the skin surface in patients with AE is higher than that of normal healthy skin. We here investigate whether different pH conditions mimicking those of AE skin and healthy skin can influence the production and release of Malassezia allergens.

Rational design of hypoallergens applied to the major cat allergen Fel d 1

Background Allergen‐specific immunotherapy is the only treatment for allergic disease providing long‐lasting symptom relief. Currently, it is mainly based on the use of crude allergen extracts. The treatment may be improved by the use of genetically engineered allergens, hypoallergens, aiming at a more effective and safer therapy.

Cloning and Characterisation of Two IgE-Binding Proteins, Homologous to Tropomyosin and α-Tubulin, from the Mite Lepidoglyphus destructor

Background: The dust mite Lepidoglyphus destructor is a major source of mite allergy in European rural environments, but it also causes allergy in urban populations around the world. We have previously cloned, sequenced and expressed several allergens from L. destructor (Lep d 2, Lep d 5, Lep d 7 and Lep d 13). The aim of this study was to identify and clone additional allergens from L. destructor, and to evaluate their IgE-binding reactivities. Methods: PCR and screening with sera from L. destructor-sensitised individuals were used to isolate new clones from a phage display L. destructor cDNA library. The complete coding sequences of the clones were determined and expressed as His6-tagged recombinant proteins in Escherichia coli. The recombinant proteins were analysed by SDS-PAGE, immunoblotting and mass spectrometry. Results: Two new clones, showing homology to tropomyosin and α-tubulin in several species, were isolated from the phage display L. destructor cDNA library. Due to its homology to group 10 dust mite allergens, the tropomyosin clone was named Lep d 10. The IgE-binding frequencies of the recombinant Lep d 10 and α-tubulin were 13% (18/136) and 12% (11/95), respectively, among subjects with IgE reactivity to mites and/or crustaceans. Conclusions: Two new allergens from L. destructor have been identified and can now be added to the repertoire of recombinant L. destructor allergens. In addition, both these allergens belong to highly conserved protein families and may be important for evaluation of allergenic cross-reactivity.

Detection of Mala f and Mala s allergen sequences within the genus Malassezia.

Malassezia species are opportunistic yeasts that are involved in the pathogenesis of a number of skin diseases including atopic eczema/dermatitis syndrome. Previously, we cloned six allergens from Malassezia sympodialis isolate ATCC 42132; these allergens are designated Mala s 1, and Mala s 5-Mala s 9. Three additional allergens, Mala f 2-Mala f 4, have been isolated from M. furfur by other investigators. The objective of the present study was to investigate the presence of these Mala sequences in seven Malassezia species. Genomic DNA amplification by PCR and sequencing showed that M. globosa, M. obtusa and M. sympodialis contain DNA sequences corresponding to all the allergens except Mala f 2 and Mala f 3. M. pachydermatis contains Mala s 1, Mala f 4, and Mala s 5-Mala s 8. M. restricta and M. slooffiae possessed Mala f 4 and Mala s 6. M. furfur was seen to possess Mala f 2-Mala f 4 as well as Mala s 5-Mala s 7. Our data from reverse-transcriptase PCR showed a more species-specific pattern of amplification. M. furfur evidenced expression of Mala f 2-Mala f 4. M. globosa and M. obtusa appeared to express only Mala s 6. M. pachydermatis expressed Mala f 4, Mala s 6, and Mala s 8, while M. restricta and M. slooffiae expressed Mala f 4 and Mala s 6. M. sympodialis expressed all the allergens except Mala f 2 and Mala f 3. Different Malassezia species appear to contain both common and species-specific allergen sequences.

Mala s 12 is a major allergen in patients with atopic eczema and has sequence similarities to the GMC oxidoreductase family

Background:  Atopic eczema (AE) is a chronic inflammatory skin disorder, characterized by impaired skin barrier and itch. The yeast Malassezia belongs to the normal human skin microflora and can induce IgE‐ and T‐cell‐mediated allergic reactions in AE patients. Previously, we have identified several IgE‐binding components in Malassezia sympodialis extract.

Intralipid® decreases the bacterial lipopolysaccharide induced release of oxygen radicals and lysozyme from human neutrophils

Human neutrophils were incubated either with purified cell envelope lipopolysaccharides (LPS) of salmonella or with different concentrations of LPS combined with Intralipid. Incubation of neutrophils with LPS alone increased their oxidative metabolism with increased release of oxygen radicals as measured by the nitroblue tetrazolium (NBT) test and chemiluminescence response. The amount of lysozyme released by the cells also increased during incubation with LPS. However, when the neutrophils were incubated with LPS together with Intralipid, the LPS induced stimulation of the neutrophil NBT reduction, chemiluminescence and lysozyme release was significantly decreased. Intralipid might substitute for plasma high density lipoproteins (HDL), which are known to inhibit the LPS effects on the neutrophils in the acute stage of an infection with Gram-negative bacteria.

Macrophage reaction in rabbit lung following inhalation of iron chloride

Groups of eight rabbits were inhalation-exposed to iron, 1.4 +/- 0.7 mg/m3 (low Fe), or 3.1 +/- 1.8 mg/m3 (high Fe) as FeCl3 or to filtered air (controls) for 2 months, 5 days/week and 6 hours/day. The alveolar macrophages were increased in number in both exposed groups. Noduli of granular macrophages were found in lungs of all the rabbits in the high-Fe group, in one from the low-Fe group, and in one control rabbit. Especially in the high-Fe group there were prominent changes in the macrophages such as enlarged lysosomes containing fibrous-looking structures, iron-rich inclusions, and densely packed, 5-nm electron-dense granules. The number of cells filled with surfactant-like inclusions as well as a smooth surface was increased in the high-Fe group and the macrophages had enhanced phagocytic capacity. There was an increase in the phospholipid concentration and in the volume density of type II cells in the high-Fe group but the level of phosphatidylcholines was not significantly changed. The fact that Fe3+ affected mainly the alveolar macrophages might be due to the relatively high concentration of iron in these cells caused by the precipitation of iron in their lysosomes.

Rabbit lung after combined exposure to soluble cobalt and trivalent chromium

Eight rabbits were exposed to 0.7 +/- 0.4 mg/m3 Co2+ as CoCl2 and 1.2 +/- 0.7 mg/m3 Cr3+ as Cr(NO3)3 (group Co + Cr), eight to 0.6 +/- 0.5 mg/m3 Co2+ (group Co), and eight to filtered air (control group), for 4 months, 5 days/week, and 6 hr/day. All rabbits in group Co + Cr and group Co showed nodular aggregation of alveolar epithelial type II cells. Volume density of the type II cells was significantly higher in group Co + Cr than in group Co and the control group. There was intraalveolar macrophage accumulation in seven rabbits in group Co + Cr, one in group Co, and one in the control group. In lavage fluid the numbers of macrophages and the percentage of these cells with smooth surface and intracellular surfactant-like inclusions were more increased in group Co + Cr than in group Co as were oxidative metabolic and phagocytic activities of the macrophages. Total phospholipids, phosphatidylcholines, and especially 1,2-dipalmitoylphosphatidylcholine was markedly increased in group Co + Cr whereas only 1,2-dipalmitoylphosphatidylcholine was slightly increased in group Co. One mechanism behind the high amount of surfactant phospholipids in group Co + Cr seems to be an enhanced production of surfactant by the type II cells. Another mechanism is probably that Cr3+ reduces the capacity of alveolar macrophages to catabolize surfactant. The results imply that it is important to investigate effects of combinations of cobalt and chromium in the occupational environment.