P. Usha Sarma

Surfactant proteins A and D protect mice against pulmonary hypersensitivity induced by Aspergillus fumigatus antigens and allergens

Allergic bronchopulmonary aspergillosis (ABPA) is an allergic disorder caused by an opportunistic fungal pathogen, Aspergillus fumigatus (AFU:). Lung surfactant proteins SP-A and SP-D can interact with the glycosylated antigens and allergens of AFU:, inhibit specific IgE binding to these allergens, and block histamine release from sensitized basophils. We have now examined the therapeutic effect of exogenous administration of human SP-A, SP-D, and a recombinant fragment of SP-D (rSP-D), in a murine model of pulmonary hypersensitivity induced by AFU: antigens and allergens, which resembles human ABPA immunologically. The ABPA mice exhibited high levels of AFU:-specific IgG and IgE, blood eosinophilia, extensive infiltration of lymphocytes and eosinophils in the lung sections, and a Th2 cytokine response. Treatment with SP-A, SP-D, and rSP-D lowered blood eosinophilia, pulmonary infiltration, and specific Ab levels considerably, which persisted up to 4 days in the SP-A-treated ABPA mice, and up to 16 days in the SP-D- or rSP-D-treated ABPA mice. The levels of IL-2, IL-4, and IL-5 were decreased, while the level of IFN-gamma was raised in the splenic supernatants of the treated mice, indicating a marked shift from Th2 to Th1 response. These results clearly implicate pulmonary SP-A and SP-D in the modulation of allergic reactions.

Protective Role of Lung Surfactant Protein D in a Murine Model of Invasive Pulmonary Aspergillosis

ABSTRACT The protective effects of intranasal administration of amphotericin B (AmB), human SP-A, SP-D and a 60-kDa fragment of SP-D (rSP-D) were examined in a murine model of invasive pulmonary aspergillosis (IPA). The untreated group of IPA mice showed no survival at 7 days postinfection. Treatment with AmB, SP-D, and rSP-D increased the survival rate to 80, 60, and 80%, respectively, suggesting that SP-D (and rSP-D) can protect immunosuppressed mice from an otherwise fatal challenge with Aspergillus fumigatus conidia.

Susceptibility of Mice Genetically Deficient in the Surfactant Protein (SP)-A or SP-D Gene to Pulmonary Hypersensitivity Induced by Antigens and Allergens of Aspergillus fumigatus

Lung surfactant protein A (SP-A) and D (SP-D) are innate immune molecules which are known to interact with allergens and immune cells and modulate cytokine and chemokine profiles during host hypersensitivity response. We have previously shown therapeutic effects of SP-A and SP-D using a murine model of lung hypersensitivity to Aspergillus fumigatus (Afu) allergens. In this study, we have examined the susceptibility of SP-A (AKO) or SP-D gene-deficient (DKO) mice to the Afu allergen challenge, as compared with the wild-type mice. Both AKO and DKO mice exhibited intrinsic hypereosinophilia and several-fold increase in levels of IL-5 and IL-13, and lowering of IFN-γ to IL-4 ratio in the lungs, suggesting a Th2 bias of immune response. This Th2 bias was reversible by treating AKO or DKO mice with SP-A or SP-D, respectively. The AKO and DKO mice showed distinct immune responses to Afu sensitization. DKO mice were found more susceptible than wild-type mice to pulmonary hypersensitivity induced by Afu allergens. AKO mice were found to be nearly resistant to Afu sensitization. Intranasal treatment with SP-D or rhSP-D (a recombinant fragment of human SP-D containing trimeric C-type lectin domains) was effective in rescuing the Afu-sensitized DKO mice, while SP-A-treated Afu-sensitized AKO mice showed several-fold elevated levels of IL-13 and IL-5, resulting in increased pulmonary eosinophilia and damaged lung tissue. These data reaffirm an important role for SP-A and SP-D in offering resistance to pulmonary allergenic challenge.

Distinct alleles of mannose-binding lectin (MBL) and surfactant proteins A (SP-A) in patients with chronic cavitary pulmonary aspergillosis and allergic bronchopulmonary aspergillosis.

Abstract Background: Distinct host immune status predisposes to different forms of pulmonary aspergillosis. Methods: Patients with chronic cavitary pulmonary aspergillosis (CCPA; n=15) or allergic bronchopulmonary aspergillosis (ABPA; n=7) of Caucasian origin were screened for single nucleotide polymorphisms (SNPs) in the collagen region of surfactant proteins A1 (SP-A1) and A2 (SP-A2) and mannose binding lectin (MBL). Results: The T allele at T1492C and G allele at G1649C of SP-A2 were observed at slightly higher frequencies in ABPA patients (86% and 93%) than in controls (63% and 83%), and the C alleles at position 1492 and 1649 were found in higher frequencies in CCPA patients (33% and 25%) than in ABPA patients (14% and 7%) (all p>0.05). However, the CC genotype at position 1649 of SP-A2 was significantly associated with CCPA (χ2=7.94; pcorr≤0.05). Similarly, ABPA patients showed a higher frequency of the TT genotype (71%) at 1492 of SP-A2 than controls (43%) and CCPA patients (41%) (p>0.05). In the case of MBL, the T allele (OR=3.1, range 1.2–8.9; p≤0.02) and CT genotype (χ2=6.54; pcorr≤0.05) at position 868 (codon 52) were significantly associated with CCPA, but not with ABPA. Further analysis of genotype combinations at position 1649 of SP-A2 and at 868 of MBL between patient groups showed that both CC/CC and CC/CT SP-A2/MBL were found only in CCPA patients, while GG/CT SP-A2/MBL was significantly higher in CCPA patients in comparison to ABPA patients (p≤0.05). SNPs analysed in SP-A1 did not differ between cases and controls. Conclusions: Distinct alleles, genotypes and genotype combinations of SP-A2 and MBL may contribute to differential susceptibility of the host to CCPA or ABPA. Clin Chem Lab Med 2007;45:183–6.

Protective Roles of Pulmonary Surfactant Proteins, SP-A and SP-D, Against Lung Allergy and Infection Caused by Aspergillus fumigatus

Pulmonary surfactant proteins, SP-A and SP-D, are immune molecules which can directly interact with pathogens and allergens, stimulate immune cells and manipulate cytokine and chemokine profiles during hosts immune response. Using an opportunistic fungal pathogen Aspergillus fumigatus (Afu), we have attempted to understand participation of SP-A and SP-D in the host immunity. Afu causes a systemic infection via lungs, called invasive aspergillosis (IPA) in immunocompromised subjects. In the immunocompetent subjects, it can cause an allergic disorder, called allergic bronchopulmonary aspergillosis (ABPA). Therapeutic administration of these proteins in a murine model of IPA can rescue mice from death. Treating mice, having ABPA, can suppress IgE levels, eosinophilia, pulmonary cellular infiltration and cause a marked shift from a pathogenic Th2 to a protective Th1 cytokine profile. These results highlight the potential of SP-A, SP-D and their recombinant forms, as novel therapeutics for lung allergy and infection.

Susceptibility of mice genetically deficient in SP-A or SP-D gene to invasive pulmonary aspergillosis.

Pulmonary surfactant proteins, SP-A and SP-D, are carbohydrate pattern recognition molecules of innate immunity, which significantly enhance phagocytosis and killing of Aspergillus fumigatus, a pathogenic fungus, by neutrophils and macrophages. The present study examined the susceptibility of immunosuppressed SP-A gene deficient (SP-A(-/-)) or SP-D gene deficient (SP-D(-/-)) mice to A. fumigatus conidia challenge compared to wild-type (WT) mice. A. fumigatus-challenged SP-A(-/-) (SP-A(-/-) IPA) mice showed less mortality (40%) than the WT-IPA mice (100%) and increased mortality (60%) following administration of SP-A with decreased TNF-alpha and IFN-gamma to IL-4 ratio than SP-A(-/-) IPA mice. The SP-D(-/-) IPA mice (57.14%) showed similar mortality as WT-IPA mice (60%). However, the SP-D (-/-) IPA mice (42.86% mortality on day 2) died earlier than the WT-IPA mice (20% mortality on day 2), showed a higher hyphal density and tissue injury in lungs. Treatment with SP-D or a recombinant fragment of human SP-D rhSP-D reduced the mortality to 50% and 33%, respectively, concomitant with higher IFN-gamma to IL-4 ratios in treated SP-D(-/-) mice, compared to untreated control group. The results showed that SP-D gene deficient mice are more susceptible to IPA while SP-A gene deficient mice acquire resistance to IPA.

Recombinant surfactant protein-D selectively increases apoptosis in eosinophils of allergic asthmatics and enhances uptake of apoptotic eosinophils by macrophages

Pulmonary surfactant protein-D (SP-D) is a multifunctional, pattern recognition molecule involved in resistance to allergen challenge and pulmonary inflammation. In view of therapeutic effects of exogenous SP-D or recombinant fragment of human surfactant protein-D (rhSP-D) (composed of eight Gly-X-Y collagen repeat sequences, homotrimeric neck and lectin domains) in murine models of lung allergy and hypereosinophilic SP-D gene-deficient mice, we investigated the possibility of a direct interaction of purified rhSP-D with human eosinophils derived from allergic patients and healthy donors. rhSP-D showed a sugar- and calcium-dependent binding to human eosinophils, suggesting involvement of its carbohydrate recognition domain. While eosinophils from allergic patients showed a significant increase in apoptosis, oxidative burst and CD69 expression in presence of rhSP-D, eosinophils from healthy donors showed no significant change. However, these eosinophils from healthy donors when primed with IL-5 exhibited increase in apoptosis on incubation with rhSP-D. Apoptosis mediated by rhSP-D in primed eosinophils was not affected by the antioxidant, N-acetyl-L-cysteine. There was a manifold increase in binding of rhSP-D to apoptotic eosinophils than the normal eosinophils and rhSP-D induced a significant increase in uptake of apoptotic eosinophils by J774A.1 macrophage cells. The study suggests that rhSP-D mediated preferential increase of apoptosis of primed eosinophils while not affecting the normal eosinophils and increased phagocytosis of apoptotic eosinophils may be important mechanisms of rhSP-D and plausibly SP-D-mediated resolution of allergic eosinophilic inflammation in vivo.

Biodegradable nanoparticles as a sustained release system for the antigens/allergens of Aspergillus fumigatus: preparation and characterisation

Abstract Ultrafine monodisperse polyvinylpyrrolidone nanoparticles (PVP np) entrapping allergens/antigens of A. fumigatus were prepared by reverse micelles method. The entrapment efficiency of various formulations of PVP np varied from 74 to 92%. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), enzyme linked immunosorbent assay (ELISA) and western blot analysis showed intact integrity and immunoreactivity of the allergens/antigens after entrapment in nanoparticles. The effect of various factors such as size of the particles, ratio of monomer to antigen concentration, degree of cross-linking and schedule of immunisation were examined on the in vitro release rate and production of specific IgG and IgE antibodies in the mice. The studies showed that the formulation with 1% cross-linking agent with a mean particle size of 100 nm and a monomer: antigen ratio of 0.09:1 released encapsulated antigens in 9 weeks. The same formulation showed a sustained IgG level (>0.400 Abs) for approximately 12 weeks in comparison to IgG level (>0.400 Abs) for 7 days with free antigen. Increase in particle size or change in monomer concentration resulted in accelerated release rate. A sustained level of IgG antibodies achieved with three divided doses of entrapped antigen could also be achieved with same amount of entrapped antigens administered in single dose. The A. fumigatus specific IgE levels of nanoparticle entrapped allergens/antigens were lower than obtained with free allergens/antigens. It is concluded that PVP np provide a promising delivery system for proteins as it is biocompatible, conserves the integrity and biological activities of proteins, augments antibody response and provide a sustained antibody level with single step immunisation. The formulation may also be useful for hyposensitisation therapy for various allergens.

Human surfactant protein D alters oxidative stress and HMGA1 expression to induce p53 apoptotic pathway in eosinophil leukemic cell line

Surfactant protein D (SP-D), an innate immune molecule, has an indispensable role in host defense and regulation of inflammation. Immune related functions regulated by SP-D include agglutination of pathogens, phagocytosis, oxidative burst, antigen presentation, T lymphocyte proliferation, cytokine secretion, induction of apoptosis and clearance of apoptotic cells. The present study unravels a novel ability of SP-D to reduce the viability of leukemic cells (eosinophilic leukemic cell line, AML14.3D10; acute myeloid leukemia cell line, THP-1; acute lymphoid leukemia cell lines, Jurkat, Raji; and human breast epithelial cell line, MCF-7), and explains the underlying mechanisms. SP-D and a recombinant fragment of human SP-D (rhSP-D) induced G2/M phase cell cycle arrest, and dose and time-dependent apoptosis in the AML14.3D10 eosinophilic leukemia cell line. Levels of various apoptotic markers viz. activated p53, cleaved caspase-9 and PARP, along with G2/M checkpoints (p21 and Tyr15 phosphorylation of cdc2) showed significant increase in these cells. We further attempted to elucidate the underlying mechanisms of rhSP-D induced apoptosis using proteomic analysis. This approach identified large scale molecular changes initiated by SP-D in a human cell for the first time. Among others, the proteomics analysis highlighted a decreased expression of survival related proteins such as HMGA1, overexpression of proteins to protect the cells from oxidative burst, while a drastic decrease in mitochondrial antioxidant defense system. rhSP-D mediated enhanced oxidative burst in AML14.3D10 cells was confirmed, while antioxidant, N-acetyl-L-cysteine, abrogated the rhSP-D induced apoptosis. The rhSP-D mediated reduced viability was specific to the cancer cell lines and viability of human PBMCs from healthy controls was not affected. The study suggests involvement of SP-D in host’s immunosurveillance and therapeutic potential of rhSP-D in the eosinophilic leukemia and cancers of other origins.

A rapid method for the isolation of genomic DNA from Aspergillus fumigatus

A majority of Aspergillus induced diseases are reported to be caused by Aspergillus fumigatus. In immunocompromized and post transplant cases it can lead to invasive aspergillosis. Due to this the molecular fingerprinting of aspergillus isolates by RFLP analysis and development of DNA diagnostic probes are gaining importance. Different methodologies are being adopted for extraction of the genomic DNA from fungus. The existing procedures for isolation of DNA are time consuming and range from several hours to few days. The most difficult step in the isolation of DNA from aspergillus species is to disrupt the tough chitin rich cell wall without causing damage to genomic DNA. We report here a rapid method for extraction of genomic DNA based on the cleavage of chitin with chitinase. The subsequent modification steps included are lysis and microwave treatment. The chromosomal DNA obtained by this procedure is 1.5-2.0 micrograms per mg of wet weight of mycelia and is observed to be minimally sheared. It is pure enough for restriction analysis and for use in the PCR to detect the gene coding for 18 kDa allergen which has been identified in our laboratory using western blot analysis with human patient sera.