Functional characterization of the different oligomeric forms of human surfactant protein SP-D.

Surfactant Protein D (SP-D) is a collectin protein that participates in the innate immune defense of the lungs. SP-D mediates the clearance of invading microorganisms by opsonization, aggregation or direct killing, which are lately removed by macrophages. SP-D is found as a mixture of trimers, hexamers, dodecamers and higher order oligomers, "fuzzy balls". However, it is unknown whether there are differences between these oligomeric forms in functions, activity or potency. In the present work, we have obtained fractions enriched in trimers, hexamers and fuzzy balls of full-length recombinant human (rh) SP-D by size exclusion chromatography, in a sufficient amount to perform functional assays. We have evaluated the differences in protein lectin-dependent activity relative to aggregation and binding to E. coli, one of the ligands of SP-D in vivo. Fuzzy balls are the most active oligomeric form in terms of binding and aggregation of bacteria, achieving 2-fold binding higher than hexamers and 50% bacteria aggregation at very short times. Hexamers, recently described as a defined oligomeric form of the protein, have never been isolated or tested in terms of protein activity. rhSP-D hexamers efficiently bind and aggregate bacteria, achieving 50-60% aggregation at final time point and high protein concentrations. Nevertheless, trimers are not able to aggregate bacteria, although they bind to them. Therefore, SP-D potency, in functions that relay on the C-lectin activity of the protein, is proportional to the oligomeric state of the protein.

Differential levels of surfactant protein A, surfactant protein D, and progesterone to estradiol ratio in maternal serum before and after the onset of severe early-onset preeclampsia.

PROBLEM: Preeclampsia (PE), a multifactorial disorder characterized by impaired placental development, elevated inflammatory response and dysregulated placental steroidogenesis. PE may be preventable if predicted early on. METHOD OF STUDY: The study evaluated the potential of immunomodulatory collectins, surfactant protein A (SP-A), surfactant protein D (SP-D), and mannose binding lectin (MBL), to predict PE before the disease onset, in a prospective study cohort of healthy pregnant women (n = 922). In addition, a cross-sectional study was conducted to determine the serum and placental profile of collectins in PE women after the disease onset (early-onset PE [EOPE], n = 33; late-onset PE [LOPE], n = 24); and controls [n = 75]. The serum profiles of estradiol (E2) and progesterone (P4) were evaluated to determine their correlation with collectins. RESULTS: In the prospective cohort, significantly decreased serum levels of SP-A, SP-D, P4/E2 ratio were observed in women who subsequently developed severe EOPE. Interestingly, after the disease onset, there was a significant increase in serum and placental levels of collectins in women with severe EOPE, whereas women with LOPE had significantly decreased levels of collectins. Serum P4/E2 ratio was significantly altered in severe EOPE and positively correlated with serum levels of SP-A and SP-D. CONCLUSION: Collectins are differentially expressed in the serum during progression of PE. Decreased serum levels of SP-A, SP-D, P4/E2 ratio and increased E2 during 10-20 weeks of gestation are novel plausible risk factors for early prediction of EOPE in Indian women.

Increased expression of immune-related genes in leukocytes of patients with diagnosed gestational diabetes mellitus (GDM).

Compelling evidence indicates that the immune system is linked to metabolism in gestational diabetes mellitus (GDM), but factors participating in these processes still are awaiting identification. Inducible nitric oxide synthase, encoded by the NOS2 gene, and surfactant protein D, encoded by the SFTPD gene, have been implicated in diabetes. We investigated NOS2 and SFTPD mRNA levels in leukocytes obtained from 125 pregnant women with (n = 87) or without (control group; n = 38) GDM, and, in turn, correlated their expression with clinical parameters of subjects. Leukocytes were isolated from the blood of pregnant women and NOS2 and SFTPD expression in these cells was determined by quantitative real time PCR (qRT-PCR). Univariate correlation analyses were performed to assess an association between leukocyte NOS2 and SFTPD expression and clinical characteristics of patients. qRT-PCR experiments disclosed significantly increased leukocyte NOS2 and SFTPD mRNA levels in hyperglycemic GDM patients (P < 0.05). In the entire study group, there were significant positive associations of leukocyte NOS2 and SFTPD mRNAs with C-reactive protein. Additionally, transcript level of SFTPD also correlated positively with fasting glycemia and insulin resistance. This study demonstrates that an impaired glucose metabolism in GDM may be predominant predictor of leukocyte NOS2 and SFTPD overexpression in diabetic patients. Furthermore, alterations in the expression of these genes are associated with glucose metabolism dysfunction and/or inflammation during pregnancy. In addition, these findings support the utilization of leukocytes as good experimental model to study a relationship between immune-related genes and metabolic changes in women with GDM, as well as to assess the potential mechanisms underlying these alterations.

Innate Immune Molecule Surfactant Protein D Attenuates Sepsis-induced Acute Pancreatic Injury through Modulating Apoptosis and NF-κB-mediated Inflammation.

Sepsis causes multiple-organ dysfunction including pancreatic injury, thus resulting in high mortality. Innate immune molecule surfactant protein D (SP-D) plays a critical role in host defense and regulating inflammation of infectious diseases. In this study we investigated SP-D functions in the acute pancreatic injury (API) with C57BL/6 Wild-type (WT) and SP-D knockout (KO) mice in cecal ligation and puncture (CLP) model. Our results confirm SP-D expression in pancreatic islets and intercalated ducts and are the first to explore the role of pancreatic SP-D in sepsis. CLP decreased pancreatic SP-D levels and caused severe pancreatic injury with higher serum amylase 24 h after CLP. Apoptosis and neutrophil infiltration were increased in the pancreas of septic KO mice (p < 0.05, vs septic WT mice), with lower Bcl-2 and higher caspase-3 levels in septic KO mice (p < 0.05). Molecular analysis revealed increased NF-κB-p65 and phosphorylated IκB-α levels along with higher serum levels of TNF-α and IL-6 in septic KO mice compared to septic WT mice (p < 0.01). Furthermore, in vitro islet cultures stimulated with LPS produced higher TNF-α and IL-6 (p < 0.05) from KO mice compared to WT mice. Collectively, these results demonstrate SP-D plays protective roles by inhibiting apoptosis and modulating NF-κB-mediated inflammation in CLP-induced API.

The production mechanism and immunosuppression effect of pulmonary surfactant protein D via toll like receptor 4 signaling pathway in human corneal epithelial cells during Aspergillus fumigatus infection.

OBJECTIVE: To observe the production mechanism of surfactant protein D (SP-D) in human corneal epithelial cells (HCECs) when infected by Aspergillus fumigatus (A. fumigatus) hyphae, and explore whether SP-D can inhibit the cell activations through toll-like receptor 4 signaling pathway during fungal infection. METHODS: mRNA and protein expressions of SP-D were evaluated in HCECs after stimulation by A. fumigatus, with or without pretreatment of TLR4 inhibitor (CLI-095) by real time PCR and Western blot. The expression levels of inflammatory cytokines IL-1ß and IL-8 evaluated when pretreated with SP-D antibody or recombinant human SP-D in fungi-stimulated HCECs by real time PCR and ELISA, IL-1ß and IL-8 expressions were also detected in A. fumigatus-stimulated HCECs that pretreated with CLI095 or MyD88 inhibitor (Pepinh-MYD) and recombinant human SP-D. RESULTS: mRNA and protein levels of SP-D increased after stimulation of A. fumigatus for 16h and 20h respectively. The upregulation of SP-D could be inhibited by CLI-095. mRNA and protein expressions of IL-1ß and IL-8 decreased significantly when pretreated HCECs with recombinant human SP-D for 4h before A. fumigatus stimulation, while IL-1ß and IL-8 increased when pretreated with SP-D antibody for 1h. Pretreatment of CLI095 or Pepinh-MYD can increase the expressions of IL-1ß and IL-8 mRNA and protein in HCECs induced by recombinant human SP-D and A. fumigatus. CONCLUSIONS: SP-D can be stimulated by TLR4 during A. fumigatus infection. Recombinant human SP-D can inhibit the expression of inflammatory cytokines through TLR4 signaling pathway.

Ovarian Hormones Regulate SP-D Expression in the Mouse Uterus During Estrous Cycle and Early Pregnancy.

PROBLEM: Differential expression of SP-D in the cycling human and mouse endometrium suggests its regulation by ovarian hormones. METHOD OF STUDY: SP-D expression in the mouse uterus was analyzed across the estrous cycle and during early pregnancy. Effect of exogenous ovarian hormones on the uterine expression of SP-D was analyzed. RESULTS: SP-D expression varied across the estrous cycle and peaked in the estrous phase. SP-D transcript levels increased by fourfold in the uteri of estrogen-treated mice while co-administration of estrogen and progesterone enhanced SP-D levels by ninefold. However, treatment with progesterone alone significantly downregulated SP-D expression. Diethylstilbestrol enhanced SP-D transcript levels in the uteri of immature mice by 10-fold. During pregnancy, SP-D levels declined rapidly from 0.5 dpc to 6.5 dpc. In silico analysis predicted the presence of two potential ERE and 1 PRE in the mouse SP-D gene promoter region. CONCLUSION: Estrogen positively regulates expression of SP-D in the mouse uterus. Progesterone, along with estrogen synergizes SP-D expression, however, when administered alone results in negative regulation.

Dectin-2 deficiency promotes Th2 response and mucin production in the lungs after pulmonary infection with Cryptococcus neoformans.

Dectin-2 is a C-type lectin receptor that recognizes high mannose polysaccharides. Cryptococcus neoformans, a yeast-form fungal pathogen, is rich in polysaccharides in its cell wall and capsule. In the present study, we analyzed the role of Dectin-2 in the host defense against C. neoformans infection. In Dectin-2 gene-disrupted (knockout) (Dectin-2KO) mice, the clearance of this fungus and the inflammatory response, as shown by histological analysis and accumulation of leukocytes in infected lungs, were comparable to those in wild-type (WT) mice. The production of type 2 helper T (Th2) cytokines in lungs was higher in Dectin-2KO mice than in WT mice after infection, whereas there was no difference in the levels of production of Th1, Th17, and proinflammatory cytokines between these mice. Mucin production was significantly increased in Dectin-2KO mice, and this increase was reversed by administration of anti-interleukin 4 (IL-4) monoclonal antibody (MAb). The levels of expression of ß1-defensin, cathelicidin, surfactant protein A (Sp-A), and Sp-D in infected lungs were comparable between these mice. In in vitro experiments, IL-12p40 and tumor necrosis factor alpha (TNF-α) production and expression of CD86 and major histocompatibility complex (MHC) class II by bone marrow-derived dendritic cells and alveolar macrophages were completely abrogated in Dectin-2KO mice. Finally, the disrupted lysates of C. neoformans, but not of whole yeast cells, activated Dectin-2-triggered signaling in an assay with nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cells expressing this receptor. These results suggest that Dectin-2 may oppose the Th2 response and IL-4-dependent mucin production in the lungs after infection with C. neoformans, and it may not be required for the production of Th1, Th17, and proinflammatory cytokines or for clearance of this fungal pathogen.

Purification of surfactant protein D (SP-D) from pooled amniotic fluid and bronchoalveolar lavage.

Surfactant protein SP-D is a multimeric collagenous lectin, called collectin. SP-D is a multifunctional, pattern recognition innate immune molecule, which binds in a calcium dependent manner to an array of carbohydrates and lipids, thus offering resistance to invading pathogens, allergen challenge, and pulmonary inflammation. SP-D is predominantly found in the endoplasmic reticulum of type 2 pneumocytes and in the secretory granules of Clara or non-ciliated bronchiolar cells. The highest expression of SP-D is observed in the distal airways and alveoli. There is also an extra pulmonary existence of SP-D. The common sources of native full-length human SP-D are bronchoalveolar lavage (BAL) washings from normal or preferably patients suffering from alveolar proteinosis who overproduce SP-D in the lungs. Amniotic fluid collected at the term during parturition is another reasonable source. Here, we describe a simple and rapid method of purifying native SP-D away from SP-A which is also present in the same source. We also describe procedures of expressing and purifying a recombinant fragment of human SP-D (rhSP-D) comprising trimeric neck and carbohydrate recognition domains that has been shown to have therapeutic effects in murine models of allergy and infection.

Increased prevalence of low oligomeric state surfactant protein D with restricted lectin activity in bronchoalveolar lavage fluid from preterm infants.

BACKGROUND: Surfactant protein D (SP-D) is a soluble oligomeric C-type lectin known to protect against lipopolysaccharide and ventilator-induced lung injury in preterm lambs. Here we assess the expression and functional status of SP-D in bronchoalveolar lavage fluid (BALF) from preterm infants at risk of chronic lung disease (CLD) of prematurity and term controls. This is the first systematic evaluation of SP-D function in any clinical cohort. METHODS: SP-D was quantified in BALF from 28 ventilated preterm infants and five ventilated term infants. SP-D lectin activity was tested in a zymosan binding assay followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot in BALF from the same infants. SP-D lectin activity was also tested towards maltose-agarose and mannan for selected BALF samples. RESULTS: SP-D expression was lower on day 1 in those preterm infants who subsequently developed CLD but increased over the first 5 days of life in term and preterm neonates. The percentage of neonatal SP-D capable of binding zymosan rarely exceeded 50% in any BALF sample and was 3.5 times lower in preterm infants than term infants on day 1 of life. Similar binding defects were observed towards maltose-agarose and mannan. SDS-PAGE analysis revealed that zymosan-bound SP-D was more highly oligomerised (≥12-mers) than unbound SP-D, which was composed primarily of trimers and lower oligomeric forms. CONCLUSIONS: Substantial and functionally relevant variation in the expression and oligomeric distribution of SP-D exists between preterm and term neonatal lung secretions. This has implications for proposed SP-D replacement therapy in this population.

Prenatal iodine deficiency results in structurally and functionally immature lungs in neonatal rats.

Maternal hypothyroidism affects postnatal lung structure. High prevalence of hypothyroxinemia (low T4, normal T3) in iodine-deficient pregnant women and associated risk for neuropsychological development along with high infant/neonatal mortality ascribed to respiratory distress prompted us to study the effects of maternal hypothyroxinemia on postnatal lung development. Female Sprague Dawley rats were given a low-iodine diet (LID) with 1% KClO(4) in drinking water for 10 days, to minimize thyroid hormone differences. Half of these rats were continued on iodine-deficient diet; ID (LID with 0.005% KClO(4)) for 3 mo, whereas the rest were switched to an iodine-sufficient diet; IS [LID + potassium iodide (10 µg iodine/20 g of diet + normal drinking water)]. Pups born to ID mothers were compared with age-matched pups from IS mothers at postnatal days 8 (P8) and 16 (P16) (n = 6-8/group). ID pups had normal circulating T3 but significantly low T4 levels (P < 0.05) and concomitantly approximately sixfold higher thyroid hormone receptor-ß mRNA in alveolar epithelium. Lung histology revealed larger and irregularly shaped alveoli in ID pups relative to controls. Lung function was assessed at P16 using a double-chambered plethysmograph and observed reduced tidal volume, peak inspiratory and expiratory flow, and dynamic lung compliance in ID pups compared with IS pups. Significant lowering of surfactant protein (SP)-B and SP-C mRNA and protein found in ID pups at P16. ID pups had 16-fold lower matrix metalloproteinase-9 mRNA levels in their alveolar epithelium. In addition, mRNA levels of thyroid transcription factor-1 and SP-D were significantly higher (3-fold) compared with IS pups. At P16, significantly lower levels of SP-B and SP-C found in ID pups may be responsible for immature lung development and reduced lung compliance. Our data suggest that maternal hypothyroxinemia may result in the development of immature lungs that, through respiratory distress, could contribute to the observed high infant mortality in ID neonates.

Decreased expression of surfactant protein D mRNA in human lungs in fatal cases of H5N1 avian influenza.

Microarray analysis of gene expression profile of lungs from two fatal H5N1 influenza cases identified 3,435 genes with higher than twofold changes in mRNA levels as compared to those of normal lung. One thousand nineteen genes and 2,416 genes were up-regulated and down-regulated commonly, respectively. Gene ontology analysis identified several ontology terms with significant association with these genes, most of which are related to cellular metabolism and regulation of cellular process including apoptosis and chemotaxis. Pulmonary surfactant protein D (SP-D) was found to be down-regulated. Quantitative RT-PCR confirmed the levels of SP-D mRNA in the lungs infected with H5N1 to be lower than those of normal lungs and lungs from patients with acute respiratory distress syndrome. SP-D plays multiple roles in respiratory innate defense against various pathogens, regulation of inflammatory responses, and maintenance of alveolar integrity. Reduction of SP-D in H5N1 influenza may play important roles in the pathogenesis of the disease.

A young woman with interstitial pneumonia and coexisting deposition of surfactant proteins.

We report a 23-year-old woman with interstitial inflammation and fibrosis associated with deposition of surfactant proteins in the airways. Biopsy of the lung showed multiple focal lesions containing dilated airways with inflammation and fibrosis. Pale eosinophilic amorphous material, dense eosinophilic bodies and numerous cholesterin clefts were observed in the airways. Surfactant protein A (SP-A) was expressed in the amorphous material. SP-D was identified in the dense eosinophilic bodies and around the acicular clefts. The finding that surfactant depositions were anatomically coincident with the interstitial pneumonia suggests that surfactant depositions were closely related to the interstitial pneumonia.

Identification of markers for newly formed beta-cells in the perinatal period: a time of recognized beta-cell immaturity.

Markers of beta-cell maturity would be useful in staging the differentiation of stem/progenitor cells to beta-cells whether in vivo or in vitro. We previously identified markers for newly formed beta-cells in regenerating rat pancreases after 90% partial pancreatectomy. To test the generality of these markers of newly formed beta-cells, we examined their expression during the perinatal period, a time of recognized beta-cell immaturity. We show by semiquantitative RT-PCR and immunostaining over the time course from embryonic day 18/20 to birth, 1 day, 2 days, 3 days, 7 days, and adult that MMP-2, CK-19, and SPD are truly markers of new and immature beta-cells and that their expression transiently peaks in the perinatal period and is not entirely synchronous. The shared expression of these markers among fetal, newborn, and newly regenerated beta-cells, but not adult, strongly supports their use as potential markers for new beta-cells in the assessment of both the maturity of stem cell-derived insulin-producing cells and the presence of newly formed islets (neogenesis) in the adult pancreas.

Molecular mechanisms of lipopolysaccharide-caused induction of surfactant protein-A gene expression in human alveolar epithelial A549 cells.

Surfactant proteins (SPs) participate in the physiological and pathophysiological regulation of sepsis-induced acute lung injury. Lipopolysaccharide (LPS), a gram-negative bacterial outer membrane component, is one of the major causes of septic shock. This study was designed to evaluate the effects of LPS on the regulation of SP-A and SP-D gene expressions in human alveolar epithelial A549 cells. Exposure of A549 cells to LPS increased SP-A mRNA synthesis in concentration and time-dependent manners without affecting SP-D mRNA production. LPS selectively enhanced translocation of transcription factor c-Jun from the cytoplasm to nuclei, but not nuclear factor kappa-B. In parallel, the DNA-binding activity of AP-1 was increased by LPS. Pretreatment of A549 cells with SP600125, an inhibitor of c-Jun N-terminal kinase, decreased c-Jun translocation, and significantly ameliorated LPS-induced SP-A mRNA production. Levels of toll-like receptor (TLR2) mRNA in A549 cells were time-dependently induced following LPS treatment. Application of TLR2 small interference (si)RNA into A549 cells significantly knocked-down the translation of this receptor, and simultaneously alleviated LPS-induced SP-A synthesis. Taken together, this study has shown that LPS selectively induces SP-A gene expression possibly through TLR2-mediated activation of c-Jun in human alveolar epithelial A549 cells.

Surfactant protein D protects against acute hyperoxic lung injury.

RATIONALE: Surfactant protein D (SP-D) is a member of the collectin family of soluble, innate, host defense molecules with demonstrated immunomodulatory properties in vitro. Constitutive absence of SP-D in mice is associated with lung inflammation, alteration in surfactant lipid homeostasis, and increased oxidative-nitrative stress. OBJECTIVES: To test the hypothesis that SP-D would protect against acute lung injury from hyperoxia in vivo. METHODS: Transgenic mice overexpressing rat SP-D constitutively (SP-D OE) or conditionally via regulation with doxycycline (SP-D Dox-on) were subjected to continuous hyperoxic challenge for up to 14 days. MEASUREMENTS AND MAIN RESULTS: Compared with littermate control mice (wild-type [WT]), SP-D OE mice exposed to 80% O(2) demonstrated substantially increased survival accompanied by significant reductions in wet to dry lung ratios and bronchoalveolar lavage (BAL) protein. Although SP-D OE and WT mice exhibited a twofold increase in total BAL cells and neutrophilia in response to hyperoxia, the SP-D OE group had lower levels of BAL proinflammatory cytokines and chemokines, including IL-6, tumor necrosis factor-alpha, and monocyte chemotactic protein-1; increased mRNA levels of the transcription factor NF-E2 related factor-2 (NRF-2) and phase 2 antioxidants hemoxygenase-1 (HO-1), glutathione peroxidase-2 (GPx-2) and NAD(P)H quinone oxidoreductase-1 (Nqo-1); and decreases in lung tissue thiobarbituric acid-reactive substances. As proof of principle, the protective role of SP-D on hyperoxic injury was confirmed as SP-D Dox-on mice exposed to 85% O(2) demonstrated increased mortality upon withdrawal of doxycycline. CONCLUSIONS: Local expression of SP-D protects against hyperoxic lung injury through modulation of proinflammatory cytokines and antioxidant enzymatic scavenger systems.

ErbB4 deletion leads to changes in lung function and structure similar to bronchopulmonary dysplasia.

Neuregulin is an important growth factor in fetal surfactant synthesis, and downregulation of its receptor, ErbB4, impairs fetal surfactant synthesis. We hypothesized that pulmonary ErbB4 deletion will affect the developing lung leading to an abnormal postnatal lung function. ErbB4-deleted lungs of 11- to 14-wk-old adult HER4heart mice, rescued from their lethal cardiac defects, were studied for the effect on lung function, alveolarization, and the surfactant system. ErbB4 deletion impairs lung function and structure in HER4heart mice resulting in a hyperreactive airway system and alveolar simplification, as seen in preterm infants with bronchopulmonary dysplasia. It also leads to a downregulation of surfactant protein D expression and an underlying chronic inflammation in these lungs. Our findings suggest that this animal model could be used to further study the pathogenesis of bronchopulmonary dysplasia and might help design protective interventions.

Expression sites of the collectin SP-D suggest its importance in first line host defence: power of combining in situ hybridisation, RT-PCR and immunohistochemistry.

Surfactant proteins A and D are pattern recognition molecules that play a role in pulmonary host defence. In this paper, we describe for the first time the expression and localisation of both collectins in various porcine tissues using a combination of in situ hybridisation (ISH), RT-PCR and immunohistochemistry (IHC). SP-D was expressed in several tissues including lung, tongue, intestinal tract, thymus, skin, gall bladder and lacrimal gland. Focal SP-D expression was detected in oesophagus, stomach, kidney, liver, prostate and spleen with both histological techniques. These tissues tested negative with RT-PCR. In contrast, SP-A expression was limited to the lung as measured by ISH and IHC. Interestingly, analysis by RT-PCR showed that thymus, trachea, jejunum and duodenum are positive for the presence of SP-A mRNA. We conclude that the combination of different methods can be advantageous if tissue-specific expression is studied. The importance of SP-D in innate immune defence of the pig is underlined by its expression at the potential ports of entry of pathogens.

Expression and localization of surfactant proteins in human nasal epithelium.

Surfactant proteins (SPs), designated SP-A, SP-B, SP-C, and SP-D, play an important role in surfactant metabolism and host defense mechanisms in the lung. This study investigates expression of the different SP types in human nasal mucosa and cultured normal human nasal epithelial (NHNE) cells and whether the expression of SP mRNA is influenced by the degree of mucociliary differentiation. RT-PCR was performed with mRNA from cultured NHNE cells and nasal mucosa. Immunohistochemical staining for SPs was performed on nasal mucosa specimens. Western blot analysis was performed on cell lysates from cultured NHNE cells. SP-A2, SP-B, and SP-D mRNAs were expressed in normal NHNE cells and human nasal mucosa. SPs were localized in ciliated cells of the surface epithelium and serous acini of the submucosal glands. SP-A, SP-B, and SP-D proteins were expressed in cultured NHNE cells. The degree of mucociliary differentiation influenced expression of the SP gene. We demonstrate that SP-A, SP-B, and SP-D are expressed in human nasal mucosa and cultured NHNE cells. Further study of the functional role of SPs in the upper airway is required.

Surfactant protein d expression in chronic rhinosinusitis patients and immune responses in vitro to Aspergillus and alternaria in a nasal explant model.

OBJECTIVES/HYPOTHESIS: Common fungi have been implicated in the pathogenesis of chronic rhinosinusitis (CRS) with eosinophilic mucus (EMCRS). Surfactant protein (SP)-D plays an important role in the immune response to Aspergillus fumigatus in the lungs. We sought to determine whether SP-D is expressed in nasal mucosa and investigated the response of SP-D in vitro to fungal allergens. STUDY DESIGN AND METHODS: 1) Nasal biopsies from 59 CRS and EMCRS patients, stratified into allergic fungal sinusitis (AFS), nonallergic fungal eosinophilic sinusitis (NAFES), and nonallergic nonfungal eosinophilic sinusitis (NANFES) were studied by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), immunostaining and enzyme-linked immunosorbent assay (ELISA). 2) Nasal tissue from three CRS and three NANFES patients was cultured with fungal allergens in a nasal explant in vitro model for 24 hours at increasing concentrations and mRNA SP-D secreted SP-D protein levels in response to the fungi determined by qRT-PCR and ELISA. RESULTS: Staining for SP-D was detected in the submucosal glands from the nasal biopsies in all patient groups except for AFS. By ELISA, SP-D was undetectable in AFS and decreased in NAFES, NANFES, and CRS compared with controls. CRS patients in vitro cultured with Aspergillus and Alternaria allergens in a nasal tissue explant model induced up-regulation of SP-D by qRT-PCR. In contrast, NANFES nasal tissue explants cultured with Aspergillus allergens induced down-regulation of SP-D. CONCLUSIONS: We report for the first time the expression of SP-D in both diseased and normal nasal mucosa. SP-D expression in CRS patients is up-regulated by fungal allergens in an in vitro model. These results may provide potential novel therapy for treatment of CRS.

A distinctive set of genes is upregulated during the inflammation-carcinoma sequence in mouse stomach infected by Helicobacter felis.

Helicobacter pylori infects over half the population worldwide and is a leading cause of chronic gastritis and gastric cancer. However, the mechanism by which this organism induces inflammation and carcinogenesis is not fully understood. In the present study we used insulin-gastrin (INS-GAS) transgenic mice that fully develop gastric adenocarcinoma after infection of H. pylori-related Helicobacter felis. Histological examination revealed that more than half of those mice developed invasive adenocarcinoma after 8 months of infection. These carcinomas were stained by NCC-ST-439 and HECA-452 that recognize 6-sulfated and non-sulfated sialyl Lewis X. Lymphocytic infiltration predominantly to submucosa was observed in most H. felis-infected mice, and this was associated with the formation of peripheral lymph node addressin (PNAd) on high endothelial venule (HEV)-like vessels detected by MECA-79. Time-course analysis of gene expression by using gene microarray revealed upregulation of several inflammation-associated genes including chemokines, adhesion molecules, surfactant protein D (SP-D), and CD74 in the infected stomach. Immunohistochemical analysis demonstrated that SP-D is expressed in hyperplasia and adenocarcinoma whereas CD74 is expressed in adenocarcinoma in situ and invasive carcinoma. These results as a whole indicate that H. felis induces HEV-like vessels and inflammation-associated chemokines and chemokine receptors, followed by adenocarcinoma formation.