Spontaneous eosinophilic nasal inflammation in a genetically-mutant mouse: comparative study with an allergic inflammation model

Background

Eosinophilic inflammation is a hallmark of chronic rhinosinusitis with nasal polyps. To model this disease process experimentally, nasal sensitization of mice with ovalbumin or aspergillus has been described. Here, we describe a genetically mutant mouse that develops robust spontaneous nasal eosinophilic inflammation. These mice lack the enzyme SHP-1 that down-regulates the IL-4Rα/stat6 signaling pathway. We compared nasal inflammation and inflammatory mediators in SHP-1 deficient mice (mev) and an ovalbumin-induced nasal allergy model.

Methods

A novel technique of trans-pharyngeal nasal lavage was developed to obtain samples of inflammatory cells from the nasal passages of allergic and mev mice. Total and differential cell counts were performed on cytospin preparations. Expression of tissue mRNA for IL-4, IL-13, and mouse beta-defensin-1 (MBD-1) was determined by quantitative PCR. Eotaxin in the lavage fluid was assessed by ELISA.

Results

Allergic and mev mice had increased total cells and eosinophils compared with controls. Expression of IL-4 was similarly increased in both allergic and mev mice, but expression of IL-13 and eotaxin was significantly greater in the allergic mice than mev mice. Eotaxin was significantly up-regulated in both allergic rhinitis and mev mice. In both models of eosinophilic inflammation, down-regulation of the innate immune marker MBD-1 was observed.

Conclusions

The mev mice display spontaneous chronic nasal eosinophilic inflammation with potential utility for chronic rhinosinusitis with nasal polyps research. The eosinophilic infiltrate is more robust in the mev mice than allergic mice, but Th2 cytokine expression is not as pronounced. Decreased MBD-1 expression in both models supports the concept that Th2-cytokines down-regulate sinonasal innate immunity in humans, and suggests a role for mouse models in investigating the interaction between adaptive and innate immunity in the sinonasal mucosa.     

Germline Mutations in the Polyposis-Associated Genes BMPR1A,SMAD4, PTEN,MUTYH and GREM1 Are Not Common in Individuals with Serrated Polyposis Syndrome

Background

Recent reports have observed that individuals with serrated polyps, some of whom meet the clinical diagnostic criteria for Serrated Polyposis Syndrome (SPS), are among those who carry germline mutations in genes associated with polyposis syndromes including; (1) genes known to underlie hamartomatous polyposes (SMAD4, BMPR1A, and PTEN), (2) MUTYH-associated polyposis and (3) GREM1 in Hereditary Mixed Polyposis Syndrome (HMPS). The aim of this study was to characterise individuals fulfilling the current WHO criteria for SPS for germline mutations in these polyposis-associated genes.

Methods

A total of 65 individuals with SPS (fulfilling WHO criteria 1 or 3), were recruited to the Genetics of Serrated Neoplasia study between 2000 and 2012, through multiple Genetics or Family Cancer Clinics within Australia, or from the New Zealand Familial Gastrointestinal Cancer Service. Individuals with SPS were tested for coding mutations and large deletions in the PTEN, SMAD4, and BMPR1A genes, for the MUTYH variants in exons 7 (Y179C) and 13 (G396D), and for the duplication upstream of GREM1.

Results

We found no variants that were likely to be deleterious germline mutations in the SPS cases in the PTEN, SMAD4, and BMPR1A genes. A novel variant in intron 2 (c.164+223T>C) of PTEN was identified in one individual and was predicted by in silico analysis to have no functional consequences. One further individual with SPS was found to be mono-allelic for the MUTYH G396D mutation. No individuals carried the recently reported duplication within GREM1.

Conclusions

Genes involved in the gastrointestinal hamartomatous polyposis, Hereditary Mixed Polyposis Syndrome and MUTYH-associated polyposis syndromes are not commonly altered in individuals with SPS.     

Subinhibitory concentrations of perilla oil affect the expression of secreted virulence factor genes in Staphylococcus aureus

Background

The pathogenicity of staphylococcus aureus is dependent largely upon its ability to secrete a number of virulence factors, therefore, anti-virulence strategy to combat S. aureus-mediated infections is now gaining great interest. It is widely recognized that some plant essential oils could affect the production of staphylococcal exotoxins when used at subinhibitory concentrations. Perilla [Perilla frutescens (L.) Britton], a natural medicine found in eastern Asia, is primarily used as both a medicinal and culinary herb. Its essential oil (perilla oil) has been previously demonstrated to be active against S. aureus. However, there are no data on the influence of perilla oil on the production of S. aureus exotoxins.

Methodology/Principal Findings

A broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of perilla oil against S. aureus strains. Hemolysis, tumour necrosis factor (TNF) release, Western blot, and real-time RT-PCR assays were performed to evaluate the effects of subinhibitory concentrations of perilla oil on exotoxins production in S. aureus. The data presented here show that perilla oil dose-dependently decreased the production of α-toxin, enterotoxins A and B (the major staphylococcal enterotoxins), and toxic shock syndrome toxin 1 (TSST-1) in both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA).

Conclusions/Significance

The production of α-toxin, SEA, SEB, and TSST-1 in S. aureus was decreased by perilla oil. These data suggest that perilla oil may be useful for the treatment of S. aureus infections when used in combination with β-lactam antibiotics, which can increase exotoxins production by S. aureus at subinhibitory concentrations. Furthermore, perilla oil could be rationally applied in food systems as a novel food preservative both to inhibit the growth of S. aureus and to repress the production of exotoxins, particularly staphylococcal enterotoxins.     

Plasma cells within granulomatous inflammation display signs pointing to autoreactivity and destruction in granulomatosis with polyangiitis

Introduction

Plasma cells residing in inflamed tissues produce antibodies in chronic inflammatory and systemic autoimmune diseases. This study examined if plasma cells, located within inflamed nasal tissue in granulomatosis with polyangiitis (GPA), express features potentially associated with the autoimmune and destructive character of this disease.

Methods

Ig gene mutation patterns of individual tissue-derived plasma cells from GPA (n = 5) were analyzed, by using laser-assisted microdissection followed by semi-nested polymerase chain reaction (PCR). Signs of B-lymphocyte maturation (ectopic lymphoid structures, ELS) and survival (a proliferation-inducing ligand, APRIL; B-cell maturation antigen, BCMA; transmembrane-activator and calcium modulator and cyclophilin interactor, TACI; receptor activator of nuclear factor κB ligand, RANKL) were examined in nasal tissues or serum, respectively, by using immunohistochemistry/fluorescence and enzyme-linked immunosorbent assay, ELISA.

Results

Plasma-cell derived Ig genes (light- and heavy-chain pairs, n = 4; heavy chains, n = 33) resembled mutation patterns seen in other autoimmune diseases, predominantly displaying selection against replacement mutations within the framework region of Ig genes (10 of 15), which is responsible for structural integrity. Ectopic lymphoid structures were similar between GPA and a disease control (that is, unspecific chronic rhinosinusitis. However, histomorphologic features distinguishing GPA from rhinosinusitis (that is, neutrophilic microabscess and granuloma) expressed considerable amounts of membrane-associated and secreted APRIL, respectively. The latter was co-localized with CD138 and found in close proximity to cells expressing IgG, TACI, and BCMA. Interestingly, plasma cells strongly expressed receptor activator of nuclear factor κB ligand (RANKL), apart from fibroblast-like cells.

Conclusions

Plasma cells within granulomatous inflammation appear to display features that might be required for autoreactivity and, possibly, RANKL-mediated destruction in GPA.     

Association of Mucosal Organisms with Patterns of Inflammation in Chronic Rhinosinusitis

Background

Chronic rhinosinusitis is a multifactorial process disease in which bacterial infection or colonization may play an important role in the initiation or persistence of inflammatory response. The association between mucosal bacteria presence and inflammatory patterns has only been partially explored.

Objective

To demonstrate specific mucosal microorganisms possible association with inflammatory patterns.

Methods

We collected nasal polyps or sinus tissues from a clinical selection of six patient groups with defined sinus disease using tissue biomarkers. In the tissues, we detected bacteria using peptide nucleic acid fluorescence in situ hybridization (PNA-FISH).

Results

After reviewing a total of 115 samples (15–20 samples per group), the mucosal presence of Staphylococcus aureus was correlated with IL-5 and SE-IgE positive chronic rhinosinusitis with nasal polyps and nasal polyps from cystic fibrosis patients. Chronic rhinosinusitis without nasal polyps with TNFα >20 pg/ml was associated with the mucosal presence of Pseudomonas aeruginosa.

Conclusion

This study identifies the relationship between intramucosal microbes and inflammatory patterns, suggesting that bacteria may affect the type of inflammation in chronic rhinosinusitis. Additional investigation is needed to further identify the nature of the relationship.     

Deprived TLR9 Expression in Apparently Healthy Nasal Mucosa Might Trigger Polyp-Growth in Chronic Rhinosinusitis Patients

Background

The origin of nasal polyps in chronic rhinosinusitis is unknown, but the role of viral infections in polyp growth is clinically well established. Toll-like receptors (TLRs) have recently emerged as key players in our local airway defense against microbes. Among these, TLR9 has gained special interest in viral diseases. Many studies on chronic rhinosinusitis with nasal polyps (CRSwNP) compare polyp tissue with nasal mucosa from polyp-free individuals. Knowledge about changes in the turbinate tissue bordering the polyp tissue is limited.

Objectives

To analyse the role of TLR9 mediated microbial defense in tissue bordering the polyp.

Methods

Nasal polyps and turbinate tissue from 11 patients with CRSwNP and turbinate tissue from 11 healthy controls in total were used. Five biopsies from either group were analysed immediately with flow cytometry regarding receptor expression and 6 biopsies were used for in vitro stimulation with a TLR9 agonist, CpG. Cytokine release was analysed using Luminex. Eight patients with CRSwNP in total were intranasally challenged with CpG/placebo 24 hours before surgery and the biopsies were collected and analysed as above.

Results

TLR9 expression was detected on turbinate epithelial cells from healthy controls and polyp epithelial cells from patients, whereas TLR9 was absent in turbinate epithelial cells from patients. CpG stimulation increased the percentage cells expressing TLR9 and decreased percentage cells expressing VEGFR2 in turbinate tissue from patients. After CpG stimulation the elevated levels of IL-6, G-CSF and MIP-1β in the turbinate tissue from patients were reduced towards the levels demonstrated in healthy controls.

Conclusion

Defects in the TLR9 mediated microbial defense in the mucosa adjacent to the anatomic origin of the polyp might explain virus induced polyp growth. CpG stimulation decreased VEGFR2, suggesting a role for CpG in polyp formation. The focus on turbinate tissue in patients with CRSwNP opens new perspectives in CRSwNP-research.     

Pathophysiological classification of chronic rhinosinusitis

Background

Recent consensus statements demonstrate the breadth of the chronic rhinosinusitis (CRS) differential diagnosis. However, the classification and mechanisms of different CRS phenotypes remains problematic.

Method

Statistical patterns of subjective and objective findings were assessed by retrospective chart review.

Results

CRS patients were readily divided into those with (50/99) and without (49/99) polyposis. Aspirin sensitivity was limited to 17/50 polyp subjects. They had peripheral blood eosinophilia and small airways obstruction. Allergy skin tests were positive in 71% of the remaining polyp subjects. IgE was<10 IU/ml in 8/38 polyp and 20/45 nonpolyp subjects (p = 0.015, Fisher''s Exact test). CT scans of the CRS without polyp group showed sinus mucosal thickening (probable glandular hypertrophy) in 28/49, and nasal osteomeatal disease in 21/49. Immunoglobulin isotype deficiencies were more prevalent in nonpolyp than polyp subjects (p < 0.05).

Conclusion

CRS subjects were retrospectively classified in to 4 categories using the algorithm of (1) polyp vs. nonpolyp disease, (2) aspirin sensitivity in polyposis, and (3) sinus mucosal thickening vs. nasal osteomeatal disease (CT scan extent of disease) for nonpolypoid subjects. We propose that the pathogenic mechanisms responsible for polyposis, aspirin sensitivity, humoral immunodeficiency, glandular hypertrophy, eosinophilia and atopy are primary mechanisms underlying these CRS phenotypes. The influence of microbial disease and other factors remain to be examined in this framework. We predict that future clinical studies and treatment decisions will be more logical when these interactive disease mechanisms are used to stratify CRS patients.     

Multi-symptom asthma is closely related to nasal blockage,rhinorrhea and symptoms of chronic rhinosinusitis-evidence from the West Sweden Asthma Study

Background

We have previously shown that approximately 25% of those with asthma in West Sweden have multiple asthma symptoms, which may describe a group of patients with more severe disease. Furthermore, asthma is associated with several co-morbid diseases, including rhinitis and chronic rhinosinusitis. The aim of this study was to determine whether multi-symptom asthma is related to signs of severe asthma, and to investigate the association between multi-symptom asthma and different symptoms of allergic and chronic rhinosinusitis.

Methods

This study analyzed data on asthma symptoms, rhinitis, and chronic rhinosinusitis from the 2008 West Sweden Asthma Study, which is an epidemiologically based study using the OLIN and GA²LEN respiratory and allergy focused questionnaires.

Results

Multi-symptom asthma was present in 2.1% of the general population. Subjects with multi-symptom asthma had more than double the risk of having night-time awakenings caused by asthma compared with those with fewer asthma symptoms (P < 0.001). The prevalence of allergic rhinitis was similar in the fewer- and multi-symptom asthma groups, but nasal blockage and rhinorrhea were significantly increased in those with multi- versus fewer-symptom asthma (odds ratio 2.21; 95% confidence interval 1.64-2.97, versus 1.49; 1.10-2.02, respectively). Having any, or one to four symptoms of chronic rhinosinusitis significantly increased the risk of having multi- versus fewer-symptom asthma (P < 0.01).

Conclusion

An epidemiologically identified group of individuals with multiple asthma symptoms harbour to greater extent those with signs of severe asthma. The degree of rhinitis, described by the presence of symptoms of nasal blockage or rhinorrhea, as well as the presence of any or several signs of chronic rhinosinusitis, significantly increases the risk of having multi-symptom asthma.     

Molecular Typing and Virulence Characteristic of Methicillin-Resistant Staphylococcus Aureus Isolates from Pediatric Patients in Bucaramanga,Colombia

Background

Staphylococcus aureus is among the most common global nosocomial pathogens. The emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA) is a public health problem worldwide that causes nosocomial and community infections. The goals of this study were to establish the clonal complexes (CC) of the isolates of MRSA obtained from pediatric patients in a university hospital in Colombia and to investigate its molecular characteristics based on the virulence genes and the genes of staphylococcal toxins and adhesins.

Methods

A total of 53 MRSA isolates from pediatric patients with local or systemic infections were collected. The MRSA isolates were typed based on the SCCmec, MLST, spa and agr genes. The molecular characterization included the detection of Panton-Valentine Leukocidin, superantigenic and exfoliative toxins, and adhesin genes. The correlation between the molecular types identified and the profile of virulence factors was determined for all isolates.

Results

Four CC were identified, including CC8, CC5, CC80 and CC78. The ST8-MRSA-IVc-agrI was the predominant clone among the isolates, followed by the ST5-MRSA-I-agrII and ST5-MRSA-IVc-agrII clones. Twelve spa types were identified, of which t10796 and t10799 were new repeat sequences. The isolates were carriers of toxin genes, and hlg (100%), sek (92%) and pvl (88%) were the most frequent. Ten toxin gene profiles were observed, and the most frequent were seq-sek-hlg (22.6%), sek-hlg (22.6%), seb-seq-sek-hlg (18.9%) and seb-sek-hlg (15.1%). The adhesion genes were present in most of the MRSA isolates, including the following: clf-A (89%), clf-B (87%), fnb-A (83%) and ica (83%). The majority of the strains carried SCCmec-IVc and were identified as causing nosocomial infection. No significant association between a molecular type and the virulence factors was found.

Conclusion

Four major MRSA clone complexes were identified among the isolates. ST8-MRSA-IVc-agrI pvl+ (USA300-LV) was the most frequent, confirming the presence of community-associated MRSA in Colombian hospitals.     

Difference in agr Dysfunction and Reduced Vancomycin Susceptibility between MRSA Bacteremia Involving SCCmec Types IV/IVa and I–III

Background

Dysfunction of agr, with reduced susceptibility or hetero-resistance to vancomycin, is thought to be associated with a worse outcome of methicillin-resistant Staphylococcus aureus (MRSA) bacteremia (MRSAB). However, the difference in agr dysfunction according to the SCCmec type in MRSA infection is undetermined. We compared the prevalence of agr dysfunction, reduced vancomycin susceptibility and the outcomes of SCCmec IV/IVa and I–III MRSAB.

Methods

The study included 307 cases of MRSAB. SCCmec types were determined by multiplex PCR. The clinical and microbiological features and outcomes of 58 SCCmec IV/IVa MRSAB were compared with those of 249 SCCmec I–III MRSAB.

Results

Compared with SCCmec I–III MRSAB, SCCmec IV/IVa MRSAB was associated with lower rates of agr dysfunction (3% vs. 43%), vancomycin minimum inhibitory concentration (MIC) = 2 µg/mL (3% vs. 15%), and hetero-resistance to vancomycin (0% vs. 8%) (all P<0.05). However, the 30-day and S. aureus-related mortality in patients with SCCmec IV/IVa MRSAB were not different from those in patients with SCCmec I–III MRSAB in multivariate analyses (HR 1.168, 95% CI 0.705–1.938; HR 1.025, 95% CI 0.556–1.889).

Conclusions

SCCmec IV/IVa MRSAB was associated with lower rates of agr dysfunction and hetero-resistance to vancomycin and a lower vancomycin MIC, compared with SCCmec I–III MRSAB. However, the outcomes of SCCmec IV/IVa MRSAB did not differ from those of SCCmec I–III MRSAB.     

Characterisation of Australian MRSA strains ST75- and ST883-MRSA-IV and analysis of their accessory gene regulator locus

Background

Community-acquired methicillin-resistant Staphylococcus aureus have become a major problem in Australia. These strains have now been isolated throughout Australia including remote Indigenous communities that have had minimal exposure to healthcare facilities. Some of these strains, belonging to sequence types ST75 and ST883, have previously been reported to harbour highly divergent alleles of the housekeeping genes used in multilocus sequence typing.

Methodology/Principal Findings

ST75-MRSA-IV and ST883-MRSA-IV isolates were characterised in detail. Morphological features as well as 16S sequences were identical to other S. aureus strains. Although a partial rnpB gene sequence was not identical to previously known S. aureus sequences, it was found to be more closely related to S. aureus than to other staphylococci. Isolates also were screened using diagnostic DNA microarrays. These isolates yielded hybridisation results atypical for S. aureus. Primer directed amplification assays failed to detect species markers (femA, katA, sbi, spa). However, arbitrarily primed amplification indicated the presence of unknown alleles of these genes. Isolates could not be assigned to capsule types 1, 5 or 8. The allelic group of the accessory gene regulator (agr) locus was not determinable. Sequencing of a region of agrB, agrC and agrD (approximately 2,100 bp) revealed a divergent sequence. However, this sequence is more related to S. aureus agr alleles I and IV than to agr sequences from other Staphylococcus species. The predicted auto-inducing peptide (AIP) sequence of ST75 was identical to that of agr group I, while the predicted AIP sequence of ST883 was identical to agr group IV.

Conclusions/Significance

The genetic properties of ST75/ST883-MRSA may be due to a series of evolutionary events in ancient insulated S. aureus strains including a convergent evolution leading to agr group I- or IV-like AIP sequences and a recent acquisition of SCCmec IV elements.     

Aberrant cytokine pattern of the nasal mucosa in granulomatosis with polyangiitis

Introduction

In granulomatosis with polyangiitis (GPA), a complex autoimmune small-vessel vasculitis frequently associated with chronic necrotizing inflammation of the nasal mucosa, elevated nasal Staphylococcus (S.) aureus carrier rates are a risk factor for relapse. As cytokines are primarily involved in the regulation of defense against potentially pathogenic microorganisms, the aim of this study was to compare healthy individuals and GPA patients with respect to their baseline cytokine expression of nasal epithelial cells (NEC), which form the first barrier against such triggers. The ability of S. aureus to influence the nasal microenvironment''s cytokine secretion was assessed by exemplary stimulation experiments.

Methods

Baseline expression of 19 cytokines of primary NEC of GPA patients and normal controls (NC) was quantified by a multiplex cytokine assay. Stimulation experiments were performed with supernatants of S. aureus and expression of interleukin-8 was determined by ELISA.

Results

In GPA, an altered pattern of baseline cytokine expression with significantly up-regulated G-CSF and reduced interleukin (IL)-8 concentrations was observed. Both NEC of GPA patients and NC responded to stimulation with S. aureus, but GPA patients displayed a significantly lower IL-8 secretion and a diminished dynamic range of response towards the stimulus.

Conclusions

The data presented underline the hypothesis of a disturbed epithelial nasal barrier function in GPA. The dysregulated baseline expression of G-CSF and IL-8 and the reduced response to microbial stimulation may facilitate changes in the composition of the nasal flora and favour an imbalanced inflammatory response, which might be relevant for the disease course.     

Potential associations between severity of infection and the presence of virulence-associated genes in clinical strains of Staphylococcus aureus

Background

The clinical spectrum of Staphylococcus aureus infection ranges from asymptomatic nasal carriage to osteomyelitis, infective endocarditis (IE) and death. In this study, we evaluate potential association between the presence of specific genes in a collection of prospectively characterized S. aureus clinical isolates and clinical outcome.

Methodology/Principal Findings

Two hundred thirty-nine S. aureus isolates (121 methicillin-resistant S. aureus [MRSA] and 118 methicillin-susceptible S. aureus [MSSA]) were screened by array comparative genomic hybridization (aCGH) to identify genes implicated in complicated infections. After adjustment for multiple tests, 226 genes were significantly associated with severity of infection. Of these 226 genes, 185 were not in the SCCmec element. Within the 185 non-SCCmec genes, 171 were less common and 14 more common in the complicated infection group. Among the 41 genes in the SCCmec element, 37 were more common and 4 were less common in the complicated group. A total of 51 of the 2014 sequences evaluated, 14 non-SCCmec and 37 SCCmec, were identified as genes of interest.

Conclusions/Significance

Of the 171 genes less common in complicated infections, 152 are of unknown function and may contribute to attenuation of virulence. The 14 non-SCCmec genes more common in complicated infections include bacteriophage-encoded genes such as regulatory factors and autolysins with potential roles in tissue adhesion or biofilm formation.     

The Impact of spgM,rpfF, rmlA Gene Distribution on Biofilm Formation in Stenotrophomonas maltophilia

Background

Stenotrophomonas maltophilia is emerging as one of the most frequently found bacteria in chronic pulmonary infection. Biofilm is increasingly recognized as a contributing factor to disease pathogenesis. In the present study, a total of 37 isolates of S. maltophilia obtained from chronic pulmonary infection patients were evaluated to the relationship between biofilm production and the relative genes expression.

Methods

The clonal relatedness of isolates was determined by pulse-field gel electrophoresis. Biofilm formation assays were performed by crystal violet assay, and confirmed by Electron microscopy analysis and CLSM analysis. PCR was employed to learn gene distribution and expression.

Results

Twenty-four pulsotypes were designated for 37 S. maltophilia isolates, and these 24 pulsotypes exhibited various levels of biofilm production, 8 strong biofilm-producing S. maltophilia strains with OD492 value above 0.6, 14 middle biofilm-producing strains with OD492 average value of 0.4 and 2 weak biofilm-producing strains with OD492 average value of 0.19. CLSM analysis showed that the isolates from the early stage of chronic infection enable to form more highly structured and multilayered biofim than those in the late stage. The prevalence of spgM, rmlA, and rpfF genes was 83.3%, 87.5%, and 50.0% in 24 S. maltophilia strains, respectively, and the presence of rmlA, spgM or rpfF had a close relationship with biofilm formation but did not significantly affect the mean amount of biofilm. Significant mutations of spgM and rmlA were found in both strong and weak biofilm-producing strains.

Conclusion

Mutations in spgM and rmlA may be relevant to biofilm formation in the clinical isolates of S. maltophilia.     

Expression profiles of hydrophobic surfactant proteins in children with diffuse chronic lung disease

Background

Abnormalities of the intracellular metabolism of the hydrophobic surfactant proteins SP-B and SP-C and their precursors may be causally linked to chronic childhood diffuse lung diseases. The profile of these proteins in the alveolar space is unknown in such subjects.

Methods

We analyzed bronchoalveolar lavage fluid by Western blotting for SP-B, SP-C and their proforms in children with pulmonary alveolar proteinosis (PAP, n = 15), children with no SP-B (n = 6), children with chronic respiratory distress of unknown cause (cRD, n = 7), in comparison to children without lung disease (n = 15) or chronic obstructive bronchitis (n = 19).

Results

Pro-SP-B of 25–26 kD was commonly abundant in all groups of subjects, suggesting that their presence is not of diagnostic value for processing defects. In contrast, pro-SP-B peptides cleaved off during intracellular processing of SP-B and smaller than 19–21 kD, were exclusively found in PAP and cRD. In 4 of 6 children with no SP-B, mutations of SFTPB or SPTPC genes were found. Pro-SP-C forms were identified at very low frequency. Their presence was clearly, but not exclusively associated with mutations of the SFTPB and SPTPC genes, impeding their usage as candidates for diagnostic screening.

Conclusion

Immuno-analysis of the hydrophobic surfactant proteins and their precursor forms in bronchoalveolar lavage is minimally invasive and can give valuable clues for the involvement of processing abnormalities in pediatric pulmonary disorders.