Bronchoalveolar lavage cell pattern from healthy human lung.

Bronchoalveolar lavage (BAL) is widely accepted as a key diagnostic procedure in interstitial lung diseases (ILD). We performed a study to obtain reference intervals of differential cell patterns in BAL fluid with special attention to the origin of lavage fluid, e.g. bronchial/alveolar, to atopy and smoking status and to age of the healthy people. We performed bronchoalveolar lavage in 55 healthy subjects with known atopy status (age: 18-64 years, non-smokers/smokers: 34/21) and determined differential cell counts and lymphocyte subsets in BAL fluid and blood. Moreover, in a subgroup of non-smoking healthy individuals we measured the expression of the regulatory T cell marker forkhead box protein 3 (FoxP3) on blood and BAL fluid lymphocytes in addition to a comprehensive set of activation markers. Differential cell counts from the alveolar lavage fraction differed significantly from calculated pooled fractions (n = 11). In contrast, marginal differences were found between atopic and non-atopic subjects. Interestingly, the BAL fluid CD4(+) /CD8(+) ratio correlated strongly with age (r(2) = 0·50, P < 0·0001). We consider the bronchial and alveolar fraction to be lavage fluid from fundamentally different compartments and recommend analysis of the alveolar fraction in diagnostic work-up of ILD. In addition, our data suggest that age corrected BAL fluid CD4(+) /CD8(+) ratios should be used in the clinical evaluation of patients with interstitial lung diseases.

Mannose-binding lectin deficiency linked to cytomegalovirus (CMV) reactivation and survival in lung transplantation.

Despite the use of immunosuppressives mainly influencing T and B cell responses, the prevalence of the bronchiolitis obliterans syndrome (BOS) after lung transplantation is high. Mannose-binding lectin (MBL) is a pattern recognition molecule of complement and an important component of the innate immunity. MBL is associated with rejection, infection and survival in other solid organ transplantations. In this study the relation between functional MBL levels and cytomegalovirus (CMV) reactivations and the development of BOS and survival after lung transplantation was investigated. MBL levels were measured in 85 patients before and in 57 of these patients after lung transplantation. The relation of MBL on survival, CMV reactivation and the development of BOS were investigated with Kaplan-Meier (log-rank) survival analysis. MBL levels decreased on average by 20% (P < 0·001) after transplantation and eventually returned to pretransplant levels. Fourteen of the 85 patients had deficient pretransplant MBL levels and these patients had a tendency towards a better survival compared to those with normal MBL levels (P = 0·08). Although no correlation was found between MBL deficiency and the development of BOS, more CMV reactivations occurred in recipients with deficient versus normal levels of MBL (P = 0·03). Our results suggest that MBL deficiency is associated with CMV reactivations and a longer overall survival, but not with the development of BOS.

Lung transplantation affects expression of the chemokine receptor type 4 on specific T cell subsets.

Alloreactive T cells that infiltrate the graft after lung transplantation (LTx) play a role in chronic rejection. Chemokines such as thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC) and monocyte chemotactic protein-1 (MCP-1) are produced locally in the lung and attract T cells via chemokine receptor 4 (CCR4). In a TARC gradient, cells expressing CCR4(++) migrate more efficiently than CCR4(+) -expressing cells. In this study, we compared the CCR4 expression of T cells in blood from 20 lung transplant recipients to healthy controls. We then examined whether CCR4 expression is associated with the occurrence of chronic rejection. The CCR4(++) expression was decreased on CD4 T cells from LTx patients (P < 0·0001) when compared to healthy controls. The analysis of CD4 T cell subsets showed that this decrease was present on central memory, effector memory and terminally differentiated T cells (P = 0·0007, P < 0·0001 and P = 0·05, respectively), while a trend was found for naive CD4 T cells (P = 0·06). Also, the expression of CCR4(+) on regulatory T cells (T(regs) ) was decreased in LTx patients when compared to healthy controls (P = 0·02). Interestingly, the CCR4(++) expression on CD4 effector memory T cells was decreased in patients developing chronic rejection sometimes more than a year before the clinical diagnosis when compared to patients who did not (P = 0·04). The analysis of CD8 T cell subsets only showed the CCR4(+) expression to be increased significantly on effector memory and terminally differentiated CD8 T cells (P = 0·02, P = 0·03, respectively) in LTx patients, but no relation was found in chronic rejection. In conclusion, the expression of CCR4 on T cell subsets was altered after LTx and appears to be related to chronic rejection.

Genetic variability in the IL1RN gene and the balance between interleukin (IL)-1 receptor agonist and IL-1ß in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a rapidly progressive interstitial lung disease of unknown aetiology. Interleukin (IL)-1ß plays an important role in inflammation and has been associated with fibrotic remodelling. We investigated the balance between IL-1ß and IL-1 receptor antagonist (IL-1Ra) in bronchoalveolar lavage fluid (BALF) and serum as well as the influence of genetic variability in the IL1B and IL1RN gene on disease susceptibility and cytokine levels. In 77 IPF patients and 349 healthy controls, single nucleotide polymorphisms (SNPs) in the IL1RN and IL1B genes were determined. Serum and BALF IL-1Ra and IL-1ß levels were measured using a multiplex suspension bead array system and were correlated with genotypes. Both in serum and BALF a significantly decreased IL-1Ra/IL-1ß ratio was found in IPF patients compared to healthy controls. In the IL1RN gene, one SNP was associated with both the susceptibility to IPF and reduced IL-1Ra/IL-1ß ratios in BALF. Our results show that genetic variability in the IL1RN gene may play a role in the pathogenesis of IPF and that this role may be more important than thought until recently. The imbalance between IL-1Ra and IL-1ß might contribute to a proinflammatory and pro-fibrotic environment in their lungs.

Genetic variation in GREM1 is a risk factor for fibrosis in pulmonary sarcoidosis.

Sarcoidosis is a granulomatous systemic disorder most often affecting the lung. Pulmonary fibrosis develops in approximately 10%-15% of patients with sarcoidosis. The human gene GREM1 encodes gremlin, a member of the bone morphogenetic protein antagonist family. Bone morphogenetic proteins are essential for the maintenance of tissue homeostasis and regeneration after injury. We examined associations between genetic variation in GREM1 and pulmonary disease outcome in patients with pulmonary sarcoidosis. Four common tag single nucleotide polymorphisms spanning GREM1 were genotyped in 483 controls and in 237 sarcoidosis patients with radiographic data at pulmonary disease outcome, defined by chest X-ray after a minimum of 4 years follow-up. Highly significant differences were found between GREM1 genotype frequencies in sarcoidosis patients without chest X-ray abnormalities (stage 0) (n = 116) versus patients who had fibrosis on chest X-ray (stage IV) (n = 59) at pulmonary disease outcome. The most significant association was with GREM1 rs1919364. The recessive model resulted in an increased risk of fibrosis development for homozygous carriers of the C allele at GREM1 rs1919364 versus carriers of the G allele [P = 9.3 × 10⁻7, χ² = 24.1, odds ratio (OR) = 6.37 (2.89-14.1)]. This study is the first to suggest that genetic variation of GREM1 predisposes to pulmonary fibrosis in sarcoidosis patients. Carriers of the GREM1 CC genotype at position rs1919364 were at 6.4 times greater risk for developing fibrosis.

T-cell activation profiles in different granulomatous interstitial lung diseases--a role for CD8+CD28(null) cells?

Lymphocytes play a crucial role in lung inflammation. Different interstitial lung diseases may show distinct lymphocyte activation profiles. The aim of this study was to examine the expression of a variety of activation markers on T lymphocyte subsets from blood and bronchoalveolar lavage fluid (BALF) of patients with different granulomatous interstitial lung diseases and healthy controls. Bronchoalveolar lavage cells and blood cells from 23 sarcoidosis patients, seven patients with hypersensitivity pneumonitis and 24 healthy controls were analysed. Lymphocyte activation status was determined by flow cytometry. Lymphocytes were stained with antibodies against CD3, CD4, CD8, CD25, CD28, CD69, very late antigen-1 (VLA)-1, VLA-4 and human leucocyte antigen D-related (HLA-DR). In general, CD28, CD69 and VLA-1 expression on BALF CD4+ lymphocytes and HLA-DR expression on BALF CD8+ lymphocytes was different in patients with hypersensitivity pneumonitis and sarcoidosis patients with parenchymal involvement. This BALF lymphocyte phenotype correlated with carbon monoxide diffusing lung capacity (Dlco) values across interstitial lung diseases (ILD) (r2 = 0.48, P = 0.0002). In sarcoidosis patients, CD8+CD28(null) blood lymphocytes correlated with lower Dlco values (r = -0.66, P = 0.004), chronic BALF lymphocyte activation phenotype (r2 = 0.65, P < 0.0001), radiographic staging (stage I versus stage II and higher, P = 0.006) and with the need for corticosteroid treatment (P = 0.001). Higher expression of CD69, VLA-1 and HLA-DR and lower expression of CD28 on BALF lymphocytes suggests prolonged stimulation and chronic lymphocyte activation in patients with ILD. In sarcoidosis, blood CD8+CD28(null) cells might be a new biomarker for disease severity but needs further investigation.

Toll-like receptor (TLR)-9 genetics and function in sarcoidosis.

Sarcoidosis is a systemic disorder characterized by the formation of non-caseating granulomas in variable organs. Toll-like receptor (TLR)-9 is important in the innate immune response against both Mycobacterium tuberculosis and Propionibacterium acnes, candidate causative agents in sarcoidosis. The aim of our study was to investigate possible genetic and functional differences in TLR-9 between patients and controls. TLR-9 single nucleotide polymorphisms were genotyped in 533 patients and divided into a study cohort and validation cohort and 185 healthy controls. Furthermore, part of the promotor as well as the entire coding region of the TLR-9 gene were sequenced in 20 patients in order to detect new mutations. No genetic differences were found between patients and controls. In order to test TLR-9 function, peripheral blood mononuclear cells (PBMCs) of 12 healthy controls and 12 sarcoidosis patients were stimulated with a TLR-9 agonist and the induction of interleukin (IL)-6, interferon (IFN)-γ and IL-23 was measured. Sarcoidosis patients produce significantly less IFN-γ upon stimulation with different stimuli. Regarding IL-23 production, a significant difference between patients and controls was found only after stimulation with the TLR-9 agonist. In conclusion, we did not find genetic differences in the TLR-9 gene between sarcoidosis patients and controls. Sarcoidosis patients produce less IFN-γ regardless of the stimulating agent, probably reflecting the anergic state often seen in their peripheral blood T lymphocytes. The differences in TLR-9-induced IL-23 production could indicate that functional defects in the TLR-9 pathway of sarcoidosis patients play a role in disease susceptibility or evolution.

MRP14 is elevated in the bronchoalveolar lavage fluid of fibrosing interstitial lung diseases.

Pulmonary fibrosis is defined by an overgrowth of fibroblasts and extracellular matrix deposition, and results in respiratory dysfunction that is often fatal. It is the end stage in many chronic inflammatory interstitial lung diseases (ILD) such as sarcoidosis and idiopathic pulmonary fibrosis (IPF). The myeloid-related proteins (MRPs) belong to the S100 family of calcium-binding proteins and are highly expressed by neutrophils, macrophages and epithelial cells during chronic inflammation. MRP14 stimulates fibroblast proliferation in vitro and is expressed in granulomas from sarcoidosis patients. We hypothesized that MRP14 may be a biomarker for fibrotic interstitial lung diseases. The objective of this study was to investigate whether levels of MRP14 in the bronchoalveolar lavage fluid (BALF) of patients with sarcoidosis and IPF correlate with clinical parameters. We used an enzyme-linked immunosorbent assay (ELISA) to measure MRP14 in BALF of 74 sarcoidosis patients, 54 IPF patients and 19 controls. Mean BALF levels of MRP14 were elevated significantly in IPF (P < 0.001) and sarcoidosis (P < 0.05) patients compared to controls. MRP14 levels were associated linearly with sarcoidosis disease severity based on chest radiographic stage. Moreover, BALF MRP14 levels were correlated inversely with diffusion capacity and forced vital capacity in sarcoidosis patients. In IPF patients, a correlation with BALF neutrophil percentage was found. In conclusion, BALF MRP14 levels are elevated in IPF and sarcoidosis and are associated with disease severity in sarcoidosis. The results support the need for further studies into the role of MRP14 in the pathogenesis of lung fibrosis.

Predicting prognosis in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a parenchymal lung disease characterized by progressive interstitial fibrosis. In 2002, the ATS/ERS published new criteria that significantly changed the definition of IPF, resulting in a more homogeneous group of patients. IPF has a poor prognosis with a median of 2.5-3.5 years, but varying from a few months to a decade. In order to predict survival at diagnosis or during follow-up, a considerable number of studies were conducted identifying promising prognostic biomarkers. However, many had been performed before the new ATS/ERS consensus and included patients who would not meet current IPF criteria. This review provides an overview of prognostic markers of survival in IPF after the ATS/ERS consensus statement in 2002. Molecular biomarkers in serum, especially so-called pneumoproteins are relatively easy to obtain and have been independently replicated as predictors of prognosis. Cellular constituents of bronchoalveolar lavage (BAL) have been investigated as predictors of survival, but results remain contradictory. Further, a robust marker of prognosis is the change in lung function over time. However, calculating change in lung function is usually only possible over a 6-12 months period, and is therefore not useful at first presentation. The extent of fibrosis on HRCT scan and the number of fibroblast foci on lung biopsy can be measured at presentation and correlate with prognosis, but the applicability of these markers is being hampered by the lack of user- and patient friendliness. In conclusion, a number of biomarkers are potential candidates for an individualised prognosis of IPF, of which so-called pneumoproteins appear most promising and should be a major focus of fu-

CA 15-3 as an alternative marker for KL-6 in fibrotic lung diseases.

BACKGROUND: KL-6 is a mucin that is increased in interstitial lung diseases (ILD), and in some malignancies. CA 15-3, a tumor marker for breast cancer, refers to the same mucin but utilizes antibodies against different epitopes. OBJECTIVE: The aim of our study was to evaluate CA 15-3 as a viable alternative to KL-6 as a for ILDs with and without fibrosis. DESIGN: Serum from 242 patients with ILDs and from 327 healthy controls were included and KL-6 and CA 15-3 were measured in all subjects. Regression analyses and ROC curves were used to compare the performances of both markers. RESULTS: KL-6 and CA 15-3 levels were both significantly higher in the ILD patients compared to the controls (p < 0.0001). A weak yet significant correlation was found between serum KL-6 and CA 15-3 levels in the controls (R = 0.39, p < 0.0001), but showed a much higher correlation in the patient group (R = 0.85, p < 0.0001). CA 15-3 correlated best with KL-6 in patients with fibrotic ILDs (R = 0.83, p < 0.0001). KL-6 performed better as a marker compared to CA 15-3 in most ILDs. Both markers performed best in identifying idiopathic pulmonary fibrosis (IPF) and were equally able to differentiate between ILDs with and without fibrosis: (sensitivity and specificity %): 100/97, 95/92, and 90/72, respectively. CONCLUSION: CA 15-3 and KL-6 are equally sensitive and specific in terms of differentiating between ILDs with and without fibrosis. The wide availability, ease of use, and cost effectiveness, make CA 15-3 a viable alternative for KL-6 as a possible marker for pulmonary fibrosis.

Variation in IL7R predisposes to sarcoid inflammation.

Sarcoidosis is a chronic granulomatous disorder characterized by a massive influx of Th1 lymphocytes. Both naive and memory T cells express high levels of interleukin 7 receptor-alpha (IL7R alpha), encoded by the IL7R gene. The purpose of this study was to investigate the role of the IL7R gene region in susceptibility to sarcoidosis. Six common single-nucleotide polymorphisms (SNPs) spanning IL7R were genotyped and analyzed in 475 sarcoidosis patients and 465 healthy controls. Replication of one significant associated SNP was carried out in 206 independent sarcoidosis patients, 127 controls and 126 patients with Löfgren's disease. The rs10213865 SNP was associated with sarcoidosis (P=0.008), and in silico analysis showed a complete linkage (r(2)=1, D'=1) with a functional nonsynonymous coding SNP in exon 6 (rs6897932, T244I). Combined analysis of 663 individuals with sarcoidosis and 586 controls (homozygous carriers of risk allele, P=5 x 10(-4), odds ratio=1.49 (1.19-1.86)) provided strong statistical support for a genuine association of IL7R with the risk of sarcoidosis. In addition, we report the same trend between variation in the IL7R gene and patients with Löfgren's disease, suggesting that variation in IL7R may confer general risk for developing granulomatous lung disease.

Effect of variation in ITGAE on risk of sarcoidosis, CD103 expression, and chest radiography.

The integrin alpha(E)beta(7) is believed to play a key role in retention of lymphocytes in mucosal tissues of gut, urogenital tract and lung. Five common single nucleotide polymorphisms spanning ITGAE, the gene encoding the alpha(E) (CD103) unit, were genotyped in 556 sarcoidosis patients and 465 controls. The -1088 A/G polymorphism was associated with sarcoidosis (P=0.004). An increased risk of disease was found for homozygous carriers of the A allele vs. carriers of the G allele (P=0.001, odds ratio=1.63 [1.22-2.17]). Analysis of lymphocytes from bronchoalveolar lavage and in vitro functional tests showed higher percentages of CD103+CD4+ T cells for the sarcoidosis risk genotype. Radiographic staging at disease outcome revealed prevalence of -1088 AA genotype in patients with fibrosis (P=0.01). A higher proportion of CD103+CD4+ T cells and ITGAE -1088 AA genotype might be associated with fibrosis formation in pulmonary sarcoidosis.

Lung function in idiopathic pulmonary fibrosis--extended analyses of the IFIGENIA trial.

BACKGROUND: The randomized placebo-controlled IFIGENIA-trial demonstrated that therapy with high-dose N-acetylcysteine (NAC) given for one year, added to prednisone and azathioprine, significantly ameliorates (i.e. slows down) disease progression in terms of vital capacity (VC) (+9%) and diffusing capacity (DLco) (+24%) in idiopathic pulmonary fibrosis (IPF). To better understand the clinical implications of these findings we performed additional, explorative analyses of the IFGENIA data set. METHODS: We analysed effects of NAC on VC, DLco, a composite physiologic index (CPI), and mortality in the 155 study-patients. RESULTS: In trial completers the functional indices did not change significantly with NAC, whereas most indices deteriorated with placebo; in non-completers the majority of indices worsened but decline was generally less pronounced in most indices with NAC than with placebo. Most categorical analyses of VC, DLco and CPI also showed favourable changes with NAC. The effects of NAC on VC, DLco and CPI were significantly better if the baseline CPI was 50 points or lower. CONCLUSION: This descriptive analysis confirms and extends the favourable effects of NAC on lung function in IPF and emphasizes the usefulness of VC, DLco, and the CPI for the evaluation of a therapeutic effect. Most importantly, less progressed disease as indicated by a CPI of 50 points or lower at baseline was more responsive to therapy in this study.

Surfactant protein-D predicts survival in patients with idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis is a progressive interstitial lung disease with a high mortality rate. As lung transplantation is the only therapeutic option, it is important to predict survival. OBJECTIVE: This study evaluates the clinical value of surfactant protein-D as a marker of prognosis in patients with idiopathic pulmonary fibrosis. DESIGN: Surfactant protein-D was measured in serum of 72 patients and 305 healthy controls. The optimal cut-off level to define unfavourable prognosis was determined using a ROC analysis. A Cox's proportional Hazards model was used to evaluate variables that were significant predictors of survival. RESULTS: Serum levels of surfactant protein-D were significantly higher in patients than in controls. ROC analysis showed 460 ng/ml to be the optimal cut-off level to discriminate survivor from non-survivors after 1 year. Patients with high levels (> 460 ng/ml) had a median survival time of 13 months, compared to 67 months in the group with low levels (< 460 ng/ml). Surfactant protein-D showed to be a significant predictor of prognosis, even when corrected for age, sex, smoking, and lung function. CONCLUSION: The measurement of surfactant protein-D in serum of patients with idiopathic pulmonary fibrosis might be a clinically relevant tool to predict survival.

Rituximab in life threatening antisynthetase syndrome.

This case study reports a patient with severe interstitial pneumonitis, mild polyarthritis and polymyositis, accompanied by the presence of anti-Jo-1 antibodies diagnosed as antisynthetase syndrome. The concurrence of anti-Jo-1 with anti-Ro/SSA antibodies leads to a more severe form of interstitial lung disease. This patient was referred to our hospital because of life threatening respiratory failure. He was refractory to glucocorticoids and cyclophosphamide, but was successfully treated with two sequential infusions of rituximab. Clinical condition improved very rapidly. Response to treatment was well correlated with a fall of levels of serum soluble IL2-receptor. A decrease in pulmonary disease activity was visualized on PET-scans before and after two sequential rituximab infusions.

Serum thymus and activation regulated chemokine levels post-lung transplantation as a predictor for the bronchiolitis obliterans syndrome.

The main reason for mortality after lung transplantation is the bronchiolitis obliterans syndrome (BOS), which represents chronic rejection. As soluble CD30, which is produced mainly by activated T helper 2 (Th2) cells, was shown to be related to development of BOS, we aimed to investigate the relation between development of BOS and Th2 chemoattractant thymus and activation regulated chemokine (TARC/CCL17). In 54 patients we measured serum TARC levels prior to transplantation by enzyme-linked immunosorbent assay, and in 44 of these patients sera were analysed at months 1, 2 and 3 after lung transplantation. In addition, longitudinal measurements were performed in sera from eight healthy controls and 14 patients, the latter taken over a period of 2 years post-transplantation from seven patients developing BOS plus seven clinically matched BOS-free patients. Median serum TARC levels post-transplantation of patients who developed BOS were significantly lower than those of the matched BOS-free patients (P = 0.05). A receiver operating characteristics analysis (area under the curve 0.77), together with a Kaplan-Meyer analysis, showed that serum TARC levels below 325 pg/ml in the first month post-transplantation can predict development of BOS post-transplantation (P = 0.001). In contrast, pretransplant serum TARC levels were not significantly different between patients developing BOS, BOS-free patients or healthy controls. In conclusion, pretransplantation serum TARC levels do not predict the development of BOS post-transplantation, but measurement of the serum TARC levels in the first month directly after transplantation can provide us with a tool to identify the group at risk of developing BOS.

18F-FDG PET in sarcoidosis: an observational study in 12 patients treated with infliximab.

BACKGROUND: 18F-FDG PET is a promising technique in sarcoidosis imaging, although it is not incorporated in routine activity assessment. The purpose of this study was to correlate 18F-FDG PET with standard sarcoidosis activity parameters during infliximab treatment. METHODS: Twelve patients with refractory sarcoidosis were treated with 6 cycles of infliximab. Pre- and post-therapy 18F-FDG PET was visually evaluated and SUVmax was measured. In addition, the effect of infliximab was evaluated by changes in symptoms, angiotensin converting enzyme (ACE), soluble interleukin-2 receptor (sIL-2R), vital capacity (VC), diffusion capacity of the lung for carbon monoxide (DLCO) and chest radiography. SUVmax and conventional parameters were correlated. RESULTS: Clinical improvement as judged by conventional parameters was seen in all patients, though with a minor response in one. Symptoms improved in 11/12 patients while chest radiographic stages did not change. The decrease in ACE was 39% and in sIL-2R 47% (p<0.01). Improvement of VC and DLCO was 5.4% and 3.3% (p<0.05), respectively. 18F-FDG PET revealed either improvement or normalization in 11/12 (92%) clinically responding patients. The overall decrease in SUVmax was 55% (p<0.01); the patient with a limited response showed a 34% increase. A decrease in SUVmax of the lung parenchyma correlated with an improvement of VC (r=-0.75, p<0.01). No significant correlation between SUVmax and other parameters was found. CONCLUSION: Changes imaged by 18F-FDG PET during infliximab treatment in sarcoidosis patients correlate with signs of clinical improvement to a considerate extent, which supports the hypothesis that 18F-FDG uptake represents disease activity.

[Pulmonary alveolar proteinosis: a disease caused by surfactant accumulation, and new treatment with sargramostim]. / Pulmonale alveolaire proteïnose: ziekte door stapeling van surfactans en nieuwe behandeling met sargramostim.

The acquired form of pulmonary alveolar proteinosis was determined in 3 patients, a woman of 31 and 2 men of48 and 38 years, respectively. Their symptoms consisted of progressive dyspnoea, with or without coughing and a tight feeling in the chest. Bronchoscopy with bronchoalveolar lavage yielded milky white, frothy material, and high resolution CT revealed parenchymal densification. All 3 patients were successfully treated with recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF; sargramostim); in addition, the first and last patient underwent total pulmonary lavage. During the pregnancy of the woman, the GM-CSF treatment was suspended; this was resumed after parturition, which took place via caesarean section. Pulmonary alveolar proteinosis is a rare disease characterised by accumulation of surfactant in the alveoli. Until recently, the treatment consisted only of total lung lavage under general anaesthesia. It has recently been discovered that IgG autoantibodies play an important role in the development ofthe disease, namely in the accumulation of surfactant in the alveoli. IgG autoantibodies appear to neutralise the biological activity of natural GM-CSF, which leads to accumulation of used surfactant in the alveoli and a decrease of the pulmonary diffusion capacity. These cases and other publications from the past few years underline the important role of GM-CSF, in addition to a total lung lavage, in the treatment of pulmonary alveolar proteinosis.

Nocardiosis: a case series and a mini review of clinical and microbiological features.

Infections caused by Nocardia species are uncommon and have a wide variety of clinical manifestations in immunocompetent and immunocompromised patients. The diagnosis of nocardiosis can easily be missed because there are no characteristic symptoms. We present one case of a Nocardia infection in detail and give a brief description of eight other cases, including a relatively unique type of Nocardia veterana, diagnosed in our hospital during a five-year period. The diversity of clinical manifestations, microbiological identification and general principles of treatment of nocardiosis are reviewed.

Pattern of diffusion disturbance related to clinical diagnosis: The K(CO) has no diagnostic value next to the DL(CO).

AIM OF THE STUDY: The diffusion capacity of the lung for carbon monoxide (DL(CO)) is an important tool in the diagnosis and follow-up of patients with pulmonary diseases. In case of a decreased DL(CO) the K(CO), defined as DL(CO)/V(A) (V(A) is alveolar volume), can differentiate between normal alveolocapillary membrane (normal K(CO)) and abnormal alveolocapillary membrane (low K(CO)). The latter category consists of decreased surface of the membrane, increased thickness or decreased perfusion of ventilated alveoli. The V(A)/TLC (TLC is total lung capacity determined by whole body plethysmography) can partially differentiate between these categories. The aim of this study was to investigate the diagnostic value of the specific diffusion disturbances, which can be constructed by combining the DL(CO), K(CO) and V(A)/TLC. METHODS: In 460 patients the diagnosis made by clinicians were fitted into five diagnostic categories: asthma, chronic obstructive pulmonary disease (COPD), treatment effects of haematologic malignancies, heart failure and diffuse parenchymal lung diseases (DPLD). These categories were linked to the pattern of diffusion disturbance. RESULTS: Almost all patients with asthma have a normal DL(CO), most patients in the other groups do not have the expected pattern of diffusion disturbance, especially in the group with DPLD a bad match is observed. CONCLUSION: In this study the pattern of diffusion disturbance is of limited use in establishing a diagnosis. The use of the K(CO) next to the DL(CO) has no additional diagnostic value. Regional ventilation-perfusion inequality probably forms an important underlying mechanism of decreased DL(CO).