Induced sputum deposition improves diagnostic yields of pulmonary alveolar proteinosis: A clinicopathological and methodological study of 17 cases.

Pulmonary alveolar proteinosis (PAP) is a rare diffuse lung disease characterized by the accumulation of intra-alveolar lipoprotein-like surfactants. Lung core biopsy and bronchoalveolar lavage (BAL) fluid are currently the two major sources of sampling for diagnosis. In the present study, we assessed the value of induced sputum in diagnosing PAP by transmission electron microscopy and examined the PAP 2-year death rate in Asians. Transmission electron microscopy was performed on the samples from 17 patients with PAP, 13 patients with inflammatory lung diseases, and 13 healthy adults. The PAP patients were followed up for 3-156 months, and inflammatory lung diseases patients or healthy adults for 12-36 months. The ultrastructural features including diagnostic lamellar bodies of induced sputum deposition (ISD) samples were similar to that of the BAL fluid sediment. However, the rates of lamellar bodies were 73.7% in the ISD group, significantly higher than the spontaneous sputum deposition (SSD) group (42.1%, P < .0487) and similar to the BAL sediment (76.2%) and the lung biopsy (54.5%) groups. The overall 2-year death rate of our PAP patients was 17.6% (3/17), not statistically different from the healthy adults and patients with inflammatory diseases (0/13, P = .237 for both). ISD may be the preferred non-invasive sampling method for diagnosing PAP by electronic microscopy because of the higher diagnostic yield than SSD. The diagnostic yields of this noninvasive method were similar to that of lung core biopsy and BAL.

Gene expression profile analysis for different idiopathic interstitial pneumonias subtypes.

OBJECTIVE: Idiopathic interstitial pneumonias (IIPs) are a group of diffuse parenchymal lung diseases of unknown etiology characterized by the presence of various degrees of inflammation and fibrosis. We aimed to screen the differences among IIPs subtypes in the gene level by using the microarray expression profiles of normal lung tissue and IIPs tissue for the key genes associated with early diagnosis and treatment of IIPs. METHODS: The gene expression profile of six kinds of IIPs (GSE 32537) subtypes tissue and normal lung tissues were downloaded. The differentially expressed genes (DEGs) in different IIPs subtypes were selected by using the expression profiling. In addition, the screened DEGs were further analyzed by function annotation, pathway analysis, and interaction network analysis to reveal the differences among these subtypes. RESULTS: The gene expression analysis showed that nine genes including SERPINA3, IL1R2, CBS, MGAM, SLCO4A1, S100A12, FPR1, SDR16C5, and MT1X in six subtypes of IIPs were significantly increased. There were significant differences in DEGs among six subtypes of IIPs, and the DEGs of some IIPs subtypes involved in immune, inflammatory response and cell adhesion processes. Moreover, the PPI network analysis indicated that SERPINA3 played an important role in the molecular mechanisms of IIPs. CONCLUSION: This comprehensive description of altered gene expression in different subtypes of IIPs underscores the complex biological processes characteristic of different subtypes of IIPs and may provide a foundation for future research into this devastating disease.

[The expressions and meanings of BMP-7 and TGF-ß in idiopathic pulmonary fibrosis and idiopathic nonspecific interstitial pneumonia].

OBJECTIVE: To investigate the expressions of cytokines in idiopathic pulmonary fibrosis (IPF) and in idiopathic nonspecific interstitial pneumonia (INSIP); To discuss expressions and meanings of bone morphogenetic protein 7 (BMP-7) and transforming growth factor beta (TGF-ß) in IPF and IPF. METHODS: Selected 47 cases of idiopathic interstitial pneumonia (IIP), which were diagnosed by clinical-radiologic-pathologic (CRP), and classified into two groups which were group IPF (25 IPF) and group INSIP (22 INSIP, including 6 cellular pattern and 16 fibrosing pattern). The normal lung tissues were collected as the control group: The fresh tissues were made to detect more than 114 kinds of cytokines' expressions via Oligo GEArray gene microarray technology. Made a tissue microarray which applied EnVision immunohistochemistry technology to detect the expressions of BMP-7 and TGF-ß in both kinds of IIPs. The two groups of patients were followed-up visited around 5 to 8 years and the survival curves were evaluated by Kaplan-Meier method. RESULTS: According to gene microarray results, these two groups were up-expression in TGF family,IL family and TNF family. Most of BMP members were down-expression, in comparison with the control group, except BMP-5,BMP-8B and BMP-15. As the tissue microarray results demonstrated, compared with normal lung tissues,BMP-7 expressed decreasingly in IPF and INSIP groups (t1 = 27.618, P < 0.001; t2 = -12.404, P < 0.001). The expression of IPF were lower than INSIP (t = 5.387, P < 0.05); In INSIP group, patients of cellular pattern expressed BMP-7 more than fibrosing pattern's (t = -5.341, P < 0.001). There were dramatically increasing expressions of TGF-ß in IPF and INSIP, when compared with the control group (t1 = 23.393, P < 0.001; t2 = -13.445, P < 0.001) and it presented negative correlation with BMP-7(group IPF: r = -0.771, P < 0.001; group INSIP: r = -0.729, P < 0.001). (3) Clinical follow-up data showed, the stability(improvement), deterioration and death rates of the group IPF and the group INSIP were, respectively, 0(0%), 2 (8%), 23 (92%) and 15 (68.1%), 3 (13.6%), 4 (18.2%). The results were statistically significant (all P < 0.05). The median survival time of the part with higher BMP-7 expression and the part with relatively lower BMP-7 expression, in the group IPF, were 110.8 and 66.4 months (t = -2.686, P < 0.05); In the group INSIP, were 146.4 and 74.9 months (t = -3.037, P < 0.05). CONCLUSIONS: Cellular cytokines presented different expression profiles in IPF and INSIP patients. Differently with highly activated TGF-ß, BMP-7 was inhibited in IIP patients, which would remind the degree of fibrosis and prognosis of IIP. BMP-7 would be expected to be a novel target for IIP pathogenesis and prognostic research.

[Effect of Wnt signaling suppression on gefitinib in non small cell lung cancer cell lines].

OBJECTIVE: To explore the effect of Wnt signaling suppression on proliferation of non small cell lung cancer to gefitinib, and its related mechanisms. METHODS: PC9 and PC9/AB2 cells of both gefitinib sensitive and resistant were treated with different concentrations of gefitinib, and the proliferation index was measured using CCK8 kit. The members of Wnt signaling pathway were detected by Western blot. Dual luciferase reportor gene assay (TOP Flash) was used to document the transcriptional level of ß-catenin. ß-catenin siRNA was transfected into PC9/AB2 cells to suppress the Wnt signaling transcription, followed by treatment with different concentrations of gefitinib. Western blot was then used to detect the expression of EGFR and its downstream signaling after inhibit the expression of ß-catenin. RESULTS: Treating with different concentrations of gefitinib, the resistance of PC9/AB2 cells to gefitinib was significantly increased (P < 0.05). The members of Wnt signaling expressed at higher level in PC9/AB2 cells than in PC9 cells (t = 24.590, P = 0.000). TOP Flash examination showed that the endogenous transcriptional activity of Wnt signaling was higher in PC9/AB2 cell than that in PC9 cell (t = 4.983, P = 0.008). Compared with the negative control group, apoptotic rate and sensitivity to gefitinib significantly increased in interfered group (P < 0.05). The expression of p-ERK1/2 significantly decreased after Wnt signaling suppression, although other proteins showed no significant alterations. CONCLUSION: Suppressing the activity of Wnt signaling can partly reverse the celluar resistance to gefitinib in non small cell lung cancer.

Transmission electron microscopy of sputum deposition in the diagnosis of pulmonary alveolar proteinosis.

OBJECTIVE: To clarify the diagnostic value of sputum in pulmonary alveolar proteinosis (PAP) through transmission electron microscopy (TEM) of sputum deposition (SD). METHODS: Eleven SD samples and 9 bronchoalveolar lavage (BAL) sediments from a PAP group including 11 patients were observed by TEM and compared with sputum direct smear, BAL cytology, and lung biopsy histopathology. Eleven healthy adults were chosen as controls. RESULTS: The 11 sputum smears from the PAP group showed no diagnostic component, but TEM of SD revealed 7 of 11 samples had many myelin-like lamellar bodies with degeneration in the cytoplasm of macrophages, alveolar epithelial cells, and extracellular spaces, which suggested PAP. Especially, 2 patients on whom lung biopsy could not be performed and who failed to be diagnosed by BAL fluid were finally diagnosed by TEM of SD. TEM of BAL sediments showed 7 of 9 cases had diagnostic myelin-like lamellar bodies. No statistical significance was found between BAL fluid and SD by TEM. The control group didn't show diagnostic components by cytology or TEM of SD. CONCLUSION: TEM of SD is an important noninvasive diagnostic method especially for patients against lung biopsy and BAL.

[The ultrastructural features of sputum deposition and its value in the diagnosis of pulmonary alveolar proteinosis].

OBJECTIVE: To investigate the ultrastructural features of sputum deposition (SD) and its value in the diagnosis of pulmonary alveolar proteinosis (PAP). METHODS: Seven patients with PAP diagnosed by lung biopsy and cytology were enrolled in this study. The patients consisted of 5 men and 2 women, whose median age was 48 years (range 36 to 73). SD and bronchoalveolar lavage fluid (BALF) sediment were made into ultrathin sections and observed under transmission electron microscope (TEM), respectively. Seven cases of control group composed of 4 men and 3 women whose median age was 49 years (range 39 to 68) including 3 cases of bacterial pneumonia, two cases of COPD and 2 cases of exudative pulmonary tuberculosis. Each SD was made into ultrathin section, and compared with the experimental group. RESULTS: In PAP group, Periodic acid-schiff (PAS) staining was performed on 7 sputum smears and none of them was tested positive for any components with diagnostic interest. Four cases from the 7 paraffin-embed sections of BALF sediment by microscopic examination suggested PAP. Under TEM, BALF sediment showed that many lamellar bodies existed in and outside alveolar epithelial cells, and 5 specimens were consistent with PAP diagnosis. Compared with BALF sediment, SD had apparent degeneration with more myelin phagosomes in the cytoplasm of macrophages, more lamellar bodies in alveolar epithelial cells, and lots of lamellar bodies in the shape of concentric circle in the extracellular spaces. Four from the 7 SD samples were consistent with the diagnosis of PAP. No significant difference was found between SD and BALF in the diagnosis of PAP by electronic examination (P > 0.05). In the 7 cases of control group no drifting osmiophilic lamellar bodies in extracellular space were detected. CONCLUSIONS: The osmiophilic lamellar bodies with diagnostic value were found in SD and BALF of patients with PAP. TEM of SD in combination with clinical manifestations and radiologic findings can make a definitive diagnosis of PAP, especially for those patients who have contraindications to lung biopsy and lung lavage.

Magnetic resonance imaging contrast agent: cRGD-ferric oxide nanometer particle and its role in the diagnosis of tumor.

To construct tumor-targeted nanometer particles as a negative magnetic resonance imaging (MRI) contrast agent. Ultra-small superparamagnetic iron oxide (USPIO) nanometer particles were prepared by one-step chemical precipitation. The covalent bond between cyclic RGD (cRGD) containing an Arg-Gly-Asp sequence targeting integrin-alphavbeta3, and USPIO was conducted by chemical crosslinking. The physico-chemical property of cRGD-USPIO was detected. Prussian blue staining was applied to detect the specific binding capacity of cRGD-USPIO and USPIO to human pulmonary adenocarcinoma A549 cells and human umbilical vein endothelial cells. Subsequently, A549 xenografts in nude mice were established, and intravenous injections of USPIO and cRGD-USPIO into the vena caudalis were performed. The enhancement of cRGD-USPIO against tumor MRI signal was evaluated. The mean hydrodynamic diameter of cRGD-USPIO was 43.97 +/- 10.10 nm and the size of the ferric oxide core was 5-10 nm. The specific saturation magnetization was 59.94 A x m2 x Kg(-1). The cell conjugation assay results indicated that the positive staining of the cRGD-USPIO group was significantly enhanced. The in vivo MRI diagnosis indicated that the cRGD-USPIO tumor signal was significantly reduced compared to that of the USPIO group (P < 0.01). The targeted superparamagnetic iron oxide nanometer particle can be a novel MRI negative contrast agent for more specific tumor early diagnosis.

Clinicopathologic and ultrastructural study of non-HIV-related primary pulmonary cryptococcosis in China: report of 43 cases.

OBJECTIVE: To clarify the clinicopathologic and ultrastructural features of primary pulmonary cryptococcosis (PC). METHODS: 43 cases of PC were observed by light microscopy and histochemical staining, including mucicarmine (MC), Alcian blue (AB), periodic acid-Schiff (PAS), and Grocott methenamine-silver (GMS). Transmission electron microscopy (TEM) was performed on 11 fresh and 8 formalin-fixed specimens. RESULTS: The detective rate of Cryptococcus neoformans (CN) by MC, AB, PAS, GMS staining, and TEM was 61.3% (19/31), 62.2% (23/37), 85.7% (30/35), 79.1% (34/43), and 89.5% (17/19), respectively. All CN detected by TEM had a capsule. Most of them possessed simple structure with undeveloped cellular organelles. CONCLUSION: Electron microscopy has a high rate of detecting CN. A combination of histochemical staining and electron microscopy can make an accurate diagnosis of PC.

[A study on the efficacy of glucocorticoid therapy for idiopathic nonspecific interstitial pneumonia].

OBJECTIVE: To investigate the efficacy of glucocorticoid therapy in idiopathic nonspecific interstitial pneumonia (INSIP). METHODS: Twenty-nine cases of INSIP confirmed by clinical- radiological-pathological (CRP) diagnosis were collected and classified into 2 groups according to the degree of fibrosis: INSIP-1 (including 9 cases of cellular type) and INSIP-2 (20 cases of mixed and fibrotic type). Thirty cases of usual interstitial pneumonia (UIP) confirmed by CRP diagnosis served as the control. Clinical and pathological features, therapeutic effects of glucocorticoids and the follow-up results were retrospectively analyzed and the survival curves were evaluated by Kaplan-Meier method. RESULTS: The mean age at onset of INSIP-1 group [(48 ± 5) years] was significantly younger than INSIP-2 group [(52 ± 11) years] and the UIP group [(57 ± 14) years]. The course of disease in INSIP-1 group [(60 ± 28) months] was longer than that in INSIP-2 group [(48 ± 33) months] and that in the UIP group [(44 ± 23) months], but the differences were not statistically significant (F = 1.22, all P > 0.05). The efficacy rate of glucocorticoid treatment in INSIP-1 group (9/9) was higher than that in INSIP-2 group (11/20) and that in the UIP group (2/30), the differences being statistically significant (all P < 0.05). The follow-up period for INSIP-1 group [(56 ± 27) months] was significantly longer than for INSIP-2 group [(23 ± 18) months] and for the UIP group [(25 ± 17) months], and the rate of significant improvement (6/9) was higher than that of the INSIP-2 group (9/20) and the UIP group (0/30), the differences being statistically significant (F = 9.224, all P < 0.05). The mortality of INSIP-1 group (0/9) was lower than that in INSIP-2 group (4/20) and the UIP group (16/30), the difference being statistically significant (exact probability value 0.000 - 0.005, P < 0.05). CONCLUSIONS: The degree of fibrosis of INSIP is closely correlated with the effect of glucocorticoid therapy and prognosis. The cellular type has a favorable reaction to glucocorticoid therapy and a better prognosis as compared to the fibrotic type.

Lower expression of platelet derived growth factor is associated with better overall survival rate of patients with idiopathic nonspecific interstitial pneumonia.

BACKGROUND: Idiopathic nonspecific interstitial pneumonia (INSIP) presents with varying degrees of interstitial inflammation and fibrosis exhibiting a uniform appearance. Lack of knowledge on the underlying mechanisms of INSIP has contributed to few effective treatment strategies. Our study is designed to explore aberrantly expressed cytokines involvement in INSIP development. METHODS: Oligo GEArray was employed to detect the expression of cytokines in INSIP patients, and idiopathic pulmonary fibrosis (IPF) was setup as isotype control. Real-time PCR and immunohistochemistry analysis were used to further confirm the expression of abnormally expressed cytokines. The correlationship between cytokines expression and overall survival rate of patients with IPF and INSIP were analyzed. RESULTS: From microarray detection, transforming growth factor-beta-1 (TGF-ß1), fibroblast growth factor 10 (FGF10), and platelet derived growth factor (PDGF) were predominantly up-regulated in patients with INSIP. Real-time PCR and immunohistochemistry also showed these cytokines was abnormally expressed in INSIP. In addition to, the clinical relevance analysis demonstrated relatively lower expression of PDGF patients had longer overall survival rate than those with higher expression of PDGF. CONCLUSIONS: Our study suggests that TGF-ß1, FGF10, and PDGF are required for the pathogenesis of INSIP, and may therefore be ideal targets in INSIP treatment. Moreover, INSIP patients with lower expression of PDGF had better survival rate.

A noninvasive examination for the diagnosis of pulmonary alveolar proteinosis: induced sputum in conjunction with transmission electron microscopy.

Transmission electron microscopy (TEM) of sputum deposition (SD) is an important method to assist in the diagnosis of pulmonary alveolar proteinosis (PAP). However, the low positive rate and poor quality of slices restrict the application of sputum samples in the diagnosis of PAP. Furthermore, it can be more difficult to obtain a sufficient amount of sample for TEM if the patients have little or no sputum. In this paper, we successfully diagnosed a patient with PAP using induced sputum deposition (ISD) with TEM, which is a novel and noninvasive method for PAP diagnosis. Therefore, ISD combined with TEM can be an effective method for PAP diagnosis, especially when a lung biopsy and bronchoalveolar lavage (BAL) cannot be performed, or little or no sputum can be obtained.

Usual interstitial pneumonia coexisted with nonspecific interstitial pneumonia, What's the diagnosis?

The differential diagnosis between idiopathic nonspecific interstitial pneumonia(INSIP) and idiopathic pulmonary fibrosis(IPF)/usual interstitial pneumonia(UIP)is tough in both clinicians and pathologists. In this study, we analyzed the lesions of right lung removed from a 58-year-old patient by gross and microscopy. The results showed that the pathological appearance of nonspecific interstitial pneumonia (NSIP) and UIP coexisted in his upper lobe. Besides, because of severe fibrosis in middle and lower lobes, it was hard to distinguish the lesions of NSIP fibrotic pattern (NSIP-F) or UIP. Based on clinic-radiologic-pathological data, the diagnosis of INSIP-F was made for this patient finally. Our study suggests that UIP is not always an accurate diagnosis when the NSIP and UIP coexist, and NSIP can have regions of UIP. VIRTUAL SLIDE: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2573531681608730.