Asthmatic patients with high serum amyloid A have proinflammatory HDL: Implications for augmented systemic and airway inflammation.

RATIONALE: Serum amyloid A (SAA) is bound to high-density lipoproteins (HDL) in blood. Although SAA is increased in the blood of patients with asthma, it is not known whether this modifies asthma severity. OBJECTIVE: We sought to define the clinical characteristics of patients with asthma who have high SAA levels and assess whether HDL from SAA-high patients with asthma is proinflammatory. METHODS: SAA levels in serum from subjects with and without asthma were quantified by ELISA. HDLs isolated from subjects with asthma and high SAA levels were used to stimulate human monocytes and were intravenously administered to BALB/c mice. RESULTS: An SAA level greater than or equal to 108.8 µg/mL was defined as the threshold to identify 11% of an asthmatic cohort (n = 146) as being SAA-high. SAA-high patients with asthma were characterized by increased serum C-reactive protein, IL-6, and TNF-α; older age; and an increased prevalence of obesity and severe asthma. HDL isolated from SAA-high patients with asthma (SAA-high HDL) had an increased content of SAA as compared with HDL from SAA-low patients with asthma and induced the secretion of IL-6, IL-1ß, and TNF-α from human monocytes via a formyl peptide receptor 2/ATP/P2X purinoceptor 7 axis. Intravenous administration to mice of SAA-high HDL, but not normal HDL, induced systemic inflammation and amplified allergen-induced neutrophilic airway inflammation and goblet cell metaplasia. CONCLUSIONS: SAA-high patients with asthma are characterized by systemic inflammation, older age, and an increased prevalence of obesity and severe asthma. HDL from SAA-high patients with asthma is proinflammatory and, when intravenously administered to mice, induces systemic inflammation, and amplifies allergen-induced neutrophilic airway inflammation. This suggests that systemic inflammation induced by SAA-high HDL may augment disease severity in asthma.

Apolipoprotein E Signals via TLR4 to Induce CXCL5 Secretion by Asthmatic Airway Epithelial Cells.

The primary function of APOE (apolipoprotein E) is to mediate the transport of cholesterol- and lipid-containing lipoprotein particles into cells by receptor-mediated endocytosis. APOE also has pro- and antiinflammatory effects, which are both context and concentration dependent. For example, Apoe-/- mice exhibit enhanced airway remodeling and hyperreactivity in experimental asthma, whereas increased APOE levels in lung epithelial lining fluid induce IL-1ß secretion from human asthmatic alveolar macrophages. However, APOE-mediated airway epithelial cell inflammatory responses and signaling pathways have not been defined. Here, RNA sequencing of human asthmatic bronchial brushing cells stimulated with APOE identified increased expression of mRNA transcripts encoding multiple proinflammatory genes, including CXCL5 (C-X-C motif chemokine ligand 5), an epithelial-derived chemokine that promotes neutrophil activation and chemotaxis. We subsequently characterized the APOE signaling pathway that induces CXCL5 secretion by human asthmatic small airway epithelial cells (SAECs). Neutralizing antibodies directed against TLR4 (Toll-like receptor 4), but not TLR2, attenuated APOE-mediated CXCL5 secretion by human asthmatic SAECs. Inhibition of TAK1 (transforming growth factor-ß-activated kinase 1), IκKß (inhibitor of nuclear factor κ B kinase subunit ß), TPL2 (tumor progression locus 2), and JNK (c-Jun N-terminal kinase), but not p38 MAPK (mitogen-activated protein kinase) or MEK1/2 (MAPK kinase 1/2), attenuated APOE-mediated CXCL5 secretion. The roles of TAK1, IκKß, TPL2, and JNK in APOE-mediated CXCL5 secretion were verified by RNA interference. Furthermore, RNA interference showed that after APOE stimulation, both NF-κB p65 and TPL2 were downstream of TAK1 and IκKß, whereas JNK was downstream of TPL2. In summary, elevated levels of APOE in the airway may activate a TLR4/TAK1/IκKß/NF-κB/TPL2/JNK signaling pathway that induces CXCL5 secretion by human asthmatic SAECs. These findings identify new roles for TLR4 and TPL2 in APOE-mediated proinflammatory responses in asthma.

Apolipoprotein E is a concentration-dependent pulmonary danger signal that activates the NLRP3 inflammasome and IL-1ß secretion by bronchoalveolar fluid macrophages from asthmatic subjects.

BACKGROUND: House dust mite (HDM)-challenged Apoe-/- mice display enhanced airway hyperreactivity and mucous cell metaplasia. OBJECTIVE: We sought to characterize the pathways that induce apolipoprotein E (APOE) expression by bronchoalveolar lavage fluid (BALF) macrophages from asthmatic subjects and identify how APOE regulates IL-1ß secretion. METHODS: Macrophages were isolated from asthmatic BALF and derived from THP-1 cells and human monocytes. RESULTS: HDM-derived cysteine and serine proteases induced APOE secretion from BALF macrophages through protease-activated receptor 2. APOE at concentrations of less than 2.5 nmol/L, which are similar to levels found in epithelial lining fluid from healthy adults, did not induce IL-1ß release from BALF macrophages. In contrast, APOE at concentrations of 25 nmol/L or greater induced nucleotide-binding oligomerization domain, leucine-rich repeat-containing protein (NLRP) 3 and pro-IL-1ß expression by BALF macrophages, as well as the caspase-1-mediated generation of mature IL-1ß secreted from cells. HDM acted synergistically with APOE to both prime and activate the NLRP3 inflammasome. In a murine model of neutrophilic airway inflammation induced by HDM and polyinosinic-polycytidylic acid, APOE reached a concentration of 32 nmol/L in epithelial lining fluid, with associated increases in BALF IL-1ß levels. APOE-dependent NLRP3 inflammasome activation in macrophages was primarily mediated through a potassium efflux-dependent mechanism. CONCLUSION: APOE can function as an endogenous, concentration-dependent pulmonary danger signal that primes and activates the NLPR3 inflammasome in BALF macrophages from asthmatic subjects to secrete IL-1ß. This might represent a mechanism through which APOE amplifies pulmonary inflammatory responses when concentrations in the lung are increased to greater than normal levels, which can occur during viral exacerbations of HDM-induced asthma characterized by neutrophilic airway inflammation.

ATP-binding cassette transporter 1 attenuates ovalbumin-induced neutrophilic airway inflammation.

Apolipoprotein A-I (apoA-I) is an important component of high-density lipoprotein particles that mediates reverse cholesterol transport out of cells by interacting with the ATP-binding cassette transporter 1 (ABCA1). apoA-I has also been shown to attenuate neutrophilic airway inflammation in experimental ovalbumin (OVA)-induced asthma by reducing the expression of granulocyte colony-stimulating factor (G-CSF). Here, we hypothesized that overexpression of the ABCA1 transporter might similarly attenuate OVA-induced neutrophilic airway inflammation. Tie2-human ABCA1 (hABCA1) mice expressing human ABCA1 under the control of the Tie2 promoter, which is primarily expressed by vascular endothelial cells, but can also be expressed by macrophages, received daily intranasal OVA challenges, 5 d/wk for 5 weeks. OVA-challenged Tie2-hABCA1 mice had significant reductions in total bronchoalveolar lavage fluid (BALF) cells that reflected a decrease in neutrophils, as well as reductions in peribronchial inflammation, OVA-specific IgE levels, and airway epithelial thickness. The reduced airway neutrophilia in OVA-challenged Tie2-hABCA1 mice was associated with significant decreases in G-CSF protein levels in pulmonary vascular endothelial cells, alveolar macrophages, and BALF. Intranasal administration of recombinant murine G-CSF to OVA-challenged Tie2-hABCA1 mice for 5 days increased BALF neutrophils to a level comparable to that of OVA-challenged wild-type mice. We conclude that ABCA1 suppresses OVA-induced airway neutrophilia by reducing G-CSF production by vascular endothelial cells and alveolar macrophages. These findings suggest that ABCA1 expressed by vascular endothelial cells and alveolar macrophages may play important roles in attenuating the severity of neutrophilic airway inflammation in asthma.

Peptidoglycan recognition protein 1 promotes house dust mite-induced airway inflammation in mice.

Peptidoglycan recognition protein (Pglyrp) 1 is a pattern-recognition protein that mediates antibacterial host defense. Because we had previously shown that Pglyrp1 expression is increased in the lungs of house dust mite (HDM)-challenged mice, we hypothesized that it might modulate the pathogenesis of asthma. Wild-type and Pglyrp1(-/-) mice on a BALB/c background received intranasal HDM or saline, 5 days/week for 3 weeks. HDM-challenged Pglyrp1(-/-) mice showed decreases in bronchoalveolar lavage fluid eosinophils and lymphocytes, serum IgE, and mucous cell metaplasia, whereas airway hyperresponsiveness was not changed when compared with wild-type mice. T helper type 2 (Th2) cytokines were reduced in the lungs of HDM-challenged Pglyrp1(-/-) mice, which reflected a decreased number of CD4(+) Th2 cells. There was also a reduction in C-C chemokines in bronchoalveolar lavage fluid and lung homogenates from HDM-challenged Pglyrp1(-/-) mice. Furthermore, secretion of CCL17, CCL22, and CCL24 by alveolar macrophages from HDM-challenged Pglyrp1(-/-) mice was markedly reduced. As both inflammatory cells and airway epithelial cells express Pglyrp1, bone marrow transplantation was performed to generate chimeric mice and assess which cell type promotes HDM-induced airway inflammation. Chimeric mice lacking Pglyrp1 on hematopoietic cells, not structural cells, showed a reduction in HDM-induced eosinophilic and lymphocytic airway inflammation. We conclude that Pglyrp1 expressed by hematopoietic cells, such as alveolar macrophages, mediates HDM-induced airway inflammation by up-regulating the production of C-C chemokines that recruit eosinophils and Th2 cells to the lung. This identifies a new family of innate immune response proteins that promotes HDM-induced airway inflammation in asthma.

Human apolipoprotein E genotypes differentially modify house dust mite-induced airway disease in mice.

Apolipoprotein E (apoE) is an endogenous negative regulator of airway hyperreactivity (AHR) and mucous cell metaplasia in experimental models of house dust mite (HDM)-induced airway disease. The gene encoding human apoE is polymorphic, with three common alleles (ε2, ε3, and ε4) reflecting single amino acid substitutions at amino acids 112 and 158. The objective of this study was to assess whether the human apoE alleles modify airway responses to repeated nasal HDM challenges. Mice expressing the human apoE ε2 (huApoE2), ε3 (huApoE3), or ε4 (huApoE4) alleles received nasal HDM challenges, and airway responses were compared with mice expressing the endogenous murine apoE gene (muApoE). huApoE3 mice displayed significant reductions in AHR, mucous cell metaplasia, and airway inflammation compared with muApoE mice. The attenuated severity of airway inflammation in huApoE3 mice was associated with reductions in lung mRNA levels of Th2 and Th17 cytokines, as well as chemokines (CCL7, CCL11, CCL24). huApoE4 mice had an intermediate phenotype, with attenuated AHR and IgE production, compared with muApoE mice, whereas airway inflammation and mucous cell metaplasia were not reduced. In contrast, HDM-induced airway responses were not modified in mice expressing the huApoE2 allele. We conclude that the polymorphic huApoE alleles differentially modulate HDM-induced airway disease, which can be stratified, in rank order of increasing disease severity, ε3 < ε4 < ε2. These results raise the possibility that the polymorphic apoE alleles may modify disease severity in human asthma.

Antioxidant components of naturally-occurring oils exhibit marked anti-inflammatory activity in epithelial cells of the human upper respiratory system.

BACKGROUND: The upper respiratory tract functions to protect lower respiratory structures from chemical and biological agents in inspired air. Cellular oxidative stress leading to acute and chronic inflammation contributes to the resultant pathology in many of these exposures and is typical of allergic disease, chronic sinusitis, pollutant exposure, and bacterial and viral infections. Little is known about the effective means by which topical treatment of the nose can strengthen its antioxidant and anti-inflammatory defenses. The present study was undertaken to determine if naturally-occurring plant oils with reported antioxidant activity can provide mechanisms through which upper respiratory protection might occur. METHODS: Controlled exposure of the upper respiratory system to ozone and nasal biopsy were carried out in healthy human subjects to assess mitigation of the ozone-induced inflammatory response and to assess gene expression in the nasal mucosa induced by a mixture of five naturally-occurring antioxidant oils--aloe, coconut, orange, peppermint and vitamin E. Cells of the BEAS-2B and NCI-H23 epithelial cell lines were used to investigate the source and potential intracellular mechanisms of action responsible for oil-induced anti-inflammatory activity. RESULTS: Aerosolized pretreatment with the mixed oil preparation significantly attenuated ozone-induced nasal inflammation. Although most oil components may reduce oxidant stress by undergoing reduction, orange oil was demonstrated to have the ability to induce long-lasting gene expression of several antioxidant enzymes linked to Nrf2, including HO-1, NQO1, GCLm and GCLc, and to mitigate the pro-inflammatory signaling of endotoxin in cell culture systems. Nrf2 activation was demonstrated. Treatment with the aerosolized oil preparation increased baseline levels of nasal mucosal HO-1 expression in 9 of 12 subjects. CONCLUSIONS: These data indicate that selected oil-based antioxidant preparations can effectively reduce inflammation associated with oxidant stress-related challenge to the nasal mucosa. The potential for some oils to activate intracellular antioxidant pathways may provide a powerful mechanism through which effective and persistent cytoprotection against airborne environmental exposures can be provided in the upper respiratory mucosa.

Three-Drug Regimens Containing Integrase Inhibitor Show Good Efficacy and Safety in Treatment-Naive Patients With HIV-1: A Bayesian Analysis.

Introduction: The extensive utilisation of antiretroviral therapy has greatly improved the survival rates of those infected with human immunodeficiency virus (HIV). The objective of this study was to compare 3-drug regimens containing non-nucleoside reverse transcriptase inhibitor with 3-drug regimens containing integrase inhibitor (INI) regarding efficacy and safety in treatment-naive HIV-1-infected adults at 48 and 96 weeks, respectively. Methods: This study was a network meta-analysis using a Bayesian methodology. On January 8, 2020, we searched databases and other sources for randomized controlled trials conducted in treatment-naive HIV-1 adults and compared multiple 3-drug antiretroviral regimens containing INI, efavirenz (EFV), or rilpivirine (RPV). We extracted data on the following outcomes: virologic suppression, CD4+ cell recovery, discontinuations, deaths, adverse events, serious adverse events, deaths related to study drugs, and drug-related adverse events. We conducted calculations within a Bayesian framework using R software. Results: The network contained 15 randomized controlled trials including 9,745 patients. For efficacy outcomes, regimens containing INI, especially dolutegravir (DTG), were generally superior to other regimens. For virologic suppression at 48 weeks, odds ratios (95% credible intervals) were 0.6 (0.43, 0.82) for EFV+ tenofovir disoproxil fumarate (TDF)+emtricitabine (FTC) versus DTG+ abacavir+ lamivudine (3TC) and 0.52 (0.36, 0.75) for EFV+TDF+FTC vs. DTG+TDF+FTC/3TC. For safety outcomes, regimens containing INI tended to be safer relative to regimens without INI. Outcomes associated with death were unsuitable for network meta-analysis due to low event rates. Conclusion: 3-drug regimens containing INI demonstrate better efficacy and safety than those containing RPV or EFV.

Transmitted Drug Resistance in Antiretroviral Therapy-Naive Persons With Acute/Early/Primary HIV Infection: A Systematic Review and Meta-Analysis.

Background: The widespread use of antiretroviral therapy (ART) has raised concerns about the emergence of HIV transmitted drug resistance (TDR). Acute HIV infection (AHI) was the most appropriate time to detect the spread of TDR. In this meta-analysis, our purpose was to evaluate the level of TDR in ART-naive patients with primary HIV infection (PHI)/AHI/early HIV infection (EHI) and to describe the critical drug-resistant mutations. Methods: We systematically searched the literature between January 1, 2008, and April 30, 2021, in PubMed, Web of Science, Embase, and the Cochrane Library. To evaluate the overall prevalence of TDR, we extracted raw data and analyzed prevalence estimates using Stata SE. Results: The data of this meta-analysis come from 12 observational studies, covering 3,558 ART-naive individuals with PHI, AHI, or EHI. The overall prevalence of HIV-TDR is 9.3% (95% CI: 6.8%-11.8%, I2 = 81.1%, in 11 studies). The prevalence of resistance by drug class is the highest for the nonnucleoside reverse transcriptase inhibitors (NNRTIs) at 5.7% (95% CI: 2.9%-8.5%, I2 = 96.6%, in 11 studies), followed by nucleoside reverse transcriptase inhibitors (NRTIs) at 3.4% (95% CI: 1.8%-5.0%, I2 = 86.3%, in 10 studies) and protease inhibitors (PIs) at 3.3% (95% CI: 2.7%-3.9%, I2 = 15.6%, in 10 studies). The prevalence of TDR to integrase inhibitors (INIs) is 0.3% (95% CI: 0.1%-0.7%, I2 = 95.9%, in three studies), which is the lowest among all antiretroviral drugs. Conclusion: The overall prevalence of TDR is at a moderate level among AHI patients who have never received ART. This emphasizes the importance of baseline drug resistance testing for public health surveillance and guiding the choice of ART. In addition, the prevalence of TDR to NNRTIs is the highest, while the TDR to INIs is the lowest. This may guide the selection of clinical antiretroviral drugs.

Factors Associated with Medication Adherence for People Living with Acute HIV Infection in a Tertiary Chinese Hospital in Beijing.

Both the management and caregiving intervention of people living with HIV (PLWH), especially during acute HIV-1 infection, represent a public health issue and a form of social support. This current study analyzed the demographic and clinical factors associated with antiretroviral therapy (ART) adherence of PLWH from positive HIV diagnosis to ART initiation in a tertiary Chinese hospital in Beijing. A total of 200 participants diagnosed with acute HIV-1 infection were enrolled in this study. We collected demographic and clinical data by the use of a self-reported questionnaire. Bivariate and multivariate logistic regressions were used to determine associations between potential variables and outcomes. We found that medication adherence was impacted by years of ART and number of reminders (all P < 0.05). In addition, medication adherence was associated with viral load at 48 weeks (P = 0.035). Future studies are needed to investigate effective interventions that could facilitate ART adherence.

Adjunct Therapy for CD4+ T-Cell Recovery, Inflammation and Immune Activation in People Living With HIV: A Systematic Review and Meta-Analysis.

Background: HIV infection results in immune homeostasis perturbations, which is characterized by CD4+ T-cell depletion, immune activation, and inflammation. Effective antiretroviral therapy (ART) does not fully restore immunologic and clinical health in people living with HIV (PLWH). Various drugs have been used to improve their immune status and CD4+ T-cell counts, but no measures have been tested effective. Here we conduct a systematic review and meta-analysis of existing clinical studies on improving CD4+ T-cell count while decreasing inflammation and immune activation. Methods: We retrieved possible relevant publications from a total of five electronic databases and selected eligible studies, which dealt with outcomes of medical therapy for CD4+ T-cell count recovery, inflammation, and immune activation with or without ART. We paid particular attention to immunologic non-responders with a favorable treatment regimen. Results: Thirty-three articles were included in the systematic review and meta-analysis. However, there were no safe and effective medications specific for improving CD4+ T-cell reconstitution. The immunological benefits or adverse events mainly depend on the safety, dosage, and duration of the candidate medication use, as well as whether it is combined with ART. Conclusion: Under the "safe, combined, adequate and long (SCAL)" principles, alternative approaches are needed to accelerate the recovery of CD4+ T-cells, and to prevent adverse long-term outcomes in PLWH with standard ART treatment.

Problem-Based Learning Could Tackle the Issue of Insufficient Education and Adherence in People Living With HIV/AIDS.

Background: Poor medication adherence is still the main cause of antiretroviral therapy (ART) failure among people living with HIV/AIDS (PLWHA). Effective behavioral interventions are needed to improve HIV awareness and medication adherence. Methods: In this retrospective cohort study, we assessed the effect of problem-based learning (PBL) approaches to HIV-related education and adherence outcomes among PLWHA and a college student sample. In our study, compared with 309 demography-matched control participants using conventional counseling methods (109 PLWHA and 200 college students), 321 subjects (111 PLWHA and 210 college students) chose to learn HIV-related knowledge via PBL-integrated methods. Co-primary outcomes were self-administered questionnaire after HIV-related education by all participants and self-reported medication adherence by newly diagnosed PLWHA, measured in terms of the number of missed doses in the past week at each of the seven visits during a 1-year period. Multivariate regression models adjusting different covariates were used to test the robustness of HIV awareness and adherence association. Mediation model was used to investigate the relationship among PBL training, awareness of HIV, and ART adherence. Results: The knowledge scores of participants in the PBL group were higher than those in the controls (P = 0.001), especially the subgroup of newly diagnosed PLWHA in the PBL group (P = 0.001). The HIV-related health scores of the PBL college students were also higher than those of subjects exposed to conventional education (P < 0.001). There was no significant difference between the two by newly diagnosed PLWHA groups in the number of missed doses during the past week at each visit except at the first follow-up visit (P = 0.018). The indirect effect of PBL-integrated education on ART adherence at the 2-week visit through HIV awareness had a point estimate of 0.0349 and a 95% bias-corrected bootstrap confidence interval of 0.0061∼0.0874 in newly diagnosed PLWHA. Conclusions: PLWHA and college students using PBL showed improved awareness of HIV and higher levels of recent ART adherence; however, there was no change in long-term ART adherence in newly diagnosed PLWHA.

Analysis of the cellular centrosome in fine-needle aspirations of the breast.

BACKGROUND: The purpose of the present investigation is to determine whether centrosome amplifications are present in breast tumor cells, whether there are differences of centrosome amplification between benign breast lesions and breast carcinomas, and whether centrosomal analysis can be of value in the diagnosis and prognosis of breast carcinoma. METHODS: Using immunofluorescence analysis with an antibody against gamma-tubulin, we analyzed centrosome abnormalities in fine-needle aspirations of 100 breast lesions (25 cases with benign lesions and 75 cases with carcinomas). RESULTS: We found that centrosome amplifications, including numerical centrosome amplification and structural centrosome amplification, were present in most breast tumors. Cells with numerical centrosome amplification were found in 23 of 25 benign lesions, and in all 75 cases of breast carcinomas. Cells with structural centrosome amplification were found in three of 25 benign lesions, and in 69 of 75 breast carcinomas. The breast carcinomas showed a mean percentage of cells with numerical centrosome amplification of 4.86% and a mean percentage of cells with structural centrosome amplification of 3.98%. These percentages were significantly higher than those in benign lesions, with a numerical centrosome amplification of 2.77% and a structural centrosome amplification of 0.10%. Furthermore, the mean percentage of cells with structural centrosome amplification was significantly associated with HER2/neu overexpression (P < 0.05) and with negative estrogen receptor status (P < 0.05), and had a borderline association with negative progesterone receptor status (P = 0.056) in breast carcinomas. CONCLUSION: Structural centrosome amplification may bear a close relationship with breast carcinoma and may be a potential biomarker for diagnosis and prognosis of breast carcinoma.

Overexpression of osteopontin is associated with more aggressive phenotypes in human non-small cell lung cancer.

PURPOSE: The extracellular matrix (ECM) molecule osteopontin is implicated in many pathologic processes, including inflammation, cell proliferation, ECM invasion, tumor progression, and metastasis. The present study evaluated the clinical and biological importance of osteopontin in human lung cancer. EXPERIMENTAL DESIGN AND RESULTS: Tissue microarrays derived from non-small cell lung cancer (NSCLC) patients were analyzed immunohistochemically. Osteopontin protein expression was observed in 64.5% (205 of 318) of primary tumors and 75.5% (108 of 143) of lymph node metastases, but in only 27.9% (12 of 43) of normal-appearing bronchial epithelial and pulmonary tissues. Osteopontin expression was associated with tumor growth, tumor staging, and lymph node invasion. In vitro osteopontin enhanced ECM invasion of NSCLC cells, and an osteopontin antibody abolished this effect. We further analyzed osteopontin levels in circulating plasma derived from 158 patients with NSCLC, 54 patients of benign pulmonary disease, and 25 healthy donors, and found that the median osteopontin levels for the three groups were 319.1, 161.6, and 17.9 ng/mL, respectively. CONCLUSIONS: Overexpression of osteopontin is common in primary NSCLC and may be important in the development and progression of the cancer. Osteopontin levels in the plasma may serve as a biomarker for diagnosing or monitoring patients with NSCLC.

An approach to studying lung cancer-related proteins in human blood.

Early stage lung cancer detection is the first step toward successful clinical therapy and increased patient survival. Clinicians monitor cancer progression by profiling tumor cell proteins in the blood plasma of afflicted patients. Blood plasma, however, is a difficult cancer protein assessment medium because it is rich in albumins and heterogeneous protein species. We report herein a method to detect the proteins released into the circulatory system by tumor cells. Initially we analyzed the protein components in the conditioned medium (CM) of lung cancer primary cell or organ cultures and in the adjacent normal bronchus using one-dimensional PAGE and nano-ESI-MS/MS. We identified 299 proteins involved in key cellular process such as cell growth, organogenesis, and signal transduction. We selected 13 interesting proteins from this list and analyzed them in 628 blood plasma samples using ELISA. We detected 11 of these 13 proteins in the plasma of lung cancer patients and non-patient controls. Our results showed that plasma matrix metalloproteinase 1 levels were elevated significantly in late stage lung cancer patients and that the plasma levels of 14-3-3 sigma, beta, and eta in the lung cancer patients were significantly lower than those in the control subjects. To our knowledge, this is the first time that fascin, ezrin, CD98, annexin A4, 14-3-3 sigma, 14-3-3 beta, and 14-3-3 eta proteins have been detected in human plasma by ELISA. The preliminary results showed that a combination of CD98, fascin, polymeric immunoglobulin receptor/secretory component and 14-3-3 eta had a higher sensitivity and specificity than any single marker. In conclusion, we report a method to detect proteins released into blood by lung cancer. This pilot approach may lead to the identification of novel protein markers in blood and provide a new method of identifying tumor biomarker profiles for guiding both early detection and therapy of human cancer.