Antibodies In Bronchoalveolar Lavage Fluid Research Articles

A quadrivalent norovirus vaccine based on a chimpanzee adenovirus vector induces potent immunity in mice

Norovirus (NoV) infection is a major cause of gastroenteritis worldwide. The virus poses great challenges in developing vaccines with broad immune protection due to its genetic and antigenic diversity. To date, there are no approved NoV vaccines for clinical use. Here, we aimed to develop a broad-acting quadrivalent NoV vaccine based on a chimpanzee adenovirus vector, AdC68, carrying the major capsid protein (VP1) of noroviral GI and GII genotypes. Compared to intramuscular (i.m.), intranasal (i.n.), or other prime-boost immunization regimens (i.m. ​+ ​i.m., i.m. ​+ ​i.n., i.n. ​+ ​i.m.), AdC68-GI.1-GII.3 (E1)-GII.4-GII.17 (E3), administered via i.n. ​+ ​i.n. induced higher titers of serum IgG antibodies and higher IgA antibodies in bronchoalveolar lavage fluid (BALF) and saliva against the four homologous VP1s in mice. It also significantly stimulated the production of blocking antibodies against the four genotypes. In response to re-stimulation with virus-like particles (VLP)-GI.1, VLP-GII.3, VLP-GII.4, and VLP-GII.17, the quadrivalent vaccine administered according to the i.n. ​+ ​i.n. regimen effectively triggered specific cell-mediated immune responses, primarily characterized by IFN-γ secretion. Furthermore, the preparation of this novel quadrivalent NoV vaccine requires only a single recombinant adenovirus to provide broad preventive immunity against the major GI/GII epidemic strains, making it a promising vaccine candidate for further development.

A Nanoparticle-Poly(I:C) Combination Adjuvant Enhances the Breadth of the Immune Response to Inactivated Influenza Virus Vaccine in Pigs.

Intranasal vaccination elicits secretory IgA (SIgA) antibodies in the airways, which is required for cross-protection against influenza. To enhance the breadth of immunity induced by a killed swine influenza virus antigen (KAg) or conserved T cell and B cell peptides, we adsorbed the antigens together with the TLR3 agonist poly(I:C) electrostatically onto cationic alpha-D-glucan nanoparticles (Nano-11) resulting in Nano-11-KAg-poly(I:C) and Nano-11-peptides-poly(I:C) vaccines. In vitro, increased TNF-α and IL-1ß cytokine mRNA expression was observed in Nano-11-KAg-poly(I:C)-treated porcine monocyte-derived dendritic cells. Nano-11-KAg-poly(I:C), but not Nano-11-peptides-poly(I:C), delivered intranasally in pigs induced high levels of cross-reactive virus-specific SIgA antibodies secretion in the nasal passage and lungs compared to a multivalent commercial influenza virus vaccine administered intramuscularly. The commercial and Nano-11-KAg-poly(I:C) vaccinations increased the frequency of IFNγ secreting T cells. The poly(I:C) adjuvanted Nano-11-based vaccines increased various cytokine mRNA expressions in lymph nodes compared to the commercial vaccine. In addition, Nano-11-KAg-poly(I:C) vaccine elicited high levels of virus neutralizing antibodies in bronchoalveolar lavage fluid. Microscopic lung lesions and challenge virus load were partially reduced in poly(I:C) adjuvanted Nano-11 and commercial influenza vaccinates. In conclusion, compared to our earlier study with Nano-11-KAg vaccine, addition of poly(I:C) to the formulation improved cross-protective antibody and cytokine response.

Induction of mycoplasmal pneumonia in experimentally infected pigs by means of different inoculation routes.

The purpose of this study was to assess the effect of three different inoculation routes into mycoplasmal pneumonia (MP) in pigs challenged with Mycoplasma hyopneumoniae (M. hyopneumoniae). Thirty six-week-old M. hyopneumoniae seronegative piglets were randomly assigned to four groups: three challenged groups with experimentally inoculated pigs by either the endotracheal (ET; n = 8), intranasal (IN; n = 8) or aerosol (AE; n = 8) routes and one uninfected group (Control; n = 6). Blood samples were collected 1 day before challenge and at necropsy, 28 days post-inoculation (dpi), to assess seroconversion. Laryngeal swabs were collected at −1, 7, 14, 21 and 28 dpi in order to evaluate colonization. At necropsy, lung lesions were scored and lung tissue was collected for histopathological studies and M. hyopneumoniae DNA detection. Broncho-alveolar lavage fluid (BALF) was also obtained to detect M. hyopneumoniae DNA, specific IgA antibodies and cytokines. MP was observed in all inoculated groups, but the ET group displayed a significantly higher number of animals affected by MP as well as a higher mean lung lesion score. These results were paralleled with an earlier seroconversion and upper respiratory tract colonization of M. hyopneumoniae. Additionally, in the ET group, higher levels of pro-inflammatory cytokines and specific IgA antibodies in BALF were found. Under the conditions of the present study, MP was reproduced by the three evaluated inoculation routes. Obtained results suggest that the ET route is the most effective in order to induce MP in pigs experimentally challenged with M. hyopneumoniae.

Increased antifungal antibodies in bronchoalveolar lavage fluid and serum in pulmonary sarcoidosis.

The recent studies suggest a role of fungi in development of sarcoidosis. Moreover, the immune response in sarcoidosis and fungal infection shows a striking similarity. We formulated a hypothesis of the possible increase in antifungal antibodies in bronchoalveolar lavage fluid (BALF) and serum in pulmonary sarcoidosis. BALF and serum levels of IgG-, IgM- and IgA-specific antibodies against the cell wall β-D-glucan and mannan of Candida albicans and Saccharomyces cerevisiae were tested in 47 patients (29 pulmonary sarcoidosis patients and 18 patients with other interstitial lung diseases (ILD - control group)) and 170 healthy controls. Our results proved: (1) an increase in IgG-, IgM- and IgA-specific antifungal antibodies in BALF in pulmonary sarcoidosis compared with the control group (C. albicans: IgG: P = 0.0329, IgM: P = 0.0076, IgA: P = 0.0156; S. cerevisiae: IgG: P = 0.0062, IgM: P = 0.0367, IgA: P = 0.0095) and (2) elevated levels of serum antifungal antibodies in pulmonary sarcoidosis compared with healthy controls (C. albicans: IgG: P = 0.0329, IgM: P = 0.0076, IgA: P = 0.0156; S. cerevisiae: IgG: P > 0.05, IgM: P < 0.05, IgA: P < 0.001). The study showed increased serum and BALF levels of antifungal antibodies in pulmonary sarcoidosis. The hypothesis that fungal infection is one of the possible aetiologic agents of sarcoidosis is interesting and deserves further attention.

Utility of immunological tests for bird-related hypersensitivity pneumonitis.

The reaction of specific antibodies and sensitized lymphocytes to antigens is important in hypersensitivity pneumonitis (HP). However, there are no known studies evaluating the utility of the lymphocyte proliferation test (LPT) or specific antibodies to avian antigens in diagnosing bird-related HP. In this study, we examined the sensitivity and specificity of these two tests. Patients with acute bird-related HP (n=10), chronic bird-related HP (n=35), acute summer-type HP (n=14), and other interstitial pneumonia (IP) (n=76) were evaluated. The optimal cutoff values were determined by receiver operating curve (ROC) analyses of specific antibodies in serum and bronchoalveolar lavage fluid (BALF), and by conducting the LPT on mononuclear cells in peripheral blood and BALF. The sensitivity and specificity of the antibodies were 80-100% and 92-100% in acute bird-related HP, and 26-79% and 73-93% in chronic bird-related HP, respectively. The sensitivity and specificity of the LPT were 50-100% and 100% in acute bird-related HP, and 46% and 91% in chronic bird-related HP, respectively. Specific antibodies and the LPT are quite useful for diagnosing acute bird-related HP. The presence of specific antibodies in BALF and the results of LPT with peripheral blood mononuclear cells are particularly useful for diagnosing chronic bird-related HP.

Type V Collagen–induced Tolerance Prevents Airway Hyperresponsiveness

Type V Collagen–induced Tolerance Prevents Airway Hyperresponsiveness

IL-17 induces type V collagen overexpression and EMT via TGF-β-dependent pathways in obliterative bronchiolitis.

Obliterative bronchiolitis (OB), a fibrotic airway lesion, is the leading cause of death after lung transplantation. Type V collagen [col(V)] overexpression and IL-17-mediated anti-col(V) immunity are key contributors to OB pathogenesis. Here, we report a previously undefined role of IL-17 in inducing col(V) overexpression, leading to epithelial mesenchymal transition (EMT) and subsequent OB. We observed IL-17-mediated induction of col(V) α1 chains [α1 (V)] in normal airway epithelial cells in vitro and detected α1 (V)-specific antibodies in bronchoalveolar lavage fluid of lung transplant patients. Overexpression of IL-17 and col(V) was detected in OB lesions in patient lung biopsies and in a murine OB model. IL-17 is shown to induce EMT, TGF-β mRNA expression, and SMAD3 activation, whereas downregulating SMAD7 expression in vitro. Pharmacological inhibition of TGF-βRI tyrosine kinase, p38 MAPK, or focal adhesion kinase prevented col(V) overexpression and EMT. In murine orthotopic lung transplants, neutralizing IL-17 significantly decreased TGF-β mRNA and protein expression and prevented epithelial repair/OB. Our findings highlight a feed-forward loop between IL-17 and TGF-β, leading to induction of col(V) and associated epithelial repair, thus providing one possible link between autoimmunity and OB after lung transplantation.

Long-term oral administration of Gynostemma pentaphyllum extract attenuates airway inflammation and Th2 cell activities in ovalbumin-sensitized mice

Our previous report demonstrated that the oral administration of short-term high dose Gynostemma pentaphyllum extract (5g/kg per day for 7days) decreased allergic reactions in ovalbumin (OVA)-sensitized mice. The aim of this study was to determine whether long-term oral administration of G. pentaphyllum attenuated airway inflammation in OVA-sensitized mice.Mice were sensitized and challenged with normal saline or OVA. OVA-sensitized mice were fed with 1.75g/kg (low dose, GPL) or 5g/kg (high dose, GPH) G. pentaphyllum extract, five days a week for 4weeks. The airway hyperresponsiveness (AHR) and eosinophilia in bronchoalveolar lavage fluid (BALF) were examined. The cytokine levels or antibodies in BALF, serum and spleen cell culture supernatants were also determined. Both high and low dose extracts reduced AHR, serum OVA–IgE, and Th2-associated cytokine levels in spleen cell supernatants and BALF in OVA-sensitized mice. These results show that long-term orally administered G. pentaphyllum extract reduced allergic reactions in OVA-sensitized mice.

Detection of disease specific sialoglycoconjugate specific antibodies in bronchoalveolar lavage fluid of non-small cell lung cancer patients

In this study, we report the presence of significantly higher level of GM3 specific IgG antibodies (IgG(TL)) in the bronchoalveolar lavage fluid obtained from tumor bearing lung of non-small cell lung cancer (NSCLC) patients as compared to other non-neoplastic controls. The antibodies were isolated using DEAE-cellulose anion exchange chromatography and molecular weight of the subunits of IgG(TL) was confirmed in SDS-PAGE. IgG(TL) revealed high specificity to GM3 and the IgG distribution was confined to IgG1. Furthermore, IgG(TL) showed strong reactivity with NSCLC cell lines as well as the tissue biopsies and cells obtained from fine needle aspirations of NSCLC patients. A 66 kDa membrane glycoprotein of NSCLC cell lines was found to interact specifically with IgG(TL), the intensity of which was drastically reduced in presence of GM3. Further, binding of Maackia amurensis agglutinin [specific for NeuAcalpha(2-->3)Gal unit , the same disaccharide unit also known to be present in GM3] to the 66 kDa band confirmed it to be a sialoglycoprotein in nature. IgG(TL) could not show any reactivity to alkaline borohydrate treated or periodate oxidised membrane fractions, suggesting the probable involvement of the carbohydrate moiety of the 66 kDa glycoprotein in the interaction with IgG(TL). Thus, the 66 kDa sialoglycoprotein seems to be the NSCLC specific sialoglycoconjugate. Taken together, IgG(TL) antibodies may have the potential to serve as a unique probe for detail investigation of NSCLC specific cell surface sialoglycoconjugate. Further, due to high specificity of IgG(TL) to GM3, it may be possible to develop a simple alternative diagnostic approach (GM3-ELISA) for NSCLC.

A FAMILIAL CASE OF VISCERAL LARVA MIGRANS AFTER INGESTION OF RAW CHICKEN LIVERS: APPEARANCE OF SPECIFIC ANTIBODY IN BRONCHOALVEOLAR LAVAGE FLUID OF THE PATIENTS

We report a familial case of visceral larva migrans (VLM) caused by Toxocara canis larvae. Patient 1 was a 45-year-old man who presented to our university hospital complaining of mild fever, general fatigue, and headache. Patient 2 was a 71-year-old man and was the father of Patient 1; he presented complaining of cough and hyper-viscous white sputum. Laboratory data from both patients showed extensive eosinophilia, their chest X-ray findings revealed multiple pulmonary infiltrates, and their bronchoalveolar lavage fluid (BALF) showed an elevated eosinophil count. The diagnosis of VLM was made based on a positive result in a serological test using T. canis larval excretory-secretory both in the serum and BALF. T. canis larvae were identified in meat that was prepared from chicken taken from the same source as that ingested. This is the first report to identify antibodies in BALF in patients with VLM.

Antigen-specific IgE and IgA antibodies in bronchoalveolar lavage fluid are associated with stronger antigen-induced late phase reactions.

The mechanism(s) leading to the development of late phase allergic reactions is (are) unknown. Previous studies have indicated that a relationship between serum IgE and the late phase exists. To explore the relationships between allergen-specific immunoglobulins in bronchoalveolar lavage (BAL) fluids and the magnitude of airflow limitation during the late phase response to inhaled allergen. Ragweed-specific IgE, IgA, secretory IgA (sIgA) and IgG were measured in BAL fluid and in the serum 1-5 weeks before whole lung antigen challenge with ragweed extract, in 16 ragweed allergic asthmatics. In addition, BAL and serum eosinophil cationic protein (ECP) and BAL fibrinogen levels were determined and BAL cells counted and differentiated. The latter procedures were repeated in a second BAL performed 24 h after the end of the ragweed challenge. After the challenge, lung function was monitored hourly for 8 h, to record the magnitude of airflow limitation. Ragweed-specific immunoglobulins were detected in 25% to 37.5% of BAL samples. Compared to the subjects with undetectable BAL fluid ragweed-specific IgE levels at baseline, those with detectable antibodies had stronger late phase reactions as determined by the nadir of FEV1 between hours 4 and 8 after the ragweed inhalation challenge (P = 0.0007). Allergen-induced changes in BAL ECP and fibrinogen levels were also higher in those subjects with detectable ragweed-specific IgE in baseline fluids (P = 0.03 and P = 0.005, respectively). Significant relationships between BAL antigen-specific IgA, serum ragweed-specific IgE and IgA and the late phase reaction were also found. The results of this study point towards the possibility that allergen-specific IgE and IgA may be independently involved in the pathogenesis of the late phase reaction. This notion merits further exploration.

Inhibition of adenovirus-mediated gene transfer by bronchoalveolar lavage fluid.

Human and animal trials with recombinant adenovirus have been discouraging, since the level of recombinant gene expression was low. Nonspecific and specific immune response mediated by, for example, macrophages, T cells and immunoglobulins may prevent infection or cause death of infected cells. We analyzed the effect of bronchoalveolar lavage fluid (BAL) on the efficiency of adenoviral infection in vitro. A total of 26 BAL samples of randomly selected patients was examined. Adenovirus-mediated gene transfer was quantified using AdCMV. Null, a recombinant adenovirus, in a modified titer assay based on immunocytochemical detection of infected 293 cells. In addition, the concentration of anti-adenovirus-type 5 IgA, IgG and IgM antibodies in BAL was determined by ELISA. 53.8% of the BAL samples (14 out of 26) reduced adenoviral infectivity by at least 50% (factor of inhibition > or = 2). All BAL samples effecting, a reduction in adenoviral infectivity contained detectable amounts of anti-adenovirus-type 5-IgA antibodies. However, the correlation between the concentration of IgA antibody and the strength of inhibition was weak (r = 0.336). Even high levels of anti-adenovirus-type 5-IgM, IgG or IgA antibodies did not influence adenoviral infectivity consistently. This observation indicates that BAL contains (a) anti-adenovirus-type 5 antibodies which are not directed against adenoviral epitopes responsible for the viral adherence and uptake process; and/or (b) inhibitors of viral infectivity different from antibodies.

Pulmonary involvement in patients with human T lymphotropic virus type 1-associated myelopathy: the presence of specific IgA antibody in bronchoalveolar lavage fluid.

It has been shown that T lymphocyte alveolitis occurs in patients with human T lymphotropic virus type-1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP). To determine whether HTLV-1-specific antibodies were present in bronchoalveolar lavage (BAL) fluid, we investigated 15 patients with HAM/TSP, five HTLV-1 carriers without HAM/TSP, and 10 normal control subjects seronegative for HTLV-1. Studies of BAL showed that 12 of 15 patients with HAM/TSP were associated with bronchoalveolar T lymphocytosis. Such abnormalities of the lung were not found in three other patients with HAM/TSP, non-HAM/TSP carriers, and normal control subjects. Using Western blots, specific IgA antibodies were detected only in BAL fluid from 12 patients with HAM/TSP and with pulmonary involvement. On the other hand, specific IgG antibodies in BAL fluid were positive not only in 12 patients with HAM/TSP and with pulmonary involvement, but also in two of three patients with HAM/TSP and three of five non-HAM/TSP carriers, both of whom showed normal BAL findings. Specific IgM class antibodies in BAL fluid were not detected in any subjects in these three groups. Specific IgA antibodies were found in the sera of 12 patients with HAM/TSP and with BAL lymphocytosis, but not in three patients with HAM/TSP and without BAL lymphocytosis, and five non-HAM/TSP carriers. These results suggest that the production of HTLV-1-specific IgA antibodies is closely related to pulmonary involvement in patients with HAM/TSP.