Broncheoalveolar Lavage Research Articles

Usefulness of immune monitoring in lung transplantation using adenosine triphosphate production in activated lymphocytes

The ImmuKnow (Cylex Inc, Columbia, MD) assay measures the amount of adenosine triphosphate (ATP) produced by helper CD4(+) cells after stimulation with a T-cell mitogen. We hypothesized that this assay can be used to assess the immune function of lung transplant recipients and identify those at risk of developing acute cellular rejection and respiratory infection. Lung transplant recipients at University of California Los Angeles between January 1, 2006 and December 31, 2009 received a bronchoscopy with broncheoalveolar lavage, transbronchial biopsy and ImmuKnow values drawn at regular intervals as well as during episodes of clinical deterioration. The recipient's clinical condition at each time-point was classified as healthy, acute cellular rejection, or respiratory infection. Mixed-effects models were used to compare the ATP levels among these groups, and odds ratios for rejection and infection were calculated. The mean ATP level was 431 ± 189 ng/ml for the rejection group vs 377 ± 187 ng/ml for the healthy group (p = 0.10). A recipient with an ATP level > 525 ng/ml was 2.1 times more likely to have acute cellular rejection (95% confidence interval [CI] 1.1-3.8). Similarly, the mean ATP level was 323 ± 169 ng/ml for the infection group vs 377 ± 187 ng/ml for the healthy group (p = 0.03). A recipient with an ATP level < 225 ng/ml was 1.9 times more likely to have respiratory infection (95% CI, 1.1-3.3). However, the test was associated with poor performance characteristics. It had low sensitivity, specificity with an area under the receiver operating characteristic curve of only 0.61 to diagnose rejection and 0.59 to diagnose infection. The ImmuKnow assay appears to have some ability to assess the overall immune function of lung transplant recipients. However, this study does not support its use as a reliable predictor of episodes of acute cellular rejection or respiratory infection.

Intranasal Administration of poly(I:C) and LPS in BALB/c Mice Induces Airway Hyperresponsiveness and Inflammation via Different Pathways

BackgroundBacterial and viral infections are known to promote airway hyperresponsiveness (AHR) in asthmatic patients. The mechanism behind this reaction is poorly understood, but pattern recognizing Toll-like receptors (TLRs) have recently been suggested to play a role.Materials and MethodsTo explore the relation between infection-induced airway inflammation and the development of AHR, poly(I:C) activating TLR3 and LPS triggering TLR4, were chosen to represent viral and bacterial induced interactions, respectively. Female BALB/c or MyD88-deficient C57BL/6 mice were treated intranasally with either poly(I:C), LPS or PBS (vehicle for the control group), once a day, during 4 consecutive days.ResultsWhen methacholine challenge was performed on day 5, BALB/c mice responded with an increase in airway resistance. The maximal resistance was higher in the poly(I:C) and LPS treated groups than among the controls, indicating development of AHR in response to repeated TLR activation. The proportion of lymphocytes in broncheoalveolar lavage fluid (BALF) increased after poly(I:C) treatment whereas LPS enhanced the amount of neutrophils. A similar cellular pattern was seen in lung tissue. Analysis of 21 inflammatory mediators in BALF revealed that the TLR response was receptor-specific. MyD88-deficient C57BL/6 mice responded to poly (I:C) with an influx of lymphocytes, whereas LPS caused no inflammation.Conclusion In vivo activation of TLR3 and TLR4 in BALB/c mice both caused AHR in conjunction with a local inflammatory reaction. The AHR appeared to be identical regardless of which TLR that was activated, whereas the inflammation exhibited a receptor specific profile in terms of both recruited cells and inflammatory mediators. The inflammatory response caused by LPS appeared to be dependent on MyD88 pathway. Altogether the presented data indicate that the development of AHR and the induction of local inflammation might be the result of two parallel events, rather than one leading to another.

Nanotitanium dioxide toxicity in mouse lung is reduced in sanding dust from paint.

BackgroundLittle is known of how the toxicity of nanoparticles is affected by the incorporation in complex matrices. We compared the toxic effects of the titanium dioxide nanoparticle UV-Titan L181 (NanoTiO2), pure or embedded in a paint matrix. We also compared the effects of the same paint with and without NanoTiO2.MethodsMice received a single intratracheal instillation of 18, 54 and 162 μg of NanoTiO2 or 54, 162 and 486 μg of the sanding dust from paint with and without NanoTiO2. DNA damage in broncheoalveolar lavage cells and liver, lung inflammation and liver histology were evaluated 1, 3 and 28 days after intratracheal instillation. Printex 90 was included as positive control.ResultsThere was no additive effect of adding NanoTiO2 to paints: Therefore the toxicity of NanoTiO2 was reduced by inclusion into a paint matrix. NanoTiO2 induced inflammation in mice with severity similar to Printex 90. The inflammatory response of NanoTiO2 and Printex 90 correlated with the instilled surface area. None of the materials, except of Printex 90, induced DNA damage in lung lining fluid cells. The highest dose of NanoTiO2 caused DNA damage in hepatic tissue 1 day after intratracheal instillation. Exposure of mice to the dust from paints with and without TiO2 was not associated with hepatic histopathological changes. Exposure to NanoTiO2 or to Printex 90 caused slight histopathological changes in the liver in some of the mice at different time points.ConclusionsPulmonary inflammation and DNA damage and hepatic histopathology were not changed in mice instilled with sanding dust from NanoTiO2 paint compared to paint without NanoTiO2. However, pure NanoTiO2 caused greater inflammation than NanoTiO2 embedded in the paint matrix.

Macrophage-Specific Targeting of Isoniazid Through Mannosylated Gelatin Microspheres

Active targeting of drug molecules can be achieved by effective attachment of suitable ligands to the surface of carriers. The present work was attempted to prepare mannosylated gelatin microspheres (m-GMs) so as to achieve targeted delivery of isoniazid (INH) to alveolar macrophages (AMs) and maintain its therapeutic concentration for prolonged period of time. Microspheres were prepared by emulsification solvent extraction method and evaluated for physicochemical characteristics, drug release, ex vivo drug uptake by AMs and pharmacokinetic characteristics. Fourier transform infrared spectroscopy and nuclear magnetic resonance spectral analysis confirmed that mannosylation took place through Schiff base formation between aldehyde and amino groups of mannose and gelatin, respectively. Prepared microspheres offered suitable physicochemical characteristics for their delivery to AMs. Their average size was about 4 μm and drug entrapment efficiency of 56% was achieved with them. Ex vivo uptake results indicated that in comparison to plain microspheres, m-GMs were selectively uptaken and were found to be associated with phago-lysosomal vesicles of AMs. Pharmacokinetic studies showed the formulation could maintain the therapeutic concentration of INH for prolonged period of time even with a reduced clinical dose. m-GMs were found to be stable in broncheo-alveolar lavage fluid. The study concluded that ligand decorated carriers could be a potential strategy to improve the therapeutic properties of INH.

Utilization of lipid‐laden macrophage index in evaluation of aerodigestive disorders

The utility of the lipid-laden macrophage index (LLMI) in the evaluation of airway inflammatory processes remains controversial. There is a paucity of normative data in both pediatric and adult populations, and there is wide variability in the reported cases. The goal of this project was to review the LLMI levels in a large series of patients with a wide range of well-documented pulmonary and airway diseases (cystic fibrosis, aspiration, tracheo/bronchomalacia, recurrent pneumonia, asthma, immunosuppressed conditions, and laryngeal clefts) to develop a better understanding of the clinical utility of the LLMI. This study is a retrospective analysis of patients who underwent initial bronchoscopy with broncheoalveolar lavage (BAL) and had specimens sent for determination of LLMI. This study was performed at a major pediatric tertiary care medical center from April 12, 2006 to July 13, 2009. Medical charts were reviewed for major diagnoses, indications for bronchoscopy, the side of lung (right versus left), documented aspiration, suspected aspiration, neutrophil counts, and patient age. These qualitative and quantitative variables were considered in relation to the LLMI. The cohort of patients examined were 4.9 ± 4.3 years (4 days to 18 years of age). Forty-two percent were female and 58% were male. The highest median LLMI was in immunocompromised patients with a median of 78. Recurrent pneumonia, aspiration, tracheo/bronchomalacia, and cystic fibrosis had median LLMI levels of 55, 49, 40, and 50, respectively. There was a wide range of LLMI within each primary diagnosis. There was no significant correlation between neutrophils percentage in the BAL and LLMI. There were no significant differences between BAL specimens obtained from the right or left side. To our knowledge, this is the largest series of patients reported in the literature to have an LLMI compared with underlying diagnoses. Based on our data and analyses, there is wide variability between the range of LLMI and the primary diagnosis. As such, the diagnostic utility of the LLMI is limited and should be interpreted with caution.

Vaccination against nicotine alters the distribution of nicotine delivered via cigarette smoke inhalation to rats

Preclinical models of nicotine vaccine pharmacology have relied on i.v. or s.c. administration of nicotine. Models using cigarette smoke inhalation might more accurately simulate nicotine exposure in smokers. Nicotine vaccine effects were examined in rats using two cigarette smoke exposure models: a 10 min nose-only exposure (NSE) producing serum nicotine levels equivalent to the nicotine boost from 1 cigarette in a smoker, and a 2 h whole-body exposure (WBE) producing serum nicotine levels similar to those associated with regular mid-day smoking. Vaccination prior to 10 min smoke NSE reduced nicotine distribution to brain by 90%, comparable to its effect on nicotine administered i.v. Vaccination prior to 2 h smoke WBE reduced nicotine distribution to brain by 35%. The nicotine concentration in broncheoalveolar lavage (BAL) fluid obtained after 2 h WBE was increased by 230% in vaccinated rats but was also increased in rats passively immunized with a nicotine-specific monoclonal antibody, and so was likely due to transfer of antibody from serum rather than local production at the pulmonary mucosa. Nicotine-specific IgA was not detectable in BAL fluid, but titers in serum were appreciable at 21–25% of the IgG titer and could contribute to vaccine efficacy. Both vaccination and passive immunization are effective in reducing nicotine distribution to brain in rats when nicotine is delivered via inhaled cigarette smoke. These data validate results previously obtained in rodents for nicotine vaccines using i.v. or s.c. nicotine dosing and provide a quantitative method for studying aspects of nicotine exposure which are unique to cigarette smoke inhalation.

CpG oligodeoxynucleotide synergizes innate defense regulator peptide for enhancing the systemic and mucosal immune responses to pseudorabies attenuated virus vaccine in piglets in vivo

Oligonucleotides containing CpG motifs (CpG ODN) are strong adjuvants for humoral and cellular immune responses in mice, and innate defense-regulator peptides (IDRs) are known to facilitate the uptake of antigens into antigen presenting cells (APCs), but data on synergistic effects of CpG and IDRs in piglets are scarce. In this report, the combination of porcine-specific CpG ODN and HH2 (a kind of IDR which was selected for its better synergy with CpG ODN) was used as immunoadjuvant to enhance the immune responses of the newborn piglets to Pseudorabies attenuated virus (PRV) vaccine. The titers of specific antibodies and serum IgG1/IgG2 subtypes to PRV vaccine, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), IL-12 and IL-4 were examined to identify the immune responses of the newborn piglets. The results showed that piglets immunized intranasally (IN) and subcutaneously (SC) with PRV vaccine and CpG–HH2 complex both presented high titers of PRV-specific antibodies and IgG2 isotype, a Th1-dominated (IFN-γ and IL-12) cytokine profiles, high levels of IgA in saliva, broncheoalveolar lavage (BAL) and intestinal washings. The results suggested that, CpG–HH2 complex augmented systemic (IgG in serum) and mucosal (IgA in saliva, BAL and intestinal washings) immune responses against antigen. CpG–HH2 complex stimulated both T-helper type1 (Th1) (IgG2) and Th2 (IgA) responses when delivered IN, and IN route could induce stronger mucosal immune responses than SC route. All these data indicate that CpG–HH2 complex is a potential effective adjuvant for the PRV vaccine in newborn piglets.

Toxicity and Clearance of Intratracheally Administered Multiwalled Carbon Nanotubes from Murine Lung

Carbon nanotubes (CNT) are known to have widespread industrial applications; however, several reports indicated that these compounds may be associated with adverse effects in humans. In this study, multiwalled carbon nanotubes were administered to murine lungs intratracheally to determine whether acute and chronic pulmonary toxicity occurred. In particular, pristine multiwalled carbon nanotubes (PMWCNT) and acid-treated multiwalled carbon nanotubes (TMWCNT) were used in this study. In broncheoalveolar lavage fluid (BALF) cell analysis, PMWCNT induced more severe acute inflammatory cell recruitment than TMWCNT. Histopathologically, both PMWCNT and TMWCNT induced multifocal inflammatory granulomas in a dose-dependent manner. The observed granulomas were reversible, with TMWCNT-induced granulomas diminishing faster than PMWCNT-induced granulomas. Although the area of granuloma reduced with time, hyperplasia and dysplastic characteristics such as mitotic figures, anisokaryosis, and anisocytosis were still observed. These findings demonstrate that MWCNT induces granulomatous inflammation, and the duration and pattern of inflammation seem to vary depending upon the types of MWCNT to which mice are exposed. Therefore, toxicity studies on various types of CNT are needed as the responsiveness to these compounds differs.

The impact of the H1N1 pandemic in pregnancy – a district General Hospital Perspective

IntroductionSwine flu (Influenza A H1N1) has been implicated in 289 deaths in England since it reached pandemic levels in April 2009. At least 2 pregnant women in the UK are...

Indications of IL1β, IL18 and VEGF cascade in maintenance of homeostasis following transient pulmonary inflammation due to carbon nanoparticle (CNP) challenge in mice

Aims: Carbonaceous nanoparticles possess an emerging source of human exposure. The pulmonary inflammation caused by deposited nanoparticles is a key event for their adverse health effects. Epidemiological studies suggest that individuals with favourable lung physiology are at lower risk for particulate matter associated respiratory diseases. We selected a mouse strain C3H/HeJ (C3) with robust lung physiology and exposed it to moderately toxic carbon nanoparticles (CNP) to study the elicited pulmonary inflammation and its resolution. Methods: 5µg, 20µg and 50µg CNP were intratracheally (i.t.) instilled in C3 mice. Inflammation was assessed using histology, broncheoalveolar lavage (BAL) analysis and by a panel of 62 protein markers to analyze the molecular response. Results: 24h after instillation of CNP, C3 mice exhibited a typical dose response, with the lowest dose (5µg) representing the 'no effect level' as reflected by polymorphonuclear leucocyte (PMN) influx, and BAL/lung concentrations of pro-inflammatory cytokines and proteins. Accordingly time course assessment of the inflammatory response was performed after 3 and 7days with 20µg CNP instilled animals. Compared to 24h, BAL PMN counts were significantly decreased at day 3 and by day 7 complete resolution of PMN influx was observed. We have identified protein markers related to acute response and also for time course response in lung and BAL. After complete resolution of PMN influx on day7, protein concentrations of FGA, CRP, APCS, CCL5, CCL9, FGF2, CCL11, vWF, XCL1, CXCL5, THPO, VEGFA, IL1B, IL18, CSF1, EDN1, CCL3, CCL4, F3 and CD40L were significantly elevated in the lung and BAL. These factors are involved in a closely regulated molecular cascade with IL1β/IL18at the upstream and FGF2/EDN1/VEGF at the downstream. BAL-protein concentration and histological analysis did not reveal any evidence of tissue injury in 20µg CNP instilled animals. Conclusion: Considering the role of VEGF, FGF2 and EDN1 in lung development and morphogenesis together with the lack of any evident tissue damage we suggest a protective/homeostatic machinery is activated to counter the CNP challenge as a precautionary measure.

Sirolimus-induced pneumonitis

A 15 year-old girl with reflux nephropathy ESKD underwent a related renal transplant in March 2000. Initial immunoupprressants administered to the patient included cyclosporine, azathioprine, and prednisolone. In January 2003, she had acute allograft dysfunction due to CNI toxicity with chronic allograft nephropathy (CAN) detected by biopsy. Thus, cyclosporine and azathioprine were withdrawn and replaced with rapamycin and mycophenolate mofetil. The serum creatinine level at the time of the change was 2.2 mg/dL, urine routine microscopy showed Trace proteinuria. Sirolimus levels were monitored and kept between 5 and 10 ng/mL. With the withdrawal of cyclosporine, renal parameters showed significant improvement and the serum creatinine level stabilized at 1.6 mg/dL. In April 2007, after approximately four years of the change, the patient presented with progressive breathlessness without any other systemic manifestations or expectoration. Computerised tomographic (CT) of the chest showed bilateral, mosaic ground-glass opacification and thickened interlobular septae [Figure 1]. Bronchoscopy, broncheoalveolar lavage, and pulmonary biopsy excluded infective etiology such as pneumocystis carnii, pulmonary Koch's, and fungal pneumonia [Figure 2]. By exclusion, the etiology was narrowed down to rapamycin-induced parenchymal lung lesions. Thus, rapamycin was withdrawn and the patient was serially followed on two drugs, mycophenolate mofetil and prednesolone. She improved clinically in twelve weeks and radiologically over six months. The followup CT of the chest showed significant clearing of the reticular and ground glass opacities [Figure 3]. Figure 1 Sirolimus-induced pneumonitis Figure 2 Lung biopsy showing parenchymal lung lesion. Transbronchial lung biopsy shows normal bronchial epithelium, bronchial cartilage, and surrounding lung tissue. Alveoli contain a granular proteinaceous material (arrows). This material is also seen lying loosely ... Figure 3 Resolved Sirolimus-induced pneumonitis Sirolimus (rapamycin) is an immunosuppressive agent that was approved by the Food and Drug Administration (FDA) in 1999 for use in renal transplantation. The macrocyclic triene antibiotic is produced by the actinomycete, Streptomyces hygroscopicus. Sirolimus acts by arresting the progression of cells from the G1 phase to the S phase by interaction with at least two intracellular proteins. Forty-three cases of pulmonary toxicity due to Sirolimus (rapamycin) have been reported including cases of interstitial pneumonitis, fibrosing alveolitis, and pulmonary hemorrhages.[1] The majority of these cases have occurred in renal transplant recipients within the initial six months of iniciation of the drug and lesions have generally responded to the lowering or withdrawal of the drug dosage. Most of the patients present with progressive breathlessness, weakness, cough, and bilateral lower zone pulmonary opacities, and over half of the cases present with fever. Our patient fulfilled all these criteria, except for the delayed presentation. The mechanism of pulmonary toxicity of Sirolimus (rapamycin) is not clear; direct toxicity, immunomediated toxicity, or both have been proposed as the mechanism.[1] The presence of lymphocytes in lung biopsies suggests an immunomediated mechanism. Possibly, some cryptic pulmonary antigens induce an autoimmune response. However, the rapid clearance of lung lesions with the discontinuation of the drugor dose reduction without steroids, favors direct toxicity.

Características citológicas del lavado bronqueoalveolar y respuesta inmune humoral frente a Aspergillus fumigatus en caballos Chilotes con obstrucción recurrente de las vías aéreas

SUMMARY In this study, the presence and characteristics of the recurrent airway obstruction (RAO), in Chilote horses were investigated. The Chilote horse is a breed of small and very rustic horses, descendent from the horses that were brought by the spaniards during the colony and remained isolated in the Chiloe island, south of Chile, for a long time. Twenty two animals, all from the same farm and living under similar conditions, were physically examined. In all cases broncheoalveolar lavage (BALF) was performed and the recovered resident cells from the respiratory tract were studied in smears stained with May Grewald – Giemsa; the level of antibodies against Aspergillus fumigatus was determined by ELISA. The results indicate that out of the total population of animals under investigation, 13 showed clinical signs compatible with RAO and 9 were normal. In all cases, the animals that were considered RAO positive showed a higher percentage of polymorphonuclear neutrophils in the BALF: furthermore, a clear positive correlation between the relative amount of neutrophilic cells present in the airways and the severity of the signs observed during the physical examination exists. All of the animals, healthy and RAO positive horses, showed high titres of antibodies against A. fumigatus, a well known allergen, commonly distributed in the region and present in the hay/straw. Our results show that Chilote horses, even though they are a rustic bread, are very susceptible to become RAO positive with similar characteristics to those described in other breeds, moreover, all animals were sensitized to A. fumigatus antigens showing the immunogenicity and the important distribution of this fungal allergen.

Occult pleural cryptococcosis in an immunocompromised patient

Sir: Cryptococcosis is a rare opportunistic, invasive, fungal, lifethreatening infection, which occurs in both immunocompetent and immunocompromised patients. In immunocompromised patients, the signs of cryptococcosis can easily be confused by the symptoms of the primary disease, which may delay adequate treatment [1]. We recently treated a 47-year-old woman with a thymoma WHO type B1 in the left hemithorax treated with three cycles of cisplatin/etoposide. Simultaneously, she developed a recurrent right-sided pleural effusion, which was drained four times and tested by an India ink and gramstained smear, bacterial culturing, Cryptococcus serum and pleural punctuate antigen testing and a cytological stain, which were all negative for microorganisms including Cryptococcus neoformans. The broncheoalveolar lavage (BAL) material was also negative. The transudate contained lymphocytes and mesothelial cells. Eight days following the last cycle of chemotherapy, the patient was admitted because of fever, headache, neck pain, and cognitive changes during chemotherapyinduced pancytopenia. Three months after pleural effusion development, C. neoformans meningitis was diagnosed in the cerebral spinal fluid. The infection was successfully treated with amphotericin B (0.7 mg/kg/day) and flucytosine (100 mg/kg/day) for 2 weeks, followed by 8 weeks amfotericin B (0.7 mg/kg/day) with flucanozole (800 mg/kg/day). Despite the diagnosis of meningeal cryptococcosis, pleural abnormalities remained attributed to the thymoma. Two weeks after cryptococcosis diagnosis, the patient underwent surgery for staging. Histology of the right middle lobe and pleura revealed a granulomatous necrotizing infection with C. neoformans (Fig. 1). One month later, a thymectomy was performed with partial pericardectomy, followed by ICU admission for 1 month. In summary, our patient showed pulmonary cryptococcosis which, due to negative bacteriological testing, was not recognized as such. Pulmonary signs were, therefore, attributed to her thymoma. Despite repeated testing for fungi of the pleural effusion and BAL, these tests remained negative during a substantial part of the disease progression. Whether the initial pleural transuduate was caused by cryptococcal infection or that secondary infection of pre-existent pleural fluid occurred, remains unclear. Microbiologic examination of specimens for cryptococci is not always conclusive: the appearance of cryptococci may vary considerably

Differential Th17 CD4 T-cell depletion in pathogenic and nonpathogenic lentiviral infections

AbstractAcute HIV infection is characterized by massive loss of CD4 T cells from the gastrointestinal (GI) tract. Th17 cells are critical in the defense against microbes, particularly at mucosal surfaces. Here we analyzed Th17 cells in the blood, GI tract, and broncheoalveolar lavage of HIV-infected and uninfected humans, and SIV-infected and uninfected sooty mangabeys. We found that (1) human Th17 cells are specific for extracellular bacterial and fungal antigens, but not common viral antigens; (2) Th17 cells are infected by HIV in vivo, but not preferentially so; (3) CD4 T cells in blood of HIV-infected patients are skewed away from a Th17 phenotype toward a Th1 phenotype with cellular maturation; (4) there is significant loss of Th17 cells in the GI tract of HIV-infected patients; (5) Th17 cells are not preferentially lost from the broncheoalveolar lavage of HIV-infected patients; and (6) SIV-infected sooty mangabeys maintain healthy frequencies of Th17 cells in the blood and GI tract. These observations further elucidate the immunodeficiency of HIV disease and may provide a mechanistic basis for the mucosal barrier breakdown that characterizes HIV infection. Finally, these data may help account for the nonprogressive nature of nonpathogenic SIV infection in sooty mangabeys.

Inflammation but no DNA (deoxyribonucleic acid) damage in mice exposed to airborne dust from a biofuel plant

Particles in ambient air are associated with such health effects as lung diseases and cancer of the lung. Exposure to bioaerosols has been found to be associated with respiratory symptoms. The toxic properties of exposure to combustion and bioaerosol particles from biofuel plants have not been studied in detail. This study investigated whether exposure to dust from biofuel plants induces DNA (deoxyribonucleic acid) damage and inflammation in exposed mice. DNA damage and inflammation were evaluated in mice exposed through the intratracheal installation of airborne dust collected at a biofuel plant at the straw storage hall and in the boiler room. The mice were given either a single dose of dust (18 or 54 microg) or four doses of 54 microg on each of four consecutive days. Control mice were exposed to a 0.9% sodium chloride solution. In the mice exposed to 4 x 54 microg of dust, the lung tissue mRNA (messenger ribonucleic acid) levels of interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2) were increased more than 10-fold if the dust was from the boiler room and 30- to 60-fold if the dust came from the straw storage hall. The levels of DNA strand breaks in broncheoalveolar lavage (BAL) cells from the mice exposed to dust did not differ from those in the control samples. The results indicate that the instillation of dust from a biofuel plant, at doses corresponding to 2 weeks of exposure to human endotoxins, results in a strong inflammatory response without detectable DNA damage in BAL cells. The dust from the straw storage hall induced the strongest inflammatory response and had the highest concentration of most microbial components.

Pneumococcal Necrotizing Pneumonia in Utah: Does Serotype Matter?

Streptococcus pneumoniae is the most common cause of bacterial pneumonia in children. Despite the use of the 7-valent pneumococcal conjugate vaccine, the incidence of pneumococcal necrotizing pneumonia (PNP) has been increasing. Our objectives were to describe temporal trends in PNP and to evaluate pneumococcal serotypes associated with PNP in Utah. We performed a retrospective review of all children <18 years of age who were cared for at a tertiary care children's hospital and who had blood, lung tissue, broncheoalveolar lavage, or pleural fluid cultures that grew S. pneumoniae, as well as radiographic evidence of pneumonia, from January 1997 through March 2006. All S. pneumoniae isolates were typed. A total of 124 children with pneumococcal pneumonia were identified, and 33 (27%) of these children had radiographic evidence of PNP. During the period 1997-2000, 5 (13%) of 39 cases of culture-confirmed pneumococcal pneumonia were associated with PNP. In contrast, during the period 2001-2006, 28 (33%) of 85 pneumococcal pneumonia cases were complicated by PNP (odds ratio, 3.34; 95% confidence interval, 1.11-12.03). Non-7-valent pneumococcal conjugate vaccine serotypes comprised 49% of the isolates during 1997-2000 and 88% of isolates during 2001-2006 (odds ratio, 7.89; 95% confidence interval, 2.91-21.90). Pneumonia due to serotype 3 was most often associated with PNP. Eleven (79%) of 14 cases of serotype 3-associated pneumonia were associated with PNP. When compared with all other serotypes, serotype 3 was strongly associated with necrosis (odds ratio, 14.67; 95% confidence interval, 3.39-86.25). PNP is a serious and increasingly common complication of S. pneumoniae pneumonia in Utah. Infection with serotype 3 is associated with an increased risk of developing PNP. The increase in the incidence of infection due to nonvaccine serotypes reported worldwide and the changing epidemiology of invasive pneumococcal disease should be considered when developing vaccine strategies.

The B Cell Superantigen Peptostreptococcus Magnus (Pm) Protein L Induces Lung Inflammation and Airway Hyperreactivity Through a MyD88-Dependent Mechanism

Pm protein L functions as a B cell superantigen (SAg) by interacting with V kappa light chain determinants on many human and mouse immunoglobulins. Inhalation of protein L elicits inflammatory cell infiltration and cytokine/chemokine release in the broncheoalveolar lavage fluid (BALF) through a mast cell-independent process. Surprisingly, this reaction occurs in the absence of immunoglobulins and requires MyD88. To further investigate the effects of protein L on the airways, C57 BL/6 mice were administered either protein L or BSA intratracheally. BALF was collected at various time points and analyzed for neutrophil counts, MIP-2, KC, TNF-α, and IL6. Lung tissue was harvested for histological examination. In some experiments, alveolar macrophages were depleted via tracheal insufflation of liposome-encapsulated dicloromethylene diphosphonate (Lipo-clod). Airway response to inhaled methacholine was measured using the Flexivent system in anesthetized, cannulated and ventilated mice. Protein L-treated mice accumulated neutrophils in their BALF as early as 4 hours, showed an alveolar and peribronchial infiltrate of inflammatory cells peaking between 8 and 24 hours, and displayed significant airway hyperresponsiveness to MCh (3-25 mg/ml) at 24 hours. MIP-2, KC, TNF-α, and IL6 levels in protein L challenged mice peaked at 4 hours. These responses were not dampened in macrophage depleted mice. Protein L induces lung inflammation and increased airway resistance via a MyD88 pathway independently of immunoglobulins, mast cells, and macrophages. The results reveal a new mechanism by which a microbial protein induces airway inflammation and hyperreactivity and suggest future mechanistic studies focus on a toll-like receptor on airway epithelial cells.

Endogenous excess cortisol production and diabetes mellitus as predisposing factors for pulmonary cryptococcosis: A case report and literature review

Pulmonary cryptococcosis usually occurs as an opportunistic infection in immunocompromised patients. Endogenous Cushing's syndrome is associated with cortisol excess and can predispose to development of cryptococcal infections. We report a case of diabetic patient with ACTH secreting pituitary tumour who developed a cavitating lung mass. Computed tomography-guided biopsy of the lesion revealed mucicarminophilic budding forms of cryptococcus. Broncheoalveolar lavage culture grew Cryptococcus neoformans. There was radiological response to treatment with liposomal Amphotericin, but patient ultimately succumbed to septicemia and multiorgan failure. Opportunistic infections with organisms like Cryptococcus neoformans, should be considered in patients with endogenous Cushing's syndrome and a pulmonary infiltrate.

Continuous monitoring of the bronchial epithelial lining fluid by microdialysis

BackgroundContents of the epithelial lining fluid (ELF) of the bronchi are of central interest in lung diseases, acute lung injury and pharmacology. The most commonly used technique broncheoalveolar lavage is invasive and may cause lung injury. Microdialysis (MD) is a method for continuous sampling of extracellular molecules in the immediate surroundings of the catheter. Urea is used as an endogenous marker of dilution in samples collected from the ELF. The aim of this study was to evaluate bronchial MD as a continuous monitor of the ELF.MethodsMicrodialysis catheters were introduced into the right main stem bronchus and into the right subclavian artery of five anesthetized and normoventilated pigs. The flowrate was 2 μl/min and the sampling interval was 60 minutes. Lactate and fluorescein-isothiocyanate-dextran 4 kDa (FD-4) infusions were performed to obtain two levels of steady-state concentrations in blood. Accuracy was defined as [bronchial-MD] divided by [arterial-MD] in percent. Data presented as mean ± 95 percent confidence interval.ResultsThe accuracy of bronchial MD was calculated with and without correction by the arteriobronchial urea gradient. The arteriobronchial lactate gradient was 1.2 ± 0.1 and FD-4 gradient was 4.0 ± 1.2. Accuracy of bronchial MD with a continuous lactate infusion was mean 25.5% (range 5.7–59.6%) with a coefficient of variation (CV) of 62.6%. With correction by the arteriobronchial urea gradient accuracy was mean 79.0% (57.3–108.1%) with a CV of 17.0%.ConclusionUrea as a marker of catheter functioning enhances bronchial MD and makes it useful for monitoring substantial changes in the composition of the ELF.

LYSINE TRUNCATION OF ALPHA1-PROTEINASE INHIBITOR UPON DIFFUSION FROM THE VASCULATURE INTO BRONCHEOALVEOLAR LAVAGE

PURPOSE: Recently the characteristic pattern of A1PI seen on high resolution isoelectric focusing (IEF) gels was elucidated by the description of a new A1PI isoform missing the C-terminal amino acid, lysine. As this modification of A1PI could be induced in vitro by the action of basic carboxypeptidases we were interested in whether it can also be found in vivo. After intravenous application of intact human A1PI in rats, we determined the IEF pattern of A1PI in broncheoalveolar lavage (BAL) and checked for the presence of the C-terminal truncated peptide using mass spectrometry (MS).