Killing of Streptococcus pneumoniae by capsular polysaccharide-specific polymeric IgA, complement, and phagocytes.

The role of IgA in the control of invasive mucosal pathogens such as Streptococcus pneumoniae is poorly understood. We demonstrate that human pneumococcal capsular polysaccharide-specific IgA initiated dose-dependent killing of S. pneumoniae with complement and phagocytes. The majority of specific IgA in serum was of the polymeric form (pIgA), and the efficiency of pIgA-initiated killing exceeded that of monomeric IgA-initiated killing. In the absence of complement, specific IgA induced minimal bacterial adherence, uptake, and killing. Killing of S. pneumoniae by resting phagocytes with immune IgA required complement, predominantly via the C2-independent alternative pathway, which requires factor B, but not calcium. Both S. pneumoniae-bound IgA and complement were involved, as demonstrated by a 50% decrease in killing with blocking of Fcalpha receptor (CD89) and CR1/CR3 (CD35/CD11b). However, IgA-mediated killing by phagocytes could be reproduced in the absence of opsonic complement by pre-activating phagocytes with the inflammatory products C5a and TNF-alpha. Thus, S. pneumoniae capsule-specific IgA may show distinct roles in effecting clearance of S. pneumoniae in the presence or absence of inflammation. These data suggest mechanisms whereby pIgA may serve to control pneumococcal infections locally and upon the pathogen's entry into the bloodstream.

Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein.

The purpose of this study was to characterize the surface receptor for toxin A, the enterotoxin from Clostridium difficile, on rabbit intestinal brush borders (BB) and on rat basophilic leukemia (RBL) cells. Purified toxin A was radiolabeled using a modified Bolton-Hunter method to sp act 2 microCi/micrograms, with retention of full biologic activity. 3H-Toxin A bound specifically to a single class of receptors on rabbit BB and on RBL cells with dissociation constants of 5.4 x 10(-8) and 3.5 x 10(-8) M, respectively. RBL cells were highly sensitive to toxin A (cell rounding) and had 180,000 specific binding sites per cell, whereas IMR-90 fibroblasts were far less sensitive to toxin A and lacked detectable specific binding sites. Exposure of BB to trypsin or chymotrypsin significantly reduced 3H-toxin A specific binding. Preincubation of BB with Bandeirea simplicifolia (BS-1) lectin also reduced specific binding, and CHAPS-solubilized receptors could be immobilized with WGA-agarose. The addition of 100 nM toxin A accelerated the association of 35S-GTP gamma S with rabbit ileal BB, and preincubation of BB with the GTP analogues GTP gamma S or Gpp(NH)p, significantly reduced 3H-toxin A specific binding. Our data indicate that the membrane receptor for toxin A is a galactose and N-acetyl-glucosamine-containing glycoprotein which appears to be coupled to a G protein.

Dual function of pneumolysin in the early pathogenesis of murine pneumococcal pneumonia.

Streptococcus pneumoniae is one of the most common etiologic agents of community-acquired pneumonia, particularly bacteremic pneumonia. Pneumolysin, a multifunctional cytotoxin, is a putative virulence factor for S. pneumoniae; however, a direct role for pneumolysin in the early pathogenesis of pneumococcal pneumonia has not been confirmed in vivo. We compared the growth of a pneumolysin-deficient (PLY[-]) type 2 S. pneumoniae strain with its isogenic wild-type strain (PLY[+]) after direct endotracheal instillation of bacteria into murine lungs. Compared with PLY(-) bacteria, infection with PLY(+) bacteria produced greater injury to the alveolar-capillary barrier, as assayed by albumin concentrations in alveolar lavage, and substantially greater numbers of PLY(+) bacteria were recovered in alveolar lavages and lung homogenates at 3 and 6 h after infection. The presence of pneumolysin also contributed to the development of bacteremia, which was detected at 3 h after intratracheal instillation of PLY(+) bacteria. The direct effects of pneumolysin on lung injury and on the ability of pneumococci to evade local lung defenses was confirmed by addition of purified recombinant pneumolysin to inocula of PLY(-) pneumococci, which promoted growth of PLY(-) bacteria in the lung to levels comparable to those seen with the PLY(+) strain. We further demonstrated the contributions of both the cytolytic and the complement-activating properties of pneumolysin on enhanced bacterial growth in murine lungs using genetically modified pneumolysin congeners and genetically complement-deficient mice. Thus, pneumolysin facilitates intraalveolar replication of pneumococci, penetration of bacteria from alveoli into the interstitium of the lung, and dissemination of pneumococci into the bloodstream during experimental pneumonia. Moreover, both the cytotoxic and the complement-activating activities of pneumolysin may contribute independently to the acute pulmonary injury and the high rates of bacteremia which characterize pneumococcal pneumonia.

Rb and p16INK4a expression in resected non-small cell lung tumors.

Inactivation of the cyclin-dependent kinase inhibitor p16INK4a (CDKN2/MTS1) is documented in a wide variety of cancer cell lines and tumors. We have shown that loss of p16INK4a protein expression is a common event in early stage non-small cell lung cancer (NSCLC), correlates with a significantly worse survival, and is more common in higher stage disease. One hundred NSCLC tumors from patients undergoing definitive thoracotomies at a single institution were examined for p16INK4a and retinoblastoma protein (pRB) expression. Abnormal pRB staining was identified in 15% of the tumors, whereas 51% possessed aberrant p16INK4a protein expression. Tumors with aberrant expression of p16INK4a by immunohistochemistry were associated with a significantly worse survival (P=0.04). Additionally, the inverse correlation of pRB and p16INK4a expression previously noted in lung cancer cell lines and tumors was confirmed in this large cohort of patients, with 65% of the tumors demonstrating inverse expression of pRB and p16INK4a (p=0.00019). A statistically significant increase in aberrant p16INK4a expression, as well as inverse expression of p16INK4a and pRB, was seen with increasing pathological stage of disease. These findings establish the prognostic significance (of the absence of p16INK4, in resected NSCLC and confirm the critical importance of disrupting the pathway of cyclin-dependent kinase-mediated phosphorylation of pRB in the molecular oncogenesis and progression of NSCLC.

Subcellular distribution and characterization of GTP-binding proteins in human neutrophils.

The subcellular distribution of GTP binding proteins in human neutrophils and their functional coupling to the N-formylmethionylleucylphenylalanine (FMLP) receptor was characterized to provide insight into mechanisms of cellular activation. Human neutrophils were nitrogen cavitated and fractionated on discontinuous Percoll gradients. Four subcellular fractions were obtained: cytosol, light membranes enriched for plasma membranes, specific granules and azurophilic granules. ADP-ribosylation catalyzed by pertussis toxin (PT) revealed a major substrate of 40 kDa only in plasma membrane and cytosol, and antiserum specific for Gi alpha confirmed the presence of neutrophil Gi alpha in plasma membrane and cytosol and its absence from specific granules. The cytosolic PT substrate was shown to be mostly in monomeric form by molecular sieve chromatography. The rate of the ribosyltransferase reaction was several-fold lower in cytosol compared to plasma membranes, and the extent of ADP-ribosylation was greatly augmented by supplementation with beta gamma subunits in cytosol. ADP-ribosylation catalyzed by cholera toxin (CT) revealed substrates of 52, 43 and 40 kDa in plasma membrane alone. FMLP receptors in plasma membrane were shown to be coupled to the 40 kDa substrate for CT by ligand-modulation of ADP-ribosylation, while FMLP added to specific granules did not induce ribosylation of this substrate even though FMLP receptors were found in high density in this compartment. Both 24 and 26 kDa [32P]GTP binding proteins were found to codistribute with FMLP receptors in specific granules and plasma membranes. Functional evidence for the coupling of GTP binding proteins to the FMLP receptor in specific granules was obtained by modulating [3H]FMLP binding with GTP gamma S, and by accelerating [35S]GTP gamma S binding with FMLP.

Gene therapy of established mesothelioma xenografts with recombinant p16INK4a adenovirus.

The absence of expression of the p16INK4a gene product is observed in virtually all mesothelioma tumors and cell lines, whereas wild-type pRB expression is maintained. We have examined the potential therapeutic role of re-expressing the p16INK4a gene product in mice with established human mesothelioma xenografts. Experiments using Adp16 treatments in mesothelioma xenografts demonstrated prolonged survival and potential cure following treatment with p16INK4a-based gene therapy. These results demonstrate that p16INK4a gene transfer may play a therapeutic role in the treatment of mesothelioma.

Immune activation and virologic response to immunization in recent HIV type 1 seroconverters.

Antigenic stimulation from invasive bacterial infections, and the vaccines designed to prevent them, may promote T cell activation and enhancement of HIV-1 replication. Changes in viral load have been correlated with antigen-specific responses. We prospectively determined the impact of immunization with 23-valent pneumococcal vaccine (PVAX) and Haemophilus influenzae type b (Hib)-modified diphtheria toxoid CRM197 (DT) vaccine on HIV-1 replication in recent HIV-1 seroconverters (n = 14; median, 5.5 months from infection; median CD4+ T cells, 535 microl), and correlated results with vaccine-related immune activation. Specific antibody responses, markers of CD4+ T cell activation (transferrin and interleukin 2 receptors), and viral burden were measured at weeks -2 (pre), 0, 1, 2, 6, and 12 after immunization. By week 2, levels of IgG had increased significantly over baseline in both HIV-1-infected patients and HIV-1-seronegative control subjects (n = 9) for each antigen (geometric mean fold rise: PVAX, 10.1 versus 5.3; Hib, 16.0 versus 11.7; and DT, 26.2 versus 24.5, respectively). Despite these vigorous responses to both polysaccharide and protein antigens, HIV-1-infected patients showed limited evidence of CD4+ T cell activation at 1 week, no consistent rise in HIV-1 burden at any point, and no decline in CD4+ T cell number over time. We conclude that recent HIV-1 seroconverters show vigorous humoral responses to vaccine antigens and limited early evidence of T cell activation, but no substantial or sustained increase in viral replication or decline in CD4+ T cell number. Thus, respiratory bacterial vaccines appear immunogenic and safe early in HIV-1 infection.

Temporal trends in the prevalence of malignancy in resected solitary pulmonary lesions.

STUDY OBJECTIVE: To determine whether there has been an increase in the prevalence of malignancy among resected, indeterminate solitary pulmonary lesions (SPL) over the past 14 years. DESIGN: A retrospective review of all thoracotomies for indeterminate SPLs from 1981 through 1994. SETTING: A university-affiliated VA Medical Center. PATIENTS: Three-hundred seventy resected indeterminate SPLs (all < or = 6 cm) in 360 patients. MEASUREMENTS AND RESULTS: Virtually all patients were men with an average age of 63 +/- 9 years. The average lesion size was 2.5 +/- 1.4 cm; 71% were 3 cm or less. Overall, 79% of resected lesions were malignant; 94% of these were bronchogenic carcinomas. Granulomas accounted for more than 50% of benign lesions. The proportion of malignant diagnoses increased from 55 to 60% in 1981 to 1983 to 90 to 100% in 1990 to 1994 (p < 0.005). The increasing proportion of malignancy over time was independent of age at time of operation and lesion size. There was no significant difference in survival among patients with a malignant lesion resected in 1981 to 1983 compared with 1990 to 1994. CONCLUSION: We conclude that there has been a striking increase in the prevalence of malignancy among resected indeterminate SPLs over the past 14 years in our institution. We suspect that this trend reflects improvements in our ability to diagnose benign SPLs preoperatively, primarily through the use of CT. Our results should prompt other institutions to review their recent experience with the diagnosis of indeterminate SPLs to provide more timely information to physicians and their patients who are contemplating resection of SPLs.

Etiology and prognostic significance of eosinophilic pleural effusions. A prospective study.

STUDY OBJECTIVE: To determine the diagnostic and prognostic significance of eosinophilic pleural effusions. DESIGN: A prospective cohort study of patients undergoing thoracentesis between September 1990 and September 1995. SETTING: A university-affiliated VA Medical Center. PATIENTS: Four hundred seventy-six consecutive patients. MEASUREMENTS AND RESULTS: Eosinophilic pleural effusions were identified in 44 of the 476 patients (9.2%). Malignancy was diagnosed as frequently in eosinophilic as in noneosinophilic effusions (20.5% vs 20.1%). The only diagnoses that were significantly associated with eosinophilic effusions were idiopathic (25% vs 8%; p = 0.001) and postthoracic surgery (11% vs 3%; p = 0.023). Median survival was 7.7 months for those with a noneosinophilic effusion compared to 16.8 months for those with eosinophilia (p = 0.017). This difference in survival persisted after adjustment for age and diagnosis. CONCLUSIONS: We conclude that malignancy is as prevalent among eosinophilic as noneosinophilic pleural effusions. However, the survival of patients with pleural fluid eosinophilia may be better than that of patients with noneosinophilic effusions.

Serum carcinoembryonic antigen as an adjunct to preoperative staging of lung cancer.

OBJECTIVE: To determine whether measurement of preoperative serum carcinoembryonic antigen concentrations adds useful prognostic data to current preoperative staging of lung cancer by computed tomography, bronchoscopy, and mediastinoscopy. METHODS: A prospective cohort study of 130 consecutive patients was evaluated for suspected lung cancer from July 1991 through December 1992 at a university-affiliated Veterans Affairs Medical Center. Serum concentrations of carcinoembryonic antigen were measured before diagnosis, staging, or resection of cancer. RESULTS: Malignant disease was diagnosed by bronchoscopy, needle biopsy, mediastinoscopy, or resection in 111 of 130 patients. In the 50 patients undergoing resection with curative intent, multivariate analysis indicated that carcinoembryonic antigen was a significant predictor of survival independent of patient age, pathologic stage, histologic type, and tumor size (P=.0357). CONCLUSIONS: Elevated preoperative serum concentrations of carcinoembryonic antigen predict a poor prognosis for lung cancer independent of other conventional staging parameters and have an adjunctive role in the staging of lung cancer.

Origins of breath nitric oxide in humans.

STUDY OBJECTIVES: Nitric oxide (NO) exists in the human breath, but little is known about its site of origin or enzyme source. The aims of this study were to locate the main site of NO release into human breath and to decide whether the inducible isoform of NO synthase (iNOS) and nasal bacteria contribute to breath NO. DESIGN: Using a chemiluminescence assay, NO levels were measured in air exhaled from the nose, mouth, trachea, and distal airway. The susceptibility of breath NO to treatment with a topical corticosteroid (to inhibit iNOS; intranasal beclomethasone dipropionate for 2 weeks) and with antibiotics (systemic amoxicillin plus clavulanic acid and intranasal bacitracin zinc, 5 to 10 days) was also tested. PARTICIPANTS: Twenty-one healthy subjects, 9 intubated patients, and 7 patients undergoing bronchoscopy. All subjects were nonsmokers free of pneumonia, rhinitis, and bronchitis. MEASUREMENTS AND RESULTS: Breath NO levels, collected in the gas sampling bags, were greater (p < 0.05) in the nose (25 +/- 2 parts per billion [ppb]) than in the mouth (6 +/- 1 ppb), trachea (3 +/- 1 ppb), or distal airway (1 +/- 2 ppb). Similar results were obtained when NO was sampled directly by cannula from nose or mouth during resting breathing. Nasal breath NO signal increased sharply during 30 s of breath-holding. Beclomethasone, but not antibiotics, decreased nasal NO levels without changing oral breath NO. CONCLUSIONS: Most NO in normal human breath derives locally from the nose where it can reach high levels during breath-holding. NO is synthesized, at least in part, by a steroid-inhibitable, nonbacterial, NO synthase, presumably iNOS.

Temporal trends in survival after surgical resection of localized non-small cell lung cancer.

To test whether modern preoperative staging modalities and perioperative care improve survival after resection of localized non-small cell lung cancer (NSCLC), we retrospectively reviewed outcomes of 454 patients with NSCLC resected from 1947 through 1969 (designated pre-1970 cases), and 540 patients with cancers resected from 1981 through 1994 (designated post-1980 cases). Mean ages, histological subtypes, surgical stages, and types of surgical procedures differed significantly between the two groups. Compared with pre-1970 cases, post-1980 cases were older, had more adenocarcinoma and less squamous cell carcinoma, and had lesser proportions of advanced stage disease. Postoperative (day 30) mortality was significantly higher for resections of localized (stages 1 and 2) NSCLC prior to 1970. For patients surviving at least 30 days after surgery, subsequent survival after resection of localized NSCLC differed minimally between pre-1970 and post-1980 groups. We conclude that perioperative mortality after resection of localized NSCLC improved, but subsequent postoperative survival for these patients did not significantly improve over the 45-year period studied.

Mechanisms of G1 checkpoint loss in resected early stage non-small cell lung cancer.

Loss of the G1 checkpoint appears to be extremely common among virtually all neoplasms. A variety of genetic and epigenetic mechanisms have been demonstrated to play significant roles in this process. In a consecutive series of early stage non-small cell lung cancer (NSCLC), we have established the loss of expression of the G1 Cdk inhibitors p15INK4b) and p16INK4a by DNA methylation is very common (37%), and methylation of p16INK4a is tightly correlated with loss of expression of p16INK4a protein (P = 0.0018). Furthermore, methylation of p15INK4b and p16INK4a appear inversely correlated, although methylation of p15INK4b is an infrequent event in this cohort (4%). Methylation was detected in all stages of NSCLC equally, and did not correlate with survival in these patients. Evidence for methylation was more frequent in squamous cell carcinomas in comparison to other tumor histologies (P = 0.0156). In addition, over-expression of cyclin D1 was found to be tightly restricted (P = 0.0032) to those tumors that had retained wild-type expression of pRB, and did not correlate with methylation or expression of p16INK4a gene product. Although loss of p16INK4a function remains tightly correlated with pRB expression, loss of other regulatory elements in NSCLC such as p53 mutation and cyclin D1 over-expression appear independent of loss of the p16INK4a gene product.

Invasive pneumococcal disease in the immunocompromised host.

A normal constituent of the human upper respiratory flora, Streptococcus pneumoniae also produces respiratory tract infections that progress to invasive disease at high rates in specific risk groups. The individual factors that contribute to the development of invasive pneumococcal disease in this distinct minority of persons, include immune (both specific and innate), genetic, and environmental elements. Specific defects in host responses may involve age, deficiencies in levels of antibodies and complement factors, and splenic dysfunction. Combinations of these immune defects contribute to the increased rates of invasive pneumococcal disease in patients with sickle cell disease, nephrotic syndrome, neoplasms, and underlying medical conditions such as diabetes and alcoholic liver disease. The number of risk factors are greatest and the rates of invasive disease are highest in patients with HIV-1 infection, which has emerged as a major risk factor for serious S. pneumoniae infection worldwide.

Pneumococcal disease in the elderly: what is preventing vaccine efficacy?

The effective prevention of Streptococcus pneumoniae infection has a renewed priority in an era in which the emergence of antibacterial-resistant strains has the potential to further compromise efforts to reduce early mortality from invasive pneumococcal infection. Although the 23-valent pneumococcal polysaccharide (PPS) vaccine was approved in the US to prevent respiratory and invasive infection in the elderly and other high-risk populations, the protective efficacy of this vaccine for the growing population of adults aged >65 years remains controversial. The apparent effectiveness of pneumococcal immunisation in clinical studies of elderly adults has varied depending upon whether a reduction in pneumococcal colonisation, pneumonia, bacteraemia or death was used as an outcome. Clinical studies of vaccine efficacy to date suggest that the current pneumococcal vaccine is 56 to 81% effective at preventing invasive pneumococcal infection, and may have additive benefit to influenza vaccine in preventing community-acquired pneumonia, particularly in elderly adults with an increased risk of serious pneumonia requiring hospitalisation. Possible reasons for incomplete protection from pneumococcal infection after immunisation include infection with non-vaccine serotypes, inadequate or ineffective antibody responses, waning of antibody responses, or compromised nonhumoral host defences. Further studies are needed to determine whether: (i) elderly adults who respond poorly to the 23-valent pneumococcal vaccine can be identified prior to immunisation and targeted for study with improved pneumococcal vaccines; (ii) specific nutrient deficiencies can be identified and corrected to improve the immune responsiveness of elderly adults to the PPS vaccine; (iii) newer protein-conjugate or DNA pneumococcal vaccines may be more uniformly immunogenic for elderly adults; and (iv) whether smoking cessation reduces the risk of invasive pneumococcal infection in elderly adults.

Effect of anesthetics on pathogenesis of experimentally induced murine pneumococcal pneumonia.

BACKGROUND AND PURPOSE: To define the effects of three commonly used anesthetic agents--sodium pentobarbital given intraperitoneally, and inhaled halothane and methoxyflurane--on the pathogenesis of pneumococcal pneumonia and bacteremia in an experimental murine model. METHODS: Swiss outbred mice were anesthetized with either sodium pentobarbital, halothane, or methoxyflurane before intranasal infection with Streptococcus pneumonia. At defined times after infection, bacterial numbers in lungs and blood, markers of acute lung injury, and lung cytokine levels were compared. RESULTS: Mice anesthetized with inhaled halothane or methoxyflurane prior to intranasal inoculation with type-2 Streptococcus pneumoniae developed pneumonia and bacteremia distinctly different from that in mice anesthetized by intraperitoneal (IP) administration of sodium pentobarbital. Mice having brief exposure to inhaled halothane or methoxyflurane had significantly greater numbers of bacteria in lungs and blood 48 hours after inoculation, compared with mice anesthetized by IP administration of pentobarbital. Also, mice inhaling halothane had significantly decreased activities of pro-inflammatory cytokines interleukin 6 and tumor necrosis factor-alpha in lung homogenates at 24 hours after inoculation, compared with those given pentobarbital IP. CONCLUSION: Effects of anesthesia on murine models of pneumonia should be considered in the design and interpretation of studies of pneumococcal pathogenesis.

Diagnosis of venous thromboembolism. Step-by-step approach to a still lethal disease.

Rapid diagnosis of VTE is vital in reducing the significant morbidity and mortality rates associated with this disease. Although angiographic studies remain the "gold standard" for diagnosis, many noninvasive diagnostic procedures are available and are appropriate for evaluation in clinically stable patients. The algorithm presented in this article facilitates the practical and efficient use of available resources in diagnosing and treating VTE.

Practical management of pleural effusions. When and how should fluid accumulations be drained?

Management of pleural effusions requires recognition of the cause and initiation of disease-specific therapy. However, it is important to be forewarned that breathing problems do not always improve in these patients, since other disease processes may be contributing to dyspnea. Many options are available for draining effusions and preventing recurrence, but decisions often need to be made quickly so the process does not progress to the point at which intervention becomes much more difficult.

Evaluating pleural effusions. How should you go about finding the cause?

Although many pulmonary and systemic diseases are known to cause pleural effusions, analysis of the pleural fluid pinpoints the cause in most cases. Distinguishing pleural transudates from exudates is an important step. Transudate effusions are caused by a small, well-defined group of illnesses (e.g., cirrhosis, congestive heart failure). Exudative effusions, on the other hand, are associated with a wide variety of causes, including pneumonia, malignancy, TB, drug-induced reactions, and many others. Some effusions remain unexplained despite extensive tests. Surgical approaches may be appropriate for some of these patients, but the risks must be carefully weighed against the benefits.

Community-acquired pneumonia. Tailoring management of adult patients according to risk category.

A cost-conscious evaluation of CAP in the adult patient requires an initial assessment of the clinical severity and the risks of complicated pneumonia. Initially, most patients should have chest radiography; some authorities also recommend sputum Gram staining and culture, but additional blood testing, culture, and diagnostic procedures are indicated only for patients who have chronically impaired health or clinical evidence of sever infection. Initial empirical antibiotic therapy varies depending on the setting (outpatient, hospitalized patient, critically ill patient). Failure of the patient to respond to empirical antibiotic therapy within 72 hours should direct the clinician to consider unusual or resistant organisms or noninfectious causes of pneumonitis.