PREVIEWAccuracy in the diagnosis of venous thrombophlebitis is often dependent on the degree of clinical suspicion before testing. In this article, the authors examine important risk factors for this often deadly disease and describe the invasive and noninvasive tests available. They also define the circumstances in which each procedure is appropriate.
Mastoparan, a tetradecapeptide component of wasp venom, is a potent activator of secretion in a variety of cell types, and has been shown to activate purified G-proteins reconstituted into phospholipid vesicles with a preferential activation of Gi over Gs (Higashijima, T., Uzu, S., Nakajima, T., and Ross, E. R. (1988) J. Biol. Chem. 263, 6491-6494). To identify the biochemical activities of mastoparan in a cellular system, we characterized the effects of mastoparan on signal transduction pathways in rat pulmonary alveolar type 2 epithelial cells, which synthesize and secrete pulmonary surfactant. Mastoparan inhibited adenylylcyclase activity in a manner that was dose-dependent (IC50 = 30 microM), but sensitive to neither guanine nucleotide nor pertussis toxin (PT). Mastoparan induced a PT-sensitive increase in cellular inositol trisphosphate and a rapid rise in cytosolic calcium released from intracellular stores; the time to onset of the calcium rise, but neither the rate nor the amplitude of the rise, were PT-sensitive. Mastoparan also caused a dose- (EC50 = 16 microM) and time-dependent activation of arachidonic acid release that was completely insensitive to pretreatment with PT. Secretion of pulmonary surfactant was increased by mastoparan approximately 8-fold over constitutive levels at 1 h with an EC50 = 20 microM, and mastoparan-stimulated secretion was partially sensitive to PT at late time points and to inhibitors of arachidonic acid metabolism, but not to the protein kinase C inhibitor H7. These findings are consistent with the activation of Gi proteins in type 2 cells by mastoparan, although the lack of predicted triphosphoguanine nucleotide and PT sensitivity for some activities indicates that mastoparan does not act in a manner strictly analogous to liganded receptors or that some activities are not mediated by activation of Gi. While mastoparan is a potent secretagogue in several cell types, its secretory activity appears to have only a limited dependence on the activation of Gi proteins in type 2 cells.
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.
We determined the immunogenicity and safety of reimmunization with the 23-valent polysaccharide pneumococcal vaccine in patients infected with human immunodeficiency virus type 1 (HIV-1). Patients immunized >5 years earlier (initially within 1 year of HIV-1 seroconversion) were randomized to receive vaccine (n=57) or placebo (n=30). Persons with recent HIV-1 seroconversion (n=14) were immunized for the first time. Preimmunization levels of capsule-specific immunoglobulin G were similar in all groups. Reimmunized patients showed a significantly lower frequency and magnitude of antibody responses compared with persons with recent HIV-1 seroconversion. Reimmunized patients did not show adverse virologic or immunologic changes, but some reported local discomfort (15%) or fever (8%). Thus, the limited responses after reimmunization of HIV-1-infected patients with the current 23-valent vaccine mandates the need for a more effective reimmunization schedule, more immunogenic vaccines, or other behavioral and therapeutic interventions.
Recent evidence from genetic experiments in yeast and from studies using guanosine triphosphate (GTP) analogues in mammalian cells suggests a key role for low-molecular-mass GTP-binding proteins (LMM-GBPs) (Mr, 19 to 28 kD) in processes of intracellular vesicular sorting and secretion. Assembly and exocytosis of the lamellar body (LB), the secretory organelle of the pulmonary alveolar type 2 pneumocyte, may be regulated by LMM-GBPs. We used [α-32P]GTP binding to Western blotted proteins, ultraviolet crosslinking of [α-32P]GTP to membrane proteins, immunoblotting with specific antisera, and botulinum exoenzyme C3-catalyzed ADP ribosylation to detect LMM-GBPs in LB. With the first two techniques, we have identified six LMM-GBPs of approximately 27, 25.5, 24.5, 23, 22, and 21 kD that are enriched in a highly purified LB fraction compared with type 2 pneumocyte homogenate, crude membranes, and cytosol. Further characterization of the LB LMM-GBPs by immunoblotting revealed that ras p21 is greatly enriched in the LB fraction compared with other type 2 pneumocyte fractions. In addition, botulinum exoenzyme C3 catalyzed the ADP ribosylation of 20- to 21-kD proteins that were similarly enriched in the LB fraction. In contrast, a monospecific antibody to ADP-ribosylation factor reacted with a 19-kD protein only in the type 2 pneumocyte homogenate and cytosol fractions. Monospecific antibodies to yeast Sec4 protein and to rab 3A did not react with any type 2 pneumocyte proteins. The LMM-GBPs specifically associated with LB may participate in intracellular events required for surfactant packaging and secretion.
Bacterial immunoglobulin A1 (IgA1) proteases may sabotage the protective effects of IgA. In vitro, both exogenous and endogenously produced IgA1 protease inhibited phagocytic killing of Streptococcus pneumoniae by capsule-specific IgA1 human monoclonal antibodies (hMAbs) but not IgA2. These IgA1 proteases cleaved and reduced binding of the the effector Fcα1 heavy chain but not the antigen-binding F(ab)/light chain to pneumococcal surfaces. In vivo, IgA1 protease-resistant IgA2, but not IgA1 protease-sensitive IgA1, supported 60% survival in mice infected with wild-type S. pneumoniae. IgA1 hMAbs protected mice against IgA1 protease-deficient but not -producing pneumococci. Parallel mouse sera with human IgA2 showed more efficient complement-mediated reductions in pneumococci with neutrophils than did IgA1, particularly with protease-producing organisms. After natural human pneumococcal bacteremia, purified serum IgG inhibited IgA1 protease activity in 7 of 11 patients (64%). These observations provide the first evidence in vivo that IgA1 protease can circumvent killing of S. pneumoniae by human IgA. Acquisition of IgA1 protease-neutralizing IgG after infection directs attention to IgA1 protease both as a determinant of successful colonization and infection and as a potential vaccine candidate.
Malignant mesothelioma causes profound morbidity and nearly universal mortality that is refractory to conventional treatment with aggressive surgery, radiotherapy, or chemotherapy. We report that pharmacologic concentrations of lovastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor, induced apoptosis in human malignant mesothelioma cell lines. Mesothelioma cell viability was decreased in a dose-dependent manner by lovastatin (5 to 30 μ M). These effects were not reversed by exogenous growth factors or cholesterol, but were reversed by addition of 100 μ M mevalonate, confirming that lovastatin affected mesothelioma viability by inhibiting mevalonate synthesis. Lovastatin appeared to decrease mesothelioma viability by inducing apoptosis, as indicated by morphologic changes, histologic evidence of nuclear condensation and degeneration, and flow-cytometric analysis of DNA content. Lovastatin's effects on cell viability were partially reversed in the presence of farnesol, and treatment of mesothelioma cells with a specific farnesyl-protein transferase (FTP) inhibitor decreased cell viability and induced morphologic changes indistinguishable from those caused by lovastatin. In addition, lovastatin-treated cells showed translocation of ras guanosine triphosphate (GTP)-binding proteins from membrane to cytosolic fractions on Western blots, suggesting that lovastatin's effects on mesothelioma were mediated in part by disrupting acylation of GTP-binding proteins. Thus, lovastatin is a commercially available and clinically well-tolerated agent that reduces viability and induces apoptosis of mesothelioma cells, and may provide the basis for adjunctive treatments of patients with mesothelioma.
Patients with non–small-cell lung cancer (NSCLC) survive for variable lengths of time, even when adjustment is made for pathological stage. Numerous reports suggest that biological markers predict survival in patients undergoing surgery for NSCLC with curative intent, but many of these claims are unconfirmed or conflicting. We postulated that the use of multiple putative markers might provide greater power in predicting survival. We studied 101 consecutive patients with NSCLC who underwent exploratory thoracotomy and who were followed for at least 2 yr. We assessed mutations in the p53 tumor suppressor gene (exons 5-8) and the K-ras oncogene (codons 12 and 13) by polymerase chain reaction amplification and single strand conformation polymorphism of the product. We identified 19 K-ras mutations (all adenocarcinomas except for two) and 40 p53 mutations among the 101 cases. We also evaluated p53 protein, bcl-2 protein, c-erbB-1 protein, c-erbB-2 protein, and MIA-15-5 antigen by standard immunocytochemical techniques, and we found that all of these antigens were variably expressed. As expected, we found a strong inverse association between surgical tumor stage and survival. Of the molecular markers studied, only MIA-15-5 antigen expression correlated strongly with survival by univariate analysis (p = 0.001) and it remained a significant predictor by multivariate analysis (p = 0.01). However, in this study, overexpression of MIA-15-5 antigen predicted an improved survival, whereas the original report showed a worse prognosis (N. Engl. J. Med. 1992;327:14). We conclude the multiple cell markers are not clinically useful in predicting survival among patients undergoing surgery for NSCLC. Differences between our results and prior reports may be due to chance, to true population differences, or to other factors.
