Differential clearance and induction of host responses by various administered or released lipopolysaccharides.

The clearance and activity of different types of lipopolysaccharide (LPS) released during infection with Gram-negative bacteria were investigated. When highly purified preparations differing in their specific endotoxin activity were administered intravenously to mice, the clearance of rough (R)-form LPS preparations from Salmonella minnesota and Escherichia coli was much faster than that of a smooth (S)-form LPS preparation from Salmonella abortus equi, but slower than that of lipo-oligosaccharides (LOS) preparations from Bordetella pertussis and Helicobacter pylori. After intraperitoneal infection with 10(7) and 10(8) CFU E. coli O111:B4, relatively high levels of LPS were detected dose-dependently in the plasma of infected mice and persisted for a long time. In addition, plasma sCD14 levels in infected mice were higher than in LPS-administered mice. These results indicate that continuously higher levels of plasma LPS followed by stronger host responses occur during infection and suggest that these differences between LPS-administered and infected mice should be taken into consideration when analyzing host responses induced by LPS.

Mycobacterial cord factor, but not sulfolipid, causes depletion of NKT cells and upregulation of CD1d1 on murine macrophages.

Trehalose 6,6'-dimycolate (TDM, cord factor) has frequently been used as an adjuvant to stimulate antibody production. Although it also induces cellular immunity, detailed studies about the underlying events do not exist. To determine the kinetics of TDM-specific changes promoting a T helper 1 (Th1) response, we injected mice with TDM or 2,3,6,6'-tetraacyl trehalose 2'-sulfate (SL, sulfolipid), another mycobacterial trehalose-containing glycolipid without mycolic acid. TDM, but not SL, caused a strong increase in serum interferon-gamma (IFN-gamma) levels 2 days later, accompanied by expansion of natural killer (NK) cells. Subsequent TDM effects included depletion of normal-density CD4(+) NK1.1(+) TCRalpha/beta(intermediate) cells from day 7 on, upregulation of MHC class II and CD1d1 on macrophages (peaking on day 21), and an increased proportion of Th1 cells evident after 3 weeks. TDM, but not a similar glycolipid without mycolic acid, can therefore initiate a cascade of events starting with strong release of IFN-gamma and NK cell expansion, resulting in the appearance of macrophages activated for antigen presentation. Our data therefore provide the basis for optimized immunization schedules with TDM as the adjuvant component of a Th1 vaccine.

Immunological properties of trehalose dimycolate (cord factor) and other mycolic acid-containing glycolipids--a review.

Mycolic acids are characteristic fatty acids of Mycobacteria and are responsible for the wax-like consistence of these microorganisms. Decades of research revealed that mycolic acid-containing glycolipids, in particular trehalose-6,6'-dimycolate (TDM, cord factor) as their best-studied representative, exert a number of immunomodifying effects. They are able to stimulate innate, early adaptive and both humoral and cellular adaptive immunity. Most functions can be associated with their ability to induce a wide range of chemokines (MCP-1, MIP-1alpha, IL-8) and cytokines (e.g., IL-12, IFN-gamma, TNF-alpha, IL-4, IL-6, IL-10). This review tries to link well-known properties of mycolic acid-containing glycolipids, e.g., stimulation of cellular and humoral immunity, granuloma formation and anti-tumor activity, with recent findings in molecular immunology and to give an outlook on potential practical applications.

Identification of cDNAs encoding two novel nuclear proteins, IMUP-1 and IMUP-2, upregulated in SV40-immortalized human fibroblasts.

Using a model system of young, senescent and SV40-immortalized WI-38 fibroblasts, we identified two mRNAs upregulated in immortalized cells (imup-1, immortalization-upregulated protein 1, and imup-2). Compared to normal tissues, both genes were more frequently expressed in cancer cells. The open reading frame of imup-1 spans 321bp, coding for a 10.9 kDa protein of 106 amino acids, while an insertion of 59bp in the otherwise identical mRNA of imup-2 leads to a frameshift, resulting in an 8.5 kDa protein of 85 amino acids. Database searches identified these genes on chromosome 19, which could account for the cloned imup-1 and imup-2 transcripts by alternative splicing. Southern blot analysis of digested genomic DNA confirmed that both transcripts are derived from a single locus. The expressed proteins IMUP-1 and IMUP-2 share 46 identical N-terminal amino acids, whereas the C-termini are unrelated. Green fluorescent protein-fusions of both IMUP-1 and IMUP-2 accumulated in the nucleus of HeLa cells. The C-terminus of IMUP-1 contains a bipartite nuclear localization signal, the deletion of which impaired nuclear translocation. In-vitro translated proteins bound to poly(rG), but did not interact with single-stranded DNA or double-stranded DNA. The nuclear localization of IMUP-1 and IMUP-2 as well as the upregulation of both underlying mRNAs in immortalized cells suggest a function in immortalization.

Roles of PilC and PilE proteins in pilus-mediated adherence of Neisseria gonorrhoeae and Neisseria meningitidis to human erythrocytes and endothelial and epithelial cells.

Unlike other type 4 pili, the neisserial pili consist of at least two distinct proteins, the highly variable major subunit PilE forming the pilus fiber and the tip-associated adhesin PilC. PilC protein purified either from gonococci or from Escherichia coli interacted with different human epithelial cell lines, primary epithelial and endothelial cells. The binding of PilC protein efficiently prevented the attachment of piliated Neisseria gonorrhoeae and Neisseria meningitidis to these cell types. Fluorescent beads coated with pili prepared from piliated wild-type N. gonorrhoeae also adhered to these cells, in contrast to beads coated with pili prepared from a piliated PilC-deficient mutant. In the latter case, the binding of fluorescent beads was restored after pretreatment of the pilus-loaded beads with purified PilC. Piliated wild-type N. gonorrhoeae, the piliated PilC-deficient mutant, and N. gonorrhoeae pili assembled in Pseudomonas aeruginosa agglutinated human erythrocytes, while nonpiliated gonococci did not. Consistently, purified PilC did not agglutinate or bind to human erythrocytes, suggesting that N. gonorrhoeae PilE is responsible for pilus-mediated hemagglutination.

Transformation competence and type-4 pilus biogenesis in Neisseria gonorrhoeae--a review.

In Neisseria gonorrhoea (Ngo), the processes of type-4 pilus biogenesis and DNA transformation are functionally linked and play a pivotal role in the life style of this strictly human pathogen. The assembly of pili from its main subunit pilin (PilE) is a prerequisite for gonococcal infection since it allows the first contact to epithelial cells in conjunction with the pilus tip-associated PilC protein. While the components of the pilus and its assembly machinery are either directly or indirectly involved in the transport of DNA across the outer membrane, other factors unrelated to pilus biogenesis appear to facilitate further DNA transfer across the murein layer (ComL, Tpc) and the inner membrane (ComA) before the transforming DNA is rescued in the recipient bacterial chromosome in a RecA-dependent manner. Interestingly, PilE is essential for the first step of transformation, i.e., DNA uptake, and is itself also subject to transformation-mediated phase and antigenic variation. This short-term adaptive mechanism allows Ngo to cope with changing micro-environments in the host as well as to escape the immune response during the course of infection. Given the fact that Ngo has no ecological niche other than man, horizontal genetic exchange is essential for a successful co-evolution with the host. Horizontal exchange gives rise to heterogeneous populations harboring clones which better withstand selective forces within the host. Such extended horizontal exchange is reflected by a high genome plasticity, the existence of mosaic genes and a low linkage disequilibrium of genetic loci within the neisserial population. This led to the concept that rather than regarding individual Neisseria species as independent traits, they comprise a collective of species interconnected via horizontal exchange and relying on a common gene pool.

PilC of Neisseria meningitidis is involved in class II pilus formation and restores pilus assembly, natural transformation competence and adherence to epithelial cells in PilC-deficient gonococci.

Type 4 pili produced by the pathogenic Neisseria species constitute primary determinants for the adherence to host tissues. In addition to the major pilin subunit (PilE), neisserial pili contain the variable PilC proteins represented by two variant gene copies in most pathogenic Neisseria isolates. Based upon structural differences in the conserved regions of PilE, two pilus classes can be distinguished in Neisseria meningitidis. For class I pili found in both Neisseria gonorrhoeae and N. meningitidis, PilC proteins have been implicated in pilus assembly, natural transformation competence and adherence to epithelial cells. In this study, we used primers specific for the pilC2 gene of N.gonorrhoeae strain MS11 to amplify, by the polymerase chain reaction, and clone a homologous pilC gene from N. meningitidis strain A1493 which produces class II pili. This gene was sequenced and the deduced amino acid sequence showed 75.4% and 73.8% identity with the gonococcal PilC1 and PilC2, respectively. These values match the identity value of 74.1% calculated for the two N. gonorrhoeae MS11 PilC proteins, indicating a horizontal relationship between the N. gonorrhoeae and N. meningitidis pilC genes. We provide evidence that PilC functions in meningococcal class II pilus assembly and adherence. Furthermore, expression of the cloned N. meningitidis pilC gene in a gonococcal pilC1,2 mutant restores pilus assembly, adherence to ME-180 epithelial cells, and transformation competence to the wild-type level. Thus, PilC proteins exhibit indistinguishable functions in the context of class I and class II pili.

Do neuron-specific enolase levels discriminate between small-cell lung cancer and mediastinal tumors?

The capacity of pretherapeutically assessed neuron-specific enolase (NSE) to differentiate between small cell lung cancer (SCLC) and mediastinal tumors was investigated retrospectively in a series of 320 patients. NSE was found to be increased in 95/130 (73.1%) patients with SCLC, in 4/62 (6.5%) patients with Hodgkin's disease, in 10/58 (17.2%) patients with non-Hodgkin's lymphoma, in 5/16 (31.3%) patients with teratoma, and in 6/54 (11.1%) patients with thymoma. The cut-off value, defined as the 95% percentile of a reference population suffering from benign pulmonary disorders (n = 192), was set at 13.8 ng/ml. When this discrimination level was increased to 26.4 ng/ml, which corresponds to a 95% specificity versus the total group with mediastinal tumors, SCLC was recognized with a detection rate of only 49.2%. In conclusion, increased NSE concentrations in a patient with a hilar mass and/or mediastinal widening on X-ray are not always diagnostic of SCLC due to the high rate of elevated NSE values associated with mediastinal tumors. However, in a patient who presents with a hilar mass and a high NSE level, bronchoscopy is always indicated to obtain adequate specimens for histology in order to plan an appropriate therapeutic regimen.