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Entamoeba histolytica is known to cause intestinal and extra-intestinal disease while the other Entamoeba species are not considered to be pathogenic. However, all Entamoeba spp. should be reported when identified in clinical samples. Entamoeba polecki, Entamoeba coli, and Entamoeba hartmanii can be differentiated morphologically from E. histolytica, but some of their diagnostic morphologic features overlap. E. histolytica, Entamoeba dispar, and Entamoeba moshkovskii are morphologically identical but can be differentiated using molecular tools. We developed a polymerase chain reaction (PCR) procedure followed by DNA sequencing of specific regions of 18S rRNA gene to differentiate the Entamoeba spp. commonly found in human stools. This approach was used to analyze 45 samples from cases evaluated for the presence of Entamoeba spp. by microscopy and a real-time PCR method capable of differential detection of E. histolytica and E. dispar. Our results demonstrated an agreement of approximately 98% (45/44) between the real-time PCR for E. histolytica and E. dispar and the 18S rRNA analysis described here. Five previously negative samples by microscopy revealed the presence of E. dispar, E. hartmanii, or E. coli DNA. In addition, we were able to detect E. hartmanii in a stool sample that had been previously reported as negative for Entamoeba spp. by microscopy. Further microscopic evaluation of this sample revealed the presence of E. hartmanii cysts, which went undetected during the first microscopic evaluation. This PCR followed by DNA sequencing will be useful to refine the diagnostic detection of Entamoeba spp. in stool and other clinical specimens.

Background: Observational studies of patients with primary colorectal cancer have identified KRAS mutation as a marker of poor prognosis. To examine more directly whether KRAS mutations are associated with accelerated metastatic progression, we evaluated KRAS mutation as well as Ki-67 expression in patients with colorectal liver metastases not treated with cetuximab., Methods: KRAS mutation status was assessed in a series of resected or sampled colorectal liver metastases. In a subset of these tumors, Ki-67 antigen expression was assessed by immunohistochemical stains. Median follow-up after liver resection or biopsy was 2.3 years., Results: KRAS mutation in the liver metastasis was detected in 27% of the 188 patients. High Ki-67 expression in the liver metastasis was identified in 62% of 124 patients analyzed. Both markers were associated with multiple liver metastases and shorter time interval to their detection. KRAS mutation and high Ki-67 expression were independent predictors of poor survival after colon resection (hazard ratio [HR] 1.9 [95% confidence interval (95% CI), 1.1-3.4], HR 2.6 [95% CI, 1.4-4.8], respectively). Tumors with high Ki-67 expression were less likely to be selected for liver resection, and KRAS mutation was independently associated with poor survival after liver resection (HR 2.4 [95% CI, 1.4-4.0])., Conclusions: KRAS mutation is associated with more rapid and aggressive metastatic behavior of colorectal liver metastases. These data suggest an important role for KRAS activation in colorectal cancer progression and support continued efforts to develop KRAS pathway inhibitors for this disease.

Introduction: We examined two genetic markers established early in colorectal tumor development, microsatellite instability (MSI) and mutation of the KRAS proto-oncogene, to see if these genetic changes influence metastatic disease progression and survival., Patients and Methods: MSI and KRAS mutation status were assessed in 532 primary adenocarcinomas (stage I-IV) from patients treated by colon resection. Median follow-up was 4.1 years (range 0-13.3 years) overall, 5.4 years for survivors., Results: MSI and KRAS mutation were detected in 12 and 36% of cases, respectively. MSI was more common in early-stage disease (I, 15%; II, 21%; III, 10%; IV, 2%; P = 0.0001). Prevalence of KRAS mutation did not vary with stage (I, 36%; II, 34%; III, 35%; IV, 40%; P = ns). Disease-specific survival was far superior for MSI tumors than for microsatellite stability (MSS) tumors (5-year survival 92 vs. 59%, P < 0.0001). KRAS mutation was a marker of poor survival (5-year survival 55 vs. 68%, P = 0.0002). Using Cox regression analysis MSI, KRAS mutation, and stage were strong independent predictors of survival in the entire patient population. A high-mortality group with MSS/KRAS-mutant tumors was identified within the stage I and II cohort., Conclusions: MSI and KRAS mutation provide fundamental genetic signatures influencing tumor behavior across patient subsets and stages of tumor development.

Background: Biologically active natural products continue to be useful in the exploration and control of intracellular signaling processes. For example, the sesquiterpene lactone parthenolide from the anti-inflammatory medicinal herb Feverfew (Tanacetum parthenium) appears to inhibit the pro-inflammatory signaling pathway. Parthenolide's direct molecular target, however, remains unknown. We set out to identify the molecular mechanisms of parthenolide's anti-inflammatory activity., Results: A parthenolide affinity reagent was synthesized and shown to bind directly to and inhibit IkappaB kinase beta (IKKbeta), the kinase subunit known to play a critical role in cytokine-mediated signaling. Mutation of cysteine 179 in the activation loop of IKKbeta abolished sensitivity towards parthenolide. Moreover, we showed that parthenolide's in vitro and in vivo anti-inflammatory activity is mediated through the alpha-methylene gamma-lactone moiety shared by other sesquiterpene lactones., Conclusions: In recent years, the multi-subunit IKK complex has been shown to be responsible for cytokine-mediated stimulation of genes involved in inflammation and as such represents an attractive target for pharmaceutical intervention. Our finding that parthenolide targets this kinase complex provides a possible molecular basis for the anti-inflammatory properties of parthenolide. In addition, these results may be useful in the development of additional anti-inflammatory agents.

In the standard model of cytokine-induced signal transducer and activator of transcription (STAT) protein family signaling to the cell nucleus, it is assumed that STAT3 is recruited to the cytoplasmic side of the cell surface receptor complex from within a cytosolic monomer pool. By using Superose-6 gel-filtration chromatography, we have discovered that there is little monomeric STAT3 (91 kDa) in the cytosol of liver cells (human hepatoma Hep3B cell line and rat liver). The bulk of STAT3 (and STAT1, STAT5a, and -b) was present in the cytosol as high molecular mass complexes in two broad distributions in the size range 200-400 kDa ("statosome I") and 1-2 MDa ("statosome II"). Upon treatment of Hep3B cells with interleukin-6 (IL-6) for 30 min (i) cytosolic tyrosine-phosphorylated STAT3 was found to be in complexes of size ranging from 200-400 kDa to 1-2 MDa; (ii) a small pool of monomeric STAT3 and tyrosine-phosphorylated STAT3 eluting at 80-100 kDa was observed, and (iii) most of the cytoplasmic DNA-binding competent STAT3 (the so-called SIF-A "homodimer") co-eluted with catalase at 230 kDa. In order to identify the protein components of the 200-400-kDa statosome I cytosolic complexes, we used the novel technique of antibody-subtracted differential protein display using anti-STAT3 antibody. Eight polypeptides in the size range from 20 to 114 kDa co-shifted with STAT3; three of these (p60, p20a, and p20b) were co-shifted in an IL-6-dependent manner. In-gel tryptic fragmentation and mass spectroscopy identified the major IL-6-dependent STAT3-co-shifted p60 protein as the chaperone GRP58/ER-60/ERp57. Taken together, these data (i) emphasize the absence of a detectable STAT3 monomer pool in the cytosol of cytokine-free liver cells as posited by the standard model, and (ii) suggest an alternative model for STAT signaling in which STAT3 proteins function in the cytoplasm as heteromeric complexes with accessory scaffolding proteins, including the chaperone GRP58.

The influence of p53 on cytokine-triggered Janus kinase-STAT signaling was investigated in human hepatoma Hep3B cell lines engineered to constitutively express the temperature-sensitive Val135 mutant of p53. In comparison to the parental p53-free Hep3B cells, these p53-Val135-containing Hep3B cell lines displayed a reduced response to IL-6 at the wild-type-like p53 temperature (32.5 degrees C). In these cells, IL-6 induced a marked reduction in the immunologic accessibility of cytoplasmic and nuclear STAT3 and STAT5 within 20 to 30 min that lasted 2 to 4 h (STAT-masking) provided that the cells had been previously cultured at 32.5 degrees C for at least 18 to 20 h. The onset of IL-6-induced STAT-masking required protein tyrosine kinase, protein tyrosine phosphatase, proteasomal, phospholipase C, and mitogen-activated protein kinase kinase 1 activities. The maintenance of IL-6-induced STAT-masking was dependent on continued signaling through the phosphatidylinositol-dependent phospholipase C pathway. Despite a reduction in IL-6-induced STAT3 DNA binding activity in the nuclear compartment during STAT-masking, there was increased and prolonged accumulation of tyrosine-phosphorylated STAT3 in both the cytoplasmic and nuclear compartments, indicating that the capacity of tyrosine-phosphorylated STAT3 to bind DNA was reduced during STAT-masking. Thus, IL-6-induced STAT-masking, as dramatically evident on immunomicroscopy, is a visible consequence of a novel cellular process by which a p53-Val135-induced gene product(s) regulates the association of masking protein(s) with and the DNA-binding capacity of STAT3.

Transport of IL-6 in blood is fundamental to the biology of this cytokine. In the present study, IL-6 transport, immunological reactivity, and biological availability were investigated in blood from melanoma patients subjected to different active specific immunization regimens (an anti-idiotypic mAb immunization protocol (mAb-keyhole limpet hemocyanin (KLH)-Calmette-Guerin bacillus (BCG), an autologous anti-cancer vaccine protocol (AAAP), or both). Sera were subjected to Sephadex G-200 gel filtration chromatography, and the structure and biological activity of IL-6 complexes in the eluate fractions were probed using five IL-6 ELISAs and two bioassays. Sera from patients administered mAb-KLH+BCG followed by AAAP contained three distinct classes of IL-6 eluting at 30, 200, and 450 kDa, each with its characteristic ELISA reactivity and bioactivity: the 30- and 450-kDa complexes were bioactive in the B9 and Hep3B assays, but the 200-kDa complex was not. The 30- and 450-kDa IL-6 complexes were preferentially reactive in the 7IL6/5IL6 ELISA, the 200-kDa IL-6 complexes were preferentially reactive in the 4IL6/5IL6 ELISA, while the three commercial ELISAs (R&D, Endogen, and Genzyme) detected essentially only the 30-kDa IL-6. In contrast, 1) sera from AAAP patients contained biologically active 30- and 450-kDa IL-6 complexes, while 2) sera from mAb-KLH+BCG patients contained 200-kDa IL-6 complexes inactive in ex vivo bioassays. Both the 450- and 200-kDa complexes included soluble IL-6R, with the 200-kDa complexes additionally containing ligand-occupied anti-IL-6 and anti-soluble IL-6R IgG. The data indicate the existence of specific mechanisms that regulate the transport and function of IL-6 in vivo.

There is increasing evidence that many, perhaps all, cytokines have a soluble form of their receptor in the systemic circulation at all times. There is also evidence that endogenous antibodies to some cytokines, including IL-6, are also found in blood. Initially these findings were evaluated in vitro, and associated with inhibiting the respective effects of those cytokines. However, it is now becoming clear that the in vivo effects are paradoxically the reverse of what is seen in vitro. As we have explained here for IL-6 it is evident that many or all of these molecules that bind and/or associate with IL-6 maintain this molecule in the systemic circulation and constitute a reservoir of masked, but potentially active IL-6. The mode of regulation of the biological activity of these IL-6-associated complexes remains unknown, but needs to be uncovered in order to pharmacologically exploit many of the potentially beneficial effects or to prevent any potential pathological effects.